Experimentally induced
radiation syndrome in
laboratory animal
Aim of the practicals
• Basics of radiobiology
• Model of acute radiation syndrome
- blood form
• Presentation of experiment (data
obtained during practice on a
model of acute radiation
syndrome)
Ionizing radiation
• The radiation emitted by radioactive
nuclides
• Electromagnetic or corpuscular radiation
which, when penetrating mass, causes
ionization (must have sufficiently high
energy).
• Energy is in the range of keV-MeV
• At the same time, the atoms and
molecules of the environment are excited
Types of ionizing radiation
• Corpuscular α, β,
neutrons
• electromagnetic γ
The energy spectrum of
radiation
Ionization vs. excitation
M M+ + eM
Mexcit
• Both types of interactions are very fast
• Formed in a ratio of 1:2
•Radiation is not limited to radioactive nuclides,
but also behaves the same way with X-rays,
particles from accelerators and cosmic rays
Units
D = dE / dm
(J.kg-1) - Gray (Gy)
• MeV, a unit of energy
• Roentgen, a unit of exposure [C /
kg dry air]
• Becquerel, activity [s-1] ≈ Curie
• Gray, dose [J / kg] ≈ rad
• Sievert, dose equivalent [J / kg] ≈
rem
Units
• Dose equivalent = dose *
constant
• γ, β, X = 1
• neutrons = 10
• α = 20
Units
Sources of ionizing
radiation?
• Natural
– space
• exposure increases with
altitude
– solar
– terrestrial sources
• natural radioactive decay of
radioisotopes (soil and
rock)
– Radon
• gas, there is a decay of
radium-226 (Uranium)
• Artificial
– medicine
• diagnosis, therapy,
sterilization
– industrial
• nuclear energy
Sources of ionizing
radiation
Chemical effect of ionizing
radiation
Oxygen effect!!
• Radiation-chemical
reactions in liquids
are best studied
(less so in gases and
solids)
• If water contains
dissolved oxygen,
the following
reaction takes place:
O2 + H HO2
O2 + e- O2
-.
Biological effects of ionizing
radiation
• Direct ionizing = direct destruction
of biomacromolecules
• Indirectly (nondirect) effects =
production of free radicals
(radiolysis of water)
DNA damage
• Very serious process
• DNA damage is
reflected in the
synthesis of damaged
proteins
• DNA repair
mechanisms
• Reproductive ability of
cells
Mechanisms of DNA damage
Repair mechanisms
• Direct repair
• Excision repair
• Mismatch repair
• SSB repair
• (DSB repair)
Effects of ionizing
radiation on the human
body
• Deterministic effects
• Stochastic effects
Deterministic effects
• There are those that will take
effect after total body irradiation,
or a tissue once
• The dependence of the probability
of occurrence of damage to an
equivalent dose have the sigmoid
character
Deterministic effects
• Acute radiation syndrome
(radiation syndrome)
• Local acute skin damage
• Damage to the fetus
• Infertility
• Lens damage
Stochastic effects
• They result from damage to a
small number of cells
• They can occur after a single
exposure of sub-threshold dose or
chronic radiation tissue or whole
body
Character of biological effects
• Deterministic
– severity dependent
(“determined”) on the dose
– manifestation is specific
– effect only when exposure
exceeded threshold
– damage of large amount of
cells
– onset rather close to the
exposure (short latency)
– types:
acute radiation syndrome
chronic post-radiation
syndrome
cataract, radiation dermatitis,
damage of the foetus in utero
sterility
• Stochastic
– probability increases with
the dose (not the severity!)
– manifestation non-specific
– gradual increase of the risk
without “safe” threshold
– damage of the single cell
enough to cause effect
– manifestation delayed
(typically years)
– types:
somatic mutation - cancer
leukemias, thyroid, lung,
breast, bones
germinative mutation (oocyte,
sperm cell)
Acute radiation syndrome
• affecting the
hematopoietic,
gastrointestinal system
and cerebrovascular
– timing, extent and severity
graded according to the
dose - deterministic effect!!
• from several hours to
several months after
exposure
Acute radiation syndrome
• Haematopoetic syndrome (> 1GY)
• GI syndrome (> 10Gy)
– early (hours) - nausea, vomiting, diarrhea
– late (days) - loss of intestinal integrity
• malabsorption, dehydration, toxemia / sepsis, ileus, bleeding
• Cerebrovascular syndrome (tens of Gy)
– headache, cognitive impairment, disorientation,
ataxia, convulsions, fatigue and hypotension
• Cutaneous
– erythema, burns, edema, impaired wound healing
Hematopoetic syndrome
• bone marrow irradiation (>
1Gy) leads to an exponential
cell death - haematological
crisis
– marrow hypoplasia to aplasia +
peripheral pancytopenia
(infection, bleeding)
• subpopulation of stem bb is
selectively more
radioresistant, (probably due
to predominance bb. stage in
Go)
– necessary for regeneration
• anemia is the result of late
(erythrocytes ~ 120 days)!
• massive stress response
(glucocorticoids) contribute to
lymphopenia (cytolytic effect)
and paradoxically delay the
onset of granulocytopenia
(release stocks. granulocytes
from the spleen)
Therapeutic effects of
ionizing radiation
• Teleterapy (60Co)
• Contact therapy (32P, 90Sr)
• Brachytherapy (60Co, 137Cs)
• Endotherapy (Na131I)
• Radioimunotherapy
• to demonstrate the deterministic effects of ionizing
radiation on hematological parameters
• to observe the dynamics of peripheral blood count
changes resulting from the changes in the blood
marrow
• acute radiation syndrome is a model situation,
helping us to understand the hematopoiesis
AIMS of experiment
Practicals I - design
Praktikum I – operační
postup
Praktikum I - hodnocení
0,2 mm
0,05 mm
Výška = 0,1 mm
• The stained smear will first be viewed at a low
magnification and an area where white cells are
not overlaid by red cells will be selected.
• Using an immersion objective, a total of 100
WBC will be registered and identified as to their
individual type.
• The viewing field will be moved in order to
count the prescribed number of leucocytes.
Viewing of smear
Hemopoesis
Erythropoiesis
http://www.noblesmedart.com/morph.mov
Erythropoiesis
• Erythropoiesis is the development of mature red blood cells
(erythrocytes).
• Like all blood cells, erythroid cells begin as pluripotential stem cells.
• The first cell that is recognizable as specifically leading down the red cell
pathway is the proerythroblast .
• As development progresses, the nucleus becomes somewhat smaller and
the cytoplasm becomes more basophilic, due to the presence of
ribosomes. In this stage the cell is called a basophilic erythroblast .
• The cell will continue to become smaller throughout development. As the
cell begins to produce hemoglobin, the cytoplasm attracts both basic and
eosin stains, and is called a polychromatophilic erythroblast .
• The cytoplasm eventually becomes more eosinophilic, and the cell is called
an orthochromatic erythroblast .
• This orthochromatic erythroblast will then extrude its nucleus and enter
the circulation as a b . Reticulocytes are so named because these cells
contain reticular networks of polyribosomes. As reticulocytes loose their
polyribosomes they become mature red blood cells.
Reticulocyte
Reticulocytes
• Reticulocytes are immature red blood
cells, typically composing about 1% of the
red cells in the human body.
• Reticulocytes develop and mature in the
red bone marrow and then circulate for
about a day in the blood stream before
developing into mature red blood cells.
• Like mature red blood cells, reticulocytes
do not have a cell nucleus.
• They are called reticulocytes because of a
reticular (mesh-like) network of ribosomal
RNA that becomes visible under a
microscope with certain stains such as
new methylene blue.
Classification of reticulocyte
counts
• Counting with imersion, magnification 100
• Out of 1000 RBC in the moving viewing field,
the number of RET will be counted
• Normal counts RAT: app. 20 %o RET
• Normal counts MAN: app 0.5-1.5% (5-15%o) RET
 number: increasing bloodforming (regeneration)
 number: inhibition of erythropoiesis
Differential leucocyte counts
• Changes in WBC number (%)
– Indication of infection, poisoning, leukemia,
chemotherapy, alergic reaction
• Normal WBC counts in man:
– neutrophils 60-70% (incr. in bacterial infection)
– lymphocytes 20-25% (incr. in viral infection)
– monocytes 3-8 % (incr. in fungal/viral infection)
– eozinophils 2-4 % (incr. in alergic reaction and
parasital infect.)
– bazophils <1% (incr. in alergic reaction)
Normal counts of WBC in rat
• Number of WBC............cca 12.5 tis.mm-3
- neutrophil granulocytes 18 - 36%
- eozinophil granulocytes 1 - 4%
- bazophil granulocytes 0 - 1%
- lymphocytes 62 - 75%
- monocytes 1 - 6%
Blood smear
• neutrophil
„band“
• neutrophil
„segment“
• monocyte
• lymphocyte
• eozinophil
• bazophil
• trombocyte