Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Oct 24.
Published in final edited form as: Radiat Res. 2015 Oct 23;184(5):545–553. doi: 10.1667/RR14191.1

Total Body Irradiation in the “Hematopoietic” Dose Range Induces Substantial Intestinal Injury in Non-Human Primates

Junru Wang a,1, Lijian Shao a,1,2, Howard P Hendrickson a, Liya Liu a, Jianhui Chang a, Yi Luo a, John Seng a, Mylene Pouliot b, Simon Authier b, Daohong Zhou a, William Allaben a, Martin Hauer-Jensen a,c
PMCID: PMC6812554  NIHMSID: NIHMS736831  PMID: 26495870

Abstract

The non-human primate has been a useful model for studies of human acute radiation syndrome (ARS). However, to date structural changes in various parts of the intestine after total body irradiation (TBI) have not been systematically studied in this model. Here we report on our current study of TBI-induced intestinal structural injury in the nonhuman primate after doses typically associated with hematopoietic ARS. Twenty-four non-human primates were divided into three groups: sham-irradiated control group; and total body cobalt-60 (60Co) 6.7 Gy gamma-irradiated group; and total body 60Co 7.4 Gy gamma-irradiated group. After animals were euthanized at day 4, 7 and 12 postirradiation, sections of small intestine (duodenum, proximal jejunum, distal jejunum and ileum) were collected and fixed in 10% formalin. The intestinal mucosal surface length, villus height and crypt depths were assessed by computer-assisted image analysis. Plasma citrulline levels were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total bone marrow cells were counted and hematopoietic stem/progenitor cells in bone marrow were analyzed by flow cytometer. Histopathologically, all segments exhibited conspicuous disappearance of plicae circulares and prominent atrophy of crypts and villi. Intestinal mucosal surface length was significantly decreased in all intestinal segments on day 4, 7 and 12 after irradiation (P < 0.02–P < 0.001). Villus height was significantly reduced in all segments on day 4 and 7 (P = 0.02–0.005), whereas it had recovered by day 12 (P > 0.05). Crypt depth was also significantly reduced in all segments on day 4, 7 and 12 after irradiation (P < 0.04–P < 0.001). Plasma citrulline levels were dramatically reduced after irradiation, consistent with intestinal mucosal injury. Both 6.7 and 7.4 Gy TBI reduced total number of bone marrow cells. And further analysis showed that the number and function of CD45+CD34+ hematopoietic stem/progenitors in bone marrow decreased significantly. In summary, TBI in the hematopoietic ARS dose range induces substantial intestinal injury in all segments of the small bowel. These findings underscore the importance of maintaining the mucosal barrier that separates the gut microbiome from the body’s interior after TBI.

INTRODUCTION

The risk of exposure to ionizing radiation due to nuclear accidents or terrorist scenarios is widely thought to be increasing. One likely outcome of exposure in such scenarios is the development of acute radiation syndrome (ARS). Among the organs that are primarily involved in ARS are the hematopoietic system/bone marrow (BM) and gastrointestinal (GI) tract (1, 2). Hematopoietic cells are highly sensitive to radiation-induced damage and relatively low levels of total body irradiation (TBI) (e.g., 1–6 Gy) can induce hematopoietic acute radiation syndrome (H-ARS), resulting from bone marrow ablation that leads to hemorrhage or infections (1–3). However, higher TBI doses (e.g., 6–8 Gy) can result in gastrointestinal acute radiation syndrome (GI-ARS), primarily as a result of the enterocyte depletion caused by the death of intestinal mucosal stem cells. GI-ARS is characterized by a dose-dependent onset of nausea and diarrhea followed by sepsis and electrolyte imbalance, which can lead to death (1–3).

Developing medical countermeasures against ARS requires well-characterized and validated animal models. Radiation-induced damage to the hematopoietic system has been relatively well established and animal models such as mice, canine and non-human primates have been utilized to investigate countermeasures against radiation-induced H-ARS (2, 4–8). Significant enhancement of hematopoietic regeneration has been reported in elegant efficacy studies that tested various combinations of cytokines such as interleukin-3, interleukin-6, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor and stem cell factor after total body irradiation in different animal models (9–11). Several U.S. Food and Drug Administration (FDA)-approved products, such as different forms of G-CSF including tbo-filgrastim and pegfilgrastim, are available to reduce chemotherapy-induced myelosuppression. Importantly, application of filgrastim (Neupogen®) to adult and pediatric patients, who were exposed to radiation, was recently approved by the FDA. Other biologic license applications have been submitted for several recombinant leukocyte growth factors that have demonstrated improved survival in multiple irradiated animal models including non-human primates (12).

Currently, there are no FDA-approved medical countermeasures against GI-ARS and there are no well-characterized large-animal models to assess TBI-induced GI-ARS (2). The development of animal models of GI-ARS is requisite to the successful development of medical countermeasures. A number of studies have investigated GI-ARS in non-human primates (13–17), but each study focused on different parameters such as the mortality and morbidity, clinical symptomology and pathology. In some studies, high TBI doses, such as 9.5, 10–14 and 15–75 Gy are used (14, 18, 19). However, baseline values for TBI-induced GI-ARS are not well characterized in non-human primates.

Our laboratory is currently developing a marketed, multi-receptor, somatostatin analog referred to as pasireotide diaspartate. This analog is a promising mitigating agent against radiation-induced GI toxicity. Our laboratory has demonstrated that pasireotide effectively reduces TBI-induced mortality in mice even when the administration of test material started 72 h after lethal doses of TBI (20). A possible mechanism for this improvement in animal longevity after TBI may be related in part to the ability of pasireotide to inhibit various pancreatic digestive enzymes (21).

To assess the efficacy of pasireotide and other potential drugs in mitigating the risk of ARS, further validation and characterization of large animal models are urgently needed. Therefore, the current study was designed to determine optimal TBI doses in non-human primates by establishing baseline changes in the intestinal mucosa, hematopoietic parameters and the intestinal injury biomarker citrulline in animals postirradiation. This study utilized TBI doses of 6.7 (LD70/60) and 7.4 Gy (LD90/60) to assess the intestinal mucosal injury and hematopoietic injuries at day 4, 7 and 12 after TBI, as well as the relationship of intestinal mucosal injury with plasma citrulline levels (22–24). Results from this study demonstrated that TBI doses of 6.7 and 7.4 Gy (typically associated with the H-ARS) induced significant intestinal injury. Data collected in testing this range of TBI doses in the non-human primate model may be useful in developing GI-ARS risk-mitigating agents in compliance with the FDA Guidance for Industry Product Development Under the Animal Rule.

MATERIALS AND METHODS

Animals

A total of 24 non-human primates (Macaca mulatta; 12 males and 12 females), 4.0–6.2 years old and weighing between 4.2 and 7.3 kg were used. The animal room environment was controlled (temperature 21 ± 3°C, humidity 30–70%, 12:12 h light-dark schedule, 10–15 air changes per hour), and temperature and relative humidity were monitored continuously. A standard certified commercial primate chow (Teklad Certified Hi-Fiber Primate Diet 7195C; Harlan® Laboratories, Madison, WI) was available to each non-human primate twice daily. Municipal tap water (which was exposed to ultraviolet light and purified by reverse osmosis) was provided to the animals ad libitum. All animals were fasted overnight prior to radiation exposure.

During the study, care and use of animals were conducted in accordance with principles outlined in the current Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (National Academies Press, 8th Ed., 2011). The testing facility was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were conducted per established standard operating procedures.

Irradiations and Study Design

Animals were placed into boxes and total body irradiated with a single uniform dose, from a Theratron® 1000 60Co source (Best® Theratronics Ltd., Ottawa, Canada), of 6.7 or 7.4 Gy at a dose rate of 60 cGy/min. The total dose was divided in two fractions with anteroposterior (AP) and postero-anterior (PA) exposure. Each animal was placed in a chair allowing appropriate restraining in a symmetrical position for TBI. Prior to exposures, dosimetry measurements were obtained in the same experimental conditions using a solid water phantom to confirm the treatment plan. Real-time in vivo dosimetry was monitored during animal exposure using a Farmer ionization chamber (PTW, Freiburg, Germany) subjected to an electrometer bias voltage of −300 V (model no. 35040; Keithley Instruments Inc., Cleveland, Ohio). Dosimeters (Scanned nanoDot, Landauer®, Glenwood, IL) and SuperFlab buildup material were used at the distal sternum and interscapular area to validate the treatment dose. Control animals were subjected to identical procedures without exposure to the radiation source. The animals were irradiated or sham irradiated and monitored for up to 12 days postirradiation, as detailed in Table 1.

TABLE 1.

Experimental Design

Necropsy
Group Radiation dose (Gy)
Day 4
 Day 7
 Day 12
Males Females Males Females Males Females
1 Sham 2 2 — — — —
2 6.7 (LD70/60) 2 2 2 2 1 1
3 7.4 (LD90/60) 2 2 2 2 1 1
Open in a new tab

Supportive Care

Buprenorphine was administered to all animals prophylactically and throughout the observation period. Certified non-human primate Liquidiet (Bio-Serv®, Flemington, NJ) was administered orally when body weight was <85% of baseline and continued until recovery. Hydration fluid (Ringer’s lactate solution and Ringer’s lactate solution with 5% dextrose, IV) was provided based on clinical evaluation. Blood products and antibiotics were not included in the current experiment to minimize potential interferences with experimental end points that were evaluated.

Blood, Bone Marrow and Intestinal Tissue Collection

Groups of animals were euthanized on day 4, 7 and 12 after irradiation. Blood, bone marrow and intestinal tissue collection were performed under the supervision of an American College of Veterinary Pathologists (ACVP) Board-Certified veterinary pathologist. Blood samples were collected for citrulline analysis. A target volume of 0.3 ml of blood was collected by venipuncture into tubes containing K3-EDTA as anticoagulant during the preirradiation period and on day 4, 7 and 12 after irradiation, and was centrifuged under refrigeration (4°C at 1,500g RCF) for 10 min. Plasma was recovered and stored at −70°C until analyzed. Bone marrow samples were collected from the femur for total marrow cellularity determination. Briefly, bone marrow was flushed from the bone with 5 ml of culture media [RPMI-1640 without phenol red, 20% fetal bovine serum (FBS) and penicillin streptomycin solution] and then gently passaged with a disposable 20-gauge needle to form a homogenous cellular suspension and kept at 4°C until analysis. Small intestine sections (duodenum, proximal jejunum, distal jejunum and ileum) were collected and fixed in 10% formalin, processed to paraffin blocks, sectioned and stained with hematoxylin and eosin (H&E) for histological and morphometric studies.

Hematopoietic and Intestinal Injury Assessments

Assessment of TBI-induced histological and morphometric injury

Mucosal surface length: A decrease in the surface area of the intestinal mucosa is a sensitive parameter of small bowel radiation injury. Previously, we measured mucosal surface area in mice and rats using a stereologic projection/cycloid method. Because of structural differences in the intestine of non-human primates (e.g., non-human primates have plicae circulares whereas mice and rats do not), we chose computer-assisted image analysis (Image-Pro® Plus, Media Cybernetics Inc., Silver Spring, MD) to measure the length of intestinal mucosal surface area in this animal model. All measurements were done with a 10× objective lens, and a total of 10 areas were measured from each intestinal segment.

Mucosal villus height and crypt depth: Mucosal villus height and crypt depth were measured with computer-assisted image analysis (Image-Pro Plus). All measurements were done with a 10× objective lens. Mucosal villus height was measured from the tip to the base of villus, and crypt depth was measured from crypt base to the top opening. All measurements were done with a 10× objective lens, and a total of 10 areas were measured from each intestinal part.

Assessment of TBI-induced plasma citrulline change

The plasma level of citrulline is a well-validated biomarker for functional enterocyte mass. Previous studies have shown that circulating concentrations of citrulline had a significant negative correlation to radiation dose in mice and humans (22–24). Here, we collected blood plasma for citrulline analysis at various time points postirradiation. Citrulline concentrations were determined using the high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology previously described by our group (25). Briefly, each plasma sample (5 μl) was added to each well of a 96-well protein precipitation plate and treated with 95% acetonitrile containing 0.2% formic acid (0.45 ml). The filtrate was injected (3 μl) directly onto the LC-MS/MS system. Separation on a diol column (50 × 2.1 mm) was accomplished using an acetonitrile gradient and 0.2% acetic acid, 0.1% formic acid and 0.005% trifluoroacetic acid (TFA) modifiers. The lower and upper limits of quantitation were 0.125 and 2,000 μM, respectively.

Assessment of TBI-induced hematopoietic injury

Bone marrow cells were suspended in 10.0 ml red blood cell lysis buffer (0.15 M NH4Cl, pH 7.2) and incubated for 15 min on ice. The cells were spun down at 200g for 6 min, washed once with 10.0 ml PBS with 2% FBS, and resuspended in 0.5ml PBS with 2% FBS. BM cells were counted using cell counter (Heska, Loveland, CO), and 1 × 106 BM cells were labeled with anti-CD45-FITC and anti-CD34-APC antibodies (eBio-science Inc., San Diego, CA) after incubation with anti-CD16/32 to block the Fcγ receptors (eBioscience, Inc.). After washing, the cells were resuspended in PBS containing 0.25 μg/ml propidium iodide (Sigma-Aldrich® LLC, St. Louis, MO) to exclude dead cells and analyzed using a LSRII flow cytometer (Becton Dickinson Biosciences, San Jose, CA). Quantity of different hematopoietic cell populations (CD45+ cells and CD45+CD34− cells) in each non-human primate were calculated by multiplying the total numbers of BM cells harvested from the femur and tibia of each non-human primate with the frequencies of each population in BM cells. The colony-forming unit (CFU) assay at day 4 and 7 postirradiation was performed by culturing 1 × 105 BM cells using MethoCult™ H4034 Optimum (STEMCELL Technologies, Vancouver, Canada). Colonies of burst-forming unit–erythroid (BFU-E) and CFU–granulocyte macrophage (CFU-GM) were scored on day 14 of the incubation according to the manufacturer’s protocol.

Statistical Analysis

Statistical analysis was performed with the software package NCSS 2004 for Microsoft Windows (NCSS, Kaysville, UT). Differences in end points among treatment groups were assessed by one-way analysis of variance (ANOVA), and two-way ANOVA was used to determine the interaction between radiation doses and times. Differences in end points between two groups were assessed with equal-variance t test of two samples. P values less than 0.05 were considered statistically significant.

RESULTS

TBI-Induced Histological and Morphometric Injury in Small Intestine

As reported in previous studies in mouse and rat models, exposure to radiation induced significant structural alterations in the intestinal wall. As shown in Fig. 1, all segments exhibited conspicuous disappearance of plicae circulares and prominent atrophy of crypts and villi after irradiation and there were no ulcerations observed in any segments of small intestine. In the 6.7 Gy irradiated group, the plicae circulares were partially restored at day 7 and 12 after irradiation, but not in 7.4 Gy irradiated group.

FIG. 1.

FIG. 1.

Open in a new tab

Histological images of intestines from sham-irradiated (control) and irradiated non-human primates. Control intestine exhibits normal architecture. In contrast, different parts of intestine show extensive conspicuous disappearance of plicae circulares and prominent atrophy of crypts and villi at day 4, 7 and 12 after irradiation. Scale bars, 100 μm.

According to morphometric studies, intestinal mucosal surface length was significantly decreased in all intestinal segments on day 4, 7 and 12 after irradiation (Fig. 2, P < 0.02–P < 0.0002). At day 12 after irradiation, mucosal surface length of duodenum was recovered for both 6.7 and 7.4 Gy irradiated groups (P > 0.05). Villus height was significantly reduced in all segments on day 4 and 7 in both groups (Fig. 3, P = 0.02–0.005), whereas it had recovered by day 12 (P > 0.05). Crypt depth was also significantly reduced in all segments on day 4, 7 and 12 after irradiation (Fig. 4, P < 0.04–P < 0.00004). TBI in the hematopoietic dose range induces consistent changes in several compartments and in all segments of the small bowel. These findings point to the importance of maintaining the mucosal barrier integrity that separates the gut microbiome from the body’s interior after TBI.

FIG. 2.

FIG. 2.

Open in a new tab

Effect of radiation on intestinal mucosal surface length in non-human primates. Mucosal surface length was measured in vertical stained sections of intestine using a computer-assisted image analysis. A total of 5 areas were measured with 4× objective lens. Mucosal surface length was significantly reduced at day 4, 7 and 12 after irradiation. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control (CTL).

FIG. 3.

FIG. 3.

Open in a new tab

Effect of radiation on intestinal mucosal villus height in non-human primates. Mucosal villus height was measured with computer-assisted image analysis. A total of 5 areas were measured with 4× objective lens. Mucosal villus height was dramatically decreased at day 4 and 7 after irradiation. It has a trend to recover at day 12 after irradiation. *P < 0.05; **P < 0.01 vs. control.

FIG. 4.

FIG. 4.

Open in a new tab

Effect of radiation on intestinal mucosal crypt depth in non-human primates. Mucosal crypt depth was measured with computer-assisted image analysis. A total of 10 areas were measured with 10× objective lens. Mucosal crypt depth was significantly reduced at day 4, 7 and 12 after irradiation. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

TBI-Induced Plasma Citrulline Changes

The mean plasma citrulline concentration from all pre-TBI samples was used as a control (100 ± 35 μM). On day 4, 7 and 12 citrulline levels significantly decreased relative to the control after 6.7 and 7.4 Gy TBI (Fig. 5). Four days after 6.7 and 7.4 Gy irradiation, citrulline levels were 45 ± 12 and 58 ± 23 μM (P < 0.001 for both groups), compared to the control level of 100 ± 35 μM (Fig. 5A). Seven days after 6.7 and 7.4 Gy irradiation, citrulline levels were 66 ± 5 μM (P = 0.01) and 43 ± 20 μM (P = 0.001) (Fig. 5B). Twelve days after 6.7 and 7.4 Gy irradiation, citrulline levels were 70 μM and 64 μM, respectively (Fig. 5C). Figure 5D and E show the effects of time after exposure to 6.7 and 7.4 Gy. It appears that citrulline levels continued to decrease on day 7 and started to recover on day 12 in the 7.4 Gy irradiated group, although the changes in citrulline relative to day 4 and 7 were not significant. However, citrulline levels appeared to start recovering on day 7 (P = 0.001) and continued to recover on day 12 in the 6.7 Gy irradiated group, although the change in citrulline relative to day 7 was not significant. It should be mentioned that only two animals were available on day 12 for both the 6.7 and 7.4 Gy irradiated groups. These data show that citrulline reduction can start as early as day 4 after irradiation in the non-human primate and could start to recover as early as 7 days after irradiation, depending on radiation dosage.

FIG. 5.

FIG. 5.

Open in a new tab

Effect of radiation on plasma citrulline levels in non-human primates. Panel A: Changes of plasma citrulline at different time points after irradiation. Panel B: Changes of plasma citrulline at different radiation doses. The levels of plasma citrulline were significantly decreased at day 4 and 7 postirradiation compared to that of control plasma citrulline. At day 12 postirradiation, there were a trend to increase plasma citrulline. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

TBI-Induced Hematopoietic Injury

To confirm the effects of radiation exposure on the hematopoietic system, BM cells were obtained. The results showed that 6.7 and 7.4 Gy TBI caused a rapid decrease in the number of total BM cells in a dose- and time-dependent manner, as shown in Fig. 6A. The nadir for the number of BM cells was reached 12 days after TBI (P < 0.001). We further used CD45 and CD34 markers to label hematopoietic cells and hematopoietic stem/progenitor cells (HSPCs), respectively. As shown in Fig. 6B and C, TBI induced a significant decrease in both hematopoietic cells and HSPCs (P < 0.001), which is consistent with the results of total BM cell counting. To investigate the effects of radiation on HSPC function, we used in vitro clonogenic assay to assess the function of BM HSPCs. The results showed that 6.7 and 7.4 Gy TBI significantly decreased the numbers of BFU-E and CFU-GM compared to that of the nonirradiated group at day 4 and 7 (Fig. 6D and E), indicating that the ability of HSPCs to differentiate into erythrocytes, granulocytes and monocytes was dramatically decreased in irradiated non-human primates compared to sham-irradiated non-human primates. HSPCs were insufficient at day 12 to run the clonogenic test. These data suggest that 6.7 and 7.4 Gy TBI causes acute hematopoietic damage in the non-human primate in a dose- and time-dependent manner.

FIG. 6.

FIG. 6.

Open in a new tab

Effects of radiation on hematopoietic cells in bone marrow in non-human primates. Panel A: 6.7 and 7.4 Gy TBI caused a rapid decrease in the number of total BM cells in a dose- and time-dependent manner. Panels B and C: A significant decrease occurs in both hematopoietic cells and HSPCs after 6.7 and 7.4 Gy TBI. Panels D and E: 6.7 and 7.4 Gy TBI significantly decreased the numbers of BFU-E and CFU-GM. **P < 0.01; ***P < 0.001 vs. control.

DISCUSSION

The current study showed that TBI doses of 6.7 and 7.4 Gy (levels typically associated with hematopoietic ARS) in the non-human primate induced significant intestinal injury in dose- and time-dependent manners. The conspicuous disappearance of intestinal plicae circulares, reduction of mucosal surface area and prominent atrophy of crypts and villi in all small intestinal segments (duodenum, proximal and distal jejunum, ileum) were observed. These histopathological changes correlated with the onset of radiation-induced diarrhea in this non-human primate model. Interestingly, circulating citrulline, a biomarker of intestinal injury (22–24), decreased after TBI in both the 6.7 and 7.4 Gy groups, which is consistent with the damage of intestinal mucosa such as mucosal surface area, villus height and crypt depth. As expected, 6.7 and 7.4 Gy TBI caused significant hematopoietic injury such as the reduction of BM cells, as well as the number and function of HSPCs. These results indicate that the TBI dose range of 6.7 and 7.4 Gy in the non-human primate could be a practical model to study countermeasures against GI-ARS.

Radiation-induced intestinal damage is widely documented in rat and mouse models of localized and total body irradiation (21, 26–32). Previously published studies on TBI-induced GI-ARS in non-human primates are limited (3, 14–17, 33–35), each of which focuses on different parameters such as mortality (13), clinical symptomology and descriptive pathology (14–17) and GI function (33–35). Some of those studies have used high doses of TBI, such as 9.5, 10–14 and 15–75 Gy (14, 18, 19). Recently, MacVittie et al. investigated TBI-induced GI-ARS in the non-human primate model and found that 10–11.5 Gy significantly reduces the number of crypts at day 7, 10 and 15 after TBI (18). No quantitative data from TBI-induced intestinal structural injury in the non-human primate are available after TBI doses typically associated with hematopoietic ARS. The current study would be the first to observe the intestinal structural and hematopoietic injuries, as well as circulation citrulline, a biomarker of intestinal injury in such dose ranges of TBI.

The intestine is a major dose-limiting organ during abdominal and pelvic radiotherapy and one of the critical organs for whole-body irradiation. The surface area of intestinal epithelium is 200 times larger than that of the skin (36). Thus the epithelial lining of the intestine constitutes the body’s most extensive and important barrier against the exterior environment. Normal homeostasis of intestinal epithelium is maintained by continuous and rapid replacement through replication and differentiation of epithelial cells located within the intestinal mucosal crypts. Intestinal clonogenic crypt epithelial cells are classical target cells of intestinal radiation toxicity (37). Radiation-induced damage of these cells causes enterocyte depletion, mucosal barrier breakdown and mucositis, eventually allowing bacteria to translocate into the circulation, causing sepsis. Mucosal barrier breakdown also reduces intestinal absorption, which induces diarrhea and eventual fluid and electrolyte loss typically associated with a decrease in body weight. Other secondary effects of mucosal barrier breakdown (e.g., cytokine production and inflammation) as well as effects on some other elements of the intestine (such as the immune system, microvasculature and nervous system) also contribute to acute GI syndrome (2).

Plasma citrulline is mainly derived from small intestinal absorptive epithelial cells (38) and it has been identified as a biomarker for functional small bowel enterocyte mass under various clinical and experimental conditions (22–24, 39), including radiation exposure. In a murine study (23), 8–12 Gy of TBI was shown to induce significant decreases in citrulline levels, which then tends to recover in 4 days. In our previous murine study, plasma citrulline decreased significantly at day 3.5, started to recover at day 7 and reached normal levels at day 14 after 9 Gy TBI, which is consistent with the recovery of intestinal mucosal surface area (40). No studies examining citrulline level of GI-ARS in the non-human primate have been published. In a baboon TBI study (41), however, it was found that after 2.5 Gy TBI citrulline levels decreased on the first day, reached the lowest levels on day 4 and started to recover on day 5, whereas at 5.0 Gy, the lowest level of citrulline occurred on day 5 and recovery started on day 7. This study indicated that radiation-induced intestinal injury could start with doses as low as 2.5 Gy and as early as the first day after TBI. Our current study showed that exposure to 6.7 and 7.4 Gy TBI in the non-human primate model resulted in significant reductions in plasma citrulline levels in a dose-effect manner. On day 4, plasma citrulline level was significantly reduced in both 6.7 and 7.4 Gy exposures. However, on day 7, citrulline levels appeared to start recovering in the 6.7 Gy irradiated group but decreased further in the 7.4 Gy irradiated group. On day 12, citrulline levels recovered slightly but did not reach normal levels in either dosing. These findings are consistent with our intestinal injury, such as intestinal mucosal length, mucosal villus height and crypt depth. Therefore, we propose that blood citrulline could be considered a biomarker of GI-ARS in the non-human primate model.

The primary cause of morbidity and mortality in radiation-induced hematopoietic syndrome is the significant reduction in numbers of platelets and neutrophils, which are produced by hematopoietic stem cells through progenitor differentiation. It has been suggested that direct damage to hematopoietic progenitors in bone marrow is a primary target under radiation exposure, resulting in anemia, neutropenia and thrombocytopenia (1–3). In our current studies, the radiation doses of 6.7 and 7.4 Gy dramatically decreased the number of total BM cells and CD45+ hematopoietic cells. We further used CD34 to label hematopoietic stem/progenitor cells and demonstrated that CD45+CD34+ cells were significantly reduced at day 4, 7 and 12 after irradiation. These data suggest that our non-human primate model is also suitable to investigate the effects of certain potential countermeasures on hematopoietic stem/progenitor cells under radiation conditions. We are currently exploring the mechanism of action through which pasireotide impacts hematopoietic stem/progenitor cells postirradiation.

We conclude that the pathophysiology of acute radiation syndrome in the non-human primate is similar to that previously observed in mice and rats as related to intestinal structure, plasma citrulline level and hematopoietic progenitors. The non-human primate appears to be a suitable large animal model to study the radiation-induced intestinal syndrome and to test radiation countermeasures in the near future. Meanwhile, we are working on additional characterizations of radiation-induced GI toxicity in the non-human primate.

ACKNOWLEDGMENTS

Financial support for this work was provided by the U.S. Department of Health and Human Services (DHHS)/Office of the Assistant Secretary for Preparedness and Response (ASPR)/Biomedical Advanced Research and Development Authority (BARDA) (contract no. HHSO100201100045C), National Institutes of Health award P20 GM109005 and the Veterans Administration (www.va.gov). The authors thank the Office of Grants and Scientific Publications at the University of Arkansas for Medical Science for their assistance in editing this manuscript.

REFERENCES

  • 1.Hall EJ, Giaccia AJ. Radiobiology for the radiobiologist. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. [Google Scholar]
  • 2.Williams JP, Brown SL, Georges GE, Hauer-Jensen M, Hill RP, Huser AK, et al. Animal models for medical countermeasures to radiation exposure. Radiat Res 2010; 173:557–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Mettler FA Jr, Voelz GL. Major radiation exposure-what to expect and how to respond. N Engl J Med 2002; 346:1554–61. [DOI] [PubMed] [Google Scholar]
  • 4.Dainiak N Hematologic consequences of exposure to ionizing radiation. Exp Hematol 2002; 30:513–28. [DOI] [PubMed] [Google Scholar]
  • 5.Anno GH, Young RW, Bloom RM, Mercier JR. Dose response relationships for acute ionizing-radiation lethality. Health Physics 2003; 84:565–75. [DOI] [PubMed] [Google Scholar]
  • 6.Stone HB, Moulder JE, Coleman CN, Ang KK, Anscher MS, Barcellos-Hoff MH, et al. Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI workshop. Radiat Res 2004; 162:711–28. [DOI] [PubMed] [Google Scholar]
  • 7.Augustine AD, Gondre-Lewis T, McBride W, Miller L, Pellmar TC, Rockwell S. Animal models for radiation injury, protection and therapy. Radiat Res 2005; 164:100–9. [DOI] [PubMed] [Google Scholar]
  • 8.Schuening FG, Storb R, Goehle S, Graham TC, Appelbaum FR, Hackman R, et al. Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis of normal dogs and on hematopoietic recovery after otherwise lethal total body irradiation. Blood 1989; 74:1308–13. [PubMed] [Google Scholar]
  • 9.MacVittie TJ, Farese AM, Herodin F, Grab LB, Baum CM, McKearn JP. Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor. Blood 1996; 87:4129–35. [PubMed] [Google Scholar]
  • 10.Patchen ML, Fischer R, MacVittie TJ. Effects of combined administration of interleukin-6 and granulocyte colony-stimulating factor on recovery from radiation-induced hemopoietic aplasia. Exp Hematol 1993; 21:338–44. [PubMed] [Google Scholar]
  • 11.Schuening FG, Appelbaum FR, Deeg HJ, Sullivan-Pepe M, Graham TC, Hackman R, et al. Effects of recombinant canine stem cell factor, a c-kit ligand, and recombinant granulocyte colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation. Blood 1993; 81:20–6. [PubMed] [Google Scholar]
  • 12.Singh VK, Newman VL, Seed TM. Colony-stimulating factors for the treatment of the hematopoietic component of the acute radiation syndrome (H-ARS). Cytokine 2015; 71:22–7. [DOI] [PubMed] [Google Scholar]
  • 13.Henschke UK, Morton JL. Mortality of rhesus monkeys after single total body irradiation. Am J Roentgenol Radium Ther Nucl Med 1957; 77:899–909. [PubMed] [Google Scholar]
  • 14.Wilson SG Jr. Radiation-induced gastrointestinal death in the monkey. Am J Pathol 1959; 35:1233–51. [PMC free article] [PubMed] [Google Scholar]
  • 15.Allen JR, Van Lancker JL, Wolf RC. Pathologic alterations produced by total body x-irradiation in monkeys. Am J Pathol 1966; 48:317–31. [PMC free article] [PubMed] [Google Scholar]
  • 16.Eldred E, Trowbridge W. Radiation sickness in the monkey. Radiology 1954; 62:65–73. [DOI] [PubMed] [Google Scholar]
  • 17.Haigh MV, Paterson E. Effects of a single session of whole body irradiation in the rhesus monkey. Br J Radiol 1956; 29:148–57. [DOI] [PubMed] [Google Scholar]
  • 18.MacVittie TJ, Farese AM, Bennett A, Gelfond D, Shea-Donohue T, Tudor G, et al. The acute gastrointestinal subsyndrome of the acute radiation syndrome: a rhesus macaque model. Health Phys 2012; 103:411–26. [DOI] [PubMed] [Google Scholar]
  • 19.Vigneulle RM, Rao S, Fasano A, MacVittie TJ. Structural and functional alterations of the gastrointestinal tract following radiation-induced injury in the rhesus monkey. Dig Dis Sci 2002; 47:1480–91. [DOI] [PubMed] [Google Scholar]
  • 20.Fu Q, Berbee M, Wang W, Boerma M, Wang J, Schmid HA, et al. Preclinical evaluation of Som230 as a radiation mitigator in a mouse model: postexposure time window and mechanisms of action. Radiat Res 2011; 175:728–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fu Q, Berbee M, Boerma M, Wang J, Schmid HA, Hauer-Jensen M. The somatostatin analog SOM230 (pasireotide) ameliorates injury of the intestinal mucosa and increases survival after total-body irradiation by inhibiting exocrine pancreatic secretion. Radiat Res 2009; 171:698–707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lutgens L, Lambin P. Biomarkers for radiation-induced small bowel epithelial damage: an emerging role for plasma Citrulline. World J Gastroenterol 2007; 13:3033–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lutgens LC, Deutz NE, Gueulette J, Cleutjens JP, Berger MP, Wouters BG, et al. Citrulline: a physiologic marker enabling quantitation and monitoring of epithelial radiation-induced small bowel damage. Int J Radiat Oncol Biol Phys 2003; 57:1067–74. [DOI] [PubMed] [Google Scholar]
  • 24.Crenn P, Messing B, Cynober L. Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction. Clin Nutr 2008; 27:328–39. [DOI] [PubMed] [Google Scholar]
  • 25.Gupta PK, Brown J, Biju PG, Thaden J, Deutz NE, Kumar S, et al. Development of high-throughput-put HILIC-MS/MS methodology for plasma citrulline determination in multiple species. Anal Meth 2011; 3:1759–68. [Google Scholar]
  • 26.Hauer-Jensen M, Poulakos L, Osborne JW. Effects of accelerated fractionation on radiation injury of the small intestine: a new rat model. Int J Radiat Oncol Biol Phys 1988; 14:1205–12. [DOI] [PubMed] [Google Scholar]
  • 27.Richter KK, Langberg CW, Sung CC, Hauer-Jensen M. Association of transforming growth factor beta immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy. Radiother Oncol 1996; 39:243–51. [DOI] [PubMed] [Google Scholar]
  • 28.Zheng H, Wang JR, Koteliansky VE, Gotwals PJ, Hauer-Jensen M. Recombinant soluble transforming growth factor beta type II receptor ameliorates radiation enteropathy in mice. Gastroenterology 2000; 119:1286–96. [DOI] [PubMed] [Google Scholar]
  • 29.Vriesendorp HM, Vigneulle RM, Kitto G, Pelky T, Taylor P, Smith J. Survival after total body irradiation: effects of irradiation of exteriorized small intestine. Radiother Oncol 1992; 23:160–9. [DOI] [PubMed] [Google Scholar]
  • 30.Booth C, Tudor G, Tudor J, Katz BP, MacVittie TJ. Acute gastrointestinal syndrome in high-dose irradiated mice. Health Phys 2012; 103:383–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Potten CS. A comprehensive study of the radiobiological response of the murine (BDF1) small intestine. Int J Radiat Biol 1990; 58:925–73. [DOI] [PubMed] [Google Scholar]
  • 32.Rotolo JA, Kolesnick R, Fuks Z. Timing of lethality from gastrointestinal syndrome in mice revisited. Int J Radiat Oncol Biol Phys 2009; 73:6–8. [DOI] [PubMed] [Google Scholar]
  • 33.Griffiths NM, Linard C, Dublineau I, Francois A, Esposito V, Neelis KJ, et al. Long-term effects of X-irradiation on gastrointestinal function and regulatory peptides in monkeys. Int J Radiat Biol 1999; 75:183–91. [DOI] [PubMed] [Google Scholar]
  • 34.Danquechin DE, Mueller GP, Eng RR, Durakovic A, Conklin JJ, Dubois A. Effect of ionizing radiation on gastric secretion and gastric motility in monkeys. Gastroenterology 1985; 89:374–80. [DOI] [PubMed] [Google Scholar]
  • 35.Shea-Donohue T, Danquechin-Dorval E, Montcalm E, El-Bayar H, Durakovic A, Conklin JJ, et al. Alterations in gastric mucus secretion in rhesus monkeys after exposure to ionizing radiation. Gastroenterology 1985; 88:685–90. [DOI] [PubMed] [Google Scholar]
  • 36.Mowat AM, Viney JL. The anatomical basis of intestinal immunity. Immunol Rev 1997; 156:145–66. [DOI] [PubMed] [Google Scholar]
  • 37.Withers HR, Elkind MM. Dose-survival characteristics of epithelial cells of mouse intestinal mucosa. Radiology 1968; 91:998–1000. [DOI] [PubMed] [Google Scholar]
  • 38.Wu G, Knabe DA, Flynn NE. Synthesis of citrulline from glutamine in pig enterocytes. Biochem J 1994; 299:115–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Prasanna PG, Blakely WF, Bertho JM, Chute JP, Cohen EP, Goans RE, et al. Synopsis of partial-body radiation diagnostic biomarkers and medical management of radiation injury workshop. Radiat Res 2010; 173:245–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Burnett AF, Biju PG, Lui H, Hauer-Jensen M. Oral interleukin 11 as a countermeasure to lethal total-body irradiation in a murine model. Radiat Res 2013; 180:595–602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Herodin F, Richard S, Grenier N, Arvers P, Gerome P, Bauge S, et al. Assessment of total- and partial-body irradiation in a baboon model: preliminary results of a kinetic study including clinical, physical, and biological parameters. Health Phys 2012; 103:143–9. [DOI] [PubMed] [Google Scholar]

RESOURCES