Abstract
Purpose
To describe the dose response relationship and natural history of radiation injury in the Wistar rat and its suitability for use in medical countermeasures (MCM) testing.
Materials & Methods
In two separate studies, male and female rats were exposed to partial body irradiation (PBI) with 5% bone marrow sparing. Animals were X-ray irradiated from 7 to 12 Gy at 7–10 weeks of age. Acute radiation syndrome (ARS) survival at 30 days and delayed effects of acute radiation exposure (DEARE) survival at 182 days were assessed. Radiation effects were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging of lung, whole-body plethysmography, and histopathology.
Results
Rats developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in mortality at doses ≥8Gy in males and ≥8.5 Gy in females. DEARE mortality occurred at doses ≥8Gy for both sexes. Findings indicate lung, kidney, and/or liver injury, and persistent hematological dysregulation, revealing multi-organ injury as a DEARE.
Conclusion
The Wistar rat PBI model is suitable for testing MCMs against hematopoietic and gastrointestinal ARS. DEARE multi-organ injury occurred in both sexes irradiated with 8–9Gy, also suggesting suitability for polypharmacy studies addressing the combination of ARS and DEARE injury.
Introduction
There are now several FDA-approved medical countermeasures (MCMs) against hematopoietic acute radiation syndrome (H-ARS), including Neulasta®, Neupogen®, Leukine®, and NPlate® (Rios et al. Citation2022). As a result, MCM development efforts have begun to focus on the treatment and prevention of gastrointestinal acute radiation syndrome (GI-ARS) and delayed effects of acute radiation exposure (DEARE), as there are currently no FDA approved countermeasures for such indications. The total body irradiation (TBI) model used for the MCM development against H-ARS is inadequate for the development of acute GI, and DEARE responses in tissues such as lung or kidney, because TBI/H-ARS radiation doses are often lower than those used for the development of GI-ARS or DEARE injury (Williams et al. Citation2012). As a result, models of partial body irradiation (PBI) with bone marrow shielding are becoming the model of choice for examining the entire progression of radiation syndromes from H-and GI-ARS, to delayed injury of lung (L-DEARE), kidney (K-DEARE), and other organ systems (Satyamitra et al. Citation2022).
Rodents are particularly well suited for early efficacy screening as housing and care costs are far less than for larger species and mouse models have made important contributions to the FDA approval of H-ARS countermeasures (DiCarlo et al. Citation2021). However, its well known that there are strain and species differences in how ARS and DEARE effects manifest. For example, the C57BL6/J mouse is highly susceptible to ARS mortality in TBI models with and without supportive care (Plett et al. Citation2012) and even in PBI models with minimal bone marrow sparring (Booth et al. Citation2012), which makes it well suited for testing ARS countermeasures in short term studies (typically lasting 30 days), but not well suited for the purposes of testing products that protect against delayed tissue injury because higher doses to these animals generally result in relatively few survivors beyond the ARS response period. Therefore, to study DEARE and multi-organ injury that manifests after PBI, we must look beyond the C57BL6/J ARS models.
The most widely used rat PBI model is often referred to as a ‘Leg out’ model (Fish et al. Citation2021) because it provides some degree of bone marrow sparing with the aid of lead shielding over some or all of one leg. This type of exposure allows for the delivery of a larger dose of radiation to solid organs and soft tissues of the gastrointestinal (GI) system, while avoiding mortality due to hematopoietic atrophy following TBI. In the rat PBI model, animals first experience GI-ARS associated morbidity in combination with bone marrow atrophy, which may progress to the development of DEARE as a multi-organ injury, affecting both lung and kidney (Cohen et al. Citation2016). DEARE injuries have been shown to contribute to DEARE morbidity in both NHP and rodents, which have become standard animal models used for the testing of radiation MCMs (MacVittie et al. Citation2019).
The WAG/Rij rat strain has been used for decades in radiation research (Broerse et al. Citation1983; Van Rongen et al. Citation1986). These animals were originally brought to and bred at the Medical College of Wisconsin under the direction of John Moulder and registered with the rat genome database as the WAG/RijCmcr strain (previously called WAG/Rij/MCW) (Smith et al. Citation2020). There is a wealth of literature describing radiation effects and medical countermeasure testing in TBI, PBI, and whole thorax lung irradiation (WTLI) WAG/RijCmcr models. Irradiated WAG/RijCmcr rats develop both L- and K-DEARE, and studies have shown that early ARS deaths can be overcome using combinations of countermeasures such as hematopoietic growth factors, supplemental hydration, and antibiotics (Fish et al. Citation2016; Gasperetti et al. Citation2021).
Recent experience during the COVID pandemic has shown the importance of development of multiple animal models for drug development. As an example, the shortage of rhesus macaques has greatly slowed the progress of MCM testing in NHPs, forcing many investigators to seek alternatives such as the Cynomolgus macaques. Although the radiation responses of the WAG/RijCmcr rats are well characterized, these animals are not commercially available for broad-based testing by the radiation community. While the WAG/RijCmcr PBI model is well described and is consistent with NHP models in many aspects (Fish et al. Citation2020), there are likely subtle differences in responses specific to this strain and/or exposure model, and whether they are consistent across other strains of rats such as the Wistar IGS should be determined. For example, to know if the biomarkers of injury or clinical effects of radiation in Wistars are like that of the WAG/RijCmcr, would be essential to understanding the potential utility of the Wistar PBI model.
Herein we describe the natural history of radiation injury using Wistar rats and characterize the temporal and dose-dependence relationship to mortality after partial body irradiation. The Wistar rat is widely and commercially available and is in fact, the parent strain of the WAG/RijCmcr strain. We hypothesized that the PBI Wistar rat model would be a suitable model for DEARE countermeasure testing due to its physiological and phylogenetic similarity with the WAG/RijCmcr strain. To determine this, we evaluated ARS survival at 30 days, DEARE survival between 30 and 180 days, and clinical indicators of DEARE effects by hematology, clinical chemistry, clinical observations, body weights, MR imaging of lung, and whole-body plethysmography in Wistars. Pathology exams of macroscopic and microscopic tissues were used to support clinical findings and describe the extent of organ injury in moribund animals and animals which were sacrificed at scheduled intervals up to 182 days post irradiation during both the ARS and DEARE periods.
Data were compiled from two separate studies conducted over a period of two years. In the first dose response study (Study 1), rats were irradiated at 9–10 weeks of age using a range of doses from 7 to 12 Gy to establish the 180 day DRR, 30 day ARS, and 180 day DEARE mortality. The second study (Study 2) served to document the natural history of radiation injury progression, in rats that were irradiated at a narrower dose range of 8 to 9 Gy to minimize the impact of ARS morbidity. This cohort was irradiated at slightly younger ages (i.e. seven to nine weeks for males, and eight to nine weeks for females) to maintain animals at a weight that would allow for repeated MR imaging as body size is a limiting factor to obtaining MR images.
Given that LD values at 30 and 180 days, hematology, clinical pathology, and histopathology findings are all similar across the age of animals included in the study, data are presented as combined data, except for MRI and WBP data that were only collected from animals irradiated at seven to nine weeks of age.
Methods
Animals
Male and female Wistar IGS rats, raised in a barrier facility and provided pathogen free, were obtained from Charles River (Raleigh, NC) at five to eight weeks of age and housed two animals per cage under controlled conditions. Due to the large number of animals required for the two studies described, animals were obtained and irradiated in 10 separate shipments over a 10-month period. Housing, acclimation, handling, irradiation, and husbandry activities were the same for all shipments as specified by intuitional standard operating procedures (SOPs). Animals were acclimated for ≥1 week prior to experimentation and received standard laboratory diet Envigo Teklad 2018C, and water ad libitum throughout the course of the experiment. Supplemental water softened chow and hydrogel were provided for 30 days following irradiation, or when body weights dropped ≥15% of their pre-irradiation body weight. No antibiotics, subcutaneous fluids, or other supportive care were provided. Experiments were performed under protocols approved by SRI International’s Institutional Animal Care and Use Committee (IACUC) in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility.
Irradiation
All exposures took place during the same time of day (i.e. 8 am–2 pm) to avoid circadian confounding conditions (Plett et al. Citation2012). Irradiations were performed with a customized Pantek HF320 X-ray cabinet irradiator (Branford, CT) operating at 250kVp, 5 mA, with 2 mm aluminum filtration, and dose rate of ∼1.0 Gy/min. Unanesthetized animals (7–10 weeks of age) were restrained in plastic jigs, with the left forelimb secured under ∼0.5 cm of lead, shielding approximately 5% of the bone marrow. Sham-irradiated (0 Gy) control rats received similar handling. Dosimetry was conducted within a Plastic Water® (CIRS, Norfolk, VA) rat phantom, averaging the dose rates obtained at the midpoint of the thoracic and peritoneal cavities. Dose rates were determined prior to study start and confirmed immediately before each day of irradiation using an NIST calibrated farmer-type ion chamber. During irradiations, animals were placed on a rotating platform (∼1 rpm) to minimize the effect of any non-uniformity of the radiation field. Group sizes ranging from 36 to 120 were comprised of equal numbers of males and females (). Study 1 animals received 7, 8, 9, 9.5, 10, 11, or 12 Gy. Study 2 animals received 8, 8.5, or 9 Gy PBI under identical conditions.
Table 1. Numbers of animals per dose group.
Forelimb shielding efficacy was confirmed using Landauer nanodot™ (ND) thermoluminescent dosimeters (TLDs) placed directly on top of the forelimb underneath the lead shield. The mean dose to the shielded leg of the highest dose group (12 Gy) was determined to be ≤0.28 Gy, all other dose groups received lower doses to the shielded leg.
Health monitoring
Clinical observations were recorded daily for the first 30 days post-irradiation, and twice weekly thereafter. Mortality was checked daily. Teeth were also checked during clinical observations and were trimmed if they appeared to interfere with the animal’s ability to obtain food or water. Body weights were collected before irradiation, three times weekly from days 1 to 90 (weeks 1 to 13), once weekly thereafter, and in accordance with body weight change criteria.
Clinical criteria for euthanasia were scored as slight, moderate, or extreme for the following parameters: abnormal appearance (e.g. ruffled fur, hunched posture, rough coat, head down, tucked abdomen, pallor, exudates around eye and/or nose, etc.), decreased activity, weight loss, and dehydration. Two extreme findings, or two moderate and one extreme finding, triggered unscheduled euthanasia. Additional qualifiers for euthanasia were failure to respond to stimuli, inability to stand/ambulate, respiratory distress, masses ≥4 cm in diameter or that interfered with the ability to obtain food or water, and weight loss ≥30%.
Respiratory function
Unrestrained whole-body plethysmograph (WBP; Buxco Electronics, Wilmington, NC) was used to assess changes in pulmonary function prior to irradiation and approximately every two to four weeks thereafter using FinePoint v.1 software. Bias flow rate was set at 2 L/min with rats allowed to acclimate in the chamber for approximately 20 min before data acquisition using 2 s intervals. Breath frequency, tidal volume, and minute volume were measured.
Magnetic resonance imaging (MRI)
Images were obtained prior to irradiation and at approximately 42-, 84-, 126-, and 180-days post irradiation using the Bruker (Billerica, MA) 7 T PharmaScan small animal MRI, 16 cm bore, 60 mm body coil, with ParaVision 6.0.1 software. Respiratory gating was applied during image acquisition. For MRI analysis, visual verification of areas with increased signal intensity and tissue density, indicating congestion, inflammation or fibrosis were recorded. The mean signal intensity of three separate, manually delineated lung sections captured around the mid-point of the thorax were recorded per animal using Image J. At each timepoint, group means were compared to both the pre-irradiation mean for that group and to the age matched control group for that timepoint. Two different scans were performed:
T2 TurboRARE: Rapid acquisition with relaxation enhancement (RARE) sequence with repetition time (TR) 2500 ms, RARE factor 3, echo time of (TE) 19.17 ms, echo spacing 7.8 ms, 10 − 25 ∼ 1 mm thick slices positioned coronally over the lungs, 45 × 45 mm field of view (FOV), and image matrix of 256 × 256.
Table 2. Numbers of animals per blood collection.
UTE: Ultrashort echo time (UTE) sequence with (TR) 5.63 ms, TE 0.423 ms, 15 − degree flip angle, 45 ∼ 1 mm thick slices positioned axially over the lungs, 45 × 45 mm FOV, with image matrix of 128 × 128.
Hematology parameters
Blood was collected for hematology analysis, clinical pathology, and clotting factor analysis. A minimum of 3 rats/sex/dose group were assigned to collection timepoints prior to irradiation and, when possible, at 42-, 56-, 84-, 126-, 154-, and 180-days post-irradiation (). Samples were also collected from moribund animals when possible. Data from moribund collections were pooled by dose group when possible. For scheduled collections, blood was collected from isoflurane anesthetized rats via the retroorbital sinus a maximum of three times per animal with the final collection being terminal. Standard hematology parameters were analyzed using the ADVIA 2120 Hematology System (Siemens, Munich, Ger.), clinical chemistry with the Cobas c-501 Chemistry Analyzer (Roche, Indianapolis, IN) and clotting factors/fibrinogen with the STA Compact analyzer (Stago, Parsippany, NJ).
Histology
In Study 2, tissue samples were collected at pre-determined intervals from a minimum of 3 rats/sex for Sham (0 Gy), 8, 8.5, and 9 Gy irradiated animals at 42-, 84-, 126-, and 180 days post irradiation, and from moribund animals when possible. For Study 1, histopathology was only performed on select animals found moribund during the DEARE period. Tissues collected included: gross lesions, sternum (marrow histology), cecum, colon, duodenum, esophagus, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, rectum, spleen, stomach, thymus, and bladder. Lungs were inflated with formalin at 20 cm hydrostatic pressure prior to embedding, and additional sections were stained with Masson’s trichrome for the identification of fibrotic lesions. Tissues were formalin fixed, paraffin embedded, cut to 5 µm sections, and stained with hematoxylin and eosin (H&E).
Statistics
Kaplan Meier survival plots, body weight, and clinical pathology figures were generated using GraphPad Prism 9.2.0 (GraphPad Software, San Diego, CA.). Hematology, clinical pathology, and clotting factor sample results from Study 1 and Study 2 were pooled and analyzed by rank sum test with post-hoc Benjamini–Hochberg correction for multiple comparisons, and probit dose estimates were generated using Stata SE v14.2 for Windows (StataCorp LLC, College Station, TX) Probit dose estimates were also generated using Stata SE v14.2 for Windows.
Results
Survival
Kaplan Meier survival curves for the combined results of Study 1 and Study 2 showing the sexes combined, males alone or females alone () reveal two important findings: a dose dependent decrease in survival, and two distinct periods of mortality due to either ARS between days 4 and 13 or DEARE from day 35 to 177.
Figure 1. Kaplan Meier survival for combined sexes, males and females from Study 1 and Study 2 combined.
There was no mortality among controls, except for a single female that spontaneously developed a large inguinal tumor that required euthanasia, and there was no mortality for males or females receiving 7 Gy PBI, either. For males, H- and GI- ARS associated deaths occurred between days 4 and 13 at doses ≥8 Gy, for females ARS deaths fell between days 4 and 9 at doses ≥8.5 Gy. In males, DEARE-associated deaths occurred between days 102 and 177 at doses between 8 and 9 Gy, and all male deaths occurred during the ARS period for doses ≥9.5 Gy. In females, DEARE morbidity occurred slightly earlier than males between day 35 and 176, at doses ≥8 Gy. Unlike males, some females irradiated at 9.5, 10 and 12 Gy became moribund during the DEARE period. Survival percentages following ARS associated mortality at 30 days and DEARE associated mortality at 180 days are presented in for combined sexes, males alone and females alone.
Table 3. Survival results at 30 and 180 days for Study 1, Study 2, and both studies combined.
Probit estimates () and the dose response relationship to mortality for 30- and 180-day survival for Study 1, Study 2, and Studies 1 and 2 combined, are shown in . Because animals in Study 2 were sacrificed starting on day 42, only the mortality up to day 30 for Study 2 was included in the probit analysis.
Figure 2. Probit Estimates for 30, 50, and 70% mortality and dose response relationship for males, females and combined sexes at 30 and 180 days.
Table 4. Probit estimates for 30 and 180 day survival.
Body weights
Mean body weights (BW) for male and female rats to day 182) are shown in . MRI could not accommodate the larger body size of animals irradiated at 10 weeks of age, as a result starting BW for males and females selected for MRI irradiated between 7 and 9 weeks of age (Study 2) are lower than those irradiated at 9–10 weeks of age (Study 1). Therefore, the trend of reduced weights for irradiated animals is not strictly dose dependent, with the 8, 8.5, and 9 Gy groups (in Study 2) having lower body weights than the remaining groups.
Figure 3. Mean body weights for males and females to Day 182. Note the substantial starting difference between M and F weights. Weights for irradiated groups are substantially lower than the control group. Error bars represent standard error of the mean.
In general, sham irradiated male and female rats increased in weight continuously throughout the study, and the weight difference between males and females was substantial. The mean BW for sham treated males and females reached 630 g, and 329 g, by 182 days, respectively.
Body weight losses occurred in two dose-dependent waves for irradiated males and females in both studies, regardless of age at the time of irradiation, and were statistically significantly lower (p ≤ .05) than age-matched, sham irradiated controls by day 2. The first period of weight loss occurred between days 2 and 12 (weeks 1 and 2) and coincided with ARS associated morbidity. The second period of weight loss followed between days 42 and 70 (weeks 6 to 10) and coincided with the start of female DEARE morbidity and preceded DEARE morbidity in males. The timing of this second period of weight loss also appears to coincide with clinical observations of tooth loss or oral malocclusion. Both periods of body weight loss were followed by periods of weight recovery in the surviving animals.
Clinical observations
The most common adverse clinical findings associated with radiation exposure are shown in . Of these, hunched posture, dehydration, and ruffled fur were most numerous and reported as early as four days following irradiation. Diarrhea and fecal staining were also reported as early as day 4, although this was less frequent than other findings.
Table 5. Numbers of most prevalent adverse clinical findings by dose group.
Among the animals irradiated at seven to nine weeks of age (Study 2), malocclusion was a prevalent clinical finding in both sexes after the ARS period. The number of animals increased with increasing dose, with 42 males and 36 females affected at doses ranging from 8 to 9 Gy. There was little difference in the time to appearance of malocclusion; onset occurred between days 42 and 49 for all animals. In Study 1 animals irradiated at 9–10 weeks of age malocclusion was less common. Malocclusion was observed between days 91 and 153 and was seen in only one male and five female rats at doses ranging from 7 to 10 Gy.
Observations of corneal opacity developed as early as day 2 in 63 female rats treated at 8–11 Gy (with the next finding on day 65), and in 33 male rats treated at ≥8–10 and 12 Gy, and these did not resolve by day 180. This was a common observation regardless of the age at irradiation and was not seen in any of the control groups.
Tumor growths (masses) were reported in 25 males treated at 7–10 Gy and 18 female rats at 7–9.5 Gy in various body locations (i.e. lip, lumbar, flank, forelimb, etc.). The majority did not exceed 8 mm in diameter, except for two males and two females treated at 7 or 8 Gy where tumor sizes grew to >10 mm. The first mass was reported in a male on day 95, and in the first female on day 28, with most findings occurring after day 120 in both males and females. No irradiated animals were euthanized due to the appearance of tumors. Although there were a number of small masses in the irradiated animals, it should be noted that the appearance of spontaneous neoplasms in Wistars is fairly common (Poteracki and Walsh Citation1998). There was no difference between the ages at irradiation related to the appearance of masses. It is likely that no masses were reported in the highest dose groups, due to the very high ARS mortality resulting in very few rats surviving into the DEARE period.
Hematology
Absolute values for selected hematology parameters for the two combined studies are presented in . There were no statistically significant differences between the two cohorts of animals irradiated at different ages.
Figure 4. Hematology values for males and females. Statistical significance is indicated by (*, q ≤ 0.05)) as determined by a rank sum test and Benjamini–Hockberg adjustment for multiple comparisons. There were no significant differences between irradiated groups as compared to similar aged controls for the following parameters: red blood cells, lymphocytes, monocytes. Statistically significant differences as compared to similar age controls were indicated for the following parameters: White blood cells; females, 9 Gy at 182 days. Platelets; females, 8 Gy at 182 days. Neutrophils; males, 9 Gy DEARE moribund vs 182 days control, females, 9 Gy at 182 days. Reticulocytes; males, 8.5 Gy at 182 days, females, 8 Gy and 8.5 Gy at 182 days. Basophils; males, 8 Gy and 8.5 Gy at 182 days, and 9 Gy DEARE moribund vs 182 day control, females, 9 Gy at 182 days. Eosinophils; males, 9 Gy DEARE moribund vs 182 day control, females, 8.5 and 9 Gy at 182 days.
For the animals found moribund during the ARS period from which blood was collected, white blood cells, platelets, lymphocyte, neutrophil, monocyte, reticulocyte and even eosinophil counts were markedly lower than pre-irradiation values, and this is consistent with H-ARS hemopoietic atrophy.
Following the ARS response is a general trend of increased absolute values among the irradiated males and females as compared to controls for white blood cell, neutrophil, monocyte, and reticulocyte cell counts during the scheduled collection times. In a few instances, these increased values are statistically significant, mostly at the final scheduled collection. Lymphocyte values were only slightly increased in males at scheduled collections, however in females there was a more pronounced increase in lymphocyte counts among irradiated groups. Among the male and female rats moribund during the DEARE period, neutrophil, monocyte and reticulocyte counts were also elevated. Red blood cell counts for irradiated males however, show a pattern of decrease compared to control, and for irradiated females values were either similar to, or slightly less than control at scheduled collections.
Changes in platelet, basophil and eosinophil absolute counts for the irradiated animals at scheduled collection times were more variable. In irradiated males, platelet values were slightly reduced as compared to age matched controls through day 84, and at day 182, while platelet counts for females were somewhat similar to controls through day 84, but from day 126 to 182, counts were generally lower than controls. Basophil counts in males were variable and similar to controls, except at day 182 when 8 and 8.5 Gy groups were significantly lower. In females, counts were generally higher than controls, particularly at day 182. Eosinophil counts in males were variable for the control group at scheduled collections, but the baseline, day 84 and day 182 values for controls are similar to irradiated groups. In contrast, female eosinophil counts were similar to controls through day 84, and were increased in irradiated groups thereafter. At day 182, 9 and 9.5 Gy female groups were significantly elevated as compared to controls.
Clinical chemistry
Blood values for selected parameters are presented in . Group sizes for clinical pathology analysis are the same as for hematology collections ().
Figure 5. Clinical chemistry values for males and females. Statistical significance is indicated by (*, q ≤ 0.05)) as determined by a rank sum test and Benjamini–Hockberg adjustment for multiple comparisons. There were no significant differences between irradiated groups as compared to similar aged controls for the following parameters: ALP: prothombin time and fibrinogen. The following comparisons and groups were statistically significant: BUN; males 8 Gy, 8.5 Gy, 9 Gy, and 10 Gy at 182 days, female 8 Gy, 9.5 Gy and 10 Gy at 182 days. Cre; males, 8.5 Gy and 9 Gy at 182 days. AST; males, 8 Gy and 8.5 Gy at 26 weeks, and 8.5 Gy DEARE moribund vs 182 day control. ALT; males, 9 Gy at 182 days.
Blood urea nitrogen (BUN) increased over time in irradiated groups, with a slightly more pronounced response in males. For both males and females, BUN values were initially similar to controls, and were increased in males after day 84, while in females this increase began after day 126. At day 182, BUN in males from most irradiated groups (8 Gy, 8.5 Gy, 9 Gy, and 10 Gy) were significantly elevated compared to controls, males moribund due to DEARE were also increased as compared to day 182 controls. In females this response was less pronounced, although 3 irradiated female groups (8 Gy, 9.5 Gy, and 10 Gy) achieved statistical significance at day 182.
Creatinine (Cre) values followed a nearly identical patten for irradiated males and females. Irradiated males showed increases after day 84, and at day 182, two irradiated groups (8.5 and 9 Gy) were significantly increased when compared to the age-matched controls. Cre in the males moribund due to DEARE was also increased as compared to day 182 controls. Female creatinine changes were much less robust, and irradiated groups were similar to controls at most time points.
Aspartate Aminotransferase (AST) values showed the greatest increase in irradiated males, while almost no change from controls was seen in the females. In males from day 56 to day 182, AST levels were higher than age matched controls, with several groups (8 and 8.5 Gy) achieving statistical significance at day 182.
Alanine Transaminase (ALT) also showed a greater response in irradiated males than females as compared to age matched controls. By day 42 most irradiated male groups had higher ALT than age matched controls, however in females this trend was reversed and most irradiated groups had ALT values modestly lower than age matched controls.
Alkaline Phosphatase (ALP) values were generally higher for all animals prior to irradiation (day 0) and these declined until day 42, even among control groups. From day 42 and thereafter, control values were relatively stable, whereas ALP values for irradiated groups at day 84 and thereafter were modestly higher.
There were no statistically significant differences between age matched controls and irradiated groups for the clotting factors of prothrombin time and fibrinogen at any of the scheduled collections, however there is considerable variability in the female prothrombin values. Among euthanized females (ARS and DEARE), fibrinogen values were noticeably increased.
Histopathology
Representative histopathology is shown in . Histopathological findings were similar regardless of age at irradiation. These were generally consistent across radiation dose groups and between the two sexes, and the numbers and severity of findings generally increased in a dose dependent fashion.
Figure 6. Representative histology of ARS and DEARE. GI- and H- ARS findings (9 Gy) indicate duodenal degeneration of crypts (small circle) and villi (large circle), marrow shows marked decrease in BM cellularity (star). DEARE-associated findings in kidney reveal glomerulosclerosis (upper left star), tubular degeneration/regeneration (upper right star) and tubular changes consistent with chronic progressive nephropathy (lower left star). Lung findings indicate focal pleural fibrosis (arrow) and a focal increase in alveolar macrophages (star). Liver findings are of biliary hyperplasia (lower left arrow), eosinophilic (right arrow) and clear cell (upper left arrow) focus.
The primary findings in animals euthanized due to ARS prior to day 30 were hematopoietic atrophy and intestinal necrosis associated with the sequalae of the hematopoietic and gastrointestinal acute radiation syndromes (H- and GI- ARS). Hematology values reported for these animals support the finding of hematopoietic failure.
The predominant cause for morbidity during the late effects period were renal disease or lung injury associated with DEARE. Findings of atrophy in ovaries and testicles were common in all irradiated necropsy animals. Except when noted, findings were relatively evenly distributed by sex, and the type of finding was generally consistent for each organ or tissue across all time points examined.
Histopathology findings of the seven to nine week old, irradiated animals (Study 2) for scheduled necropsies at 42-, 84-, 126-, and 182 days post-irradiation were most prevalent in kidney, liver, lung, and vasculature. Radiation-associated microscopic findings for most other tissues were sporadic and of low incidence.
For the kidney, tubular degeneration and regeneration were present at 42 days post-irradiation and appeared to progress to chronic progressive nephropathy and glomerulosclerosis by 126 days post-irradiation. In addition, at the 182-day necropsy, severity increased for fibrinoid necrosis of arterioles and focal tubular hyperplasia. An adenoma was found to be present in one male animal in the 9 Gy group.
For liver, findings of increased altered hepatocellular foci were present at 84 days post-irradiation and biliary hyperplasia was consistently present at 182 days post-irradiation. Vascular lesions were also consistently present at 182 days post-irradiation.
Radiation-induced microscopic findings in the lung included focal to multifocal alveolar fibrosis (present at 42-, 84-, 126-, and 182 days), and severity of focal pleural fibrosis (present at day 182), diffuse hemorrhage (at days 126 and 182), focal alveolar macrophages (at day 182), focal bronchoalveolar hyperplasia (at days 84 and 182) increased over time. Fibrosis (pleural and/or alveolar) was shown to effect up to 25% of the microscopic field examined, and in the more severe findings, up to 50% of the field. At the 182-day necropsy, these findings were slightly more prevalent in males. In moribund, unscheduled necropsy animals, all of these findings generally occurred at a higher incidence and severity. There were no findings of pleural effusions or fluid within the thoracic cavity reported at the time of necropsy.
Whole body plethysmography (WBP)
Unrestrained whole body plethysmograph was used to assess changes in respiratory frequency, tidal, and minute volumes prior to irradiation and approximately every two to four weeks thereafter, for (Study 2) seven to nine week old irradiated males and eight to nine week irradiated females ().
Figure 7. Male and female whole body plethysmography. (*) indicates statistically significant differences vs. age matched control by two-way ANOVA and Dunnett’s test of multiple comparisons.
Overall, males maintained a consistently lower average breath rate frequency than females, and there was considerable variation in frequency within same sex controls. Despite this, there was a general trend of increased frequency amongst controls compared to irradiated groups for each sex, although there were few statistically significant differences among 8.5 and 9 Gy male groups at weeks 4 and 8, and female groups at day 56. Minute and tidal volumes also continued the trend of increased volumes among controls as compared with irradiated groups, and this pattern was also more pronounced in males. Minute volumes were significantly lower for almost all male irradiated groups as early as 28 days after irradiation until the end of the study, and in females only the 56-day timepoint achieved statistical significance. Male tidal volumes were significantly lower in most irradiated groups between day 84 and 182, and in females statistical significance was attained at days 28, 84, and 112, with significance seen only in the highest dose group at days 28 and 112.
Magnetic Resonance imaging (MRI)
Representative images are shown in . Manual identification of areas of high SI within lung sections was consistent with histopathologic findings within those same regions of the lung, verifying that RILI could be detected by MRI. However, quantitation of lung injury using raw SI values of lung sections was problematic, and due to the broad distribution of SI within each dose group, no statistically significant differences were noted between the irradiated groups and controls or their baseline measures at any timepoint. MRI comparisons between irradiated female animals and their sex/age matched controls indicated a pattern of increased signal intensity in both T2 and UTE scans, although these increases were slight and did not appear to increase with increasing dose.
Figure 8. Representative MR scans of T2 (A and C) and UTE (B and D) images. C&D images show areas of increased MR signal intensity (circles) in a 9.5 Gy irradiated female 84 days post irradiation.
Discussion
The purpose of the studies presented were to evaluate the suitability of the Wistar Rat PBI model to be used for the testing of MCMs. Although the Wistar is a parent strain of the WAG/RijCmcr, recent literature describing ARS, DEARE, and/or the natural history of radiation injury in the PBI model is sparse and incorporates substantially greater bone marrow shielding (Boittin et al. Citation2015) than the model described here, therefore it is important to fill this knowledge gap for the purposes of assessing this readily available strain for potential use in MCM investigations.
The PBI BM5 Wistar rat model shows many characteristics common to animal models of ARS, which are consistent for certain endpoints when comparing across different species. For example, leukocytopenia, hematopoietic atrophy, and gastrointestinal necrosis are well documented effects and findings common to ARS morbidity across mouse (Booth et al. Citation2012; Plett et al. Citation2012), rat (Matsuu-Matsuyama et al. Citation2020; Fish et al. Citation2021), and rhesus macaque NHP (MacVittie et al. Citation2012; Thrall et al. Citation2015) models. However, some endpoints reveal characteristics which may be unique to the Wistar rat model.
30 Day ARS Survival: Wistar rats are susceptible to ARS associated morbidity at doses ≥8 Gy for males and ≥8.5 Gy for females. This was true despite the age ranges at the time of irradiation, and ARS associated mortality in these animals increased in a dose dependent fashion. Time to the first deaths was four days, with the last ARS associated deaths occurring on day 14. Earlier deaths prior to day 9 are likely resultant of GI-ARS and generally attributable to the combination of leukocytopenia and sepsis that results from the combination of hematopoietic atrophy and disruption of gut barrier due to radiation induced intestinal necrosis (Fish et al. Citation2021). Slight differences in 30 day survival after 8 or 9 Gy exposures were seen when comparing the 8 and 9 Gy results from the 10–11 week-old animals (from Study 1) and the 7–9 week-old animals (from Study 2). While survival for the combined sexes at 8 Gy was identical at 94% for both studies, 9 Gy survival was slightly higher in the Study 2 rats, at 71% vs 54% for Study 1 rats, indicating that ARS survival may be greater in younger rats. However, whether this difference is attributable to the differing ages at irradiation, or is partly attributable to natural variation with the Wistar population is unknown. Regardless, by 180 days, the survival difference between the 8 and 9 Gy dose groups was reversed, with survival of the 8 Gy Study 1 animals at 92% as compared to those from Study 2 at 77%, while 9 Gy 180 day survival was at 50% for both studies, which may indicate that older animals are less susceptible to DEARE mortality. In fact, this has been reported in the WAG/RijCmcr model, with geriatric rats showing the least DEARE associated morbidity as compared to adult and juvenile animals (Medhora et al. Citation2019). It may be possible that younger rats are less susceptible to ARS deaths and more susceptible to DEARE death, but based on these 2 studies alone it would be premature to conclude this for the Wistar rats since literature supporting an equivalent PBI Wistar model at other institutions are lacking. Nonetheless, the high ARS mortality observed in Study 1 at doses >9Gy, and the consistent ARS findings across both age groups and experiments indicate that the Wistar may be a suitable model for ARS MCM testing. This Wistar PBI model may be particularly valuable in developing polypharmacy strategies to deal with the combination of ARS and DEARE injuries. This can be approached by using FDA approved countermeasures to overcome ARS morbidity at doses ≥9 Gy, followed by a second, appropriately timed countermeasure treatment to combat DEARE multi-organ injury, L-, and/or K- effects. Further work will need to be done to support this approach.
180 Day DEARE Survival: Results suggest that more animals irradiated at seven to nine weeks were found moribund during the DEARE period than the older cohort at 9 Gy for both sexes, and to a lesser extent in males irradiated at 8 Gy. This is at least partly due to the selection of doses for Study 2 (seven to nine week irradiated), which ranged between 8 and 9 Gy so as to minimize the number of ARS deaths and create a larger pool of animals for the study of DEARE effects by utilizing the lower end of the dose range employed in the initial survival study (Study 1). For animals moribund after day 30, the cause of mortality was identified as either K- or L- DEARE by pathological examination. Later DEARE morbidity beginning about day 150 in the younger seven to nine week animals approximates the described period of K-DEARE in the WAG/Rij (Fish et al. Citation2021). The cause of mortality in these younger animals was identified as kidney injury (pathology) although there were also concurrent findings of minimal to mild lung fibrosis and inflammation in many of these animals. In contrast, animals irradiated at 10–12 weeks did not show a pronounced period of mortality during the DEARE period, rather the deaths were distributed between day 40 and day 165. L-DEARE morbidity associated with pneumonitis has been demonstrated between approximately days 62 and 94 in the Wag/RijCmcr model (Fish et al. Citation2016) which is consistent with mortality and histopathology findings in the Wistar PBI model. It should also be noted that kidney injury as reported in other animal models falls during the later part of this range (day 150) and the majority of animals moribund at that time also showed signs of kidney injury in histopathology samples. This may in part be due to the majority of these (10–12 week) animals were irradiated at higher doses than the 7–9 week irradiated animals so it remains unclear if older animals are more susceptible to L-DEARE. While the cause of death, and periods of DEARE morbidity are consistent with other animal models, it should be noted that DEARE associated mortality for this study was much lower than has been reported for the WAG/Rij model (Fish et al. Citation2016), and the pronounced ARS morbidity of Wistar rats without supportive care or MCM treatment limits the number of animals available for evaluation of DEARE at doses ≥9 Gy.
Clinical indicators of radiation injury can be used to evaluate radiation responses in rat models for the evaluation of countermeasures against either ARS or organ responses specific to DEARE and determine the suitability of such models to mimic the known radiation response of higher order species such as NHPs and humans.
Body weight is one example of a clinical biomarker of both ARS and DEARE effects (Fish et al. Citation2021), and weight loss increases with radiation dose, similar to mortality. In this model, immediately following irradiation is a period of acute weight loss, and weights are markedly reduced between days 2 and 12, which corresponds to the first wave of (ARS) morbidity between days 4 and 13. The second period of weight loss between days 42 and 70, corresponds to earlier DEARE mortality in females between days 35 and 176 and precedes male mortality between days 102 and 177. This same bi-phasic pattern of weight loss has also been shown in Wistar (Boittin et al. Citation2015) and WAG/RijCmcr (Fish et al. Citation2021) radiation models. Other clinical indicators appear to be relevant to either ARS or DEARE.
Clinical indicators of ARS: In addition to acute weight loss, marked leukocytopenia was shown to occur in the H-ARS and GI-ARS response. Absolute counts of white blood cells, platelets, lymphocyte, neutrophil, monocyte, reticulocyte, and eosinophil counts were markedly lower than pre-irradiation values. Clinical observations consistent with ARS were also reported; hunched posture, dehydration, and ruffled fur were most common and reported as early as four days following irradiation. Diarrhea and fecal staining, which would be consistent with GI injury were also reported as early as day 4. Examination of sternal bone marrow and GI tissues are consistent with the known pathologies of H- and GI-ARS.
Clinical indicators of DEARE: Wistars are susceptible to ARS mortality, and survivors are also susceptible to developing DEARE. There are ample clinical findings to indicate DEARE multi-organ injury, of which many have been demonstrated in the similar WAG/RijCmcr PBI model (Gasperetti et al. Citation2021). Radiation injury of the lung, kidney, and liver are the most prominent delayed effects in this Wistar model, and these occur in concert with persistent hematopoietic dysregulation indicated by long term hematological changes. The clinical indicators of DEARE in the solid organs are supported by histopathological findings from animals collected at scheduled sacrifice times, and from those collected as moribund during the DEARE period.
One interesting clinical observation which may be indicative of DEARE to follow was the onset of malocclusions, which are infrequently reported in literature. In the Wistar, malocclusions were not observed at 7 Gy wherein there was no DEARE associated morbidity. Interestingly, malocclusions alone are not responsible for the observed second period of weight loss, as animals without malocclusions also lost weight. While the potential association between DEARE effects and malocclusions is still unclear, at a minimum researchers should be aware that untreated malocclusion could impact study endpoints such as body weight for long term TBI and PBI studies. Whether or not younger irradiated animals are more prone to developing malocclusion, and if this is a herald of later DEARE injury is yet to be determined, further use of the Wistar PBI model may reveal this through additional studies.
Clinical indicators of L-DEARE can be seen in MR images, and in WBP results. Initially, we sought to verify the appearance of radiation lung injury in MR images by scanning moribund animals, and those found to have obvious lesions detected by MR were sent for histopathology. This confirmed the appearance of inflammation and fibrosis in the approximate location as detected by MRI within the lungs of these animals. Subsequently, we attempted to compare the raw signal intensity values from the lungs of irradiated and non-irradiated animals over time with the expectation that the irradiated lung would have higher signal intensity values than non-irradiated animals. However, raw signal intensity values were highly variable and there were no significant differences noted between irradiated and non-irradiated groups. WBP data reflects reduced lung function and suggests radiation-induced lung injury. This is particularly evident when examining changes in minute and tidal volumes, which were reduced in irradiated groups as compared to age matched controls. However, because the body weights of the irradiated animals were also significantly lower than controls, it is difficult to determine if, or how much of the reduced volumes are attributable to the irradiated animals lack of growth, or how much is attributable to radiation lung injury and resultant inflammation and fibrosis. Regardless, histopathological findings show that the lungs of the majority of these animals had some degree of alveolar inflammation and alveolar and/or pleural fibrosis. There were a total of 6 representative DEARE moribund animals for which radiation lung injury was determined to be the cause of death, at doses ranging from 8 to 12 Gy, all of which became moribund between days 43 and 67, which is consistent with the timing for L-DEARE reported in other rat models (Fish et al. Citation2016).
K-DEARE can be identified through increases in BUN and Cre, which have been shown in rat PBI models to be increased with radiation induced kidney injury, and BUN values have been used as a criterion for euthanasia for the WAG/RijCmcr model (Fish et al. Citation2016). In the Wistar model presented here, we see a delayed increase in BUN and Cre, which begin as early as 126 days post irradiation and reach statistical significance in most irradiated groups by day 182, but BUN values never reach the same level as reported for the WAG/Rij model. Creatinine values mirror this trend, although the pattern of increase was not as pronounced. In males, creatinine reached statistical significance at 182 days at 8.5 and 9 Gy, however the increase in females at day 182 was not statistically significant. It should also be noted that Cre values were increased in samples collected from DEARE moribund animals, with the greatest response in moribund males. These clinical indicators of kidney injury are supported by histopathology findings. Tissue collection from 10 representative moribund animals from which blood was collected indicated mortality due to radiation kidney injury at doses ranging from 8 to 9 Gy and on days between 148 and 178 is consistent with the timing observed in other rat models (Fish et al. Citation2016).
Liver DEARE may also play a role in overall DEARE mortality by contributing to radiation induced multi-organ dysfunction. Although pathological examination did not indicate liver injury to be a cause of death among moribund animals, changes in plasma AST, ALT and ALP indicate the potential for liver injury (Boone et al. Citation2005), in the Wistars. Results from our studies showed that the liver response was most pronounced in males. Both AST and ALT levels were elevated in all irradiated male groups at 84 days post irradiation and remained elevated until the end of the experiments. DEARE moribund males also showed elevated AST, which was statistically significant at 8.5 Gy. Male ALT values were also persistently elevated at day 84 post irradiation and onwards, and within the DEARE moribund males. Interestingly, these same responses were not observed in the female groups; ALT and AST remained similar to, or slightly lower than controls at most scheduled collections and in the DEARE moribund females. ALP showed a similar pattern of change for both males and females. Immediately following irradiation, we observed a decrease in ALP for all animals. This could potentially be due to a change in animal diet, when animals transitioned from the diet provided from the supplier during transit to the study diet. After the initially observed decrease in ALP at 42 days, we see the trend of increased values at 84 days for male and female irradiated groups which remain elevated throughout the study, although these increases were not statistically significant. DEARE moribund animals had similar ALP values compared to controls. These indications of liver injury were supported through histopathological examination. Focal hepatocellular vacuolation was present at in both sexes, along with altered hepatocellular foci attributable to irradiation at ≥7 Gy in males and ≥8 Gy in females. Contribution to overall mortality cannot be determined, however, there is sufficient evidence to indicate that liver injury occurs as a part of the overall multi-organ dysfunction as a long term DEARE effect.
Persistent hematopoietic dysfunction: The hematopoietic compartment, and bone marrow specifically, is known to be very sensitive to radiation injury. In this PBI model we observed reduced bone marrow cellularity in ARS moribund animals at doses as low as 8 Gy. This transient decline in marrow cellularity had resolved/repopulated when examining histopathology samples collected 42 days post irradiation. Despite this apparent regeneration, there remained persistent changes in the hematological compartment, for the same cell types which were depleted in the ARS. Absolute counts of white blood cells, neutrophils, lymphocytes, monocytes and reticulocytes were all increased in both male and female irradiated groups, generally at doses ≥ 8 Gy, across nearly all time points examined, and in many cases these parameters were found to be increased in DEARE moribund animals as well. A decrease in mature red blood cells was also observed for males across all post irradiation timepoints, and females at days 56, 84, and 126. For the males this finding, coupled with increased numbers of banded reticulocytes may indicate anemia, and a compensatory response of the bone marrow to repopulate lost erythrocytes. Whether these results are due to persistent damage of the repopulated bone marrow itself, or the result of crosstalk between persistently damaged irradiated tissues of liver, lung, kidney, and other organs, it remains unclear if hematological dysregulation contributes to the overall DEARE morbidity in this model.
Evaluation of irradiation survival up to 180 days post-exposure in this study was an important first step in establishing an institutional lethality dose (LD) profile, and Probit estimates for the LD30/180, LD50/180, LD70/180 for the Wistar rat PBI model. These were reported to be 8.6, 9.0, and 9.5 Gy, respectively, as determined in Study 1. Similar results were reproduced by a second study, despite the lower body weights and slightly younger ages of the animals at irradiation. Evidence of persistent or progressive tissue damage in lung, kidney, liver, eyes, and reproductive organs were observed upon macroscopic and microscopic evaluation. In addition, body weights and lung function never recovered to levels seen in the sham-irradiated controls, supporting progressive multi-organ injury as a DEARE in this model of radiation injury.
Evaluating the suitability of the Wistar rat model for MCM testing
There are certain advantages to the use of this rat model. For example, the Wistar rat is readily available from several animal commercial animal providers. There are also intrinsic advantages of rats having a larger body size than mice, which allows for larger blood volumes at collection and an expanded opportunity to examine multiple clinical endpoints. Larger dose volumes can be delivered as well, this may be required for some newly developed MCMs. There are also potential advantages to the use of Wistars, due to the canonical ARS responses reliably produced at doses as low as 8 Gy. This naturally lends itself to models of combined H- and GI-ARS injury for the testing of medical countermeasures, for which there is no FDA approved countermeasure. In addition, these animals show a multi-organ injury as a DEARE effect, and because of the natural sequalae of injury as animals progress from ARS to DEARE, the Wistar may be a good alternative to the WAG/RijCmcr model for testing a polypharmacy approach to overcome ARS, and mitigate multi-, L-, or K- organ injury as a DEARE effect.
The major disadvantage of PBI Wistar model is the lack of published data describing the DRR and natural progression of radiation injury, which was the main motivation for this manuscript. It is likely that the WAG/RijCmcr model will continue to be the rat model of choice for the foreseeable future for the testing of countermeasures against DEARE effects. This is deservedly so because of the abundant literature and extensive studies performed to date describing the WAG/Rij PBI model and three distinct periods of mortality due to ARS, L- and K- DEARE. Additional studies of the Wistar PBI model will be needed to define these periods of L- and K- DEARE mortality . A second advantage of the WAG/Rij PBI model is that it has been developed to the point that it can allow for the mitigation of ARS mortality with supportive care or MCMs, for polypharmacy studies against DEARE effects, although ARS mitigation has yet to be demonstrated in Wistars.
In conclusion, the Wistar PBI model represents an important step forward in animal model development, a necessary component to satisfy the FDA animal rule required for the testing of medical countermeasures. Evaluation of traditional measures of radiation injury: survival, body weight loss, clinical observation, clinical observation, and histopathology, hematology, coagulation, and clinical chemistry parameters, in general, were consistent and followed expected patterns for H- and GI-ARS and DEARE and indicate the suitability of the Wistar PBI model to be used in ARS, or polypharmacy studies addressing the combination of ARS and DEARE injury.
Acknowledgements
We would like to thank Meetha Medhora, Heather Himburg, Brian Fish, and Tracy Gasperetti at the Medical College of Wisconsin for the many discussions of the technical details regarding rat PBI models, and the toxicology support staff (TSS) at SRI for their impeccable work over the course of two years during the conductance of the studies presented here.
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No potential conflict of interest was reported by the author(s).
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
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Notes on contributors
Tyler Beach
Tyler Beach, PhD, is a Research Scientist, Study Director, and Co-investigator at SRI International focusing on the development of animal models and medical countermeasures against radiation.
James Bakke
James Bakke, BS, is a Research Scientist and Study Director at SRI International focusing on efficacy and safety toxicology studies and animal model development for advancement of radiation countermeasure candidates in a variety of animal species.
Ed Riccio
Ed Riccio, BS, is a retired Program Director for the Genetic Toxicology Testing Program at SRI International.
Harold S. Javitz
Harold S. Javitz is a Senior Biostatistician at SRI International and conducts statistical analyses for toxicology and radiation studies.
Denise Nishita
Denise Nishita, BS, is a Research Associate and Study Director in the Molecular Toxicology group at SRI International, conducting pre-clinical toxicology and efficacy studies of medical countermeasures against radiation.
Shweta Kapur
Shweta Kapur, MS, is a Research Associate in the Molecular Toxicology group at SRI International, supporting toxicology studies, animal model development, and efficacy studies of medical countermeasures against radiation.
Deborah I. Bunin
Deborah I. Bunin, PhD, is Director of Molecular Toxicology at SRI International and conducts pre-clinical toxicology and efficacy studies of medical countermeasures against radiation.
Polly Y. Chang
Polly Y. Chang, PhD, is Scientific Director of the Nonclinical Development Program in SRI’s Biosciences Division and serves as Principal or co-Investigator on a number of NIH, BARDA, NASA, and commercially sponsored grants/contracts. She is actively engaged in radiation biology research and product development of medical countermeasures against radiation.
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