9th International Symposium
on
Experimental and Clinical
Neurobiology
June 2 - 5
2024
ACKNOWLEDGMETS
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9th International Symposium
on
Experimental and Clinical
Neurobiology
June 2 - 5
2024
9th
International Symposium
on Experimental and Clinical Neurobiology
Program and Abstract Book
Edited by: RNDr. Ján Gálik, PhD
RNDr. Alexandra Kisucká, PhD
RNDr. Juraj Blaško, PhD
Printed and published by Copyvait, s.r.o.
Kosice, Slovakia
ISBN 978-80-973388-6-2
9* International Symposium
on Experimental and Clinical Neurobiology
organized by
Institute of Neurobiology of Biomedical Research Center
Slovak Academy of Sciences
&
Association for Support of Neuroscience Research
Scientific Advisory Board
Dobrota D. (Slovakia)
Filipcik P. (Slovakia)
Gdovinova Z. (Slovakia)
Ježova D. (Slovakia)
Lehotsky J. (Slovakia)
Marsala M. (USA)
Matute C. (Spain)
Motlik J. (Czech Republic)
Pastorekova S. (Slovakia)
Strosznajder (Poland)
Voitenko N. (Ukraine)
Scientific Program Committee
Marsala M. - Chairman
Galik J. Lukacova N.
Bonova P. Racekova E.
Kisucka A. Pavel J.
Vanicky I.
Organizing Committee
Galik J. - Chairman
Dobrota D.
Barkacs Z.
Bonova P.
Kisucka A.
Lukacova N.
Nementhova M.
Sevc J.
Vaničky I.
Bačova M.
Bimbova K.
Jurusova D.
Koncekova J.
Martoncikova M.
Pavel J.
Snopková J.
Vasinska A.
Bagi J.
Blasko J.
Kalincakova J
Kováčova I.
Mucha R.
Racekova E.
Szekiova E.
Zavodska M.
Symposium Venue
Catholic University, Hlavná 89, Kosice
Address of the Secretariat
Institute of Neurobiology of Biomedical Research Center
Slovak Academy of Sciences
Soltesovej 4 - 6
Kosice, Slovak Republic
Phone: +421 55 727 6201
Fax: +421 55 727 6202
WEB: http://www.neurobiology.sk/symposium
General schedule of events
Sunday, June 2, 2024
16.00 -19:00 Registration, Symposium venue, Catholic University, Hlavna 89,
Kosice
Monday, June 3, 2024
08:00- 15:00 Registration - Symposium venue
08:00- 08:50 Uploading presentations and posters installations
09:00 - 09:15 Opening of the Symposium
09:15 - 10:00 Plenary lecture
10:00- 10:55 CNS injury - mechanisms and neuroprotection I
11:00- 11:20 Coffee break, posters
11:20 - 12:25 CNS injury - mechanisms and neuroprotection II
12:30- 13:30 Lunch
13:30 - 14:00 Poster session I
14:00- 14:55 CNS injury - mechanisms and neuroprotection III
15:00- 15:20 Coffee break, posters
15:20- 16:35 Varia I
17:00- 20:00 Get together party
Tuesday, June 4, 2024
08:0 0 - 15:00 Registration - Symposium venue
08:0 0 - 08:50 Uploading presentations and posters installations
09:0 0 - 09:45 IBRO lecture
09:5 0 - 10:50 CNS injury - mechanisms and neuroprotection IV
10 5 0 - 11:10 Coffee break, posters
11 10- 12:25 Neurodegenerative Diseases & Clinical Neuroscience I
12 30- 13:30 Lunch
13:30- 14:00 Poster session II
14:00- 14:45 Neuroregeneration - cell based therapy, neurogenesis I
15:00- 20:00 Trip and wine tasting in Tokaj region
Wednesday, June 5, 2024
08:00- 10:00 Registration - Symposium venue
08:00- 08:50 Uploading presentations and posters installations
09:00- 10:25 Varia II
10:30- 10:50 Coffee break
10:50- 12:05 Varia III
12:05 - 12:30 Closing of the Symposium
12:30- 14:00 Lunch
Scientific program
Monday, June 3, 2024
09:15- 10:00 Plenary lecture
iNets - novel human neural networks to study neurodegenerative diseases.
Hruska-Plochan M.
CNS injury - mechanisms and neuroprotection I
Chairperson: Motlík J.
10:00 - 10:25 Processing of nociceptive peripheral input in spinal cord networks.
Krotov V, Agashkov K., BlashchakL, Andrianov Y., Romanenko S., Halaidych O.,
KoroidK., KeyesA.L., Safronov B., Voitenko N., Usachev Y.M., BeIan P.
10:25 - 10:40 3D printed scaffolds for repair of injured spinal cord and peripheral
nerves.
Medvediev V, Grebenyuk S., Dobropolska Y., SheremetY., Abdalla I., Ustymenko V,
Bomikhov O., Pivneva T., RangaA., BeIan P., Voitenko N.
10:40 - 10:55 Application of tubular conduit in transected rat tail nerve.
Blaško J., Mojžíšová M., Székiová E., Michalová Z., Vaničky I.
CNS injury - mechanisms and neuroprotection II
Chairperson: Strnadel J.
11:20 - 11:45 Mechanism of selective neurodegeneration after global brain ischemia.
Proteasomal versus endoplasmic reticulum stress.
Ziakova K., KovalskaM., Pilchova I., Dibdiakova K., BrodnanovaM., PokusaM.,
Kalenska D., RacayP.
11:45 — 12:00 Blood cells-derived secretome- a novel approach for the treatment of
ischemic stroke outcomes.
BonovaP., Koncekova J., Kotorova K., NemethovaM., BonaM., Gottlieb M.
12:00 - 12:25 Multiple roles for Angiogenin as a new clinical target for stroke. ONLINE
RosellA.
12:30- 13:30 Lunch
CNS injury - mechanisms and neuroprotection III
Chairperson: Pavel J.
14:00 - 14:25 Brain myelin as an energy source. ONLINE
Matute C.
14:25 - 14:40 Evaluation of the analgesic potential of recombinant GABAergic hiPSCs
and their exosomes in a model of spinal cord injury induced pain. ONLINE
Jergova S., Behnaz Rahimi B., Pressman Y., TierneyL., Sagen J.
14:40 - 14:55 Behavioral, morphological, and synaptic changes in valproate-induced
autism rat model.
BakošJ., László K., Vörös D., Havránek T., Mihalj D., Kupková K., Bogyová E., Bačova Z.
Varia I
Chairperson: Voitenko N.
15:20 - 15:35 Mirtazapine intake during pregnancy has only moderate effect on
hippocampal excitability of the offspring.
Lacinova L., Dubiel-Hoppanova L., IdunkovaA., Bukatova S., Ondacova K., Tomko M.,
Jurkovicova-Tarabova B., Dubovicky M.
15:35 - 15:50 Carotid endarterectomy - potential induction of ischemic tolerance in
clinical praxis.
Sihotsky, Mucha, Kopolovets, Furman, Nemethova, Virag
15:50 - 16:05 Clinical use of the phenomenon of ischemic tolerance.
Burda R., Némethova M., Burda J.
16:05 - 16:20 Unlike in other vertebrates, majority of cerebrospinal fluid-contacting
neurons in the spinal cord of C56B1/6N mice is present in ectopic
position.
KošuthJ., Tonelli Gombalová Z., AlexovičMatiašováA., Zrubáková J., Žežulal,
Daxnerová Z., Sevc J.
16:20 - 16:35 Emission of 50-kHz ultrasonic vocalizations in hemiparkinsonian rats as a
new preclinical approach to study the affective properties of drugs used in
the dopamine replacement therapy of Parkinson's disease.
SerraM., Marongiu J., Simola N, Costa G.
18:00 - 20:00 Get together party
Posters I - Monday, June 3, 2024
P-01 Selected genetic indicators of induced brain stroke ischemic tolerance found in
human blood: A quantitative analysis.
Furman M., Sihotsky V., Virag M., Kopolovets I., Nemethova M., Mucha R.
P-02
The impact of traumatic spinal cord injury on the expression of Angiotensin II
receptors within the hypothalamus-pituitary-adrenal axis.
Hvozdikova E., Liptakova V., Snopková J., Keller ova E., Pavel J.
P-03 Body weight-supported treadmill training in spinal cord injured rats.
Ihnátová L., Magurová M., KisuckaA., Kuruc 7'., Ileninová M., Kuchařová K, Lukačová N, GálikJ.
P-04 The tryptophan-kynurenine pathway- therapeutic strategy for neuroprotection in
tauopathies.
Khiratkar K, Majerova P., KovacA.
P-05 The impact of subpial administration of Chondroitinase ABC on glial scar
components following SCI.
KisuckáA., KissBimbová K, BačováM., IleninováM., Kuruc T., KuchařováK, IhnatováL.,
Lukáčová N.
P-06 Remote ischemic conditioning modulates the inflammation after stroke in the rat
model of hyper-inflammation.
KončekováJ., Kotorová K, Némethova M., BonováP.
P-07 Blood cell-derived secretome as an alternative stroke treatment approach for obese
individuals.
KotorováK, Konceková J., GottliebM., BonováP.
P-08 Aerobic exercise-driven brain resilience: Insights into hippocampal alterations of
obese rats.
Kuruc T., Kuchařová K, KisuckáA., Ileninová M., Ihnátová L., Kiss Bimbová K, Magurová M.,
GálikJ., Lukáčová N.
P-09 Neuroprotective and anti-inflammatory effects of neuroactive steroids in model of
perinatal focal cerebral ischemia.
Kutna V.,HolásekM.,Kudová E.,ChodounskáH.,DrugaR., Tsenov G.
P-10 The effect of Angiotensin receptor type 2 stimulation on neuroregeneration after
severe spinal cord injury.
Liptakova V., Snopková J., Hvozdikova E., PavelJ.
P-l 1 Siponimod shows greater efficacy than Methylprednisolone and Atorvastatin in
reducing neuroinflammation and promoting recovery after spinal cord injury.
LukáčováN., Kiss Bimbová K, Kisucká A., BačovaM., IleninováM., Kuruc 7'., Kuchařová K,
Ihnatová L., Magurová M., GálikJ.
P-12 Exploring combination therapy with epidural stimulation and atorvastatin for spinal
cord injury recovery in rat model.
MagurovaM., BacovaM., Papcunova S., GalikJ.
P-l3 The effect of siponimod on alpha-synuclein expression and inflammatory markers in
a spinal cord injury model.
MotylJ., WencelP., KisuckaA., CzubowiczK, LukacovaN., StrosznajderR.
P-14 Proteomic analysis of signaling pathways in the rat hippocampus after delayed
remote ischemic postconditioning.
NémethováM., Taliáni., TkáčikováS., MuchaR., KončekováJ., KotorováK, BonováP.
P-l5 The stimulation of AT2 receptors can promote an angiogenic response after severe
spinal cord trauma.
Snopková J., Liptakova V., Hvozdikova E., PavelJ.
P-l6 The role of sphingosine-1-phosphate receptor modulator (Siponimod) in spinal cord
injury.
Wencel P., KisuckaA., MotylJ., LukacovaN., Strosznajder R.
Tuesday, June 4, 2024
09:00 - 09:45 IBRO lecture
Advancing spinal cord injury repair through multidisciplinary therapeutic
approaches.
Silva N.
CNS injury - mechanisms and neuroprotection IV
Chairperson: Hruska-Plochan M.
09:50 - 10:05 Is Angiotensin II receptor type 2 involved in potentiated intrinsic
regenerative ability in injured spinal cord?
PavelJ., Liptakova V., Hatalova E., Snopková J.
10:05 - 10:20 Neonatal nervous tissue exhibits superior reparative potential compared to
the mature nervous tissue after minimal spinal cord injury.
Sevc J., Mochnacký F., Košuth J., AlexovičMatiašová A., Slovinská L., Blaško J.,
Bukhun I, Holota R., Tomori Z, Daxnerová Z.
10:20 - 10:35 Neurotrophic factor expression in different microenvironments six weeks
after thoracic spinal cord injury and carnosine treatment.
Kuchařová K, Kuruc T., Ihnátová L., GálikJ., MagurováM., IleninováM., KisuckaA.,
Lukáčová N.
10:35 - 10:50 Modulation of the gut-spinal cord axis by probiotic treatment in the Th9
compression model.
Ileninová M., Kiss Bimbová K, KisuckáA., Bačova M., Mudroňová D., Kuruc T., Gálik
J., Kuchařová K, Ihnátová L., Lukáčová N.
Neurodegenerative Diseases & Clinical Neuroscience I
Chairperson: Ježova D.
11:10-11:35 Induced pluripotency and its potential in modeling of oncologic and
neurodegenerative diseases.
Strnadel J.
11:35- 12:00 Phenotypical, genotypical and pathological characterization of the
moonwalker mouse, a model of ataxia.
Sekerková G., Kilic S., Cheng Y.H., Fredrick N., OsmaniA., Kim H, Opal P., Martina
M.
12:00 - 12:25 Detrimental role of hyperhomocysteinemia in the ischemic/reperfusion
conditions.
Lehotsky J, Baranovicova E, Hnilicova P, Kalenska D, Kovalska M, Kaplan P,
Tatarkova Z
12:30- 13:30 Lunch
Neuroregeneration - cell based therapy, neurogenesis I
Chairperson: Lehotsky J.
14:00 -14:15 Transgenic minipig models of the serious eye diseases.
MotlikJ.
14:15- 14:30 Mesenchymal stem cells conditioned medium affects neurons and glial
cells differently depending on the time of conditioning.
Székiová E., Blaško J., Michalová Z., Vaničky I.
14:30 - 14:45 Mature neurons of the olfactory neurogenic region are connected with the
striatum.
Fabiánova K, Martončíková M., Vaničky I.,Blaško J., Popovičová A.,Racek A.
Račeková E.
15:00-20:00 Trip and wine tasting in Tokaj region
Wednesday, June 5, 2024
Varia II
Chairperson: Bakos J.
09:00 - 09:25 Significance of animal models of mental disorders: experimental and
clinical point of view.
Ježova D., Hlaváčova K, Izákova L.
09:25 - 09:40 Inhibition of aldosterone synthesis during the stress-hyporesponsive
period results in negative neurobiological effects in juvenile rats.
Hlaváčova N., Hrivikova K, Graban J., Karailievova L., Ježova D.
9:40 - 9:55 Ultrastructural insight on the arrangement of ependymal cells, vasculature
and fractone bulbs in the central canal lining of rat spinal cord.
AlexovičMatiašová A., Týč J., Herranz-Pérez V., Košuth J., Malčický L., Vancová M.,
NebesářováJ., Daxnerová Z., García-Verdugo J.M., SevcJ.
9:55 - 10:10 In search for an optimal tool for detection of apoptosis in nervous tissue:
Cleaved caspase-3 is present in the majority of glial cells in the intact rat
spinal cord during postnatal life.
HolotaR., Dečmanová V, Alexovič Matiašová A., Košuth J., Slovinská L., PačutL.,
Tomori Z., Daxnerová Z., Sevc J.
10:10- 10:25 Persistent olfactory dysfunction after COVID-19: the results of remote
olfactory testing and data collection in Slovakia.
Martončíková M., Doležal P., Fabiánova K, KarhánekM., GálikJ., Racek A.,
PopovičováA., Račeková E.
Varia III
Chairperson: Kuchařova K.
10:50- 11:05 Bridging the gap: P2X7 receptor as a molecular link between a-synuclein
toxicity, Parkin dysfunction, and mitochondrial Impairment.
Wilkaniec A., Olech-Kochaňczyk G, Babiec L., Czapski G.A., Lenkiewicz A.M. Adamczyk
A.
11:05 — 11:20 Current advances in thrombolytic research - how to break the translational
block in preclinical research.
HložkováJ., ScheerP., OndrušJ., Goliášová S., Aksu DavutA., Brhelová E., Thalerová
S., Biskupič J., Kuchyňka M., MikulikR.
11:20 — 11:35 Genes expression altered in activation of ischemic tolerance through
carotid endarterectomy in human blood.
Mucha R., Furman M., Sihotsky V., Kopolovets I., ViragM., Nemethova M.
11:35 - 11:50 Genetic forms of Parkinson's Disease in Central Europe
Ostrozovicova M, Tamas G, DusekP, GrofikM, Han V, Holly P, Jech R, Kalinova K,
Klivenyi P, Kovacs N, Kulcsarova K Kurca E, Lackova A, Lee H Lewis P, Magocova V,
Marekova M, Murphy D, Necpal J, Pinter D, Rabajdova M, Ruzicka E, Serranova T,
Smilowska K Soos K Straka I, Svorenova T, Valkovic P, Zarubova K Gdovinova Z,
Houlden H RizigM, Skorvanek M.
11:50- 12:05 Heart rate variability in the assessment of autonomic dysfunction in
idiopathic REM-sleep behavior disorder.
Ventosa J.
12:05- 12:30 Closing symposium
12:30- 14:00 Lunch
Posters II - Tuesday, June 4, 2024
P-01 Oxidative stress, neuroinflammation and astrogliosis in an a-synuclein
seeding/spreading mouse model of Parkinson's disease.
Adamczyk A., Ruiz-Ortega E., Olech-Kochaňczyk G, Czapski A., CiešlikM., Gawinek E., Wilkaniec A.
P 02
High-purity extracelullar vesicles used as a brain biopsy in neurodiagnostic.
Bello O.D., Rivera L., Bustos D.M., Muhoz EM., FilipčíkP., Škrabaná R.
P-03 Diaschisis-related changes in main excitatory neurotransmitter glutamate in rat brain
following the MCAO.
BonaM., Hvizdosova N., Kollarova P., Koncekova J., Kotorova K, Bonova P.
P-04 Studying unconstrained behavior of mice in a labyrinth to explore cognitive deficits.
Gášpárová L., Ercsényiová S., Trudičová D., Stefan J.J., Hromádka T.
P-05 Negative perception of a stressful situation is not associated with salivary
concentrations of the stress hormone Cortisol in toddlers.
Vancova A., Romanova Z., Ježova D.
P-06 PPAR-a synthetic ligands and their effect on the level of mRNA expression related
to redox state, and mitochondria proteins in different parts of the brain in an animal
model of Alzheimer's disease
Žuliňska S., Czubowicz K, Strosznajder J.B.
P-07 Cell therapy for traumatic brain injury, current status and future plans - involvement
of 14-3-3£
DurgalaA., RiveraL., SkrabanováM., CenteM., ŠkrabanáR., FilipčíkP.
P-08 Behavioral and functional consequences of maternal separation of various duration
in rats.
Racek A., Žideková M., Martončíková M., Fabiánova K., Račeková E.
P-09 A model for studying long distance axonal regeneration in the rat.
Vaničky I.
P-10 Effect of early chronic stress on proliferation and migration of neuroblasts in the rat
rostral migratory stream.
Žideková M., Martončíková M., Fabiánova K, Racek A., Račeková E.
P-l 1 Are aged microglia capable of surprising? Responsiveness of microglial cells within
the pineal gland during aging.
Freites C.L., AvilaM., Muňoz EM.
Comparative study of imaging methods and therapy results in diseases of the spine in
dogs at the Veterinary University Hospital in Kosice.
Kuncova M., Zeleznik P., Torok M., LiptakT., Fuchs J.
P-13 The analysis of apoptotic and non-apoptotic roles of caspase-3 in cells of the rat
spinal cord in the postnatal period.
PačutL., Holota R., KošuthJ., Matiašová A.A., Daxnerová Z., SevcJ.
P-14 How to evaluate the efficacy of thrombolysis in vivo.
Scheer P., Hložková J., Aksu DavutA., Brhelová E., Kuchyňka M., MikulikR.
P-15 Evidence that PECAM-1 is a component of Reissner's fiber produced by cells of the
subcommisural organ in rats, but not in mice.
Malčický L., Alexovič Matiašová A., BarčákD., Košuth J., Daxnerová Z., SevcJ.
Oxidative stress, neuroinflammation and astrogliosis in an a-synuclein
seeding/spreading mouse model of Parkinson's disease
Adamczyk A., Ruiz-Ortega E., Olech-Kochanczyk G., Czapski G.A., Cieslik M., Gawinek E.,
Wilkaniec A.
Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland
A key pathological process in Parkinson's disease (PD) is the
oligomerization/aggregation of a presynaptic protein alpha-synuclein (a-syn) and its
spreading through the brain. However, the exact mechanisms of seeding, cell-to-cell
transmission and neurotoxicity of a-syn are still not fully understood. In the present study, we
used an a-synuclein seeding/spreading model of PD induced by striatal injection of
oligomeric form of a-syn. C57/BL6 mice received bilateral intrastriatal administration of
endotoxin-tested recombinant oligomeric murine a-syn (5ug of protein/5 ul/side) and were
euthanized at 3 hours, 7, 14, 30, 90 or 180 days post-injection. We performed quantitative
analysis of a-syn inclusions, dopaminergic cells degeneration, measured free radicals level
and generated proinflammatory gene expression profiles in different brain regions at different
timepoints after disease induction. A significant decrease in striatal dopamine (DA) and 3,4dihydroxyphenylacetic
acid (DOPAC) levels were observed at early time-points after a-syn
injection along with the presence of intraneuronal inclusions containing misfolded a-syn.
Moreover, we demonstrated that intrastriatal a-syn administration contributes to free radical
generation and neuroinflammation in the striatum and in the midbrain. We observed a
significant time-dependent changes in mRNA levels of proinflammatory cytokines, such as
TNF-a, IL-ip, and IL-6 in both brain structures. We also indicated that microglia and/or
astrocyte activation is region-dependent in this a-syn mouse model of PD. Our observations
indicate that oligomeric a-syn could be the major driver in the early phases of PD-like
neurodegeneration. Oxidative stress and neuroinflammation, could play an important role in
this process.
This research was funded by National Science Centre, Poland (https://www.ncn.gov.pl), by
grant number 2020/39/I/NZ4/01031 for A. A.
17
Behavioral, morphological, and synaptic changes in valproate-induced
autism rat model
u
Bakos J.,3
'4
Laszl6 K., 3 4
V6ros D., 1>2
Havranek T.,1
Mihalj D., ^upkovaK., ^ogyovaE.,
^acova Z.
institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Slovakia
2
Faculty of Medicine, Comenius University in Bratislava, Slovakia
3
Medical School, Institute of Physiology, University of Pecs, Hungary.
4
Neuroscience Center, University of Pecs, Hungary
Although the role of dopaminergic pathways in connection with motor functions,
reward processing and motivation is extensively studied, their developmental aspects are less
known. Moreover, dopaminergic pathway dysfunction is implicated in the etiology of autism
spectrum disorder (ASD). Prenatal valproate (VPA) administration to mothers can lead to
autistic symptoms in offspring, thus it is widely experimentally used to understand ASD and
investigate the disorder's neurobiological basis. The aim of the present studies was to assess 1)
the shape and growth of primary neurons isolated from dopaminergic brain areas 2) the
expression of synaptic proteins in dopaminergic brain areas on postnatal day 5 and 3) early
social communication behavior using ultrasound vocalization test of control and prenatally
VPA-treated rats. Dams received a single intraperitoneal injection of VPA or saline, thereafter,
on first postnatal day, primary neurons were isolated for morphology evaluation, or on
postnatal day 5, behavioral tests were performed followed by tissue sample collection. A
significantly lower neurite branching of neurons and a shorter length of the longest neurite
were observed in neurons isolated from the tegmentum and striatum of rats prenatally affected
by VPA administration compared to the control group. Cortical neurons isolated from
prenatally VPA-treated animals showed significantly less branching, but no statistically
significant differences were observed in the longest neurites. On postnatal day 5, a significant
decrease in synaptic protein SNAP25 and chemokine fractalkine expression was observed in
the striatum of VPA-treated females, but not in males. These findings suggest that potentially
autistic behavioral changes in the VPA-induced animal model may relate to both changes in
dopaminergic neuron morphology and alterations in synaptic proteins and chemoattractants
within projection brain areas such as the striatum. Some findings also indicate sex differences,
which require further investigation.
Supported by VEGA 2/0057/23, APVV-21-0189 and bilateral Hungarian-Slovak collaboration
HAS-SAS-2022-02.
High-purity extracellular vesicles used as a brain biopsy in
neurodiagnostics
'Bello O.D., *Rivera L., Gustos D.M., ^ u n o z E.M., 2
Filipčík P.,2
Škrabana R.
^HEM-CONICET, Universidad Nacionál de Cuyo, Mendoza, Argentina
2
Neuroimunologický ústav SAV, Bratislava, Slovenská republika
Neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by
the progressive accumulation and spread of mutated or wild type misfolded proteins, which
result in neurodegeneration and functional decline. Recent advances suggest that these
proteins can propagate pathogenicity by transferring via extracellular vesicles (EVs) that cross
cell boundaries. Our research focuses on the role of EVs, derived from microglial cells, as
carriers of these misfolded proteins, potentially serving as a "liquid biopsy" for
neurodegenerative conditions.
We have developed an approach using immortalized murine microglial BV2 cells
exposed to tau protein fibrils (dGAE T40) to simulate the cell environment of a proteinopathy.
Conditioned media from these cells undergo gradient centrifugation and flotation assays,
yielding high-purity EVs. Preliminary results demonstrate the transfer of misfolded proteins
through EVs. To evaluate the disease-propagating capacity, HEK293T sensor cells, which
have been engineered to report the nucleation and aggregation activity of pathological
proteins, are incubated with EVs loaded with dGAE T40 fibrils.
Our results hold significant promise for clinical applications, offering a less invasive
alternative with the potential for early diagnosis through blood-based biomarkers of
neurodegenerative and other brain diseases. Furthermore, understanding the dynamics of EVmediated
protein transfer could illuminate new therapeutic targets to disrupt the pathological
progression of neurodegenerative diseases.
Acknowledgement: This work was supported by Grant number 873127 InterTAU (Marie
Sklodowska-Curie Action Research and Innovation Staff Exchange Grant Agreement).
Keywords: extracellular vesicles, proteinopathies, liquid biopsy, tau protein
19
Application of tubular conduit in transected rat tail nerve
flaško J., 2
Mojžišová M., ^zékiová E., Michalová Z., Vaničky I.
institute of Neurobiology, Biomedical centre of Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice
2
Faculty of science, University of Pavol Jozef Šafárik, Košice
Peripheral nerve injury and subsequent limitation/loss of function has long been an
unsatisfactorily resolved medical issue, given the nature of the nervous system. Among the
most severe forms of nerve damage is transection (neurotmesis), which results in the complete
disruption of nerve continuity and, in some cases, the loss of entire nerve segments,
complicating following surgical intervention. In this context, tubular conduits appear as a
potentially suitable tool for bridging interrupted nerve stumps, as they can provide not only
physical support but also a conducive environment for regenerative processes. In our
experiment, we performed a transection on our established model of peripheral nerve
damage—the rat tail nerve—and subsequently applied a tubular conduit that connected the
proximal and distal nerve stumps, with the stumps placed at various distances within the
conduit. Quantification of regenerated axons after the survival period showed that the tail
nerve could grow through a gap of up to 5 mm between the stumps in the tubular conduit we
used, but it could not bridge a 10 mm gap. These results lay the groundwork for our further
experiments.
The work was supported by projects VEGA2/0109/24 and VEGA2/0120/23.
Diaschisis-related changes in main excitatory neurotransmitter glutamate
in rat brain following the MCAO.
^ona M., 2
Hvizdosova N., 3
Kollarova P., 4
Koncekova J., 4
Kotorova K., 4
Bonova P.
department of Medical Physiology, Faculty of Medicine, P J Safarik University, Kosice, Slovakia
department of Anatomy, Faculty of Medicine, P J Safarik University, Kosice, Slovakia
'Department of Pathology, Faculty of Medicine, P J Safarik University, Kosice, Slovakia
institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Kosice, Slovakia
Middle cerebral artery occlusion is the main cause of ischemic stroke in the human
population. There are lots of metabolic, biochemical and histological changes in ischemiaaffected
tissue but it influences also tissue distant from a stroke. This phenomenon is called
"Diaschisis" and originally was characterized in 1902 by Monakow as a sudden change of
function in a portion of the brain connected to a distant, but damaged, brain area. In the recent
past, diaschisis includes all changes in regions distant from impacted tissue. According to
types of fibres, 3 main types of diaschisis are defined. Cerebro-spinal, cortico-commissural
and associative, all explained by several processes, i.e. deafferentation, oedema and last but
not least spreading depression (SD). SD represents a wave of electrophysiological
hyperactivity followed by a wave of inhibition and causes excessive and abnormal excretion
of neurotransmitters in non-affected portions of the brain, connected to the infarcted area via
axons. As glutamate is considered the main excitatory neurotransmitter in the vertebrate brain,
we chose it for our analysis to evaluate the ratio of spreading depression in rat brain during
infarct maturation (up to 7 days). In the present work, we measured the glutamate
concentration in homogenate of 9 different parts of rat brain. While in parts that are directly
affected by ischemia (core and penumbra), the glutamate increase is caused by glial disability
to convert glutamate to glutamine, in unaffected parts of the ipsilateral hemisphere and the
contralateral hemisphere it is most likely caused by the SD effect. In the ipsilateral
hemisphere, the most noticeable changes distally from the ictus were detected in in prefrontal
(decrease compared to control) and occipital cortex (increase compared to control) during the
whole time of infarct maturation. Minor changes in other non-affected structures of the
ipsilateral hemisphere (cerebellum, pons and hypothalamus) were monitored as well. The
contralateral hemisphere shows a similar pattern of glutamate distribution changes in the
prefrontal and occipital cortex. Interestingly, decreasing concentration in glutamate of the
majority of structures 7 days following infarction was confirmed. Presented results show
infarct maturation changes in glutamate concentration as a manifestation of diaschisis in all
structures of the brain regardless of the type of axonal connection (cerebro-spinalis, corticocommissural
and associative).
Supported by VEGA 1/0723/21, 2/0096/22 and APVV 21-0069.
21
Blood cells-derived secretome- a novel approach for the treatment of
ischemic stroke outcomes
^onova P., ^oncekova J., Motorová K., ^emethova M., 2
Bona M., Gottlieb M .
'institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Kosice, Slovak
Republic
department of Medical Physiology, Faculty of Medicine, P J Safarik University, Kosice, Slovak Republic
The current conception of stroke patient treatment is based on the fastest start of the
therapy. Ischemic events, the most common type of stroke, are treated by clot-dissolving
drugs, or by direct removal of the clot by thrombectomy. Nevertheless, only a low percentage
of the affected meet the conditions for those therapies according to ASA.
Remote ischemic conditioning (RIC) has recently become an attractive approach for
improving or replacing current stroke therapies in preclinical and clinical trials. RIC
represents short, repeated ischemia of a distant tissue, organ, or limb, capable of inducing
tolerant phenotype, i.e., stimulating endogenous mechanisms of tissue protection against
lethal ischemia. Our previous research confirmed the essential role of blood circulation in
RIC-mediated neuroprotection. At the same time, we have proved the possibility of using this
humoral pathway to transfer tolerant phenotype to non-conditioned individuals. RIC
stimulates the blood cells (BCs) via their transcriptional/translational pattern of protein
synthesis, which ensures the induction of tolerant phenotype in the recipient following the
transfusion of those RIC-treated cells. These findings motivated us to verify changes in the
BCs paracrine activity following RIC stimulation and the possibility of using this altered cellfree
secretome to improve the stroke outcome.
Tolerant secretome was prepared by incubating RIC-stimulated BCs in artificial
plasma and tested in vitro (primary cortical neurons) and in vivo in a rat model of middle
cerebral artery occlusion (MCAO). In addition, the protein profile of the secretome was
analyzed to identify the secreted proteins and the bioactive component ensuring the induction
of the tolerant phenotype. The increased neuronal survival mediated by the tolerant secretome
was confirmed in vitro and in vivo in pre- and post-treatment of MCAO-subjected rats.
Bioinformatic-based analysis revealed higher amounts of proteins released by the tolerant
BCs; 29 proteins were recognized as secreted. More than half of them were involved in the
biological processes of the response to the stimulus (GO:0050896) and the response to
chemicals (GO:0042221). The protective phenotype was most likely mediated by the
synergistic effect of multiple identified proteins, including those unique to the tolerant
secretome (ceruloplasmin, D-3-phosphoglycerate dehydrogenase), and was promoted by the
co-participation of several reaction pathways (the most probably post-translation protein
modification, MAP2K, and MAPK activation, and platelet activation). Taken together, our
results demonstrate that properly stimulated BCs could serve as a source for cell-free-based
therapies of regenerative medicine.
Supported by APVV 21-0069, VEGA 1/0723/21 and 2/0096/22.
Clinical use of the phenomenon of ischemic tolerance
^urda R., 2
Némethová M., x
Burda J.
'Department of Trauma Surgery, Medical Faculty, University of P.J.Safarik University Hospital of L.Pasteur,
Kosice, Slovak Republic
institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Kosice, Slovak
Republic
Ischemia with reperfusion initiates a wide complex of changes of an inflammatory
nature that can worsen local conditions, but also lead to dysfunction of a distant organ or the
entire organism. Ischemia occurs in clinical practice during acute arterial occlusions (ischemic
cerebrovascular accident, myocardial infarction and limb ischemia), where reperfusion
subsequently occurs as part of treatment (thrombolytic treatment, angioplasty, surgical
revascularization, operations with tourniquet application). Also common surgical procedures
(organ transplantation, transfer of free lobes, cardio-pulmonary bypass, vascular operations,
polytrauma) lead to ischemia-reperfusion damage.
The possibility of influencing the extent of ischemic damage and the consequences of
reperfusion syndrome is still an unsolved challenge. Knowledge and the possibility of clinical
influence of ischemia-reperfusion damage is still very limited. A practical problem is the fact
that the transfer of experimental and animal results to clinical practice is still very problematic
with controversial results.
In the lecture, the authors discuss the possibilities of using ischemic tolerance in the
treatment of ischemia-reperfusion syndrome. Ischemic tolerance represents a robust internal
defense mechanism and a state in which cells are resistant to the devastating effect of
ischemia, which would itself lead to cell death. This is one of the forms of evolution. Cells
exposed to metabolic stress or sublethal ischemia (preconditioning) become temporarily
resistant to the action of subsequent lethal stress. The power of protection obtained by
ischemic tolerance is surprisingly large.
In an experimental animal model, the protective effect of pre-conditioning and postconditioning
against ischemic-reperfusion damage is clearly confirmed, this fact is confirmed
by countless experimental works. On the contrary, the number of works devoted to the clinical
use of ischemic tolerance in humans is very limited. The results of these works are often
contradictory, or clearly not confirming the potential protective effect of ischemic tolerance.
In the gerontic population, as a result of co-medication, activation of ischemic
tolerance often does not occur, but in the case of traumatic injuries in young healthy people,
the phenomenon of postconditioning can be fully utilized. In the given case, the injury can be
considered as the first stage of activation of ischemic tolerance, and the therapeutically
applied subsequent stress leads to full activation of ischemic tolerance.
The aim of the lecture is to provide a comprehensive overview of the use of the
phenomenon of ischemic tolerance in practice, while the authors, based on the results of
experimental and clinical works, state the possibility of direct application of double activated
plasma from healthy donors as the most optimal use of ischemic tolerance in practice, while
this is the only one that can lead to immediate treatment of ischemic -reperfusion damage.
23
Cell therapy for traumatic brain injury, current status and future plans involvement
of 14-3-3^
u
Durgala A., 3
RiveraL., ^krabanová M., ^enteM., škrabaná R., ^ilipčikP.
'Slovak Academy of Sciences, Institute of Neuroimmunology, Dúbravská cesta 9, 845 10 Bratislava, Slovak
Republic
2
Comenius University, Faculty of Science, Department of Molecular Biology, Mlynská dolina Ilkovičova 6, 842
15 Bratislava, Slovak Republi
'National University of Cuyo, Institute de Histológia y Embriología de Mendoza, Centra Universitario,
M5502JMA, Ciudad de Mendoza, Argentina
Traumatic brain injury (TBI) is one of the major causes of injury-related mortality and
morbidity around the world. TBI can cause cognitive and memory impairments, hearing loss
or various neuropsychiatric and neurological diseases. It can be caused by sports-related
injuries, automobile accidents, or blast-related injuries. Based on the severity of primary
damage as well as the extent of secondary damage, TBI can be categorized into mild,
moderate, and severe. Many efforts to treat TBI as a single pathophysiological condition did
not lead to the successful results. Newly tested regenerative therapy approaches TBI using
mesenchymal stem cells (MSCs) seem to be promising. Preclinical research demonstrated that
transplanting MSCs have the potential to decrease secondary neurodegeneration and
neuroinflammation, stimulate neurogenesis and angiogenesis, and enhance overall functional
outcomes in experimental animals. Recently it was revealed that 14-3-3 family proteins seem
to be closely associated with TBI. Specifically, the isoform 14-3-3^ has been found involved
in connection to the cell therapy of rat model of TBI, in which it fastened neuronal recovery
of the animals via decreasing the inflammation and apoptotic processes and prevention of
neurodegeneration.
In our project, we focused on the isolation, differentiation and "in vitro"
characterization of mice MSCs intended for treatment of TBI. In our project we isolated
adipose tissue from retroperitoneal region of mice. After harvesting of sufficient amount of
MSCs, we differentiated the cells into neuron-like cells. These cells were compared with the
control non-differentiated MSCs. After the treatment with specific compounds (melatonin,
forskolin, IBMX), we have observed significant morphological neuron-like changes.
Subsequently we analyzed the expression of neuronal markers such as Tau protein, Nestin,
and Beta-tubulin III. We found, that Tau protein and Beta-tubulin III were expressed in the
cytoplasm and Nestin expression was observed in the nucleus and cytoplasm of differentiated
MSC. Our future goal is to further improve differentiation of MSC and prepare enough cells
for the transplantation into mice after mild TBI and mice with a neurodegenerative phenotype.
Animal models have been established at our institute and will be used to test the efficacy of
the cell therapy, as well as detail roles of 14-3-3^ in animal recovery after TBI.
Acknowledgement: The experiments were supported with APVV-20-0615, InterTau project,
The Marie Sklodowska-Curie the Research and Innovation Staff Exchange (RISE), program
Horizont 2020.
Mature neurons of the olfactory neurogenic region are connected with the
striatum
Fabiánova K., Martončíková M., Vaničky L, Blaško J., Popovičová A., Racek A., Račeková E.
Institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
The subventricular zone (SVZ) on the walls of the lateral brain ventricles harbors the
largest neurogenic niche in the adult rodent brain. Neuronal precursors of the SVZ proliferate,
migrate to the olfactory bulb in a restricted pathway known as the rostral migratory stream
(RMS), and differentiate into neurons. Regulatory mechanisms of postnatal neurogenesis in
the SVZ/RMS are still not fully understood. Recent evidence suggests that neurogenesis in the
SVZ/RMS could be regulated by neurons located directly in these regions. Till now, in the
SVZ/RMS two cell populations showing morphological characteristic of mature neurons have
been identified: nitric oxide (NO) producing neurons and neurons expressing secretagogin
(SCGN). The role of both types of neurons in the regulation of neurogenesis has already been
demonstrated. The aim of our work was to map possible projections of these populations of
neurons in the SVZ and in the RMS. To test the hypothesis that NO and SCGN positive
neurons of the SVZ/RMS are connected with the adjacent striatum, we injected the retrograde
fluorescent tracer Fluoro-Gold (F-G) into this brain structure using a stereotaxic device. To
verify the identity of nitrergic neurons and SCGN expressing neurons, double
immunoflourescent labelings with anti-nNOS/anti-SCGN and anti-Fluoro-Gold antibodies
were used. Microscopic analysis revealed the presence of F-G, administered into the striatum,
in cells of the SVZ, as well as in different parts of the RMS. F-G-labeled cells in the
SVZ/RMS were identified as nitrergic neurons or SCGN expressing neurons, indicating the
existence of a neuronal circuit in which NO producing and SCGN expressing neurons are
involved. Our morphological findings on the connectivity of nitrergic/SCGN expressing
neurons of the SVZ/RMS with the striatal structure suggest the existence of neural circuit that
may be involved in the regulation of postnatal neurogenesis within the olfactory neurogenic
area.
This work was supported by: VEGA 2/0119/22, APVV-19-0279.
25
Are aged microglia capable of surprising? Responsiveness of microglial
cells within the pineal gland during aging
Freites C.L., Avila M., Mufioz E.M.
Laboratory of Basic and Translational Neurobiology, Institute of Histology and Embryology of Mendoza "Dr.
Mario H. Burgos" (IHEM)-UNCuyo-CONICET, Mendoza, Argentina
Microglia, the resident immune cells of the central nervous system, are constantly
patrolling their environment, interacting with neighboring cells, and exchanging effectors and
modulatory signals. Despite their surveillance role, deleterious signals prompt microglia to
shift into stimulus-dependent reactive phenotypes. Once the insult is resolved, microglia may
return to a basal state. In the circumventricular organs such as the pineal gland (PG), which
lack a complete blood-brain barrier, microglia are continuously maintained in activated-like
states due to persistent stimulation by diffusible mediators from the bloodstream and local
molecules. Considering that microglia undergo detrimental changes during aging, we
investigated the responsiveness of aged microglial cells within the 21-month-old (21 m) rat
PG after two consecutive intraperitoneal injections (100 ug/kg body weight each) of
lipopolysaccharide (LPS) from E. coli using fluorescent immunolabeling followed by
confocal microscopy and image analysis. We observed a slight but significant increase in the
number of IBA1+
microglia, along with the formation of many cell clusters, after LPS
treatment compared to the vehicle-injected group. Additionally, LPS appeared to enhance the
expression of the cell proliferation marker PCNA in PG cells, including IBA1+
phagocytes.
Surprisingly, LPS treatment did not significantly impact on morphological parameters, such as
cell size and shape descriptors, in these cells. Furthermore, we detected an elevated
expression of the immunomodulatory cannabinoid receptor CB2 in the treated PG, including
in IBA1+
cells. These findings demonstrate that microglial cells within the 21 m PG are still
able to orchestrate immune responses to harmful cues, such as LPS. Given the
circumventricular nature of PG, circulating molecules are likely to induce acute inflammatory
events within the gland parenchyma more rapidly than in other brain areas, leading to a faster
resolution of inflammation.
This work was supported by InterTAU Grant no. 873127, and PICT2017-0499, PICT2021-
0314: E M M , FONCYT-ANPCYT.
Selected genetic indicators of induced brain stroke ischemic tolerance found
in human blood: A quantitative analysis
furman M., 3
Sihotsky V , Virag M., 2
Kopolovets L, ^emethova M., ^ucha R.
'institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
2
Eastern Slovak Institute of Cardiovascular Diseases and Faculty of Medicine, Pavol Jozef Šafárik University,
Ondavská 8, 040 01 Košice, Slovakia
'Faculty of Medicine, Pavol Jozef Šafárik University, Trieda SNP 1, 040 11 Košice, Slovakia
Stroke stands as the second leading cause of mortality in the European Union, making
it a significant medical concern. Carotid stenosis accounts for 15% of all ischemic cerebral
strokes. However, there currently lacks a reliable screening method for carotid disease.
Utilization of DNA analysis from peripheral blood is increasingly prevalent in diagnosing
various diseases. Moreover, the potential therapeutic approach of inducing tissue tolerance to
ischemia has primarily been investigated in animal models (Furman 2023). This study aims to
investigate alterations in the expression of specific brain ischemia markers subsequent to
carotid endarterectomy, hypothesized to activate ischemic tolerance. During carotid
endarterectomy, temporary blockage of the internal carotid artery occurs. Through RT-qPCR
analysis, we identified changes in peripheral blood after sub-lethal cerebral ischemia induced
by carotid endarterectomy, focusing on early reported gene indicators of brain ischemia
(ADM, CDKN1A, GADD45G, IL6, TM4SF1). Patients undergoing the surgical procedure
were categorized into three groups: asymptomatic, symptomatic, and those undergoing carotid
endarterectomy after an acute stroke. Outcomes were compared with those of a negative
control group. Carotid endarterectomy impacted all monitored indicators. Significant
differences (p-value 0.05-0.001) were observed when comparing the groups, indicating
evidence of brain tissue's resistance to ischemic episodes. In summary, following carotid
endarterectomy, symptomatic patients displayed alterations in ADM, GADD45G, and
TM4SF1; acute patients exhibited changes in GADD45G and IL6, while the asymptomatic
group showed modifications in CDKN1A and ADM.
Supported by SK- VEGA 2/0101/24 grant.
Reference:
Furman, M. et al. (2023) 'Quantitative analysis of selected genetic markers of induced brain
stroke ischemic tolerance detected in human blood', Brain Research, 1821, p. 148590.
doi: 10.1016/j .brainres.2023.148590.
27
Studying unconstrained behavior of mice in a labyrinth to explore cognitive
deficits
Kašparová L., 2
Ercsényiová S.,3
Trudičová D., 4
Štefan J.J.,5
Hromádka T.
gymnázium, Grosslingová 18, Bratislava
2
Centrum Nadania, Bratislava
3
Škola pre mimoriadne nadané deti a Gymnázium, Bratislava
4
Filozofická fakulta, Univerzita Komenského, Bratislava
'Neuroimunologický ústav SAV, v.v.i., Bratislava
Neurodegenerative disorders represent the most common causes of dementia
worldwide. The underlying neural mechanisms linking distinct molecular changes and
morphological changes to behavioral and cognitive deficits, however, remain unknown.
Behavioral changes in early stages of neurodegenerative disorders can be subtle and
commonly used behavioral tasks struggle to reveal such changes in various animal models of
brain disorders. Studying unconstrained behavior of animals provides a rich environment for
exploring cognitive deficits associated with various disorders of the brain. Observing mice
navigate, explore, and learn freely in a complex environment allows for a more
comprehensive assessment of their cognitive abilities than using more common constrained
tasks which may limit behavioral expression. The resulting nuanced understanding of
behavioral changes would lead to a better understanding of changing neural mechanisms
between healthy and diseased brains.
To study unconstrained behavior of mice we have adapted a recently introduced
labyrinth and estimated the dynamics of learning in mice searching for rewards. The labyrinth
(60 cm x 60 cm) was made of acrylic semi-transparent to infrared light. Experiments were
conducted in a dark room, the labyrinth was illuminated by two infrared LED arrays, animal's
trajectory was tracked using an infrared camera and Bonsai software, and behavior was
monitored using a Bpod system. During the initial phase of testing, wild-type mice were
allowed to freely explore the labyrinth connected to a mouse cage during several consecutive
sessions. After learning the position of a water reward, mice quickly learned to switch
between reward ports placed at two adjacent positions separated by a labyrinth wall.
Switching between ports required learning the shortest path between them. Mice appeared to
adapt quickly even after rotation of the maze when the position of the water reward has
changed relative to the entry to the labyrinth.
Studying complex unconstrained exploration in mice offers a window into
understanding cognitive deficits in neurodegenerative disorders. We suggest that evaluating
such unconstrained behavior in wild-type mice and mouse models of neurodegenerative
disorders could reveal cognitive impairments even in early stages of the disorders, and might,
in turn, provide insights into the underlying neural mechanisms.
This work was partially supported by APVV-20-0344 and APVV-19-0585.
Inhibition of aldosterone synthesis during the stress-hyporesponsive period
results in negative neurobiological effects in juvenile rats
Hlaváčova N., Hrivikova K., Graban J., Karailievova L., Ježova D.
Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava,
Slovakia
In the developing rodent, there is a period (postnatal day (PND) 3-14), called the
stress-hyporesponsive period, when pups show reduced capacity to secrete corticosterone in
response to several stress stimuli (1). We found that rat pups show increased rather than
reduced response of the mineralocorticoid hormone aldosterone to several acute stress stimuli
throughout this developmental period (2). We tested the hypothesis that the inhibition of
aldosterone synthesis during the stress-hyporesponsive period results in altered behavior and
modified neuroendocrine response to stressors in juvenile rats. Newborn Sprague-Dawley rat
pups (males n=40, females n=41) were treated with aldosterone synthase inhibitor FAD286
(30 mg/kg per day, orally) or vehicle from PND 3 to PND 9. They were behaviorally tested in
an open field (PND 23) and elevated plus-maze (PND 29) and half of them were exposed to
restraint stress for 120 min (PND46). Results showed that in 10 days-old pups, treatment with
FAD286 resulted in increased gene expression of CYP11B2 (aldosterone synthase) and
CYP11B1 (11-beta-hydroxylase) in the adrenal glands, increased plasma levels of
corticosterone, and decreased concentrations of plasma aldosterone. Postnatal FAD286
treatment increased anxiety-like behavior in female rats, not male rats. In juvenile rats treated
with FAD286 postnatally, the rise in corticosterone concentrations in response to restraint
stress was much more pronounced than in vehicle-treated ones, regardless of sex. Obtained
results demonstrate that the lack of aldosterone in the early postnatal period as a consequence
of aldosterone synthase inhibition influences anxiety-like behavior in female, but not male
juvenile rats. Blockade of aldosterone synthesis during the stress-hyporesponsive period in
newborn rats has consequences on stress hormone release later in life.
Supported by the Scientific Grant Agency of the Ministry of Education of Slovak Republic
(grant number VEGA 2/0158/22).
References:
Sapolsky R M , Meaney MJ. (1986). Maturation of the adrenocortical stress response:
neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Research,
396(1), 64-76.
Varga J, Ferenczi S, Kovács KJ, Garafova A, Ježova D, Zelena D. (2013). Comparison of
stress-induced changes in adults and pups: is aldosterone the main adrenocortical stress
hormone during the perinatal period in rats?. PloS one, 8(9), e72313.
29
How to evaluate the efficacy of thrombolysis in vivo
u
Hlozkova J.*, u
Scheer P.*, ^oliasova S., *Aksu Davut A., u
Brhelova E., u
MikulikR.
^asaryk University Brno
2
St. Anna's Faculty Hospital - International Clinical Research Centre Brno
*Both authors contributed equally to the work
Background: If a clot is visualized in an animal, then biological effect of thrombolytic
drugs could be assessed directly by measuring thrombus size in thromboembolic animal
models. Study validates a novel systemic embolization in vivo model using microfluoroscopy
imaging for to evaluate the efficacy of thrombolysis in vivo.
Aims: 1/ To quantifying the thrombolytic efficacy of alteplase (rtPA); 21 to verify the
yield of multiple clots application; 3/ to describe the mortality rate and technical complication
of the model.
Methods: Pilot validation study. Twenty-six outbreed Wistar rats, randomized into two
groups, received three human-fibrin based clots (AC artificial clot) labelled with BaS04 into
the descending aorta. Using micro-fluoroscopy imaging for 60 minutes, the size of detected
clots, after administration of rtPA (0.9 mg/kg/hour) in 16 rats (analysed 33 ACs) or saline
solution (rats 10 analysed ACs 21) in an equivalent volume and time, were measured. For
model validation, the relative change (%) in the clot area, lysis rates (%/min), and area under
the curve (% x
min) were calculated. Validated models were used in next studies with different
dose of rtPA (rats 19, ACs 40), tenecteplase (rats 27, ACs 47), plasminogen addition (rats 49,
ACs 99), novel mutant of rtPA - ALT04 (rats 30, 54).
Results: Clots were detected in each animal (n=132), with the mean 2.136 clots per
animal. No animal died. The relative change in clot area at 60 minutes from baseline in the
treated vs. control group was -46±26% vs. -21±14%. The average lysis rate in the period 0-60
minutes was -0.9±0.5 %/min (95%CI [0.7 to 1.1]) in the treated group and -0.4±0.3 %/min
(95%CI [0.3 to 0.6]) in the control group. The area under the thrombolytic curve in the time 0
to 60 minutes was -662 % x
min in the control group (95%CI [-866,6 to -456,5]) vs. -1433 %
x
min in the treated group (95%CI [-180556 to -1232,9]).
Conclusions: The model with C-arm micro-fluoroscopy imaging visualized clots in
animals, allowed measurement of clot size and was sensitive to detect thrombolytic activity of
Alteplase. Such model holds promise to be used for translation of new thrombolytics in
preclinical research into clinical practice.
The research was supported by projects: NU21-08-00510 Biocompatible nanoparticles as
targeted drug delivery systems and theranostics for the treatment of cerebrovascular disease.
NU23-08-00499 Improving the Efficacy and Safety of Cerebral Infarction Recanalization
Using Dual Thrombolytic Therapy.
In search for an optimal tool for detection of apoptosis in nervous tissue:
Cleaved caspase-3 is present in the majority of glial cells in the intact rat
spinal cord during postnatal life
^Holota R., ^ečmanová V., Mariášová A. Alexovič, ^ošuth J.,3
'4
Slovinská L., ^ačut L.,
5
Tomori Z., Daxnerova Z., ^evc J.
'institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 04154
Košice, Slovak Republic
institute of Experimental Medicine, Czech Academy of Sciences, Vídeňská 1083, Prague 142 00, Czech
Republic
'Associated Tissue Bank, Faculty of Medicine of P. J. Šafárik University and L. Pasteur University Hospital, Tr.
SNP 1, 04011 Košice, Slovak Republic
department of Regenerative Medicine and Cell Therapy, Institute of Neurobiology Biomedical Research
Center, Slovak Academy of Sciences, Šoltésovej 4, 04001 Košice, Slovak Republic
'Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 04001 Košice, Slovak Republic
Cell death is an essential process that occurs during the development of the central
nervous system. Despite the availability of a wide range of commercially produced antibodies
against various apoptotic markers, data regarding apoptosis in intact spinal cord during
postnatal development and adulthood are mostly missing. We investigated apoptosis in rat
spinal cord at different stages of ontogenesis (postnatal days 8, 29, and 90). For this purpose,
we applied immunofluorescent detection of two widely used apoptotic markers, cleaved
caspase-3 (cC3) and cleaved poly (ADP-ribose) polymerase (cPARP). Surprisingly, we found
significant discrepancy between the number of cC3+
cells and PARP+
cells, with a ratio
between 500:1 and 5,000:1 in rat spinal cord at all postnatal time points. The majority of cC3+
cells were glial cells and did not exhibit an apoptotic phenotype. In contrast with in vivo
results, in vitro analysis of primary cell cultures derived from neonatal rat spinal cord, treated
with the apoptotic inductor staurosporine, revealed a similar onset of occurrence of both cC3
and cPARP in cells subjected to apoptosis. Gene expression analysis of spinal cord revealed
elevated expression of the Birc4 (XIAP), Bird and Birc5 (Survivin) genes, which are known
potent inhibitors of apoptosis. Our data indicate that cC3 is not an exclusive marker of
apoptosis, especially in glial cells, due its possible presence in inhibited forms and/or its
participation in other non-apoptotic roles. Therefore, cPARP appears to be more appropriate
marker to detect apoptosis.
This study was supported by grants VEGA no. 1/0760/20 and APVV-19-0279.
31
iNets - novel human neural networks to study neurodegenerative diseases
Hruska-Plochan M.
Department of Quantitative Biomedicine, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
Switzerland
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal
neurodegenerative diseases. Pathogenesis of these TDP-43 proteinopathies cannot be studied
in animal models due to human-specific TDP-43 mRNA binding targets. Therefore, we
generated human iPSC-derived, colony morphology neural stem cells (iCoMoNSCs).
Differentiated iCoMoNSCs formed self-organized long-lived neural networks consisting of
glia and synaptically connected and electrophysiologically active neurons - iNets.
Overexpression of wild-type TDP-43 in a minority of iNet neurons led to its progressive
fragmentation and aggregation, resulting in neurotoxicity. scRNA-seq revealed misregulation
of synaptic protein NPTX2, the mRNA levels of which are controlled by TDP-43 binding on
NPTX2 3'UTR. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity,
whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-ignited
neurodegeneration. Importantly, we confirmed NPTX2 misaccumulation in ALS and FTLD
patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and
NPTX2 accumulation, thereby indicating a new pathway of neurotoxicity. Next, we plan to
utilize iNets and MacNets (iNets integrated with iPSC-derived microglia) to study the
mechanisms and consequences of TDP-43 aggregation, their spread from neuron to neuron
and the link between these events and TDP-43 loss of function.
The impact of traumatic spinal cord injury on the expression of
Angiotensin II receptors within the hypothalamus-pituitary-adrenal axis
Hvozdikova E., Liptakova V., Snopkova J., Kellerova E., Pavel J.
Institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Soltesovej 4-6, 040
01 Kosice, Slovakia
Angiotensin II as an important stress hormone can modulate the activity of the
hypothalamus-pituitary-adrenal gland (HPA) axis and sympathetic nervous system through
activation of its receptors. However, traumatic spinal cord injury (SCI) can disrupt the
sympathetic innervation of adrenal gland and dysregulate HPA axis function which
subsequently contribute to altered endocrine regulation. Since Angiotensin II receptors are
expressed within organs of the HPA axis, it seems that the expression of both ATI and AT2
receptors could be significantly impacted by the disruption of the sympathetic innervation.
Therefore, the level of circulating Angiotensin II and the expression of its major receptors
within the HPA axis have been studied during 28 post-traumatic days in response to severe
spinal cord compression (40g weight for 15 min) induced at higher (Thl) and lower (Th9)
thoracic spinal level. The observed changes were correlated with alteration of plasma level of
stress hormones including corticotropin-releasing hormone, adrenocorticotropic hormone,
corticosterone, epinephrine, norepinephrine and aldosterone.
During the first three days, there was detected an elevated concentration of circulating
Angiotensin II that then gradually decreased to the control values. Angiotensin II receptors
levels within the HPA axis as well as stress hormones fluctuated dynamically during 28 days
survival period. Spinal cord trauma at higher thoracic level significantly decreased the levels
of both Angiotensin II receptors.
We can conclude that the extent of sympathetic denervation of adrenal gland caused
by SCI can affect neuroendocrine regulation and the expression of Angiotensin II receptors
within the HPA axis.
The study was supported by APVV-18-0163, APVV-22-0248 and VEGA No. 2/0123/23.
33
Body weight-supported treadmill training in spinal cord injured rats
Ihnátová L., Magurová M., Kisucká A., Kuruc T., Heninová M., Kuchařová K , Gálik J.,
Lukáčová N.
Institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Kosice, Slovakia
The goal of therapeutic interventions for spinal cord injury (SCI) is to recover both
sensory and motor function, specifically aiming to restore locomotor function. In the absence
of innovative treatment for SCI, rehabilitation exercise has shown promise in promoting
recovery and functional improvements for individuals living with SCI. While complete SCI
may not be entirely reversible, locomotor training can impact morphological, nutritional, and
synaptic changes in intact motor neurons below level of injury, thereby playing a crucial role
in promoting motor functional restoration.
This study was aimed to investigate the effect of body weight-supported treadmill
training on the recovery of locomotor functions in rats with Th9 spinal cord injury.
Rehabilitation has started 1 week after Th9 compression. The exercise schedule followed a
regimen of 20 min/daily, 5 day per week, for a total training time of 11 weeks. Treadmill
speed was 7cm/s. The motor activity of hind limbs was evaluated using the BBB motor score
(Basso-Beattie and Bresnahan locomotor rating scale) and Catwalk test. After the 11 -week
rehabilitation training, the survival and regeneration of excitatory (glutamatergic) and
inhibitory (GABAergic) neurons in the ventral horns of the spinal cord were analyzed using
immunohistochemical methods.
The results showed that systematic and targeted training on a treadmill with body
weight support can significantly contribute to the improvement of motor functions after spinal
cord injury. The BBB testing showed that rats subjected to treadmill training exhibited
significantly improved locomotor recovery starting at 15 days after SCI compared to nontraining
rats. This finding was also confirmed by the Catwalk test. Immunohistochemical
analyzes revealed an increased number of GABAergic neurons in the ventral horns of the
spinal cord of trained animals. These inhibitory neurons play a crucial role in the modulation
of motor neuron activity that ultimately excites the corresponding muscle. No changes were
observed in the number of glutamatergic neurons.
Results of this work indicate that locomotor training can be an effective therapy to
promote the recovery of motor function after spinal cord injury. It represents, in combination
with other pharmacological/non-pharmacological therapies, great potential in the treatment of
SCI. However, further research is needed to confirm these hypotheses.
Supported by Návratový grant Ihnátová Lenka and APVV-19-0324.
Modulation of the gut-spinal cord axis by probiotic treatment in the Th9
compression model
Leninova M., 1
Kiss Bimbová K., fesucká A., bačova M., 2
Mudroňová D., ^uruc T.,
1
Gálik J., sucharová K., ^hnátováL., T^ukáčováN.
'institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4-6, Košice,
040 01, Slovakia
department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy in Košice,
Komenského 73, Košice, 041 81, Slovakia
The inflammatory response that ensues after spinal cord injury (SCI) is mediated by
many factors with antagonistic pro- and anti-inflammatory properties which act
simultaneously on resident and infiltrating immune cells. One of the promising approaches in
SCI-managing could be the targeted modulation of the gut microflora and restoration of
intestinal homeostasis, which have recently been associated with neuroregeneration and
neurological function improvement. The aim of this study was to regulate the inflammatory
response early after SCI using the probiotic strain Lacticaseibacillus paracasei Ž2 (5 x 109
,
CFU/mL), which is characterized by promising immunoregulatory properties (e.g. inhibitory
activity against bacterial pathogens, biofilm formation, systemic and local intestinal immunity
stimulation). Selected pro- and anti-inflammatory transcripts were analyzed in the spinal (the
lesion site and adjacent rostro-caudal segments; 0.3 cm) and jejunal tissues of rats subjected to
Th9 compression (40 g/15 min) and daily probiotic supplementation lasting 7 and 14 days.
The results showed that markers specific for microglia/macrophages (CDlib), reactive
microglia and astrocytes (CD68, IL-6, iNOS and Clq), and for T L R / N F - k B signaling
pathway, playing a crucial role in injury-induced inflammation, were significantly
downregulated in all monitored tissues as early as the 7th
-day after probiotic treatment. In the
spinal cord, the effect of two-week probiotic therapy was region and target-dependent with an
upward trend from the lesion site to the cranial segment. The same therapy had a more
pronounced effect in the jejunum than a shorter treatment. In addition, probiotic therapy
significantly stimulated the neuroprotective milieu in both tissues (gene expression ratio in
favor of CD206, SOCS3 or Ptx3), supported functional remodeling and ameliorated
neurogenic bowel and bladder symptoms in the first days after injury. This study shows the
potential of the probiotic strain L. paracasei Z2 in managing SCI-induced inflammatory
events in both the gut and affected spinal cord tissue during the subacute phase.
This study was supported by grant APVV-19-0324, APVV 22-0248, VEGA 2/0145/21,
VEGA 2/0115/24 and FMTS: 313011V344.
35
Evaluation of the analgesic potential of recombinant GABAergic hiPSCs
and their exosomes in a model of spinal cord injury induced pain
Jergova S., Rahimi B., Pressman Y., Tierney L., Sagen J.
University of Miami, Miami Project to Cure Paralysis, Miami, Florida, USA
Spinal cord injury (SCI)-induced chronic pain presents a therapeutic challenge.
Recombinant cell-based therapy can provide sustained and targeted delivery of analgesic
compounds, while cells can functionally integrate with the host tissue. Dysfunctional
GABAergic signaling and calcium-dependent release of pain neurotransmitters are among
major pathologies underlying chronic pain. Our previous studies showed that spinal
transplantation of rat GABAergic progenitors releasing an F D A approved calcium channel
blocker conotoxin MVIIA can attenuate injury-induced hypersensitivity in rats. Human
induced pluripotent stem cells (hiPSCs) enable autologous cell derivation from readily
accessible sources like blood or skin. hiPSCs also produce modulatory factors released in the
form of exosomes, which have shown promising outcomes in attenuation of chronic pain. The
goals of the proposed study are to explore i) the analgesic and translational potential of
hiPSCs derived recombinant GABAergic cells and ii) an early intervention with hiPSCsderived
exosomes for attenuation or prevention of chronic pain development. Fibroblast and
blood-derived hiPSCs were used to generate GABAergic neuronal cells. Cells were
transduced by AAV2/8 MVIIA construct developed by our lab to generate recombinant cells.
Both naive and recombinant cells were grown in sufficient quantities to allow isolation of
exosome fractions using ultracentrifugation. The analgesic potency of naive and recombinant
GABAergic hiPSCs and their exosomal fractions were evaluated in clip compression model
of SCI pain. Sprague Dawley rats were used in the experiment. Cells were grafted
intraspinally at 4 weeks post SCI and rats were evaluated weekly for mechanical and thermal
hypersensitivity. In a separated group, exosomal fractions were injected intravenously at
24hours post SCI with a booster injection at 1 week post SCI. Our results showed GABAergic
phenotype and the presence of MVIIA in all transduced cell lines. Exosomal fractions were
isolated from selected cell lines and characterized by Nanosight. Attenuation of
hypersensitivity was detected in animals grafted with GABAergic hiPSCs with stronger effect
observed in the recombinant group. The effect was mitigated by anti MVIIA or bicuculine
injection. An early injection of exosomal fractions followed up by a booster partially
attenuated development of tactile hypersensitivity compared to a single injection. Our data
suggests the beneficial effect of recombinant GABAergic hiPSCs in the management of
chronic pain and possible attenuation of chronic pain development using exosomal infusion.
Supported by Department of Florida of Health COPBC - University of Miami.
The University of Miami and J.S. and S.J. hold rights to intellectual property used in the study
and may financially benefit from the commercialization of the intellectual property.
Significance of animal models of mental disorders: experimental and
clinical point of view
1 1 2
JezovaD., Hlaváčova D., Izákova L.
'institute of Experimental Endocrinology of the Biomedical Research Center, Slovak Academy of Sciences
department of Psychiatry, Faculty of Medicine, Comenius University and University Hospital Bratislava,
Slovakia
The translation of knowledge obtained in animal models to humans is a difficult
process. Nevertheless, animal models of human disease are very useful tools for investigating
pathophysiological mechanisms and unravelling new treatment targets that cannot be
discovered by alternative approaches (Homberg et al. 2021). However, animal models of
human disease have many limitations and they rarely mimic the human disease state
completely. This is particularly true for preclinical research devoted to neuropsychiatric
disorders, as we cannot model the disorders themselves but only some of their signs and
symptoms. The significance of the results obtained is often differentially perceived by
experimental researchers and those from clinical practice. We have analysed our own
experience from animal studies related to psychiatric disorders by confronting the opinions of
an animal physiologist and a clinical psychiatrist. We have realised that even contradictory
opinions can be explained and understood if these views are combined. We may conclude that
in the fields of psychiatry and neurology, it is desirable to create research teams with both
experimental and clinical researchers who are listening to the points of view of each other.
Supported by the Slovak Research and Development Agency (grant No. APVV-20-0202) and
VEGA (grant No. 1/0644/23).
Reference:
Homberg JR., Ježova D., Genzel L.: The continued need for animals to advance brain
research. Neuron. 109(15):2374-2379, 2021
37
The tryptophan-kynurenine pathway- therapeutic strategy for
neuroprotection in tauopathies
Khiratkar K., Kovac A., Majerova P.
Institute of Neuroimmunology of Slovak Academy of Sciences, Bratislava, Slovakia
Alzheimer's disease (AD) is a progressive neurodegenerative disease that deteriorates
cognitive functions, characterized by the intracellular accumulation of abnormal filaments of
the microtubule-associated protein tau, amyloid-P plagues (AP), and neuronal apoptosis in the
central nervous system (CNS) releasing pro-inflammatory cytokines with hyper-reactive brain
immune cells (microglia and astrocytes). Moreover, these deposits are associated with
systemic inflammation and disruption of the many signaling pathways including the
kynurenine pathway. The tryptophan-kynurenine pathway (TKP) is functionally maintained
by the catabolism of the essential amino acid, L-tryptophan. This pathway generates various
metabolites specifically the kynurenic acid (KA), a neuroprotective metabolite in the CNS. In
neuroinflammatory conditions, under the influence of inflammatory cytokines (INF-Y) and
reactive oxygen species (ROS), a key regulatory enzyme indoleamine 2,3-dioxygenase (IDO)
is induced, declining the neuroprotective metabolite levels and increasing the production of a
neurotoxic agent such as Quinolinic acid (QA). The IDO-induced QA over-stimulates the
excitatory receptor N-methyl-D-aspartate (NMDA) acting as an agonist whereas, K A acts as
an antagonist of this receptor modulating the functioning of the brain. We aim to screen
analogs of kynurenic acid having similar biological activity, to modulate the
neuroinflammation in the CNS and its functions using an in-vitro blood-brain barrier (BBB)
model as well as an in-vivo model using the transgenic rats, thereby providing the new
possibility that can lead to its effective use under neuroinflammatory conditions.
Acknowledgement: VEGA 2/0129/21, APW-21-0321, APVV-22-0313
The impact of subpial administration of Chondroitinase ABC on glial scar
components following SCI
Kisucká A., Kiss Bimbová K., Bačova M., Heninová M., Kuruc T., Kuchařová K., Ihnatová
L., Lukáčová N.
Institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Košice, Slovakia
Although the glial scar has a protective function in the early stages after spinal cord
injury, it becomes a significant issue later on. This is because it forms a physical barrier that
prevents nerve fiber overgrowth through the lesion site, hindering functional recovery and
nerve tissue sensitivity. Neuronal regeneration is inhibited by the release of various inhibitory
molecules, primarily chondroitin sulfate proteoglycans (CHSPGs). Our study aimed to
examine the behavior of glial scar components (CHSPGs, astrocytes and
microglia/macrophages) after Th9 compression (40g/15 min) and subsequent treatment with
the chondroitinase ABC enzyme (ChABC) at the lesion site and in adjacent spinal cord
sections in adults Wistar rats. A significant increase in the expression of CHSPGs (NG2,
Neurocan and Phosphacan) was observed during the initial two weeks after compression,
followed by a subsequent decline to control values. This period was identified as the optimal
time for therapeutic intervention because it coincides with the maturation, stabilization, and
physical barrier function of the glial scar. Two weeks after injury, we administered ChABC
(0.2U in lOul) subpially, ensuring accurate enzyme delivery to the injury's epicenter while
avoiding tissue damage. Twenty-four hours after ChABC enzyme treatment, there was a
significant reduction in mRNA expression of all CHSPGs compared with untreated animals in
all examined segments; the lesion site and cranial segment were the most affected. One week
after treatment, the effect of the ChABC enzyme no longer persisted. The reduction in
CHSPG expression at the lesion site 24 hours after treatment strongly correlated with the
decrease in mRNA expression of cytokine genes produced by reactive astrocytes (GFAP,
S100, C3, TNFa, Clq). Microglia responding to injury were also reduced 24 hours after
treatment at the lesion site (Iba, Cx3Crl). Simultaneously, a clear correlation was observed
between the reduction of CHSPGs and an increase in the expression of genes representing
microglia producing pro-inflammatory cytokines (II lb, iNOS), allong with a decrease in the
expression of genes characteristic of microglia producing neuroprotective cytokines (CD206,
Illrn, TGFb, I14Ra). The subpial application of the ChABC enzyme, administered at the
appropriate time, has been shown effective in modulating the levels of CSPGs and the
polarization of individual components within the glial scar post-SCI. However, a more stable
modification of enzyme is needed to make the effect last longer.
Supported by grant APVV 19-0324 and VEGA grants 2/0115/24 and 2/0117/24.
39
Remote ischemic conditioning modulates the inflammation after stroke in
the rat model of hyperinflammation
Koncekova J., Kotorova K., Nemethova M., Bonova P.
Biomedical Research Center of SAS, v.v.i., Institute of Neurobiology, Kosice, Slovak Republic
In 2019, stroke was the second leading cause of death and disability worldwide. In
present, the FDA-approved treatment of stroke in clinic is thrombolysis using recombinant
tissue plasminogen activator (rt-PA). However, efficiency of this treatment could be affected
by coagulopathy and inflammation, what is an important factor for many bacterial and viral
infections, COVID-19 including. Therefore, the increased demand for developing novel nonpharmacological
therapies to elevate the tolerance to ischemia has been paying attention.
Remote ischemic conditioning (RTPC) meets these conditions because of the ability to
stimulate the endogenous protective mechanisms ensuring resistance to stroke. Our previous
results confirmed efficiency of RIPC in reduction of infarct size, even in the presence of
hyper-inflammatory response. In the current work, we examined the effect of RIPC on the
systemic inflammation.
Hyper-inflammation was induced by lipopolysaccharide (LPS) intratracheal
administration. We induced focal ischemia after one day of LPS intoxication (development of
hyper-inflammatory reaction). RIPC was applied one hour before (pre-conditioning) and after
(post-conditioning) the brain ischemia in the form of hind limb ischemia. We investigated the
modulation of inflammation by RIPC during the early (3 hours) and delayed phase (24 hours)
of postischemic reperfusion. We monitored the effect of this therapy on the sedimentation
rate, hematocrit, clotting time and systemic oxidative stress. Moreover, we observed the
changes in plasma cytokine profile.
Our results showed that RIPC affects the inflammatory response to the LPS
administration and ischemia induction positively. The pre- or post- conditioning of stroke
animals results in almost identical systemic response to treatments showing the early (3 hours
of reperfusion) and delayed (24 hours of reperfusion) reply. We observed significantly
improved sedimentation rate and hematocrit and reduction of systemic oxidative stress. RIC
treatment transiently improved clotting time in early stage of postischemic reperfusion,
however loss of its efficiency was observed at one day. At 3 hours of postischemic
reperfusion, plasma level of hyper-acute chemokines CINC-1, LIX and RANTES increased
transiently, also in conditioned animals. However the level of all of them dropped during the
delayed phase, following the RIPC treatment significantly.
On the base of our results, we can conclude that RIPC should influence the systemic
inflammation, what predispose this approach as additional treatment of stroke in patients with
ongoing hyper-inflammation.
This work was supported by APVV-21-0069 and VEGA 2/0096/22.
Unlike in other vertebrates, majority of cerebrospinal fluid-contacting
neurons in the spinal cord of C56B1/6N mice is present in ectopic position
^ošuth J., lr
[bnelli Gombalová Z., ^atiašová A. Alexovič, ^rubáková J., 2
Žežula L,
Daxnerova Z., ^evc J.
'Faculty of Science, P. J. Šafárik University in Košice, Institute of Biology and Ecology, Košice, Slovak
Republic
2
Faculty of Science, P. J. Šafárik University in Košice, Institute of Mathematics, Košice, Slovak Republic
Cerebrospinal fluid-contacting neurons (CSF-cNs) represent a specific group of
neurons localized in close vicinity of the ependymal lining surrounding central canal of the
spinal cord and brain ventricles.
However, this fact does not apply to the mouse strain C57BL/6N. Specifically, only in
this strain we identified the CSF-cNs localized in an ectopic position, which is exceptional
compared with several mammalian species tested. The fact is even more interesting since the
genetically closely related mouse strain C57B1/6J does not share the phenotype. On the other
hand, the close genetic relationship and availability of genomic data from both strains enabled
us to reveal genetic markers associated with this trait. An in silico comparison of the both
genomes showed hundreds of variations between the both genomes. Within them, 236
variations represented homozygous sequence variants. However, only small portion - 39 (34
SNPs, 2 small indels, and 3 structural variants) targeted known protein coding D N A
sequences. Comparison with another mice strain strain (Balb/C, with standard CSF-cNs
localization) showed that only 20 variants were specific for C57B1/6N. Majority of the
polymorphisms resulted in missense mutations. The final 5 candidate genes studied in the
segregation analysis {Crbl, Cyfip2, Adamtsl2, Plkl and HerpudI) were selected according to
their expression in spinal cord, severity of mutation on protein function and conservation of
the candidate gene/protein in mice strains with standard C57B1/6N localization. Mating of two
divergent inbreed lines (C57B1/6N x Balb/C, ectopic x standard) gave rise to consistent
heterozygous F l progeny, that showed more or less wild-type CSF-cNs arrangement. Then,
according to the segregation of phenotypes in the F2 progeny we found that more than one
factor is required for development of proximal localization of CSF-cNs in the spinal cord.
Further sequencing of segregating F2 progeny showed that the only relevant genotypes
associated with increased median of distance of CSF-cNs from the wall of central canal are
Crblmm
Cyfip2mm
and Crblmm
Cyfip2mi+
.
Our study shows important role of Crbl and Cyfip2 proteins for proper localization of
CSF-cNs in spinal cord. The detected anatomical divergence in the C57BL/6N strain is also a
reminder that the choice of the used model for research should be carefully considered.
Acknowledgement: The research was supported by Slovak Research and Development
Agency under the contract No. APVV-19-0279.
41
Blood cell-derived secretome as an alternative stroke treatment approach
for obese individuals
Kotorova K., Koncekova J., Gottlieb M., Bonova P.
Institute of Neurobiology of Biomedical Research Center, SAV, v.v.i., Kosice, Slovakia
Currently, a very promising neuroprotective strategy for stroke treatment is remote
ischemic postconditioning (RIPostC). This method involves inducing ischemic tolerance by
applying sublethal ischemia to a distant organ or limb, leading to the activation of various
endogenous neuroprotective mechanisms. Despite its positive effects, RIPostC's clinical
effectiveness is limited by factors such as age, gender, or obesity - one of today's public
health problems. Therefore, current research focuses on cell therapy, specifically on the use of
cell-derived secretome, which plays a significant role in paracrine signaling. In our study, we
focused on the concept of utilizing blood cell-derived secretome as a potential therapy for
obese individuals. 12-week-old lean healthy Wistar rats underwent RIPostC consisting of
three cycles of 5-minutes of ischemia and reperfusion. Subsequently, the blood from RlPostCstimulated
Wistar rats was processed into secretome and intravenously injected into obese
ischemic rats. Our findings showed a significant reduction in infarct volume of obese rats
which was positively correlated with the neurological score improvement 24 hours after
reperfusion. In addition, injection of tolerant secretome successfully inhibited DNA damage in
the lymphocyte cells of obese animals, while also demonstrating a significant increase in
certain antioxidant enzymes. Furthermore, the typical characteristic of RIPostC mediated
ischemic tolerance - the reduction in tissue glutamate excitotoxicity along with a simultaneous
elevation in blood glutamate levels - was observed in those rats. We can conclude that the
intravenous administration of the secretome, derived from the blood cells of healthy
individuals treated by RIPostC, activates endogenous mechanisms of neuroprotection in obese
animals and fully substitutes the missing effect of limb ischemia cycles of RIPostC.
This research was funded by Slovak Research and Development Agency under the Contract
no. APVV-21-0069 and research grant VEGA 2/0096/22
Processing of nociceptive peripheral input in spinal cord networks
feotov V., ^gashkov K., ^lashchak I., ^Andrianov Y., ^omanenko S., ^alaidych O.,
^oroid K., 2
Keyes A.L.,3
SafronovB.,4 5
Voitenko N.,2
Usachev Y.M., 1 , 4
Belan P.
'Departments of Molecular Biophysics and Sensory Signaling, Bogomoletz Institute of Physiology, Kyiv 01024,
Ukraine
2
Department of Neuroscience and Pharmacology, University of Iowa, Iowa City, Iowa
3
Neuronal Networks Group, Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto,
Portugal
4
Department of Biomedicine and Neuroscience, Kyiv Academic University, Kyiv 03142, Ukraine
'Research Center, Dobrobut Academy Medical School, Kyiv 01033, Ukraine
In this work we have centered on a better understanding of processing of peripheral
afferent inputs in lamina I and X by dorsal horn networks, descending pathways and microglia
using ex-vivo electrophysiology, optogenetics and 2P imaging. Lamina I spino-parabrachial
neurons (SPNs) receive peripheral nociceptive input, process it and transmit to the supraspinal
centres. We have found that the vast majority of the SPNs received a few direct nociceptive
C-fiber inputs and generated one spike in response to saturating afferent stimulation, thus
functioning as simple transducers of painful stimulus. However, majority of afferent
stimulation-induced action potentials in the entire SPN population originated from a small
fraction of high-output neurons. These neurons amplified and integrated the nociceptive input
gradually encoding its intensity into the number of generated spikes thus far encoding an
emotional aspect of pain.
Several brain regions contain neurons projecting to lamima I and X of spinal cord (SC)
that is critical for the descending regulation of nociceptive pathways. Unfortunately, little is
known of how these projections control the peripheral nociceptive inputs and local neuronal
network in these laminae. Here, we show that activation of descending tracts controls both
primary afferent-inputs and local networks in both lamina I and X.
Recent studies suggest that neuropathic pain is associated with a robust upregulation
of complement effectors in the spinal cord, which ultimately leads to production of a highly
active complement product, C5a. Here, we have elucidated C5a-dependent spinal mechanisms
that contribute to the development of neuropathic pain induced in mice by spared nerve injury
(SNI). We have demonstrated that C5a/C5aRl signaling results in an increased intrinsic
excitability of dorsal horn neurons likely via microglial activation and plays an important role
in SNI-induced neuropathic pain. Overall, our findings demonstrate complex mechanisms
spinal nociceptive processing in both norm and pathology.
43
Neurotrophic factor expression in different microenvironments six weeks
after thoracic spinal cord injury and carnosine treatment
Kuchařová K., Kuruc T., Ihnátová L., Gálik J., Magurová M., Ileninová M., Kisucká A.,
Lukáčová N.
Institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01,
Košice, Slovakia
Traumatic spinal cord injury (SCI), along with therapeutic interventions, triggers a
cascade of pathophysiological processes that determine the fate of neuronal recovery. The
current study investigated how 6-weeks carnosine supplementation at a dose of 50 mg/kg/day
before (CBI) or after (CAI) SCI affects the cellular expressions of brain- and glial cell linederived
neurotrophic factors (BDNF, GDNF) in the neuronal and muscle microenvironments
below and above the injury. Zucker- fatty rats were divided into naive (NC) and carnosinetreated
control (CC), Th9 compression untreated (SCI), and carnosine applied before (CBI) or
after injury (CAI) groups.
Compared to both controls, the protein concentrations of BDNF and GDNF of all
injured animals, i.e., with or without treatment, were decreased in the lumbar spinal cord
segments. Unlike proteins, the gene expressions of both neurotrophic factors were increased.
However, while the highest increase in BDNF mRNA was detected in both carnosine-treated
groups, the greatest upregulation of GDNF was found in untreated rats. Because both
neurotrophic factors are known to be produced by multiple cell types, we also evaluated the
expression of cell-specific genes such as microglial CX3C-motif-chemokine receptor-1
(CX3CR1), astrocyte glial fibrillary acidic protein (GFAP), oligodendrocyte 01ig2, fibroblast
collagen 6, and neuronal nitric oxide synthesis (nNOS) mRNAs. Correlation analyzes
between neurotrophins and cell-specific genes showed strong relationships between
microglial CX3CR1, BDNF and GDNF mRNA in animals from all injured groups. But, in
contrast to untreated injured rats, no correlation was found between increased expressions of
BDNF, GDNF, astrocyte GFAP, and fibroblast collagen-6 genes in the CAI group. BDNF
protein concentration was also decreased in cervical spinal cord segments in all injured
groups. But unlike the CBI and SCI groups, GDNF protein concentration did not decrease in
CAI rats. In the gastrocnemius muscle, the decrease of BDNF and GDNF protein
concentration was stopped in the CAI group. In untreated rats, the oligodendrocyte 01ig2
mRNA was significantly decreased in lumbar and nNOS in cervical spinal cord segments
compared with control and CAI rats. The current study demonstrated the effect of carnosine
supplementation on all glial cells. Moreover, its neuroprotective role was also seen in the
cervico-lumbar nNOS connection. In the muscle microenvironment, the main treatment effect
was observed in the gastrocnemius muscles of CAI rats.
This work was supported by FMTS: 313011V344 European Regional Development Fund and
VEGA project 2/0117/24.
Comparative study of imaging methods and therapy results in diseases of
the spine in dogs at the Veterinary University Hospital in Kosice
Kuricova M., Zeleznik P., Torok M., Liptak T., Fuchs J.
University of veterinary medicine and pharmacy in Kosice, Veterinary university hospital, Small animal clinic,
Komenského 73, 041 81 Kosice
In our study, we recorded the occurrence of spine and spinal cord diseases in 3% of all
dogs admitted to our clinic during the monitored period.
In the diagnosis of neurological diseases originating from the affected spine and spinal
cord, x-ray is still the most widely used because it is broadly available. In spinal cord
compressions, use of a contrast study - myelography has higher predictive value for lesions in
the thoracolumbar segment than the lumbosacral segment. Advanced imaging is often
required to confirm and refine the complete distribution of extruded disc material within the
spinal canal and to identify noncompressive lesions. Over the years, we noticed a change in
the number of different breeds undergoing examination, as well as a higher number of MRI
performed, which is related to the increased availability and the increasing willingness of
animal owners to undergo this procedure due to the understanding of its higher diagnostic
value.
In our retrospective study, during 2015-2018, we performed myelography 3-4 times
more often than MRI. Between 2019-2022, we used MRI 2-times more often than
myelography. This led to detection of greater numbers of spinal lesions, especially
intramedullary, also to a better decision-making in terms of surgical treatment. A secondary
effect of higher availability of MRI is higher success rate of our patients' therapy.
Predisposition to disc herniation reflects biomechanical forces associated with the
dog's body structure, genetic factors influencing disc degeneration, environmental stress. The
German Shepherd, Doberman, Rottweiler, Great Dane, Basset Hound and crossbreeds are
among the most commonly affected large breeds. Thoracolumbar lesions make up the
majority of cases and small breeds are most often affected, especially Dachshund, French
Bulldog, Yorkshire Terrier, Cocker Spaniel, Shih-tzu, Lhasa Apso, Maltese, Beagle. With the
increasing availability of MRI, we have seen a higher incidence of the use of surgical therapy.
The rate of recurrence of clinical signs in surgically treated dogs averaged 25%, and 59% for
conservative therapy. This higher incidence is due to the large number of predisposed dog
breeds mentioned above, in which recurrence after surgical therapy is usually a new
herniation of another disc. When choosing a therapy, the main factors influencing the decision
are type of the disease, degree of neurological dysfunction, severity of clinical symptoms and
chronicity of the disease. With medical or conservative treatment, if clinical symptoms persist
or worsen within a few days, it is necessary to consider surgery as soon as possible.
45
Aerobic exercise-driven brain resilience: insights into hippocampal
alterations of obese rats
Kuruc T., Kuchařová K , Kisucká A., Ileninová M., Ihnátová L., Kiss Bimbová K., Magurová
M., Gálik J., Lukáčová N.
Institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4,6, 040 01,
Košice, Slovakia
Although the impact of lifestyle on overall health is extensively documented and well
managed, obesity is considered a significant concern in contemporary society. In addition to
affecting other vital organs, obesity can detrimentally alter brain function. Specifically,
hippocampal changes associated with obesity may lead to chronic low-grade inflammation,
disrupted neurogenesis, and impaired neuroplasticity, ultimately affecting cognitive abilities.
Since aerobic endurance training (ET) leads to numerous health benefits (favourable influence
on weight, glucose control, regulation of blood pressure, and overall improvement in quality
of life), it is the most recommended treatment for obesity in humans. In this study, we focused
on molecular mechanisms that could reveal the potential of 6-week ET to counteract obesityinduced
alterations in hippocampus of a rat genetic model of obesity. Zucker-fatty and lean
rats were divided into four experimental groups: lean and obese controls, as well as trained
lean and obese groups. Over a 6-week period, the rats underwent an ET program on treadmill
with gradually increasing speed (from 9 m/min to 18 m/min) and duration (from 15 min/day
to 30 min/day). Using qPCR analysis, we assessed gene expression changes related to
inflammation, activation of neurotrophin-associated signaling pathways critical for
synaptogenesis and neuroplasticity and regeneration. ET of obese rats led to a reduction in the
expression of the pro-inflammatory microglial marker Ibal and modulation of neurotrophinassociated
PLCy/PKC/CAMKII signaling. Exercised animals exhibited a preference for the
PLCy/CAMKII signaling branch downstream of PLCy, which plays a pivotal role in memory
formation and synaptic plasticity. Additionally, the concurrent increase in mRNA levels of the
presynaptic marker synaptophysin further supported the involvement of ET in promoting
synaptogenesis and neuroplasticity. Simultaneously we observed the upregulation of mRNAs
for regenerative immature/mature oligodendrocyte markers (CNP/PLP1) and neurofilaments.
Finally, a passive avoidance test has shown the improvements in cognitive functions. These
findings underline the potential of ET to mitigate the obesity-induced inflammatory milieu,
provide neuroprotection, and promote proper hippocampus functioning.
This research was supported by the Research and Development Operational Programme
funded by the European Regional Development Fund (EVITS: 313011V344), Slovak Research
and Developmental Agency (APVV-19-0324), and Grant Agency of the Ministry of
Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of
Sciences (VEGA 2/0115/24 and VEGA 2/0117/24).
Neuroprotective and anti-inflammatory effects of neuroactive steroids in
model of perinatal focal cerebral ischemia
^utna V., ^olasek M., 2
Kudova E., 2
Chodounska H., 3
Druga R., ^senov G.
'Experimental Neurobiology Division, Preclinical Research Program, National Institute of Mental Health,
Klecany, Czech Republic
institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic
'institute of Anatomy, First Faculty of Medicine, Charles University, Prague, Czech Republic
Neuroactive steroids (NAS) have been shown to impact central nervous system
function through the modulation GABAergic and glutamatergic receptors. The levels of
neuroprotective NAS are pronouncedly elevated in the perinatal period. As had been
demonstrated previously, synthetic analogues of NAS engage in their natural metabolism and
modulating GABA and glutamatergic neurotransmission without obvious adverse effects in
immature rats. Likewise, synthetic analogue of NAS had neuroprotective effect in the model
of perinatal focal cerebral ischemia (pFCI), anticonvulsive effect in model of tonic-clonic
seizures in immature rats and did not show neurotoxicity effect. Due to the importance of
other mechanism and factors in the brain insult outcome, we aimed to evaluate an effect of
3a5P-pregnanolone glutamate (synthetic analogue of NAS) on parvalbumin positive GABAergic
interneurons (PV) activated microglia (OX-47) and astrocytes (GFAP) in the model of
pFCI.
The pFCI had been induced by the infusion of the endothelin-1 (ET-1, 40pmol/lul,
0.25 ul /min) into the right dorsal hippocampus of 12-days-old male and female rats. All
procedures had been performed under 1.5-2 % isoflurane anaesthesia in the stereotaxic
apparatus. One minute after the end of infusion, anaesthesia was terminated and skin was
glued by the collodium. The 3a5P-pregnanolone glutamate (PregG) at the dose 1 mg/kg was
administrated intraperitoneally 5 min after the end of ET-1 infusion. Animals had been
returned to their dams and transcardially perfused 24h later, brains were removed,
cryoprotected, sectioned (50 um, 1 in 5 series), stained on PV, OX-47 and GFAP, and
evaluated.
Our results indicate that PregG administration following pFCI has a neuroprotective
effect of on P V +
neurons in the dentate gyrus, reduce the level of microglia activation and
reduce the level of reactive astrocytes.
This study was supported by the Grant Agency of the Czech Republic 23-05746S and M H CZ
- DRO (NUDZ, 00023752).
47
Mirtazapine intake during pregnancy has only moderate effect on
hippocampal excitability of the offspring
T^acinovaL., ^ubiel-Hoppanova L., ^dunkova A., 2
Bukatova S., ^ndacovaK., 'Tomko M.,
1 2
Jurkovicova-Tarabova B., Dubovicky M.
'Center of Biosciences, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences,
Bratislava, Slovak Republic
2
Center of Experimental Medicine, Institute of Experimental Pharmacology and Toxicology, Slovak Academy of
Sciences, Bratislava, Slovak Republic
Untreated maternal depression has a negative impact on the neurodevelopment of her
fetus. Use of antidepressants during pregnancy may have adverse effects on fetus, as well.
Therefore we tested the consequences of maternal depression and/or prenatal antidepressant
treatment on the offspring neurodevelopment in an animal model. We have used an
established model of maternal depression based on three weeks long pregestational chronic
unpredictable stress. As an antidepressant we choose mirtazapine applied starting with 10th
gestational day via biscuit. We focused on possible changes of excitability of hippocampal
neurons as hippocampus was shown to be affected by a depression. Four experimental groups
were formed: no stress + vehicle, no stress + mirtazapine, stress + vehicle, and stress +
mirtazapine. Hippocampal excitability was tested either in primary culture established at the
first postnatal day which reflects results of prenatal development, or in acute hippocampal
slices prepared on postnatal days 11 to 13. Whole cell patch clamp was used for assessment of
passive and active electrophysiological properties.
In primary hippocampal culture maternal stress resulted in significantly hyperpolarized
resting membrane potential and this effects was not relieved by mirtazapine treatment. Input
resistance was not significantly different between groups. Passive membrane properties were
not altered in hippocampal slices. Stress significantly increased a threshold for action
potential firing in primary cultures but lowered it in hippocampal slices. In primary cultures
but not in slices this change was compensated by mirtazapine treatment. Stress also increased
the percentage of neurons in a primary culture which exhibited spontaneous firing at the
postnatal days 10-13. The mean instantaneous frequency of firing was not altered in any
experimental group. Altogether, we observed moderate changes in electrical activity of
offspring hippocampal neurons in early postnatal period caused by pregestational maternal
stress. These changes were not compensated by mirtazapine treatment. Mirtazapine itself only
moderately affected offspring neuronal activity.
Supported by APVV-19-043 5 grant.
Detrimental role of hyperhomocysteinemia in the ischemic/reperfusion
conditions
Lehotsky J., Baranovicova E., Hnilicova P., Kalenska D., Kovalska M., Kaplan P., Tatarkova
Z.
Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Mala Hora, SK- 03601 Martin
Slovakia
Hyperhomocysteinemic conditions (hHcy) as result of genetic polymorphisms or high
intake of sulfur containing proteins and low vitamins B status is considered as risk factor for
acute and neurodegenerative neuronal disorders.We present here results of metabolomic,
histomorphological and biochemical analysis of brain tissue as well as of blood plasma
developed by homocysteine (Hey) intraperitoneal injections or by high Met diet. This
conditions induce hHcy status and initiates changes in plasma and hippocampal and cortical
metabolome profile, as well alterations in biochemical, histo-morphological and behavioral
patterns in rats. High Hey metabolic supply leads to the alterations in "methylation index",
changes the one C unit metabolism and tissue redox balance. This can be thought as the main
damaging and noxious effect of hHcy. Our experiments also offer basic clues into the
molecular mechanisms of how hHcy if combined with ischemic/reperfusion injury (IRI) could
aggravate development of concurrent neuropathological processes, such higher level of
neuronal loss and also presence of amyloid deposition and hyperphosphorylation of tau
protein. As hHcy is frequently manifested with hypertension in humans, preventative
interventions can have an important implications for likely development of ischemic stroke
and neurodegeneration and deserves deeper experimental and human studies.
Supported by VEGA 274/23 and APVV 15/107
49
The effect of Angiotensin receptor type 2 stimulation on neuroregeneration
after severe spinal cord injury
Liptakova V., Snopkova J., Hvozdikova E., Pavel J.
Institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Kosice, Slovakia
The renin-angiotensin system, primarily through its main effector hormone
Angiotensin II plays an important role in regulation of blood pressure and maintaining hydroelectrolytic
balance. The effects of Angiotensin II are mainly mediated by ATI, but also by
AT2 receptors. It has been shown that AT2 receptors (AT2R) have an important function in
neuroregeneration processes as well. In this study, the AT2R was stimulated in an
experimental model of severe spinal cord compression in adult Wistar rats. Spinal cord injury
is a serious medical condition that is causing profound functional impairment of limbs
movement as well as disruption of vital bodily functions, such as micturition or bowel
movement. Such functional damages can significantly impact an individual's quality of life
and therefore it is crucial to restrict the negative consequences of such an injury. Interestingly,
significantly faster motor function recovery was observed after AT2R stimulation in
comparison to untreated controls. Furthermore, the selective AT2R agonist administration
increased expression of markers for myelin and axonal regeneration. Histological staining
supported these findings and confirmed statistically significant increase of spared spinal cord
tissue, reduced microcysts and cystic cavity formation, mainly in caudal regions. These
positive effects were prevented either partially or entirely by the application of the specific
AT2R antagonist. Overall, these observations strongly support the hypothesis that stimulation
of AT2R exhibits a considerable potential as therapeutic intervention for spinal cord injuries.
This study was supported by APVV-22-0248 and VEGA 2/0123/23.
Siponimod shows greater efficacy than methylprednisolone and
Atorvastatin in reducing neuroinflammation and promoting recovery after
spinal cord injury
Lukáčová N., Kiss Bimbová K., Kisucká A., Bačova M., Ileninová M., Kuruc T., Kuchařová
K., Ihnatová L., Magurová M., Gálik J.
Institute of Neurobiology of Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4,6, 040 01,
Košice, Slovakia
The study investigated the effect of three anti-inflammatory drugs (Atorvastatin,
Methylprednisolone, and Siponimod) on spinal cord injury (SCI) and the subsequent
inflammatory response, focusing on the activation of microglia/macrophage and astrocytes.
We aimed to understand how these drugs limit the inflammatory response and influence
reactive vs. neuroprotective phenotypes of microglia and astrocytes in both subacute and
chronic phases after Th9 compression (40g/15 min). Gene expression was analyzed at the
lesion site, as well as cranially and caudally, at 2 and 6 weeks after SCI. Atorvastatin (5mg/kg;
i.p., 7d) and Siponimod (2 mg/kg, 7d, i.p.) significantly reduced the microglia/macrophage
marker (CDlib) across the cranio-caudal extent, while effect of methylprednisolone (30
mg/kg, i.p., followed 6h a 24h later bylOmg/kg) was limited to the lesion site and cranially.
All drugs decreased Ibal and iNOS expression in cranial segments two weeks after injury.
Siponimod showed the strongest effect in promoting tissue-regenerative microglia marker
(Argl) and reducing neurotoxic microglial markers. Argl mRNA was found at both time
points in the caudal segment, which is known to host a detrimental environment. Siponimod
treatment yielded the best results, suggesting that this modulator of the sphingosine
1-phosphate receptor could shift microglia/macrophages from a neurotoxic to a
neuroprotective phenotype, thus aiding axonal and neuronal regeneration. The effect of
Siponimod on neuroprotective markers such as NF-H, NF-L, PLP1, and RBFox was most
pronounced six weeks post-SCI. The impact on astrocytes was also remarkable. All three
drugs decreased markers for reactive astrocytes (C3, TNF-a, Clq) at 2 weeks, with
Siponimod demonstrating the strongest suppression of these markers in the chronic phase.
Conversely, Siponimod significantly upregulated neuroprotective astrocytes, known to upregulate
many neurotrophic factors, showing a 5- to 6-fold increase in Tgml and Ptx3
expression. This effect was more pronounced than with Atorvastatin and Methylprednisolone.
In summary, Siponimod emerged as the most effective drug in reducing inflammatory
response after SCI, promoting neuroprotective microglia and astrocytes, and supporting
axonal and neuronal regeneration. It showed significant potential to reduce reactive
astrogliosis and enhance neuroprotection in both subacute and chronic phases after SCI,
suggesting a beneficial impact on functional outcomes.
Supported by grant APVV 19-0324, VEGA grants 2/0115/24 and 2/0117/24.
51
Exploring combination therapy with epidural stimulation and atorvastatin
for spinal cord injury recovery in rat models
Magurova M., Bacova M., Papcunova S., Galik J.
Institute of Neurobiology of Biomedical Center, SAS, Soltesovej 4-6, 040 01 Kosice
The spinal cord trauma represents a serious clinical problem and improving the
condition of patients is among the priorities of many scientific teams around the world.
Despite progress in the understanding of pathogenesis the overall mechanism of the processes
taking place in the spinal cord after traumatic intervention has not been clarified yet. Our
study investigates the potential synergistic effect of regenerative therapy combining electrical
stimulation with application of anti-inflammatory drug atrovastatin (ATR) following spinal
cord injury (SCI). We induced SCI at the T9 segment using an isoflurane anesthesia with
a compression device for 15 minutes under a 40g weight. An oscillating field stimulator
(OFS) subcutaneously implanted delivered a weak electric current (50 uA), changing polarity
every 15 minutes for 6 weeks to enhance axonal growth from the site of injury. Experimental
animals (Wistar albino female rats) were randomly divided to four groups: SCI with
functional stimulator (SCI+OFS), SCI with non-functional stimulator (SCI+nOFS), and two
SCI groups that received atorvastatin in addition to stimulator for 7 days after compression
injury (SCI+OFS+ATR, SCI+nOFS+ATR). Behavioral testing (hot-plate test and BBB scale)
proved improvements in sensory and motor functions in animals receiving combination
therapy. Using Western Blot method we analyzed protein levels for astrocytes (GFAP),
neurofilaments (NF-1), newly sprouting nerve fibers (GAP-43) and oligodendrocytes
(CNPase). Protein analysis showed increased levels of neurofilaments, newly sprouting nerve
fibers and oligodendrocytes in the groups with applied combination and single therapy.
Optical density decreased in the order SCI+OFS+ATR, SCI+nOFS+ATR, SCI+OFS and
SCI+nOFS. Beyond that, the level of astrocytes proteins, as an inflammation indicator
decreased more in the SCI+OFS+ATR group compared to others. Histological analysis of
transverse sections showed a significant reduction in both white and grey matter after the SCI.
In groups with applied therapies (SCI+OFS+ATR, SCI+nOFS+ATR and SCI+OFS) we
observed an increase in cumulative white and grey matter. These findings suggest
a synergistic effect of atorvastatin and OFS stimulation in promoting neural recovery postSCI,
highlighting the potential of combination therapies in enhancing regenerative outcomes.
The study was supported by VEGA no. 2/0098/20 and doktogrant 2022 - APP0405.
Evidence that PECAM-1 is a component of Reissner's fiber produced by
cells of the subcommisural organ in rats, but not in mice
^alčický L., ^atiašová A. Alexovič,2
Barčák D., ^ošuth J., Daxnerova Z.,1
Ševc J.
'institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Slovak Republic
institute of Parasitology, Slovak Academy of Sciences, Košice, Slovak Republic
Reissner's fiber is the extracellular structure composed of glycoproteins synthesized
and released by the cells forming subcommisural organ of the brain. This structure passes
along the brain ventricular system and central canal of the spinal cord, ultimately breaking
down in the terminal region called ampulla caudalis. Evidence that developmental
abnormalities of subcommisural organ and Reissner's fiber may be associated with the body
axis deformities and hydrocephalus emphasized the demand to uncover its structure and
function, which is only partly understood in mammalian CNS. The aim of our study was to
characterize cells forming subcommisural organ and Reissner's fiber in Wistar rats and
BALB/c mice using electron microscopy, immunolabeling and gene expression analyses. In
both rodent species, subcommisural organ has been identified in postero-dorsal wall of the
third cerebral ventricle as a structure composed of polarized cells with nuclei located basally,
close to the posterior commissure. The apical surface of these cells is sparsely covered by
short cilia. Reissner's fiber was identified as a fibrous structure present on the ciliated cells of
the third brain ventricle and on the surface of ependymal cells in spinal cord. We identified
that Reissner's fiber is immunoreactive to anti-PECAM-1 antibody in rat spinal cord, which
detects glycoprotein PECAM-1 typically used as a marker of blood vessels. On the other
hand, in mice spinal cord PECAM-1 has been identified only in the vasculature, while
Reissner's fiber has not been PECAM-1 positive. To verify, if PECAM-1 could be an integral
component of Reissner's fiber, subcommisural organ of both rodent species has been
immunolabelled. We identified that PECAM-1 is present in the apical cytoplasm of cells
forming subcommisural organ in rats, but not in mice. To test, if cells forming subcommisural
organ of rats express mRNA for PECAM-1, tissue samples were isolated by laser capture
microdissection and analyzed using RT-PCR. Our results proved higher expression of
PECAM-1 in cells forming subcommisural organ compared to other studied areas of the
brain, such as ependymal lining of third brain ventricle, hippocampus and plexus choroideus
indicating that cells of the subcommisural organ actively produce PECAM-1. Our data
represent the first evidence, that PECAM-1 is present in subcommisural organ and Reissner's
fiber of rats and underline another potential difference between two rodent species often used
in neurobiological research.
Acknowledgement: This study was supported by APVV grant No. APVV-19-0279.
53
Persistent olfactory dysfunction after COVID-19: the results of remote
olfactory testing and data collection in Slovakia
^artončíková M . , 2
Doležal P., ^abianová K., 3
Karhánek M . , 1
Gálik J., ^aček A.,
^opovičová A., 1
RačekováE.
'institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
2
Clinic of Otorhinolaryngology and Head and Neck Surgery, University Hospital-St. Michal's Hospital,
Satinského 1, 811 08 Bratislava, Slovakia
'Laboratory of Bioinformatics, Biomedical Research Center, Slovak Academy of Sciences, Dúbravská cesta 9,
845 05 Bratislava, Slovakia
Smell and /or taste loss was one of the commonly occurring symptoms of SARS-CoV-
2 infections. Olfactory dysfunction (OD) was transient in most patients, but persisted for a
longer time in approximately 10% of them.
The growing number of patients with persistent OD after COVID-19 in Slovakia,
where olfactory testing in clinical practice is not established, prompted the creation of the
project "Smell and COVID-19", in which a network of otolaryngologists also participated.
The aim of the project was to test the sense of smell in post-COVID-19 patients with
persistent OD with standardized smell tests, the results of which served as an objective
indicator for otolaryngologists who provided them with medical care. Due to the pandemic,
testing was performed remotely. For this purpose, the website www.cuch.sk was created and
smell tests were sent to patients upon request. A simple six item smell test for odor
identification, the Odorized Markers Test (OMT) was used in this study. From March to June
2021, 1025 patients requested smell testing. 824 patients met the inclusion criteria, i.e. they
indicated OD due to COVID-19 and OD lasted 1 month or more. 72.6% of the participants
were women and 27.4% were men. Average ages of the women and men were 40.6 and 36.6
years, respectively. The duration of OD at the time of testing ranged from 1 month to 1 year.
Based on the data provided by the participants, OD occurred most frequently between
November 2020 and February 2021 which corresponds with occurrence of the Alpha variant
(B.l.1.7) of SARS-CoV-2 related to higher risk of OD. Using OMT, hyposmia or anosmia
was confirmed in 82.6% of participants. The remaining 17.4%, of participants, although
determined to be normosmics according to OMT, complained of parosmia and/or phantosmia.
Self-assessment of smell did not correlate with OMT outcomes of participants in all cases.
That points to the relevance of psychophysical smell testing. Our study also revealed same
correlations between persistent OD and the monitored parameters.
This work was supported by Civic Association for the Support of Neuroscience Research;
Biomedical Research Center, Slovak Academy of Sciences; VEGA 2/0119/22; APVV-19-
0279
Ultrastructural insight on the arrangement of ependymal cells, vasculature
and fractone bulbs in the central canal lining of rat spinal cord
Mariášová A. Alexovič, 2
Týč J., 3
Herranz-Pérez V , ^ošuth J., ^alčický L., 2
Vancová M.,
2 1 3 1v
Nebesářová J., Daxnerova Z., " García-Verdugo J.M., Sevc J.
'Department of Cell Biology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik
University in Košice, Slovak Republic
laboratory of Electron Microscopy, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech
Republic
3Laboratory of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology,
University of Valencia, Spain
Ventricular system of CNS is covered by ependymal cells, which are generated during
the late embryonic period and early postnatal life. Even though, the ependymal cells of spinal
cord have been intensively studied during the past decades in adult rodents, their postnatal
development and function remain poorly understood. Moreover, the data on arrangement of
ependymal cells, vasculature and fractone bulbs in spinal cord during the ontogenesis are
missing. In this work, we focused on the study of ultrastructure of ependymal cells in selected
time points during the postnatal life (8, 29 or 90 postnatal days). We identified that vimentin
positive ependymal cells of central canal lining in neonatal, as well as adult rats, form basal
processes directed toward the nearby pan-laminin positive blood vessels or pia mater of the
ventral median fissure. Ultrastructural analyses revealed that single or even two basal
processes of ependymal cells contact vascular basal lamina or its narrow extensions termed
vascular fractones. At the age of P29, pan-laminin positive globules emerge at the interface
between the ependymal cells and the surrounding neuropil. These structures corresponded to
the labyrinths of extracellular matrix termed fractone bulbs, which were identified in this
region using electron microscopy and immunogold labelling. 3D reconstructions of images
obtained by serial block face-scanning electron microscopy revealed that fractone bulbs
surround the basal processes of ependymal cells. We observed small vesicles in the cytoplasm
of ependymal cells, as well as the globules separating from the fractone bulbs, indicating
potential molecular transport between these two compartments. Since fractone bulbs are
composed by laminin, we hypothesized whether laminin is produced by the ependymal cells
and subsequently transported to the fractone bulbs. Screening of laminin subunits transcripts
proved that ependymal cells in adult rat spinal cord express mRNA encoding laminin, mainly
the a3, a5, f>2 and yl subunits. Taken together, our data indicate that ependymal cells contact
nearby vasculature in each of the studied time points and contribute to the development of
fractone bulbs, which role in the spinal cord remains unknown.
Acknowledgement: APVV-19-0279, Czech-Bioimaging LM2018129 funded by MEYS CR,
VEGA 1/760/20
Keywords: ependymal cells, vasculature, fractone bulbs, rats
55
Brain myelin as an energy source
Matute C.
Achucarro Basque Center for Neuroscience, CIBERNED and Department of Neurosciences, University of
Basque Country, 48940-Leioa, Spain
Recent discoveries demonstrate that CNS myelin acts as an intrinsic energy store and
source that supports brain metabolism in extreme conditions. It appears that neural function is
preserved in the absence of glucose transport in oligodendrocytes, and that myelin content
diminishes extensively and selectively in the brain of marathon runners at the completion of
the effort. This myelin reduction is extensive and involves white and grey matter including
primary motor and sensory cortical areas and pathways. Post-exercise rest allows myelin
levels to recover fully within weeks. These findings reveal that myelin consumption and
replenishment is a novel form of metabolic plasticity aimed at maintaining brain function
during hypoglycaemia. Mechanistic details of how myelin lipids contribute to brain energy
demand remains unknown and difficult to grasp. Obtaining this knowledge, however, may
help understanding brain function decline during ageing and neurodegenerative diseases, as
waning glial support leading to synaptic, axonal and neuronal malfunctions may cause fuel
shortage thus leading to metabolic deficits and nervous tissue damage.
These emerging findings challenge the commonly held view that neuronal function
relies exclusively on glucose and oxygen supply, as the brain has no obvious energy stores,
except for limited astrocyte glycogen.
Supported by Spanish Ministry of Science and Innovation, Basque Govern and CIBERNED.
3D Printed Scaffolds for Repair of Injured Spinal Cord and Peripheral
Nerves
1 5 6
Medvediev V.*, 2
Grebenyuk S.*, 3 7
Dobropolska Y , 4
Sheremet Y , u
Bomikhov O.,
5
Abdalla 1,6
Petriv T.,u
Ustymenko V , 3 7
PivnevaT., 2
Ranga A., 4 7
BelanP., 1 , 7
VoitenkoN.
dobrobut Academy Medical School, 03022 Kyiv, Ukraine
laboratory of Bioengineering and Morphogenesis, Department of Mechanical Engineering, KU Leuven,
Leuven, Belgium
'Department of Sensory Signaling, Bogomoletz Institute of Physiology of NAS of Ukraine, 01024 Kyiv, Ukraine
4
Department of Molecular Biophysics, Bogomoletz Institute of Physiology of NAS of Ukraine, 01024 Kyiv,
Ukraine
'Department of Neurosurgery, Bogomolets National Medical University, Kyiv, Ukraine, vavo2010@gmail.com
6
Restorative and Functional Neurosurgery Department, Romodanov Neurosurgery Institute,
Kyiv, Ukraine
department of Biomedicine and Neuroscience, Kyiv Academic University of NAS of Ukraine, 03142 Kyiv,
Ukraine
*These authors are equally contributed to this work
Injuries of peripheral nerve (PNI) and spinal cord (SCI) are a significant medical and
social problem because it is characterized by long-term limb dysfunction and a high level of
disability. Treating PNIs represents a major challenge in reconstructive surgery and
regenerative medicine. 3D printed scaffolds represent a promising approach for treatment of
these injuries. Here we suggest a biotechnological approach for 3D printing of scaffolds with
a biomimetic architecture at a spatial resolution up to a micrometer, which were used for
implantation in treatment of PNI and SCI in Wistar rats. The approach is based on 2-photon
photopolymerization of organic polymers and is scalable to lesion geometries. The scaffolds
were implemented as multiple densely packed parallel micro-tunnels (-50 um) running
through their whole length. Having thin walls between tunnels, the scaffolds are almost
hollow and simultaneously creates a large internal surface area, providing thereby a natural
spatially oriented substrate for the axonal and vascular ingrowth. We have found that the
scaffolds, implanted in the excision of the lateral half-fragment of the spinal cord at the level
of T12-T13, were well integrated into the spinal cord without the formation of a significant
gliofibrous scar. Axons surrounded by oligodendrocytes, as well as vessels were observed in
almost each tunnel. Implantation also significantly improved the motor and sensory functions
of the paretic ipsilateral limb by 4t h
week, and the achieved recovery was maintained until the
end of 5t h
month after the operation. The implantation of the scaffolds in a place of dissected
sciatic nerve also resulted in the functional recovery outperforming one for both hollow
silicon tube and neurorrhaphy. Thus, 3D oriented hollow scaffolds having a large internal
surface area creates conditions for enhancing peripheral nerve and spinal cord regeneration
and recovery of the motor and sensory function of the paretic limb.
57
Transgenic minipig models of the serious eye diseases
Motlik J.
Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Libechov, Czech Republic
The retina is the innermost, light-sensitive layer of tissue of the eye, it develops from
the embryonic diencephalon and is considered brain tissue. Neurosensory cell types split into
ciliated sensory cells, photoreceptors and retinal ganglion cells. Our experimental effort is
focused to gene mutations, till now incurable eye diseases. Usher Syndrome (USH) is
rare genetic disorder caused by a mutation in one of 11 genes resulting in hearing loss and
visual impairment. Mutation in the gene USHlc, coding protein harmonin, is responsible for
the congenital deafness. Piglets bearing homozygous mutation in USHlc have the serious
statokinetic phenotype, too. Both, stereocilia of the inner and outer hearing cells in organ of
Corti as well as stereocilia of sensory hair cells in semicircular canals are heavily
disorganized. The first symptom after birth is the circular movement of piglets that requires a
special care to learn udder feeding. Moreover, the homozygote piglets do not listen the
mother's signal for feeding. The retina phenotype develops slowly and it is first recognized by
electroretinography and later by behavioral tests, too. The codiong sequence for harmonin
(4.7 kb) is suitable for composition of AAV vectors that are used for gene therapy of vision
via surgical deposition in the subretinal space. The limited spreading of vectors among
photoreceptors represents the main disadvantage of this approach. The recently developed
vectors penetrating the inner limiting membrane are promising solution of this obstacles. The
vector introduction directly in lateral semicircular canal can improve vestibular disfunction
and partly remove deafness. The USHlb mutation, causing absence of myosin-VIIA 7a
(MY07A), also affects hearing and vision. However, 6,645-bp M Y 0 7 A cDNA is too large to
be delivered using standard AAV technology. The dual AAV vector systems, in which the
transferred gene is split into two separate vectors that recombine in the target cell, can allow
the use of AAV vectors for larger genes. Recently, the third generation of lentiviral (LV)
vectors was developed. LV vectors have a high coding capacity beyond 10 kb allowing the
delivery of the entire coding sequence of large genes in a single vector. The large animal
model is key precondition for development of safe and efficient gene therapy. Center
PIGMOD disposes with transgenic minipigs for USHER Syndrome 1C and IB and Stargardt
disease and together with a broad international cooperation it strives for the development of
preclinical models for vestibulo-cochlear and vision impairments.
The effect of Siponimod on neuronal and glial cell protein markers in the
rat spinal cord injury model
1 2 3 2 3 2
Motyl J., Wencel P., Kisucka A., Czubowicz K., Lukacova N., Strosznajder R.
'Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical
Engineering, Polish Academy of Sciences, Warsaw, Poland
laboratory of Preclinical Research and Environmental Agents, Mossakowski Medical Research Institute, Polish
Academy of Sciences, Warsaw, Poland
'institute of Neurobiology of Biomedical Research Centre of Slovak Academy of Sciences, Kosice, Slovakia
The activation of resident glial cells, i.e. astrocytes and microglia in response to spinal
cord injury (SCI) is a crucial mechanism accompanied by the secondary, sub-acute phase of
trauma, which can harm surrounding neurones, when is overactivated. Siponimod (SIPO),
currently used in Multiple Sclerosis may be a promising candidate for SCI management. SIPO
prevents lymphocyte infiltration to the central nervous system, as well as it can bind to
sphingosine-1-phosphate (SIP) receptors S1PR1 and S1PR5 expressed by neuronal and glial
cells.
This study aimed to examine the protein level of glial cell markers, as well as alphasynuclein
(a-syn), and other proteins crucial in the proper function of neurones in the rat's
spinal cord (SC) after SCI and SIPO administration.
Female Wistar Rats were divided into the three experimental groups as follows: The
control group subjected to laminectomy only, the SCI group passed the Th9 segment
compression with a weight of 40 g/15 min and rats after the SCI with post-surgical SIPO
administration (lmg/kg i.p.). SIPO was administered 30 minutes after the compression and for
six consecutive days until decapitation. Then the cranial segment (above the site of injury) of
SC was isolated for Western blot analysis.
The level of IBA1 and GFAP, being strongly and specifically expressed by microglia
and astrocytes, respectively, was increased after SCI. This up-regulation was abolished by the
SIPO administration. Similarly, we observed a raised level of a-syn, which was partially
eliminated under SIPO treatment, a-syn is a presynaptic protein crucial in neurotransmission,
but when overexpressed it can gain pathologic conformation and function, which can share
with the intact cells in a prion-like spreading mechanism. Simultaneously, we observed that
trauma can increase Akt kinase phosphorylation on Ser473, being crucial to reach its prosurvival
activity.
Current results suggest that six days after trauma in the SC both harmful processes,
like an uncontrolled inflammatory response, and a-syn overexpression, as well as some
compensatory mechanisms, may occur. Furthermore, SIPO has a suppressive effect on
the major changes observed after trauma, which makes it a potentially promising
neuroprotectant in SCI therapy.
Supported by the PAS Polish - Slovak Joint Project No.8 for the years: 2023-2024, PAS-SAS-
2022-04, APVV-19-0324, N C N grant 2019/32/C/NZ4/00455 and statutory budget of MMRI
theme No.7.
59
Genes expression altered in activation of ischemic tolerance through carotid
endarterectomy in human blood
'Mucha R., 'Furman M., 3
Sihotsky V, 'Nemethova M., 3
Kopolovets L, Virag M.
'institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
2
Eastern Slovak Institute of Cardiovascular Diseases and Faculty of Medicine, Pavol Jozef Šafárik University,
Ondavská 8, 040 01 Košice, Slovakia
'Faculty of Medicine, Pavol Jozef Šafárik University, Trieda SNP 1, 040 11 Košice, Slovakia
Ischemic stroke poses a significant health threat and ranks as the second leading cause
of death in the European Union. Among all cases of ischemic strokes, carotid stenosis
contributes to approximately 15%. Although therapeutic approaches aiming to induce tissue
tolerance to ischemia have primarily been investigated in animal models [Furman 2023], their
potential benefits in humans remain underexplored. This study aims to explore alterations in
the expression of genes associated with cerebral ischemia in the peripheral blood of human
patients affected by cerebral ischemia and subsequently treated with carotid endarterectomy
(CEA), a procedure believed to activate ischemic tolerance mechanisms. Patients undergoing
CEA were categorized into Symptomatic and Asymptomatic groups based on the presence or
absence of symptomatic atherosclerosis and stroke before the CEA procedure. Additionally, a
third group, termed the Oxymetric group, exhibited a decrease in oximetry levels below 20%
during CEA. Another group consisted of patients with leg arterial thrombosis, while the final
group served as a negative control, comprising healthy individuals without a history of stroke,
thrombosis, or CEA. Peripheral whole blood samples were collected from all study groups,
and whole transcriptome analysis was conducted on their cellular fractions. The analysis of
the Symptomatic group revealed 791 genes exhibiting a significant fold change in expression
(> ±2) compared to the negative control, with 449 genes specific to this group. In the
Asymptomatic group, 688 genes were identified, including 360 specific to this group. The
Oxymetric group showed 637 genes, with 302 being specific to this category. In the
Thrombosis group, 879 genes were detected, with 506 being unique to this group. When
compared to the negative control, several genes, among the more than 20,000 tested, emerged
as specific to our test cohorts. Upon overlapping between the groups, only a few dozen genes
with potential involvement in the pathways associated with the induction of ischemic
tolerance were identified.
Supported by SK- VEGA 2/0101/24 grant.
Reference:
Furman, M. et al. (2023) 'Quantitative analysis of selected genetic markers of induced brain
stroke ischemic tolerance detected in human blood', Brain Research, 1821, p. 148590.
doi:10.1016/j.brainres.2023.148590.
Proteomic analysis of signaling pathways in the rat hippocampus after
delayed remote ischemic postconditioning
^émethová M., 2
Talian L, 2
Tkáčiková S., ^ u c h a R., ^ončeková J., Motorová K., t o n o v á
R
'institute of Neurobiology of Biomedical Research Center of the Slovak Academy of Sciences, Šoltésovej 4,
Košice, Slovakia
department of Medical and Clinical Biophysics, Faculty of Medicine, Pavol Jozef Šafárik University in Košice,
Trieda SNP 1, Košice, Slovakia
Delayed remote ischemic postconditioning (dRIPostC) is a protective procedure for
brain damage caused by ischemia/reperfusion (IR), but the mechanism of this treatment is not
yet clear. In this study, the neuroprotective effect of dRIPostC was investigated in a rat model
of cerebral IR performed by four-vessel occlusion (4VO), and the potential mechanism was
assessed by proteomic analysis. Rats were divided into groups (n=5): Sham (surgery without
ischemia), IR (10 min of 4VO and 3 days of reperfusion) and dRIPostC (10 minutes of 4VO
and 3 days of reperfusion, interrupted by 20 minutes of ischemia caused by reversible
hindlimb strangulation after 48 hours of reperfusion). The brain tissue was extracted and
processed for proteomic analyses. The LC-MS/MS data obtained were analysed using
Scaffold software and bioinformatic analysis was performed for three comparisons: IR vs.
dRIPostC, IR vs. Sham, and Sham vs. dRIPostC. Pathway analysis results were provided
using the Reactome Analysis Service. Pathways that were differentially expressed at an
adjusted p-value <0.05 were considered significantly regulated. We detected 36 down and 6
up-regulated (p<0.05) pathways in IR vs. dRIPostC; 46 down and 1 up-regulated (p<0.05)
pathway in Sham vs. dRIPostC; and 23 down and 2 up-regulated (p<0.05) pathways in IR vs.
Sham. We were mainly interested in signaling pathways with a possible neuroprotective
effect. In the IR vs. dRIPostC comparison group, we detected 8 top down-regulated signal
transduction pathways (e.g. signaling by Rho GTPases, RhoA GTPase cycle, RhoA
activation) and 2 top down-regulated programmed cell death pathways. When comparing
Sham vs. dRIPostC, we detected pathways that correlated with IR vs. dRIPostC (4 top downregulated
signal transduction pathways). The IR vs. Sham comparison revealed the top downregulated
pathways: e. g. Rho GTPase effectors, p75NTR regulate axonogenesis and RhoA
activation. Our analysis showed that dRIPostC can down-regulate pathways involved in
programmed cell death and thus may play a beneficial role in cell survival after ischemia. In
addition, delayed postconditioning acted as a down-regulator of signaling through Rho
signaling pathways. The Rho/ROCK pathway mediated by the Rho GTPase is closely
associated with neuronal apoptosis and axonal growth inhibition. Inhibition of the Rho/ROCK
pathway can play a key role in the stroke pathogenesis and its targeting may be critical for
improving the treatment of ischemic brain injury.
This study was supported by VEGA 2/0096/22, 2/0101/24 and APW-21-0069 grants.
61
Genetic forms of Parkinson's Disease in Central Europe
U
'3
Ostrozovicova M.,4
Tamas G., 5
Dusek P., 6
Grofik M . , u
H a n V., 5
Holly P., 5
Jech R.,
7
Kalinova K., 8
Klivenyi P.,9
Kovacs N., 1 , 2 1 0
Kulcsarova K., 6
Kurca E., 1 2
Lackova A., 3
Lee H.,
3 , 1 1
Lewis P., 1 , 2
Magocova V., ^arekova M., 3
Murphy D., 12
Necpal J., 9
Pinter D., *Rabajdova
M., 5
Ruzicka E., 5
Serranova T., 13
Smilowska K., 4
Soos K., 15
Straka I., 12
Svorenova T.,
1 4 1 5
Valkovic P., 16
Zarubova K., 1 2
GdovinovaZ., 3
HouldenH., 3
RizigM., u
Skorvanek M .
'UPJS, Kosice, Slovakia
2
UNLP, Kosice, Slovakia
3
UCL Queen Sq IoN, London, UK
4
Semmelweis University, Budapest, Hungary
'1st Faculty of Medicine, Charles University and General University Hospital in Prague, Czechia
6
Jessenius Faculty of Medicine, Comenius University and University Hospital Martin, Slovakia
7
Gottfried Schatz Research Center, Medical University of Graz, Austria
8
University of Szeged, Hungary
'University of Pecs, Medical School, Hungary
1 0
MEDrPARK, UPJS, Kosice, Slovakia
n
Royal Veterinary College, London, UK
12
Zvolen Hospital, Slovakia
13
Silesian Centre of Neurology Katowice Poland
14
Comenius University in Bratislava Faculty of Medicine, University Hospital Bratislava, Slovakia
"Slovak Academy of Sciences, Bratislava, Slovakia
16
2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czechia
Introduction: Parkinson's disease (PD) is a neurodegenerative disorder with complex
genetic background, where 10% of cases show a clear Mendelian inheritance. However, most
of the genetic studies were performed in the PD cohorts of North-West parts of Europe, and
there are only a few genetic data coming from Central European region. To address this gap,
this study aimed to screen for the monogenic form of PD in Central Europe.
Methods: Multicentric cohort of 1612 PD patients from 9 tertiary centres in Central
Europe, was screened using the microarray (n=1612) or whole-exome sequencing in earlyonset
and familial cases (n=220). Variants in validated and candidate disease genes and risk
factors for PD and atypical parkinsonism were further analysed. All findings were confirmed
by Sanger sequencing.
Results: In our cohort, we identified 27 PD carriers of the GBA1 risk variant, 5 of the
LRRK2 variant, 3 of bi-allelic POLG and 1 of PRKN pathogenic variant, 1 was positive for
the ATP1A3 pathogenic variant. Within the exploratory analysis, we identified additional
carriers of the ATP1A3, CSF1R, DCTN1, GBA1, LRRK2, MAPT, PDGFB and PLA2G6 rare,
potentially pathogenic variants of unknown significance. Additionally, we detected 4 PD cases
of p.L1795F LRRK2 variant, which was recently proposed as a possible strong risk factor. All
4 cases were characterised by akinetic-rigid PD phenotype with early onset of severe motor
fluctuations, 2 receiving LCIG therapy and 2 implanted with STN DBS; all 4 cases showed
unsatisfactory effect of advanced therapies on motor fluctuations.
Conclusion: Our data suggest that GBA1 and LRRK2 variants represent the majority of
the PD monogenic cases in Central Europe, where the novel p.L1795F LRRK2 may represent
the most common currently known pathogenic LRRK2 variant. Together with the ongoing
clinical trials for LRRK2 inhibitors, this finding emphasises the urgent need for more ethnic
diversity in PD genetic research.
The analysis of apoptotic and non-apoptotic roles of caspase-3 in cells of the
rat spinal cord in the postnatal period
Pačut L., Holota R., Košuth J., Matiašová A. Alexovič, Daxnerova Z., Sevc J.
Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 04154,
Košice, Slovak Republic
Apoptosis is a process with an important role in the development, function and
pathology of nervous system. During apoptotic events, a specific group of proteases known as
caspases, plays a key role, however, there is an evidence that these enzymes may be involved
also in non-apoptotic processes. Our previous experiments proved presence of large
population of cells with cleaved caspase-3 (cC3) in the nervous tissue of postnatal rat spinal
cord. We observed a high number of cC3+
cells and a very low number of cells positive for
cleaved poly(ADP-ribose) polymerase (cPARP) representing an another marker of apoptosis.
Since these data raised questions regarding the role of cC3 in this region, we hypothesized
that activity of caspase 3 could be strictly regulated in spinal cord. One of the potential
mechanism involved in regulation of cleaved caspase-3 activity could represent inhibitors of
apoptosis, known as IAPs. Therefore, we studied expression of Birc genes (Bircl-BircT)
encoding IAPs in rat spinal cord by digital PCR. The expression was studied at three stages of
postnatal ontogenesis, at neonatal - P8, adolescent - P29, adult - P90 rats. Our data revealed
that the most abundant gene transcripts represented Birc4, Birc2 and Birc5, whose comprised
more than 90 % of the entire IAP gene family. The most expressed gene was Birc4 (XIAP),
which contributed by more than 50 % to whole IAP gene transcripts pool at P8 and gradually
rised its expression throughout the ontogenetic stages. The level of Birc2 gene expression was
stable throughout the ontogenesis, without significant changes between the studied time
points. On the other hand Birc5 transcription showed an evident decline from P8 until P90.
According to our data, the activity of caspase-3 could be regulated by proteins from the IAPs
family, especially by the protein XIAP encoded by Birc4 gene or Survivin (encoded by Birc5),
which is associated with proliferation during early ontogenetic stages. The steady presence of
Birc2 expression might be explained by the involvement of its product in multiple signalling
pathways besides apoptosis. Altogether, our findings suggest possible mechanism of caspase-
3 regulation in the nervous system and the need for further research of the complex regulation
of apoptosis and elucidate also other non-apoptotic mechanisms of function of these
proteases.
Acknowledgement: This study was supported by APVV grant No. APVV-19-0279, VEGA
1/0760/20.
63
Is Angiotensin II receptor type 2 involved in potentiated intrinsic
regenerative ability in injured spinal cord?
Pavel J., Liptakova V , Hvozdikova E., Snopkova J.
Institute of Neurobiology, Biomedical Research Center of the Slovak Academy of Sciences, Kosice, Slovakia
Axonal regeneration in the CNS is a complex process influenced by both intrinsic and
extrinsic factors. The inhibitory molecules are not only critical determinant of axonal
regeneration failure, but diminished intrinsic regenerative ability of mature CNS neurons also
plays crucial role. Angiotensin II receptor type 2 (AT2) is abundant during the prenatal period,
but its expression gradually decreases with postnatal age. Interestingly, it becomes
upregulated after pathological conditions, including CNS injuries. Therefore, we studied the
time-dependent receptor expression after severe spinal cord injury in order to accurate timing
of pharmacological intervention.
Female adult Wistar rat weighing 250-320g were used to induce severe spinal cord
compression (40g weight for 15 min) at Th9 spinal level. Injured spinal cord was studied
within four weeks survival period (on 1, 3, 7, 14, 21 and 28 day). The AT2 receptors have
been continually stimulated using selective high affinity ligand CGP42112, or blocked with
PD123319 via osmotic minipumps. Hind limb motor function, body weight, bladder function,
transcranial motor evoked potentials, spinal cord histopathology as well as the gene and
protein expression of the AT2 receptor, GAP-43, neurofilaments, myelin basic protein,
CNPase, myelin proteolipid protein, and 01ig2 have been analysed.
Delayed and transient increase of the AT2 receptor expression was observed from 14th
to 21st post-injury day. However, it normalized almost to control values 7 days later. The time
period of upregulated receptor expression was selected as most accurate interval for its
pharmacological activation. The improvement of monitored functional parameters, increased
spinal cord tissue preservation, decreased number of cysts and cavities, and upregulation of
axonal outgrowth and major structural components were detected. The AT2 receptor
antagonist prevented observed beneficial effects.
We can conclude that the stimulation of delayed and transiently expressed AT2
receptors improved the observed functional parameters and potentiated the diminished
intrinsic regenerative ability of severe injured spinal cord.
The study was supported by Grants APVV-22-0248 and VEGA No. 2/0123/23.
Behavioral and functional consequences of maternal separation of various
duration in rats
Racek A., Žideková M., Martončíková M., Fabiánova K., Račeková E.
Institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
The neurogenesis hypothesis of depression assumes that postnatal production of new
neurons in the olfactory system may be causally related to depressive behaviors. To test this
hypothesis, it is necessary to investigate neurogenesis in an animal model of depression.
Maternal separation (MS), the postnatal disruption of mother-infant interaction, is a traumatic
event that can have long-term effects on brain development and subsequent function and
behavior. The aim of this study was to investigate behavioral consequences of MS of different
duration in order to find an appropriate model of separation to induce depressive-like
behavior. Our next goal was to find out the suitability of MS for investigation the
functionality of neurons arising in the postnatal neurogenesis in the olfactory system. In the
first two groups, rat pups were separated from the mother daily for 3h from the first postnatal
day (PI) till P7 or P21. The animals from these groups were allowed to survive until
adulthood, when they were tested in the emergence test and elevated plus maze test. The third
group was designed to investigate MS-induced activation of postnatally born neurons in the
olfactory bulb (OB). In this group, pups were exposed to single maternal separation (SMS) for
2h at the P21. The activation of neurons after SMS was measured by c-Fos expression. The
results of behavioral tests showed that one week as well as three weeks of MS induced longlasting
significant changes in animals behavior. These changes indicate an increase in anxietylike
behavior, or can be a manifestation of increased risk assessment. Although changes in
anxiety-like behavior were comparable at both durations of MS, locomotor activity was
significantly reduced only after three weeks of MS. This suggests a more severe behavioral
impact of MS of longer duration. Regarding functional consequence of MS, quantification of
immunohistochemically labeled c-Fos+ cells revealed that exposure to SMS induced marked
increase of the number of Fos+ cells in the OB, the structure dedicated to odor information
processing and where expression of c-Fos protein is usually evoked by odor stimulation .
Although MS is not a typical model of odor stimulation, our results indicate an involvement
of olfactory system in mediation of the MS-evoked effects. In conclusion, our results suggest
the suitability of three-week MS to induce depression-like behavior, as well as the relevance
of two-hour SMS to investigate the functionality of newly generated OB neurons.
Supported by VEGA 2/0119/22.
65
Multiple roles for Angiogenin as a new clinical target for stroke
Rosell A.
Neurovascular Research Laboratory. Vail d'Hebron Research Institute, Barcelona
Stroke is a medical condition that abruptly reduces blood flow to the brain resulting in
brain injury affecting more than 15 million people each year worldwide, and leading to a
significant number of stroke survivors with neurological deficits. With this scenario, the only
available and approved treatments for ischemic stroke are the pharmacological reperfusion
therapies to dissolve the clot/thrombus with tissue plasminogen activator or the mechanical
approaches to evacuate the occluding thrombus (thrombectomies). Although these are
effective and saving-live therapies, in general, they can only be applied to ischemic patients
within the first hours of the symptoms onset. In this scenario, for a large number of stroke
patients the only therapeutic options are the long-term rehabilitation programs. Therefore, it is
urgent to investigate for complementary strategies supporting the current reperfusion and
rehabilitation therapies. After ischemic stroke, not all cells die immediately after the blood
supply interruption and there might be hypoperfused brain tissue at risk where the cells can be
rescued with appropriate reperfusion or neuroprotection strategies. In this context, our group
has studied the role of Angiogenin (ANG) as a potential biomarker for stroke recovery and as
new acute neuroprotective therapy. As a unique ribonuclease, ANG is a small protein that
shares 33% of its sequence with the bovine pancreatic ribonuclease A. Therefore, A N G (also
named RNase 5) has been classified as a member of the ribonuclease A superfamily for its
ribonuclease activity (the activity to catalyze the degradation of R N A into smaller
components contributing to the metabolism of nucleic acids), crucial for cell bio-functions.
This unique property determines its center roles in angiogenesis, cell proliferation and cell
survival. The neuroprotection effects of A N G have been mainly described in
neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis and Parkinson's disease,
however its role in stroke disease is nowadays unclear. It will be shown how in different
studies A N G increases in ischemic tissue both in mice and human brain, and in plasma after
rehabilitation therapy, the second one related to better outcomes. Also, apart from proangiogenic
actions of human recombinant ANG on endothelial cells in vitro, ANG treatment
increases SVZ-derived Neural Stem Cells yields in culture and is found up-regulated after
physical exercise in the ipsilateral neurogenic SVZ niche after ischemia in mice compared to
control non-ischemic brains. Finally, acute post-stroke therapy in C57B1/6 male mice
undergoing transient middle cerebral artery was occluded involving the inhibition of the
apoptosis signaling pathway. Overall, the presented data supports A N G as a potential
treatment for acute ischemic stroke in a clinically-relevant scenario and associates its
increased levels during post-stroke recovery with better outcomes.
Phenotypical, genotypical and pathological characterization of the
moonwalker mouse, a model of ataxia
'Sekerkova G., ^ i l i c S., ^heng Y.-H., 2
Fredrick N., ^smani A., fern H., 2
Opal P.,1
Martina
M .
Department of 'Neuroscience and 2
Neurology, Northwestern University, Feinberg School of Medicine, 300 E.
Superior, Chicago, IL, 60611
Spinocerebellar ataxias (SCAs) are a group of debilitating genetic diseases
characterized by cerebellar dysfunction, specifically by loss of balance and coordination. No
cure is currently available for SCAs. A critical factor in the lack of available treatment is that
the cellular mechanisms of SCAs are poorly understood. Two SCAs, SCA14 and SCA41, are
caused by transient receptor potential channel 3 (TRPC3) gain of function. Notably, a TRPC3
gain of function mutation had been identified in a mouse ENU ataxia mutagenesis screen and
dubbed the Moonwalker (MWK) mouse. In this study we undertook a detailed
characterization of the natural course of the disease, the cellular pathology, and the possible
disease mechanisms. Our analysis of the M W K phenotype shows a non-progressive ataxia
that is first detectable around P17 and characterized by a broad spectrum of signs. The M W K
mouse shows numerous behavioral symptoms including tremor, altered gait, circling behavior,
impaired motor coordination, and impaired motor learning. Cerebellar pathology is
characterized by early postnatal loss of unipolar brush cells (UBCs) and a slowly progressive,
moderate Purkinje cell (PCs) loss after 3 months of age. Interestingly, no obvious correlation
was observed between PC loss and severity of motor symptoms. This is probably due to the
fact that PC function is severely impaired much earlier than the appearance of PC loss.
Indeed, PC firing is already impaired in 3 weeks old mice. Notably, structural damage to PCs
also precedes the PCs death by several weeks. At postnatal day 40 the synaptic contacts
between PC spines and parallel fibers are severely decreased and the PC axons show signs of
degeneration. Genetic analysis of 21-day old M W K cerebella shows, among other alterations,
changes in neuronal development and in synaptic signaling. Our results indicate that the gain
of function TRPC3m w k
mutation affects the development and network integration of PCs and
UBCs and causes PC dysfunction and ultimately PC loss. Finally, our data suggest that PCs
activate specific mechanisms to defend themselves from cell death (possibly in a lobuledependent
fashion). Understanding these mechanisms might help developing innovative
strategies to treat neurodegenerative diseases.
67
Emission of 50-kHz ultrasonic vocalizations in hemiparkinsonian rats as a
new preclinical approach to study the affective properties of drugs used in
the dopamine replacement therapy of Parkinson's disease
Serra M., Marongiu J., Simola N., Costa G.
Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy
Dopamine replacement therapy used in Parkinson's disease (PD) may induce
alterations in the emotional state, which have been implicated in the manifestation of
iatrogenic psychiatric-like disturbances. Preclinical investigations in this field are currently
limited, since few reliable paradigms are available. To provide a relevant experimental tool in
this respect, we evaluated: i) the effects of dopaminomimetic drugs on the emission of 50-kHz
ultrasonic vocalizations (USVs), a behavioral marker of positive affect, in rats bearing a
unilateral lesion with 6-hydroxydopamine in the medial forebrain bundle; ii) the associated
modifications in neuronal activation in subcortical and cortical regions of the dopaminedenervated
hemisphere. A first cohort of hemiparkinsonian rats received apomorphine (2 or 4
mg/kg, i.p.), L-3,4-dihydroxyphenilalanine (L-DOPA, 6 or 12 mg/kg, i.p.), or pramipexole (2
or 4 mg/kg, i.p.) in a test cage (x 5 administrations) on alternate days. Seven days after
treatment discontinuation, rats were re-exposed to the test cage to measure conditioned calling
behavior and thereafter received a drug challenge. Hemiparkinsonian rats treated with either
apomorphine or L-DOPA, but not pramipexole, markedly vocalized during repeated treatment
and after challenge, and showed conditioned calling behavior. Moreover, apomorphine, LDOPA
and pramipexole elicited different patterns of 50-kHz USV emissions and rotational
behavior, indicating that calling behavior in hemiparkinsonian rats treated with
dopaminomimetic drugs is not a byproduct of motor activation. A second cohort of rats
received an acute or repeated (x 5, on alternate days) administration of apomorphine (2
mg/kg, i.p.) or L-3,4-dihydroxyphenilalanine (L-DOPA, 12 mg/kg, i.p.) and were evaluated
for immunoreactivity for Zif-268, a marker of neuronal activation, in the nucleus accumbens
(NAc), caudate-putamen (CPu) and medial prefrontal cortex (mPFC), which are brain regions
that regulate the affective properties of drugs. Acute and repeated treatment with either
apomorphine or L-DOPA stimulated the emission of 50-kHz USVs in hemiparkinsonian rats,
and this effect was paired with increased Zif-268 immunoreactivity in the NAc and striatum,
but not mPFC. These findings indicate that subcortical and cortical areas may differently
regulate the emission of 50-kHz USVs in hemiparkinsonian rats treated with dopaminergic
drugs used in the DRT of PD. Taken together, our behavioral and neurochemical results
suggest that measuring 50-kHz USV emissions may be a relevant experimental tool for
studying how dopaminomimetic drugs modify the affective state in parkinsonian rats.
Carotid endarterectomy - potential induction of ischemic tolerance in
clinical praxis
'Sihotsky V , 2
Mucha R., ^opolovets 1,2
Furman M., 2
Nemethova M., Virag M.
'Cardiocenter AGEL Kosice-Saca, Lucna 57, 04015 Kosice - Saca
institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
'Eastern Slovak Institute of Cardiovascular Diseases and Faculty of Medicine, Pavol Jozef Safarik University,
Ondavska 8, 040 01 Kosice, Slovakia
Stroke is the second most frequent cause of death and the most common reason for
older population disability in developed countries. Stenosis of the internal carotid artery (ICA)
can also contribute to stroke. Carotid endarterectomy (CEA) represents the prevention of
stroke in patients with ICA stenosis. CEA is indicated in patients with asymptomatic stenosis
of ICA over 60% with a presence of minimally one risk factor of stroke. In patients with
neurological symptoms, C E A is indicated when ICA stenosis is over 50%. Major
postoperative complications after C E A are stroke and death. The rate of major, severe
postoperative complications should not exceed 3% in asymptomatic patients and 6% in
symptomatic patients. CEA can be performed under general anesthesia or in locoregional
anesthesia. It is essential to ensure blood flow in the ICA during clamping of carotid arteries.
For that purpose, shunts are inserted into carotid arteries. More methods are used to monitor
blood perfusion during the clamping of carotid arteries, such as monitoring consciousness
during local anesthetics and transcranial Doppler stump pressure. Our department uses
transcranial cerebral oximetry and hasgoodexperience with it. Carotid endarterectomy can be
performed as a conventional or eversion.We prefer eversion endarterectomy.
We found eversion carotid endarterectomy in general anesthesia as the optimal
method, and transcranial cerebral oximetry was used to save monitoring of brain perfusion
during the operation.
69
Advancing spinal cord injury repair through multidisciplinary herapeutic
approaches
u
S i l v a N .
'Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga,
Portugal
2
ICVS/3B's Associate Lab, PT Government Associated Lab, 4806-909 Braga/Guimaraes, Portugal
Spinal cord injury (SCI) is a devastating neurological disorder that strongly impacts
the physiological, psychological, and social behaviour of those affected. The spinal cord
trauma, known as "primary injury", triggers a cascade of events, termed "secondary injury",
leading to further neurological damage and contributing for regeneration failure after SCI.
These include glutamate excitotoxicity, a potent inflammatory response, the release of
molecules that inhibit axonal growth and the formation of a glial scar. It is urgent to develop
new and effective therapeutic strategies. Several important contributions for the field have
been made by Silva's Lab, namely by: 1) Introducing new molecular therapies that are able to
protect the neural tissue after a SCI, leading to functional recovery of paralyzed animal
models; 2) Identifying new mechanistic basis of the dysfunctional regulation of the
inflammatory response mounted after SCI. We demonstrate that temporal changes in the
sympathetic signaling to the spleen are associated with neutrophil infiltration into the spinal
cord; 3) Pioneering the use of macrophage-derived secretome as a molecular therapy of Spinal
Cord Injury repair. We demonstrated that polarized macrophages-derived soluble factors and
extracellular vesicles are a promising therapy for SCI; and 4) Development of novel therapies
for SCI repair based on Biomaterials and Tissue Engineering. This lecture will explore recent
breakthroughs from our lab, shedding light on future directions in SCI treatment and repair.
The stimulation of AT2 receptors can promote an angiogenic response after
severe spinal cord trauma
Snopkova J., Liptakova V , Hvozdikova E., Pavel J.
Department of Neurodegeneration, Plasticity and Repair; Institute of Neurobiology, Biomedical Research Center
of the Slovak Academy of Sciences, Kosice, Slovakia
Traumatic spinal cord injury is a serious medical condition that can result in
permanent functional deficits for patients. The injury causes local disruption of vascular
integrity, leading to ischemia, inflammation, and tissue damage. Therefore, blood supply
recovery is crucial for the regeneration of the injured spinal cord tissue. This study examines
the activation of Angiotensin II type 2 receptors (AT2 receptors) and their involvement in the
formation of new blood vessels, their integration into functional vasculature, and the
evaluation of their overall impact on functional improvement. The pharmacological
stimulation of the A T 2 receptor was conducted in an experimental model of severe spinal cord
compression in adult female Wistar rats using the selective A T 2 receptor agonist CGP42112
(0.1 mg/kg per day) continuously administered by osmotic minipumps (s.c). Within four
weeks after trauma, the expression profile of the A T 2 receptor in the injured spinal cord
correlated with the increased permeability of the blood-spinal cord barrier, as analysed
through Evans blue tracing, and with the expression of vascular endothelial growth factor-A
(VEGF-A). After 28 days following the trauma, our analyses using the qRT-PCR and ELISA
showed an increase in the expression of proangiogenic markers including VEGF-A and its
receptor, angiopoietin 1 and 2, and PECAM. This was observed after the stimulation of A T 2
receptors, compared to spinal cord injury alone. These results were also consistent with
functional recovery. During the 28-day post-traumatic period, the motor function recovered
rapidly, and the improvement was more significant after A T 2 receptor stimulation compared
to spinal cord injury alone (BBB locomotor score: 10.4 points vs. 9 points). The improvement
was strongly negatively correlated (Pearson r = -0.908) with a notably shorter latency (7.03
ms vs. 10.8 ms). Many of these positive effects were partially or completely prevented by
blocking the A T 2 receptor. Our results indicate that the A T 2 receptors are likely involved in
revascularization, and their activation may promote the proangiogenic signalling pathway in
the injured spinal cord.
The study was supported by VEGA grant No. 2/0123/23 and APVV-22-0248.
71
Induced pluripotency and its potential in modeling of oncologic and
neurodegenerative diseases
Strnadel J., Mersakova S., Braný D., Chodelkova O., Hajduchova D., Suroviakova S.,
Mitruskova B., Novákova S., Skovierova H., Pokusa M., Pec M.J., Pecová R., Marcinek J.,
Nosál V , Kurca E., Plank L., Kalman M., Halasova E.
Jessenius Faculty of Medicine in Martin (JFM CU), Comenius University in Bratislava, Slovakia
Induced pluripotency is a state-of-the-art technique that allows the reprogramming of
any fully differentiated somatic cell into stem cells. This technology, developed in 2006 by
Japanese researcher Shinya Yamanaka and awarded with Nobel Prize in 2012 opened new
fascinating horizons in the field of regenerative medicine as well as in the field of in vitro
disease modeling. The research team of Cellphie lab at Jessenius Faculty of Medicine in
Martin previously utilized this technique to prepare novel cell models or cell lines for
modeling neurodegenerative diseases. The first Slovak induced pluripotent stem cell lines
modeling sporadic form of amyotrophic lateral sclerosis and later Duchenne muscular
dystrophy were already prepared by our team. The development of other cell lines modeling
neurodegenerative diseases is currently in progress in our lab. Interestingly, we found that the
reprogramming approach seems to be also applicable for the generation of special, 3D culture
cancer cell lines from different types of tumors like neuroendocrine tumors, pancreatic ductal
adenocarcinoma and colorectal cancer. Protocol designed and later modified by our team
already led to the development of two unique cancer cell lines that were accepted by the
American Tissue Culture Collection (ATCC) repository for general distribution to the
researchers. Induced pluripotency and cell reprogramming towards a less differentiated state
probably makes the hard-to-survive cancer cells isolated from tumor tissues more aggressive
and more stem cell-like, thus promoting the cancer cells to be more effectively seeded on the
plates. In conclusion, induced pluripotency is an extremely useful technique for the
preparation of cell models in the field of neurology as well as oncology.
Acknowledgment: The project described in this lecture was supported by research grant
No.APVV-21-0372 from Slovak Research and Development Agency.
Mesenchymal stem cells conditioned medium affects neurons and glial cells
differently depending on the time of conditioning
Szekiova E., Michalova Z., Blasko J., Vaničky I.
Institute of Neurobiology Biomedical Research Center, Slovak Academy of Sciences, Kosice, Slovakia
Mesenchymal stem cells (MSC) represent a source of bioactive substances capable of
inducing the survival and regeneration of nerve cells. Recent studies with MSC conditioned
medium (MSC-CM) have suggested the possibility of its use in regenerative medicine instead
of whole cells. The aim of this in vitro study was to monitor the paracrine effect of rat bone
marrow-derived mesenchymal stem cells (BMMSCs), using conditioned media prepared by
conditioning BMMSCs for 24 hours (CM24), 48 hours (CM48), and 72 hours (CM72) in
serum-free medium. We investigated the effect of these CMs on neurite length and density of
DRG neurons and the proliferative activity of D R G glial cells . Proteomic profiles of
individual secretomes were analyzed by mass spectrometry, and the concentrations of four
selected neurotrophins (BDNF, NGF, GDNF, and VEGF) were determined using ELISA.
Proteomic analysis revealed an increased number of proteins in CM72, many of which
are involved in neuroregenerative processes. ELISA documented a gradual increase in the
concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro data suggest a
different effect of BMMSC-CM on glial cell population and neurite outgrowth.
Our results indicate variability in the number and representation of identified specific
proteins, concentrations of monitored neurotrophins, and the effect of C M on individual cell
populations under in vitro conditions. Findings from the in vitro part of the study suggest that
media obtained after long-term conditioning may have a more significant impact on the
density/proliferation of glial cells, while media obtained from earlier conditioning times more
significantly affected the neurite outgrowth and density.
Fundings: VEGA 2/0109/24, VEGA 2/0120/23
73
Neonatal nervous tissue exhibits superior reparative potential compared to
the mature nervous tissue after minimal spinal cord injury
Sevc J., Mochnacký R, Košuth J., Mariášová A. Alexovič, ' Slovinská L., Blaško J.,
^ukhun L, holota R., 4
Tomori Z., Daxnerova Z.
'institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 04154
Košice, Slovak Republic
associated Tissue Bank, Faculty of Medicine, P. J. Šafárik University in Košice and L. Pasteur University
Hospital, Tr. SNP 1, 04011 Košice, Slovak Republic
'institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 04001
Košice, Slovak Republic
institute of Experimental Physics, Watsonova 43, Slovak Academy of Sciences, 04001 Košice, Slovak Republic
Spinal cord injury (SCI) resulting from trauma decreases the quality of human life.
Numerous clues indicate that the limited endogenous regenerative potential is a result of the
interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions
of immune and glial cells. Knowledge gained from comparing regeneration in adult and
juvenile animals could draw attention to factors that should be removed or added for effective
therapy in adults. Therefore, we generated a minimal spinal cord injury (mSCI) model with a
comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the
neonatal period. The mechanism of injury is based on unilateral incision with a 20 gauge
needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal
segment. The incision should harm a similar amount of gray matter on a coronal section in
each group of experimental animals. According to our results, the impact causes mild injury
with minimal adverse effects on the neurological functions of animals, but still has a
remarkable effect on nervous tissue and its cellular and humoral components. Testing the
mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory
response of immune cells and astrocytes at the lesion site, as well as increased proliferation in
the central canal lining in neonates compared with adult animals. Our results indicate that
developing nervous tissue could possess superior reparative potential and confirmed the
importance of comparative studies to advance in the field of neuroregeneration.
This study was supported by VEGA grants no. 1/0820/17 and by Slovak Research and
Development Agency under the contracts No. APVV-19-0279.
Keywords: spinal cord injury, inflammation, development, glial scar, neural stem cells
Negative perception of a stressful situation is not associated with salivary
concentrations of the stress hormone Cortisol in toddlers
^Vancova A., ^omanovaZ., ^ezovaD.
'institute of Experimental Endocrinology of the Biomedical Research Center, Slovak Academy of Sciences
department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia
A negative perception of stressful situations early in life may represent a greater risk
for short-term and long-term negative outcomes including neurological and psychiatric
disorders, throughout the lifespan. Stressor paradigms used in toddlers, such as a separation
from a caregiver or exposure to a novel object or stranger have not proven an association with
stress hormone concentrations. The present study aims to test the hypothesis that a negative
perception of a different stressor correlates with salivary concentrations of the stress hormone
Cortisol during a still-face paradigm. The sample consisted of 41 toddlers (17 girls and 24
boys) aged 7 - 9 months accompanied by their mothers. The still-face paradigm consisted of
three phases. During the first phase (3 min), the mother was playing with the baby. During the
second phase (2 min) the mother looked at the baby with her face remaining still. This phase
was followed by a reunion (3 min). The mother and her baby were left in the examination
room alone while being recorded by 2 cameras. The saliva for Cortisol measurements was
collected before the task and 20 min thereafter. The baby's negative perception of the situation
was evaluated from the videos as the time spent crying and the latency to cry using the
program H77+. The results failed to confirm the association between Cortisol concentrations
and signs of negative stress perception measured. Numerically, the boys started to cry earlier
and spent more time crying than girls, however, the difference failed to reach statistical
significance. The latter finding is supportive of the suggestion that early life stress puts males
at an increased risk of presenting with neurodevelopmental disorders.
Supported by VEGA (grant No. 1/0644/23)
75
A model for studying long distance axonal regeneration in the rat
Vaničky I.
Institute of Neurobiology, Biomedical Research Centre, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice
In clinical practice, the prognosis after segmental injuries to the peripheral nerve with
gaps longer than 3cm is unfavorable. It is known that regenerating axons are capable of
growing for long distances within the preserved distal stump. It is not clear why the growth
fails within long grafts or graft alternatives after nerve reconstruction.
For understanding this paradox, a model that allows removal of long segments of the
peripheral nerve is necessary. This can be obviously made in large animal models, but these
are expensive and laborious. In our study, we document that clinically relevant segmental
injuries can be made in small laboratory animal, as well. In the rat, tail nerves are the longest
peripheral nerves in their body. We suggest that ventral caudal nerve (VCN) may serve as a
model for studying segmental nerve injuries and long distance regeneration.
For this purpose, we have studied the anatomy and morphometry of the V C N in
control animals. 10 cm long segment of the V C N was removed, and transversal sections were
collected at 10 mm distances. The myelinated axons were counted, and the series of data were
used to characterize the craniocaudal tapering of the nerve. In a separate group of animals,
retrograde tracing with Fluorogold was used to localize and quantitate the spinal neurons
projecting their axons into the VCN.
After complete nerve transection, the time course of histopathological changes in the
distal segment was studied. The primary goal was to define the time needed for axonal
disintegration. In later periods, axonal debris removal and rearrangement of tissue elements
was documented.
After compression injury (axonotmesis), Wallerian degeneration was followed by
spontaneous regeneration of axons. We show that the growing axons will span the 10 cm
distance within 4-8 weeks. After different survival periods, the numbers of regenerating axons
were counted at 10mm distances. These data were used to characterize the dynamics of axonal
regeneration during 4 months' survival period. In the present study we show that axonal
regeneration across 10 cm distance can be studied and quantitatively analyzed in a small
laboratory animal.
Supported by VEGA 2/0120/23.
Heart Rate Variability in evaluation of autonomic dysfunction in idiopathic
REM-sleep behaviour disorder
u
Ventosa J.R., u
Kulcsárová K., 3
Mertová L., ^lejárM., 1 , 2
Škorvánek M., 1 , 2
Gdovinová Z.,
1 2
' Feketeová E.
department of Neurology, Medical Faculty, University of Pavol Jozef Safarik, Kosice
department of Neurology, Louis Pasteur University Hospital, Kosice
3
IBM Slovensko, spol. s r.o., Kosice
Introduction: Nearly 80% of people diagnosed with idiopathic R E M sleep behaviour
disorder (iRBD) via video-polysomnography (v-PSG) are expected to be in the prodromal
stage of an alpha-synucleinopathy. Signs of autonomic dysfunction can appear earlier than
motor or cognitive alpha-synucleinopathy symptoms. Heart Rate Variability (HRV) can
potentially be an objective measurement of autonomic dysfunction, and furthermore can be
obtained directly from v-PSG.
Aim: The aim of this study was to evaluate dysautonomia in iRBD subjects using
HRV obtained during different sleep stages and wakefulness from v-PSG.
Methods: Subjects positively screened by an RBD screening questionnaire (RBD-SQ)
underwent v-PSG to diagnose RBD. HRV obtained from v-PSG recordings was correlated to
dysautonomia evaluated from a Non-Motor Symptoms Scale (NMSS) questionnaire. Optimal
cut-off values of HRV parameters to predict dysautonomia were calculated using receiver
operating characteristics (ROC) - area under the curve (AUC) analysis. The effect of
confounder variables was predicted with binomial logistic regression and multiple regression
analyses.
Results: Out of 72 positively screened subjects, 29 subjects were diagnosed as iRBD
(mean age 66 ± 7.7 years) by v-PSG. Eighty-three per cent of the iRBD subjects in our cohort
were at the time of diagnosis classified as having possible or probable prodromal Parkinson's
Disease (pPD) compared to zero subjects being positively screened in the control group. The
iRBD-positive subjects showed significant inverse correlations of NMSS score, particularly to
log low-frequency (LF) component of HRV during wakefulness: r = -0.59 (p = 0.001). Based
on ROC analysis and correlation between NMSS score, log LF during wakefulness (AUC
0.74, cut-off 4.69, sensitivity 91.7%, specificity 64.7%, p = 0.028) was considered as the most
accurate predictor of dysautonomia in the iRBD group. Apnoea-hypopnoea index (AHI)
negatively predicted dysautonomia in the iRBD group. None of the HRV components was
able to predict the presence of iRBD in the full cohort. Age, gender, and PSG variables were
significant confounders of HRV prediction.
Conclusion: The presented study did not confirm the possibility of using HRV from vPSG
records of patients with iRBD to predict dysautonomia expressed by questionnaire
methods. This is probably due to several confounding factors capable of influencing HRV in
such a cohort.
Key words: RBD Screening Questionnaire; dysautonomia; heart rate variability; idiopathic
R E M sleep behaviour disorder.
77
The role of sphingosine-l-phosphate receptor modulator (Siponimod) in
spinal cord injury
'Wencel P.,2
Kisucka A., 3
Motyl J.,2
LukacovaN., 'Strosznajder R.
'Laboratory of Preclinical Research and Environmental Agents, Mossakowski Medical Research Institute, Polish
Academy of Sciences (PAS), Warsaw, Poland
institute of Neurobiology of Biomedical Research Centre of Slovak Academy of Sciences, Kosice, Slovakia
'Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical
Engineering, Polish Academy of Sciences, Warsaw, Poland
Spinal cord injury (SCI) is a pathological condition associated with excessive
activation of microglia that leads to the overproduction of proinflammatory cytokines and
causes neuronal injury. The molecular mechanisms of this process are still not fully elucidated
and therapies for SCI are marginally effective. Sphingolipids are bioactive lipids that
represent an interesting therapeutic target for SCI. Sphingosine-l-phosphate (SIP) is a
sphingolipid generated from sphingosine by sphingosine kinases (SPHK1 and -2) that plays
an important role in mediating inflammation, cell proliferation and survival.
This study aimed to examine the mRNA levels of enzymes and receptors engaged in
SIP signalling as well as pro-survival sirtuin 1 (SIRT1) in the cerebellum and spinal cord
(SC) of rats after SCI. Moreover, the effect of Siponimod (SIPO, SIP receptor (S1PR) 1&5
modulator) was evaluated.
Female Wistar Rats (weight 300g±30g) were used in the experiment and divided into
the three experimental groups: Control, Compressed (COMP) and Compressed rats that
received SIPO (i.p. lmg/kg). Control and COMP animals received only vehicle (DMSO). SCI
was induced by compression at Th9 vertebral level. The spinal cord was exposed very
carefully and compressed using a device with a weight of 40g for 15 minutes. SIPO was
administered 30 minutes after the compression. After 7 days of SIPO administration
cerebellum and cranial segment (above site of injury) of SC was isolated for qPCR analysis.
All experiments were analysed using one-way ANOVA followed by Tukey's post-hoc test
(n=4-6 rats/group).
We observed a significant reduction of Sphk2, Slpr7,-5,-5 and Sirtl gene expression in
both cerebellum and SC of COMP rats. The Sphkl mRNA levels remained unchanged in both
analysed structures. SIPO administration further downregulated Sphk2 mRNA levels in
cerebellum and upregulated its expression in SC. Moreover, SIPO showed a tendency to
elevate mRNA levels of Slprl in SC and Slpr5 in both structures.
These results indicate that genes encoding SIP signalling proteins and SIRT1 are
altered after SCI. We also showed that SIPO acts differently on Sphk2 expression and this
effect is structure/region-dependent. Obtained results suggest that modulation of SIP
receptors can be considered as a potentially important factor in the pathology of SCI.
Supported by the PAS-SAS Polish - Slovak Joint Project No.8 for the years: 2023-2024, PASSAS-2022-04,
APVV-19-0324, N C N grant 2019/32/C/NZ4/00455 and statutory budget of
MMRI PAS, theme No.7.
Bridging the Gap: P2X7 Receptor as a Molecular Link Between o>
Synuclein Toxicity, Parkin Dysfunction, and Mitochondrial Impairment
Wilkaniec A., Czapski G.A., Gawinek E., Olech-Kochanczyk G., Lenkiewicz A., Babiec L.,
Adamczyk A.
Mossakowski Medical Research Institute, Polish Academy of Sciences, Department of Cellular Signalling, 5
Pawihskiego 5, 02-106 Warsaw, Poland
While both a-Synuclein (a-Syn) and Parkin abnormalities and their associated
dysfunctions are implicated in the molecular pathways of Parkinson's disease (PD), in this
study, we investigated functional relationships between these two proteins using various in
vitro and in vivo models. Our findings reveal a direct connection between the downregulation
of Parkin and extracellular a-Syn signaling. Our in vitro study showed that a-Syn-induced
oxidative/nitrosative stress plays a crucial role in the S-nitrosylation of Parkin, leading to its
impaired function and degradation. Also in the murine model of a-synucleinopathy, the
progressive decrease in Parkin protein was observed in the midbrain. Furthermore, the
reduction in Parkin levels triggered by a-Syn treatment largely depended on activating the
purinergic P2X7 receptor. Extracellular a-Syn - evoked P2X7 receptor activation resulted in
mitochondrial impairment and the decrease of Parkin level, ultimately causing the loss of
Parkin-mediated mitophagy and accumulation of the dysfunctional mitochondria. Conversely,
elevating Parkin levels prevented mitochondrial dysfunction and oxidative stress induced by
exogenous a-Syn. These findings provide compelling evidence for the direct association of
P2X7 receptors activation, and Parkin dysfunction with extracellular a-Syn, highlighting its
critical role in the pathophysiology of PD.
Funding: Narodowe Centrum Nauki, grant 2020/39/I/NZ4/01031
79
Mechanism of Selective Neurodegeneration after Global Brain Ischemia.
Proteasomal versus Endoplasmic Reticulum Stress
'Ziakova K., 2
Kovalska M . , ^ilchova L, 3
Dibdiakova K., ^rodnanova M . , ^okusa M . ,
4
Kalenska D., 3
Racay P.
'Biomedical Center, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin
Slovakia
department of Histology and Embryology, Jessenius Faculty of Medicine in Martin, Comenius University in
Bratislava, Martin, Slovakia
'Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in
Bratislava, Martin, Slovakia
4
Department of Anatomy, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin
Slovakia
A brief period of transient global brain ischemia leads to selective ischemic
neurodegeneration associated with death of hippocampal CA1 pyramidal neurons days after
reperfusion. The mechanism of such selective and delayed neurodegeneration is still
uncertain.
Our work aimed to study the involvement of proteasomal and endoplasmic reticulum
(ER) stress in ischemic neurodegeneration. We have performed laser scanning confocal
microscopy analysis of brain slices from control and experimental animals that underwent
global brain ischemia for 15 minutes and varying times of reperfusion. We have focused on
PUMA, a proapoptotic protein of the Bcl-2 family overexpressed in response to both
proteasomal and ER stress, and p53, which controls expression of PUMA. We have also
examined the expression of HRD 1, an E3 ubiquitin ligase that was shown to be overexpressed
after ER stress. We have also examined potential crosstalk between proteasomal and ER stress
using cellular models of both proteasomal and ER stress.
We demonstrate that global brain ischemia is associated with an appearance of distinct
immunoreactivity of PUMA and p53 in pyramidal neurons of the CA1 layer of the
hippocampus 72 hours after ischemic insults. Such changes correlate with a delay and
selectivity of ischemic neurodegeneration. Immunoreactivity of HRD1 observed in all
investigated regions of rat brain was transiently absent in both CA1 and CA3 pyramidal
neurones 24 hours after ischemia in the hippocampus, which does not correlate with a delay
and selectivity of ischemic neurodegeneration. We do not document significant crosstalk
between proteasomal and ER stress.
Our results favour dysfunction of the ubiquitin proteasome system and consequent
p53-induced expression of PUMA as the main mechanisms responsible for selective and
delayed degeneration of pyramidal neurons of the hippocampal CA1 layer in response to
global brain ischemia.
This work was supported by Vedecká grantová agentúra Ministerstva školstva, vedy, výskumu
a športu Slovenskej republiky a Slovenskej akadémie vied (Grants 1/0299/20 and 1/0183/23).
Effect of early chronic stress on proliferation and migration of neuroblasts
in the rat rostral migratory stream
Žideková M., Martončíková M., Székiová E., Fabiánova K., Racek A., Račeková E.
Institute of Neurobiology, Biomedical Research Center, Slovak Academy of Sciences, Šoltésovej 4, 040 01
Košice, Slovakia
Postnatal neurogenesis in the subventricular zone (SVZ) - rostral migratory stream
(RMS) -olfactory bulb (OB) is a complex process that involves different phases of neuronal
maturation: proliferation, migration, differentiation of progenitor cells, and integration of new
neurons into existing neuronal circuits. It has been found that individual processes of
neurogenesis in the SVZ-RMS-OB can be influenced by environmental factors such as
different types of radiation, stress, enriched environment, etc. The aim of this study was to
investigate the effect of chronic early stress induced by separation of rat pups from the mother
during the first three postnatal weeks (daily for 180 min.) on proliferation and migration of
neuronal precursors in the RMS. Pups exposed to separation were allowed to survive until
adulthood. To assess cell proliferation, brain sections were processed for
immunohistochemistry, using Ki-67 antibody against an endogenous marker of proliferation.
Proliferating cells were counted by Disector software in three anatomical parts of the RMS,
the vertical arm, the elbow and the horizontal arm. For analyzing neuroblast migration, the
RMS was dissected out of the brain sections, and the RMS explants were cultured in vitro for
72 hours. Then, the explants were processed for immunohistochemistry using antibody
against neuroblasts (anti-Dcx). Neuroblast migration was quantified by measuring the area of
migration and longest migration length for each explant using ImageJ software. We found that
chronic early stress caused by maternal separation resulted in significant decrease in cell
proliferation in all parts of the RMS of adult rats in comparison with control animals.
Quantitative analysis of neuroblast migration based on explants methodology showed nonsignificant
increase in both parameters, the area of migration and longest migration length in
group of animals that underwent chronic early stress. Furthermore, in this group of rats, the
neuroblasts of the RMS explants tended to migrate individually, rather than in chains, in
contrast to control animals. We can conclude that chronic early stress caused by maternal
separation had an impact on neurogenic processes such as cell proliferation and migration in
the RMS of adult animals.
Funding: VEGA 2/0119/22, APVV-19-0279
81
PPAR-a synthetic ligands and their effect on the level of mRNA expression
related to redox state and mitochondria proteins in different parts of the
brain in an animal model of Alzheimer's disease
1 * 2 1
Zulinska S., Czubowicz K., Strosznajder J.B.
'Department of Cellular Signaling and laboratory of Preclinical Research and Environmental Agents,
Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland
Peroxisome proliferator -activated receptor a (PPAR-a) is a ligand activated
transcription factor. Recently genome-wide association studies and several novel data
indicating promising neuroprotective effect of PPAR-a.The last results suggest that PPAR-a
pharmacological ligands could be repurposed for treatment of Alzheimer's disease (AD) and
other neurological and psychiatric disorders.
Our study aimed to analyze the effect of PPAR-a synthetic ligands: Fenofibrate (FF)
and GW7647 on transcription of genes involved in amyloid P metabolism and mitochondria
function in different parts of the brain in an animal AD model.
The studies were carried out using familiar A D transgenic mice with" London"
mutation (V717I) of 3 and 12 month old treated with GW 7647 s.c. in a dose of 5mg/kg body
weight (bw) or with Fenofibrate (FF) i.p. in a dose of 30mg/kg bw during 14 days. Control
animals were treated with appropriate solvents respectively. The brain cortex and
hippocampus were used for analysis. Quantitative qPCR and immunochemical, biochemical
methods were applied.
The role of PPAR-a synthetic ligands in genes expression was evaluated on mRNA
and protein levels in the brain parts of 3 and 12 month old AD Tg mice. Our study indicated
that transcription of genes encoded enzymes involved in free radical homeostasis (such as
Sod2, Sirtl and Parpl) was significantly decreased in the brain cortex of 3-month-old AD Tg
mice. Moreover, our last data showed that expression of genes coding proteins of
mitochondria biogenesis (Ppara, Ppargcla, Nrf2 and Tfam) were down regulated in the brain
cortex of 12-month-old A D Tg mice. GW7647 enhances the expression of genes related to
mitochondria biogenesis as compared to control mice (without transgene). However, the
current study demonstrated that in the hippocampus GW7647 decreased the level of mRNA
genes engaged in mitochondria biogenesis and likely affected their turnover as well. It is
suggested that GW7647 may affect brain cortex and hippocampus differently. In the following
studies, the effect of FF in A D Tg mice was investigated and our data revealed that FF
activates selected genes involved in mitochondria dynamic {Mfnl, Opal) and also enhances
the transcription of Nrf2, Tfam, involved in mitochondria biogenesis in the brain cortex of 12
month old AD Tg mice. Moreover, FF influences expression of selected genes related to Ap
metabolism (decreases Bacel, Psen2 and activates AdamlO, Ide) in the brain cortex of AD Tg
mice. It is suggest that FF may exert effect on Ap homeostasis in the brain of AD Tg mice.
Our results indicate novel aspects of the molecular mechanism of PPAR-a agonist's
action, which may depend on the brain parts and age of AD Tg mice. However, further studies
are necessary to better understand the role of PPAR-a ligands in AD.
Supported by National Science Center (PL) Grant 2019 /35/N/NZ4 /03706
Errata
Current approach in thrombolytic therapy research - how to break the
translational block
u
Hložková J.*, u
Scheer P.*, 2 3
0ndruš J., Goliášová S., *Aksu Davut A., u
Brhelová E.,
^iskupič J., kuchyňka M., 3
Novobilský A., 1 , 2
Mikulík R.
'Masaryk University Brno
2
St. Anna's Faculty Hospital - International Clinical Research Centre Brno
'Veterinary Research Institute Brno
*Both authors contributed equally to the work
In vivo preclinical research on thrombolytic therapy of stroke is complicated in terms
that the thrombolysis efficacy and the thrombolysis treatment safety cannot be evaluated
simultaneously. Therefore, we developed two models for testing new thrombolytics and new
dual treatment methods.
The thrombolysis "efficacy model" is performed with continuous X-ray scanning of
artificial clots embolized into the systemic circulation in rats. Artificial clots are human fibrin
based and barium labelled. The data measured are the area under the thrombolytic curve
(%*min), the lysis rate (%/min) and the relative change in clot area (%) over time. The model
is time-efficient with data amplification, because three artificial clots are administered per rat.
The model has zero mortality (0 from 174) and in animal including to study is 100% success
rate (174 from 174) in minimally one clot lysis tracking/rat.
The safety study model is based on "classic" thromboembolic Middle Cerebral Artery
Occlusion and uses the same type of clot as the "efficacy model". However, accepted is all
embolization to the large cerebral vessels (anterior, middle, or posterior cerebral artery).
Thrombolysis is initiated four hours after stroke induction. After 24 hours, the brain is
impregnated for micro computed tomography (miCT) examination and haemorrhagic
transformation is detected and quantified. Brain sections can be further processed using
topographic mass spectrometry (LA-ICP-MS) and histology. Ex-vivo miCT scan allows
repetitive image analysis in post scanning time in different type picture analysis or re-analysis
in new setup (e.g., prospectively with the help of artificial intelligence). We have repeatedly
used the developed models to test novel thrombolytics or thrombolytic procedures and to
compare them with standard alteplase and/or tenecteplase stroke treatments.
The presented novel approach for thrombolytics treatment testing in stroke with two
complementary models allows for streamlining preclinical research by reducing the number of
animals used, almost 100% inclusion of experimental subjects for data processing, and
subsequent reanalysis of digital data using artificial intelligence, if beneficial.
The research was supported by projects:
NU21-08-00510 Biocompatible nanoparticles as targeted drug delivery systems and
theranostics for the treatment of cerebrovascular disease.
NU23-08-00499 Improving the Efficacy and Safety of Cerebral Infarction Recanalization
Using Dual Thrombolytic Therapy.
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ISBN 978-80-973388-6-2