Effect of Total Body X-ray Irradiation on Lymph Node in Tibet Minipig
J. Radiat. Res.
Effect of Total Body X-ray Irradiation on Lymph Node in Tibet Minipig
Shao-Jie WU 3
Yu-Jue WANG 1
Kun-Yuan GUO 3
Chi CHEN 3
Tong-Feng ZHAO 3
Mao-Ben SUN 3
Wei-Wang GU 1
Ying Ying GAO 3
Hui Juan HAN 3
Fei ZOU 0
0 School of Public Health and Tropical Medicine, Southern Medical University , 1838
1 Department of Laboratory Animal Center , Southern Medical University , 1838
2 Guangzhou North Road , 510515, Guangzhou, Guangdong, China. doi:10.1269/jrr.11077
3 Department of Hemotology, Zhujiang Hospital, Southern Medical University , 253
Tibet Minipig/X-ray irradiation/Lymphocytes/Lymph node/Apoptosis and necrosis. The purpose of this study was to determine the time-dose-effect of total body X-ray irradiation on lymphocytes in lymph nodes and peripheral blood in Tibet minipigs. Forty-eight Tibet minipigs were assigned into 6 groups including 5 experimental groups with 9 and the control group with 3. The minipigs in experimental groups were subjected to a total body X-ray irradiation of 2, 5, 8, 11, and 14 Gy respectively. Lymph nodes and peripheral blood samples were collected at 6, 24, and 72 hours after X-ray exposure and received histological microscopy examination and apoptosis analysis. Histology observation showed that the number of lymphocytes decreased within the lymph nodes with the increase of radiation doses and exposure time. The observation of transmission electron microscopy (TEM) showed typical apoptotic cells below 11 Gy while at 14 Gy necrotic cells were dominant. The apoptotic rate of lymphocytes in the lymph nodes was positively correlated with radiation dose in the range of 2-11 Gy, and reached the maximal level (39.4 ± 2.8) at 24 hours after 11 Gy irradiation, followed by a decrease in the apoptotic rate, but still higher than that of the control group. The number of lymphocytes in the peripheral blood samples was decreased significantly by increasing of the radiation dose and exposure time. We conclude that early damage of lymphocytes by total body X-ray irradiation is dose and time dependent below 11 Gy and before 24 hours post irradiation, and that the dosage of irradiation less than 11 Gy induced apoptosis, whereas the dose at 14 Gy resulted in necrosis in lymphocytes of the lymph nodes.
At present, with wide utilization of unclear technique,
radiation damage will happen at any time and anywhere.
Whereas, it is impossible to study radiation damage in
human body, so we should use biology models to understand
the characteristics of the radiation damage. Compared with
small animal models such as rodents, large animal models
are superior in many aspects for the study of human diseases
and pre-clinical therapies. The miniature pig is similar to
human in anatomy, development, physiology,
pathophysiology, and disease occurrence, etc.
(see for a review Tumbleson,
. Since the development of the Minnesota miniature
pig in 1949 at the Hormel Institute (USA), miniature pigs
have been used as a large animal model in medical studies
for scientific, economic, and ethical reasons.1–3) Although
miniature pigs as animal models have been applied in
several fields of study, it is rarely used as an animal model for
acute radiation injury. The Tibet minipigs have a character
of genetic stability, small size, early maturation, high
fecundity. Therefore, the Tibetan minipigs could be a suitable
animal model for the study of radiation diseases.
Human and experimental data have shown that severe
injury of lymphatic organs and decrease of lymhpocyte
count are being explained as important reasons to the
difficulties in the curing of acute radiation injury by large doses
radiation. It is also known that lymphocytes are one of the
most radiosensitive immunocompetent cells in lymphatic
tissues and peripheral blood.4–6) Previous studies have shown
that low doses of X-ray irradiation could cause cell
apoptosis (programmed cell death)7–9) and high doses of X-ray
irradiation cause cell necrosis10) in vitro or in vivo by local or
fractional radiation. It is critical to understand the death
modes (apoptosis or necrosis) of the lymphocytes so as to
evaluate the irradiation injury. A correlation of the radiation
dose or post-irradition time and the death modes of
lymphocytes induced by irradiation have not been studied
systematically. The aim of this study was to define the early-stage
damages to lymphocytes in the lymph node and peripheral
blood in Tibet minipigs after total body X-ray irradiation.
MATERIALS AND METHODS
Adult uncastrated male Tibet minipigs (weighing 21.16 ±
5.54 kg, supplied by Center of Laboratory Animal, Southern
Medical University, Guangzhou, china) were kept under
standard laboratory conditions with a 12 hour light and 12
hour dark cycle, and were allowed free access to feed and
water. This study was approved by the supervising state
agency (license number SCXK Yue 2006-0015) and
performed in full accordance with the state guidelines.
Fortyeight anesthetized (0.15 ml/kg Sumianxin) Tibet minipigs
were divided into one control and five experiment groups
and placed in a phantom for fixation postures to subject to
irradiation exposure by an 8 Mv X-ray (isocenter) linear
accelerator (Precise System Treatment, ELEKTA, Sweden).
The irradiation was carried at the Cancer Centers of Armed
Police Hospital of Guangdong following the decribed
elsewhere.11) Animals in control (n = 3) were not exposed to
Xray, and those in experiment group (n = 9 in each group)
received 2, 5, 8, 11, and 14 Gy doses of total body X-ray
radiation, respectively, in a single fraction irradiation. The
dose rate was 255 cGy/min in all treatment groups. The
animals were sacrificed by bloodletting after anestheticed at the
time points of 6, 24, and 72 hours post-irradiation, and
specimens were collected for histological observation and
apoptosis analysis, peripheral blood samples were collected from
veins for lymphocytes count.
The cervical lymph nodes were fixed in 10% PBS
buffered formalin solution for 48 hours. The smples tissues were
dehydrated in different grade enethyl-alcohol and embedded
in paraffin. Two-micrometer sections were cut and stained
with hematoxylin–eosin. To determine the numbers of
lymphocytes in the lymph nodes, we counted the lymphocytes
in five randomly-selected microscopic view fields for each
section from a lymph node under a magnification of 200
using an image analysis system (Image-Pro Plus 6.0, Media
Cybernetics, USA). In preparation of samples for
examination by transmission electron microscopy (TEM), the
cervical-lymph nodes were fixed in glutaraldehyde solution
(2.5%), fixed again in uranyl acetate (0.5%), followed by
dehydrated and embedment in resin. The samples were cut
into semi-thin and ultra-thin longitudinal sections, and
examined with a transmission electron microscope (JEOL 1010;
Jeol, Tokyo, Japan). Peripheral blood samples were collected
from veins of Tibet minpigs for lymphocytes count.
Lymph nodes were cut and put in 3 mL of RPMI 1640
complete medium (RPMI 1640, supplement with 10% fetal
bovine serum and 1% penstrep sloution). After filtration
with 150-μm sterile nylon mesh, the cell suspension were
washed 3 times with RPMI 1640 complete medium. The cell
pellet was treated with 3 mL RBC lytic buffer for 5 minutes
at room temperature, followed by addition of 3 mL RPMI
1640 complete medium. The treated cell suspension was
washed 3 times in RPMI 1640 complete medium and
resuspended in 3 ml of 1640 complete medium and kept at 4°C
for further analysis. To prepare for examination of apoptosis
and necrosis by flow cytometry, 200 μl of cells (about 1 ×
105 cells) were added with 300 μl of binding buffer and 10
μl of Annexin V-FITC to each samples and mixed gently,
followed by incubation at room temperature in the dark for
15 minute. Immediately before analysis by flow cytometry,
2 μL of PI were added to each sample.
The data from control and experimental groups were
statistically analysed by one-way analysis of variance (ANOVA)
with a post hoc Turkey test for all pairwise multiple
comparison procedures. Non-parametrically distributed
groups were studied by the Mann-Whitney U analysis of
variance on ranks. The parameters were expressed as a mean ±
SD. Probability (P) value < 0.05 was accepted as significance.
All animals have survived the irradiation procedures and
been maintained during the observation period after X-ray
Pathological Changes of Lymph Nodes after X-ray
Irradiation at Different Dosages and Time Points
Anatomically, in the animals from the experimental
groups, the lymph nodes have shown signs of shrinkage and
apparent peripheral hemorrhage with increase of radiation
doses. Under light microscope, the lymphocytes have shown
nuclear condensation, fragmentation, and dissolution at 6
hours after irradiation on all dosages of X-ray radiation (Fig.
1B1). The most obvious changes were found in lymphatic
nodules. The cellular debris was cleared at 24 hours
postirradiation (Fig. 1B2), and the lymphatic nodules became
empty with increase of dosages. Similarly changes were
seen at 72 hours, except for reticular cell and plasma cells
can be discernable, and no regeneration of lymphocytes can
be seen at this time point after different doses of X-rays. The
number of the lymphocytes decreased sharply in the cortex
and the cortex was atrophied, so that the cortex and medulla
of the lymph nodes became hardly distinguishable at 14 Gy
(Fig. 1C). The total number of lymphocytes decreased
significantly compared with control group (0 Gy) after different
doses and different time of X-ray, especially at 24 and 72
hours (Fig. 1D). But in the control group, the above changes
were not found (Fig. 1A).
Ultrastructural Change of Lymph Node after X-ray
Typically Morphological features of apoptotic cells were
observed in the deep-staining cell (Fig. 2B), in the early
stages, the cells showed marginal condensation of the
chromatin, followed by the formation of typical crescent and ring
chromatins and compaction of cytoplasmic organelles. In the
late stage, the cells were showing shrinkage and apoptotic
bodies were observable.12) These morphological changes of
lymphocytes could be seen throughout the observation
period from the animals in all experimental groups at all time
points. At 6 hours after 11 Gy X-ray irradiation, spotty
necrosis could be seen. More prominent necrotic lesions
were shown at 14 Gy (Fig. 2C). Phagocytosis phenomenon
of microphage, and cytoplasmic vacuolization, mitochondria
expansion in the reticular cells were observable, but absent
in the control group (Fig. 2A).
The Apoptosis Lymphocytes of Lymph Node After X-ray
Irradiation at Different Dosages and Time Points
It was found that the numbers of the apoptotic cells were
significantly increased with the increases of the radiation
strength from 2 to 11 Gy at 6, 24, and 72 hours after
irradiation, compared with that of the control group (p < 0.05),
MannWhitney U test (Fig. 3). However, at 72 hours after radiation
there was no difference in the number of apoptotic cells with
the irradiation from 11 Gy to 14 Gy (Fig. 3). When comparing
the apoptotic cells at different time points in each treatment
group, we found obvious differences in the group with 11 Gy,
while there were no differences between 6 and 72 hours within
others irradiation groups, except for 11 Gy group (Fig. 3).
Fig. 3. The average apoptotic ratio in lymph node with different
doses of irradiation at each time point. Each bar represents the
means ± SD of cell numbers. Irradiated group P < 0.05 compared
with control group. #p < 0.05 ( Mann-Whitney U).
The Effect of Irradiation on the Peripheral Blood Lymphocytes Count
The average peripheral blood lymphocyte counts was
decreasing with the increases of the irradiation doses from
2 to 14 Gy at every time points after irradiation (Fig. 4).
With the extension of observation period to 72 hours after
radiation, the decrease of peripheral blood lymphocyte
counts became more significant, when compared with the
control group (p < 0.05, post hoc Turkey test) (Fig. 4).
Pigs share many advantages for experimental studies.
They are large enough to allow varied surgical techniques to
be carried out and are suitable for numerous experimental
protocols. Pigs have particular advantages for
radiobiological studies. The pigs has been successfully used in study of
skin and renal and lung and intestinal effects of radiation
exposure.13) Results from studies in non-irradiated animals
are in agreement with reported data for different breeds of
pig13, 14-dihydroxy-retino,12,14) and these data have shown
similarities to those in man, particularly the data related to
the concentration of white blood cells, percentage of
lymphocytes and neutrophils.
This study using Tibetan minipigs has demonstrated that
the lymph node was one of the radiosensitive lymphatic
tissues. We observed cellular debrises and hyperemia and
apoptosis cells in the lymph nodes examined at 6 hours after
exposure to different dosages, that is why we did not find
difference between irradiated groups through lymphocytes
counting in this time point; and the cellular debrise was
almostly cleared at 24 hours, when lymph nodes appeared
almost hollow. We did not see lymphocytes regeneration at
72 hours after 2 Gy X-ray exposure, except that reticular
cells and plasma cells were observable, meanwhile, Kvacheva
IuE results have shown that the regeneration of bone marrow
begin in weeks after X-ray.15) Similar results were observed
in animals in all irradiated groups and the pathological
changes were more prominent when radiation doses
increased. Also, the apoptotic lymphocytes with typically
morphological features were discernable under transmission
electron microscopy (TEM), These observations were in
accordance with the results of previous studies.16,17)
It was found that the count of peripheral blood
lymphocytes decreased rapidly in the Tibetan minipigs. The
lymphocytes count reduced to a minimal level (0.065 ± 0.02) at
72 hours after 14 Gy X-rays exposure, which was also noted
by other researchers.18,19) Whereas, Azizova TV et al.
observed lymphocyte count decrease nearly to zero over 6
Gy at 72 hours in human,18) possibly due to the high
radiosensitivity of these cells.
In this study, we found that the apoptosis rate of
lymphocytes in the lymph nodes increased to a maxminnal level
(39.4 ± 2.8) at 24 h after 11 Gy X-ray exposure, and the
apoptosis rate was positively correlated with the irradiation
dosages in the range of 2–11 Gy. However, such a correlation
was not found after 14 Gy irradiation, where the apoptosis
analysis has shown a reduced apoptotic response to
irradiation, compared with 11 Gy at 24 hours (P < 0.05), that
because the apoptotic rate decreased and the necrotic rate
rose; and necrotic foci could be observed under both light
and electron microscopy at 14 Gy dosage of irradiation,
Transmission Electron Microscopy (TEM) was, therefore,
used as a gold standard to distinguish adequately two
morphobogically disinct modes of cell death: apoptosis and
necrosis. These results are consistent to other reported
results, Payne et al.20) observed the peak cell loss occurred
after high-dose irradiation (10–20 Gy), but the peak level of
apoptosis occurred after low-dose Irradiation. H. Louagie et
al.21) found that the primary necrosis occurred at 20 Gy of
gamma radiation, but no details were given in terms of the
dosages. These results suggested that apoptosis is the main
mode of death for lymphocytes following the X-ray
radiation exposure below 11 Gy.
In conclusion, in Tibetan minipig model, the early injury
of lymphocytes by the whole body X-ray irradiation is dose
and time dependent below the dosage of 11 Gy and within
24 hours following the irradiation. The dosage of irradiation
less than 11 Gy induces dominantly apoptosis in the
lymphocytes of the lymph nodes, followed by necrosis when the
dosage increased to 14 Gy. Also, the apoptotic rate of the
lymphocytes in lymph node is positively correlated with the
radiation doses at 2–11 Gy X-ray irradiation, suggesting that
apoptosis is the major way of lymph node lymphocyte death
after ≤ 11 Gy radiation.
The authors thank Jin-Jun Xue for the technical assistance
with the irradiation protocol. Yuan Bi is acknowledged for
making TEM sections and Yan-Qing Ding for processing the
electron micrographs. Guang Jin Lu for reading of
manuscript. The study supported by “The Third Phase Project of
Chinese National 211 Leading Academic Discipline”
(Project Number: C1030380).
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