Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.     

Nonuniform irradiation of the canine intestine. II. Dosimetry

An experimental model has been developed for quantitative studies of radiobiological damage to the canine small intestine following partial-body nonuniform irradiation. Animals were irradiated with 60Co gamma rays to simulate the nonuniform irradiation which do occur in victims of radiation accidents. The model used a short source-to-surface distance for unilateral irradiations to produce a dose gradient of a factor of two laterally across the canine intestinal region. The remainder of the animal's body was shielded to prevent lethal damage to the bone marrow. In situ dosimetry measurements were made using thermoluminescent dosimeters to determine the radiation dose delivered as a function of position along a segment of the small intestine. This system made it possible to correlate the radiation dose delivered at a specific point along the small intestine with the macroscopic and microscopic appearance of the intestinal mucosa at that point, as determined by direct observation and biopsy using a fiberoptic endoscope. A key feature of this model is that dosimetry data for multiple sites, which receive a graded range of radiation doses, can be correlated with biological measurements to obtain a dose-response curve. This model is being used to evaluate the efficacy of new therapeutic procedures to improve survival following nonuniform irradiation.     

Enhanced body radioresistance resulting from the transfusion of autologous blood irradiated with low doses of ionizing radiation]

Preliminary administration of autogenic blood irradiated in vitro with ionizing radiation in small doses of 0.05, 0.3 or 0.5 Gy resulted in a pronounced increase in the radioresistance of mice [correction of rats] subsequently irradiated in a dose of 9 Gy. The optimum was autotransfusion of blood irradiated in a dose of 0.3 Gy a day or 10 days prior to the total irradiation which increased the survival rate of experimental animals to 80% while, in control groups, the survival rate was only 10%.     

Effects of dexamethasone on late radiation injury following partial-body and local organ exposures.

Dexamethasone was evaluated as a treatment for radiation-induced lung, kidney, liver, and spinal cord injuries in rats. One experimental group was partial-body-irradiated (22.5 Gy) with the head, femur, and exteriorized intestine shielded to prevent acute mortality. Other animals received local irradiation to the kidney (20 Gy), liver (25 Gy), or a 1-cm segment of cervical spinal cord (18 to 40 Gy). Following irradiation half of the animals in each radiation group were given drinking water containing 188 micrograms/liter of dexamethasone. Tests were done to assess kidney function (hematocrit, plasma urea nitrogen, ethylenediaminetetraacetic acid clearance), liver function (rose bengal clearance, plasma glutamic oxaloacetic acid transaminase), or spinal cord injury (paralysis). The effectiveness of dexamethasone in preventing radiation injury was tissue specific. Dexamethasone eliminated lethal pleural fluid accumulation after partial-body irradiation and delayed development of kidney dysfunction after local kidney irradiation. As a result, dexamethasone increased the median survival time from 63 to 150 days after partial-body irradiation and from 126 to 175 days after local kidney irradiation. After whole-liver irradiation, development of hepatic functional injury was retarded by dexamethasone treatment but without significantly changing survival time. Dexamethasone had no effect on spinal cord tolerance but significantly shortened the latent period between radiation and paralysis.     

Kidney and lung injury in irradiated rats protected from acute death by partial-body shielding

Ninety-six CD-1 male rats were exposed to gamma-ray doses (0-25 Gy) in increments of 5 Gy. One femur, the surgically exteriorized GI tract, and the oral cavity were shielded during irradiation to protect against acute mortality from injury to the hematopoietic system, small intestine, and oral cavity. In addition, the thoraxes of half of the animals from each dose group were shielded. At approximately monthly intervals from 2 to 10 months after irradiation the hematocrit, plasma urea nitrogen (PUN), and 51Cr-EDTA clearance were measured. During the study 20 thorax-shielded and 19 thorax-irradiated animals died. All rats whose thoraxes received 25 Gy irradiation and three out of seven rats whose thoraxes received 20 Gy died 1 to 3 months postirradiation with massive pleural fluid accumulation. Shielding the thoraxes prevented this mode of death at these doses. Kidney injury was judged to be the primary cause of death of all thorax-shielded animals and 15- and 20-Gy thorax-irradiated animals. Animals with kidney damage had elevated PUN and reduced 51Cr-EDTA clearance and hematocrits. The relative merits of each of these end points in assessing radiation-induced kidney injury after total-body exposure are discussed.     

The inhibiting effect of a low-dose ionizing radiation on the primary humoral immunity in the early period of an postnatal ontogeny

The single exposition of 3- and 4-week mice in a dose 0.1 Gy resulted in depressing primary T-dependent humoral immune response. Unlike juvenile ones at adult 16-week animals the stimulation antibody-formation took place. As a result of the administration of immunomodulating drugs, thymogene and nucleinate of a sodium in irradiation mice of 3-week age was an augmentation of the number of antibody-producing cells relatively in 2 and 4 times. At the same time at irradiated in dose 0.1 Gy of adult mice thymogene abrogated the stimulating effect of radiation. It is suggested that the probable cause of the immunosuppressing effect of a small dose of radiation can be an inactivation of a radiosensitive subpopulation of helper cells or selective stimulation of the functional activity of neonatal suppressor cells.     

The adaptive response modifies latency for radiation-induced myeloid leukemia in CBA/H mice.

We have investigated the effect of the adaptive response on acute myeloid leukemia (AML) induced in CBA/Harwell mice by a chronic radiation exposure. Groups of mice irradiated with a total dose of 1. 0 Gy at two different chronic dose rates (0.5, 0.004 Gy/h) had similar frequencies of AML. Compared to control animals that did not develop AML, irradiation at either of these dose rates did not change the longevity of the mice that did not die of leukemia. The survival rates of irradiated mice that did develop leukemia in the two groups were not different from each other, indicating that the dose rates produced similar responses and therefore were both chronic exposures. We then tested the ability of a chronic 10-cGy (0. 5 Gy/h) exposure to ionizing radiation, mild hyperthermia (40.5 degrees C whole-body, 60 min) or treatment with interleukin-1 (1500 U i.p.) to induce an adaptive response and modify the frequency or latency of AML which resulted from a subsequent (24 h later) 1.0-Gy (0.5 Gy/h) chronic radiation exposure. The frequency of radiation-induced leukemia was not changed in mice given any of the three adapting treatments 24 h prior to the chronic 1.0-Gy dose that induced leukemia. However, the latent period for development of AML was significantly increased by both the prior low radiation dose and mild hyperthermia treatment. Injection of interleukin-1, in contrast, may have reduced the latent period. Similar to the single 1.0-Gy chronic exposure alone, none of the adapting treatments prior to that exposure influenced the survival of animals that did not develop AML. These results indicate that an earlier exposure to a small adapting dose of radiation or to a mild heat stress can influence secondary steps in radiation-induced carcinogenesis.     

Influence of protein nutrition on dose-survival relationship following rat kidney irradiation

Immediately following unilateral nephrectomy the remaining kidney of juvenile male Sprague-Dawley rats was sham irradiated or irradiated to doses of 14-30 Gy. Following irradiation the animals were placed on isocaloric diets of either 20 or 4% protein. Median life spans for the animals on the low protein diet were significantly increased compared to the median life spans on the 20% protein diet. Serum urea nitrogen (SUN) levels were periodically measured in rats from each of the experimental groups. SUN levels in the irradiated rats fed the 20% protein diet increased significantly over unirradiated controls as a function of time. In contrast animals fed the 4% protein diet showed no significant changes in SUN levels irrespective of the size of radiation dose and time post irradiation. Renal protective factors calculated as the ratio of 80% survival times for animals fed the 20% protein diet compared to animals fed the 4% protein diet can be calculated to be 2.3 at 18 Gy and 2.8 at 22 Gy. Likewise, a SUN protective factor calculated as the ratio of percentage of nonirradiated control SUN values for the two diets (SUN 20% irradiated) (SUN 20% nonirradiated) (SUN 4% irradiated) (SUN 4% nonirradiated) is 2.4 for 18 Gy and 3.9 for 22 Gy.     

Effect of Pseudomonas contamination or antibiotic decontamination of the GI tract on acute radiation lethality after neutron or gamma irradiation

The influence of antibiotic decontamination of Pseudomonas contamination of the GI tract prior to whole-body neutron or gamma irradiation was studied. It was observed that for fission neutron doses greater than 5.5 Gy, cyclotron-produced neutron doses greater than 6.7 Gy, and 137Cs gamma-ray doses greater than 14.4 Gy, the median survival time of untreated rats was relatively constant at 4.2 to 4.5 days, indicating death was due to intestinal injury. Within the dose range of 3.5 to 5.5 Gy of fission neutrons, 4.9 to 6.7 Gy of cyclotron-produced neutrons, and 9.6 to 14.4 Gy of gamma rays, median survival time of these animals was inversely related to dose and varied from 12 to 4.6 days. This change in survival time with dose reflects a transition in the mechanisms of acute radiation death from pure hematopoietic, to a combination of intestinal and hematopoietic, to pure intestinal death. Decontamination of the GI tract with antibiotics prior to irradiation increased median survival time 1 to 5 days in this transitional dose range. Contamination of the intestinal flora with Pseudomonas aeruginosa prior to irradiation reduced median survival time 1 to 5 days in the same radiation dose range. Pseudomonas-contaminated animals irradiated within this transitional dose range had maximum concentrations of total bacteria and Pseudomonas in their livers at the time of death. However, liver bacteria concentration was usually higher in gamma-irradiated animals, due to a smaller contribution of hematopoietic injury in neutron-irradiated animals. The effects of both decontamination of the GI tract and Pseudomonas contamination of the GI tract were negligible in the range of doses in which median survival time was dose independent, i.e., in the pure "intestinal death" dose range. Finally, despite the marked changes in survival time produced by decontamination or Pseudomonas contamination in the "transitional dose range," these treatments had little effect on ultimate survival after irradiation as measured by the LD50/5 day and the LD50/30 day end points. The implications of these results with respect to treatment of acute radiation injury after whole-body irradiation are discussed.     

Changes in vascular permeability following thorax irradiation in the rat

A double isotope technique was used to measure changes in the vascular permeability surface area product (PS) for albumin after irradiation. PS was measured in several tissues of the rat during the first 38 days following 11, 13.5, 18, or 25 Gy whole thorax irradiation. After 18 and 25 Gy most irradiated and nonirradiated (shielded) tissues showed elevated permeability at 1 day after radiation, which declined to control levels by Day 4. All irradiated tissues showed a second wave of increased permeability between 14 and 38 days after radiation that varied in onset and extent depending upon tissue and dose. Lung and heart showed a direct response to dose between 11 and 18 Gy during this period. Peak lung values averaged three times control values at 19 days after 18 Gy. Peak heart values averaged twice control values at the same time and dose. The double isotope technique has proven to be a reliable means of quantitatively determining vascular permeability response to radiation over time.     

Radioprotective potential of histamine on rat small intestine and uterus

The aim of this study was to improve knowledge about histamine radioprotective potential investigating its effect on reducing ionising radiation-induced injury and genotoxic damage on the rat small intestine and uterus. Forty 10-week-old male and 40 female Sprague-Dawley rats were divided into 4 groups. Histamine and histamine-5Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Histamine-5Gy and untreated-5Gy groups were irradiated with a dose of whole-body Cesium-137 irradiation. Three days after irradiation animals were sacrificed and tissues were removed, fixed, and stained with haematoxylin and eosin, and histological characteristics were evaluated. Proliferation, apoptosis and oxidative DNA markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate chromosomal damage. Histamine treatment reduced radiation-induced mucosal atrophy, oedema and vascular damage produced by ionising radiation, increasing the number of crypts per circumference (239±12 vs 160±10; P<0.01). This effect was associated with a reduction of radiation-induced intestinal crypts apoptosis. Additionally, histamine decreased the frequency of micronuclei formation and also significantly attenuated 8-OHdG immunoreactivity, a marker of DNA oxidative damage. Furthermore, radiation induced flattening of the endometrial surface, depletion of deep glands and reduced mitosis, effects that were completely blocked by histamine treatment. The expression of a proliferation marker in uterine luminal and glandular cells was markedly stimulated in histamine treated and irradiated rats.The obtained evidences indicate that histamine is a potential candidate as a safe radio-protective agent that might increase the therapeutic index of radiotherapy for intra-abdominal and pelvic cancers. However, its efficacy needs to be carefully investigated in prospective clinical trials.Key words: histamine, ionising radiation, radio-protectors, small intestine, uterus.     

Hepatic proliferation after partial liver irradiation in Sprague–Dawley rats

The liver has powerful capability to proliferate in response to various injuries, but little is known as to liver proliferation after irradiation (IR) injury. This study investigated whether liver proliferation could be stimulated in low-dose irradiated liver by partial liver IR injury with high dose radiation. Sprague–Dawley rats were irradiated by 6-MV X-ray with single dose of 25 Gy to the right-half liver, while the left-half liver was shielded (0.7 Gy) or irradiated with single doses of 3.2, 5.6, and 8.0 Gy, respectively. Hepatic proliferation in the shielded and low-dose irradiated left-half liver was evaluated by serum hepatic growth factor (HGF), proliferating cell nuclei antigen (PCNA), liver proliferation index (PI), hepatocyte mitosis index (MI). The observation time was 0 day (before IR), 30, 60, 90, and 120 days after IR. Our results showed that serum HGF and hepatocyte HGF mRNA increased after IR with HGF mRNA peak on day 30 in the shielded and low-dose irradiated left-half livers, and their values increased as the dose increased to the left-half liver. Liver PI and PCNA mRNA peaked on day 60 with stronger expressions in higher doses-irradiated livers. MI increased after IR, with the peak noted on day 60 in the shielded and on day 90 in the low-dose irradiated left-half livers. There was a 30 day delay between MI peaks in the shielded and low-dose irradiated livers. In conclusion, 25 Gy partial liver IR injury could stimulate the shielded liver and low-dose irradiated liver to proliferate. In the livers receiving a dose range of 3.2–8.0 Gy, the proliferation was stronger in higher doses-irradiated liver than the low-dose irradiated. However, IR delayed hepatocyte mitosis.     

Vascular response to radiation injury in the rat lung.

Changes in relative left-to-right lung blood flow ratios were followed as an index of vascular radiation injury in left-hemithorax-irradiated Sprague-Dawley rats. Single doses of 11 to 21 Gy gamma radiation resulted in a dose-dependent decrease in relative blood flow to the irradiated lung from 3 to 5 weeks after exposure during the development of pneumonitis. Blood flow returned to near normal by 5 weeks after lower doses (11-13.5 Gy). After a single dose of 15 Gy the left-to-right blood flow ratio recovered to 75% of normal at 12 weeks and leveled off. Following 18 Gy irradiation a second period of reduced flow began 16 weeks after exposure. After 21 Gy irradiation flow to the irradiated side remained low for 1 year after exposure. Rats that received a single dose of 18 Gy to the left hemithorax were also treated with one or two of the following drugs: captopril, cyproheptadine, dexamethasone, diethylcarbamazine, penicillamine, or theophylline. Dexamethasone was most effective at preventing the decrease in blood flow to the irradiated lung when treatment was continued through the pneumonitis period and dose was not tapered until 8 weeks after radiation exposure. All other drugs and drug combinations were, for the most part, virtually ineffective after the pneumonitis period. There was a relatively poor correlation with earlier vascular permeability surface area product studies. This suggests that endothelial damage, as well as damage to other cell types, contributes to the development of post-irradiation fibrosis in the lung.     

Radiobiology of stem cells in intestinal epithelium. Effect of single and multiple preliminary sublethal irradiation of mice on the dose dependence of the survival of stem cells in small intestine epithelium

After a single or three-fold whole body irradiation of mice with a dose of 4 Gy and the time interval for the proliferation to be restored (5 days or 3 weeks) the survival curve for stem cells of small intestine epithelium with regard to radiation dose was the same as that for non-preirradiated mice. This indicated that the proliferative potential of stem cells in these experimental conditions was not reduced.     

Nonuniform irradiation of the canine intestine. I. Effects

To investigate the effects of nonuniform irradiation on the small intestine, we prepared 24 dogs for continent isoperistaltic ileostomies under aseptic surgical conditions and general anesthesia. After a 3-week recovery period, the ileum was catheterized with a fiberoptic endoscope to observe the intestinal mucosa and to harvest mucosal biopsies. The baseline macroscopic and microscopic appearance of the intestinal mucosa was determined. Two weeks later, the ileum was catheterized with a 100-cm soft tube containing 40 groups of three thermoluminescent dosimeters placed at equally spaced intervals, and a dose of either 4.5, 8, 10, 11, or 15 Gy 60Co gamma rays was delivered to the right abdomen (nonuniform exposure). This method allowed a direct and precise assessment of the dose received at 40 sites located in the 100-cm intestinal segment. The intestinal mucosa was again evaluated 1, 4, and 6 days after irradiation. All animals exposed to 4.5 and 8 Gy survived, whereas none survived after 11 and 15 Gy. After exposure to 10 Gy, 60% of the animals died within 4-6 days and 40% survived with symptoms associated with both the intestinal and the hematopoietic syndromes. Crypt cell necrosis, blunting of villi, and reduction of the mucosal lining increased between 1 and 4 days after irradiation, and mucosal damage was correlated with intraintestinal dosimetry at Day 6. The granulocyte counts at Day 4 were significantly lower than baseline level in animals that died within 4-6 days but not in survivors. The present model appears to be realistic and clinically relevant, allowing the concurrent study of the intestinal and hematopoietic effects of high-dose nonuniform irradiation similar to that received by patients during radiation therapy as well as by radiation accident victims.     

Production of humoral factors that stimulate spleen colony-forming units in mice irradiated with moderate doses of X rays

The production of humoral factors that stimulate spleen colony-forming units (CFU-S) has been studied in irradiated mice using an in vivo diffusion chamber assay. The experiments show that a significant release of factors that stimulate CFU-S takes place in the first few days after irradiation with moderate doses of 1.5 or 5 Gy. In contrast, the release of significant amounts of these humoral factors was not seen in animals irradiated with either low (0.75 Gy) or high (10 Gy) doses of X rays. The correlation observed between the production of factors that stimulate the CFU-S and the hemopoietic regeneration kinetics of the irradiated mice suggests that these factors represent part of the physiological regulators controlling the proliferation of CFU-S.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

Acceleration of regeneration of mucosa in small intestine damaged by ionizing radiation using anabolic steroids

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.     

小剂量^16O^8＋离子预辐射减轻大剂量辐射所致小鼠睾丸组织损伤

The testes of the B6C3F1 hybrid strain mice were irradiated with 0.05 Gy of 16O8+ ion as the pre-exposure dose (D1), and were then irradiated with 2 Gy of 16O8+ ion as challenging radiation dose (D2) at 4 h after per-exposure. Testicular morphology was observed by light microscope at 35th day after radiation. The results showed that irradiation of mouse testes with 2 Gy of 16O8+ ion significantly impaired, mainly reduction of tubule diameter and decrease or loss of germ cells in various developing stages, especially spermatogenic elements. Pre-exposure to a low-dose (0.05 Gy) of 16O8+ ion significantly alleviated above mentioned damage on testicular morphology induced by subsequent a high-dose (2 Gy) radiation.     

Hepatic injury after whole-liver irradiation in the rat

Radiation-induced hepatic injury in rats, which is characterized by marked ascites accompanied by liver necrosis, fibrosis, and vein lesions, is described in this study. These adverse sequelae are produced within 30 days after irradiation if there is surgical removal of two-thirds of the liver immediately after whole-liver irradiation. The LD50/30 day and median survival time after liver irradiation and two-thirds partial hepatectomy is 24 Gy and 17 days, respectively. Death is preceded by reduction in liver function as measured by [131I]-labeled rose bengal clearance. Prior to death, liver sepsis and endotoxemia were detected in most irradiated, partially hepatectomized animals. Pretreatment of the animals with endotoxin and/or antibiotic decontamination of the GI tract, which increase the host resistance to infection and endotoxemia, resulted in increased survival time, but no irradiated, partially hepatectomized animal survived beyond 63 days. The combination of these treatments resulted in additive effects leading to 38% survival at 100 days. These treatments did not, however, prevent the eventual development of radiation-induced liver pathology. This suggests that sepsis and endotoxemia resulting from the bacteria in the intestine are the immediate cause of death after 30-Gy liver irradiation and partial hepatectomy. It is concluded that the hepatectomized rat model is an economical and scientifically manageable experimental system to study a form of radiation hepatitis that occurs in compromised human livers.