Developing and comparing models of hematopoietic-acute radiation syndrome in Göttingen and Sinclair minipigs

Abstract

Purpose

Current animal models of hematopoietic-acute radiation syndrome (H-ARS) are resource intensive and have limited translation to humans, thereby inhibiting the development of effective medical countermeasures (MCM)s for radiation exposure.

Materials and methods

To improve the MCM pipeline, we developed models of H-ARS in male Göttingen and Sinclair minipigs. Weight matched Göttingens and Sinclairs received total body irradiation (TBI; 1.50–2.10 Gy and 1.94–2.90 Gy, respectively), were observed for up to 45 days with blood collections for clinical pathology analysis, and were examined during gross necropsy.

Results

The lethal dose for 50% of the population over the course of 45 days (LD_50/45) with ‘field’ supportive care (primarily antibiotics and hydration support) and implanted vascular access ports was 1.89 and 2.53 Gy for Göttingens and Sinclairs, respectively. Both minipig strains exhibited prototypical H-ARS characteristics, experiencing thrombocytopenia and neutropenia, and nadirs approximately 14 days following irradiation, slightly varying with dose. Both strains experienced increased bruising, petechia, and signs of internal hemorrhage in the lungs, GI, heart, and skin. All observations were noted to correlate with dose more closely in Sinclairs than in Göttingens.

Conclusion

The results of this study provide a template for future MCM development in an alternate species, and support further development of the Göttingen and Sinclair minipig H-ARS models.

Keywords:

• Acute radiation syndrome (ARS)
• miniswine
• lethality
• total body irradiation (TBI)

Introduction

Civilians, medical responders, and military personnel are all at risk of radiation exposure from various sources of radiation such as inadvertent occupational exposure, nuclear energy facility accidents (i.e. Fukushima Daiichi power plant in Japan) or nuclear terrorism. For humans managed with supportive care, the lethal dose of total body irradiation for 50% of a population over the course of 60 days (LD_50/60) is approximately 3.5–4.0 Gy (Lopez 2011). Currently there are limited medical countermeasure (MCM)s available for individuals exposed to potentially lethal amounts of radiation (Singh et al. 2015). In humans, hematopoietic acute radiation syndrome (H-ARS), which consists of clinical signs including: nausea, vomiting, headache, fatigue, fever, and transient skin reddening, can develop after exposures as low as 2 Gy (Donnelly et al. 2010). Exposures above 6 Gy generally constitute a combination of H-ARS and gastrointestinal-ARS (GI-ARS) in humans. H-ARS is characterized by decreases in red blood cells, lymphocytes, and platelets (Nagayama et al. 2002; Flynn and Goans 2006; Farese et al. 2014). In addition, granulocytes such as neutrophils spike within the first three hours after exposure and then decline drastically (Dainiak et al. 2003; Moroni, Lombardini et al. 2011). As a result, thrombocytopenia (absolute platelet count < 20,000 µL⁻¹) and neutropenia (absolute neutrophil count < 500 µL⁻¹) are definitive signs of H-ARS used in research models (Bolduc et al. 2016; Farese et al. 2019). Due to the potential threat of radiation exposure, lethality, and severe clinical symptoms of ARS, and since human trials are not possible, animal models of ARS are needed to advance radiation MCM development.

Several animal species have been used in irradiation research, including mice (Chua et al. 2012), beagle dogs (Zaucha et al. 2019), non-human primates (NHP; Thrall et al. 2019), and minipigs (Moroni, Coolbaugh et al. 2011). In all investigative research, larger animals generally have more translational success due to greater similarity to humans. In addition, larger animals provide greater utility for reasons such as sufficient size for repeat blood sampling over an extended time course and visual evaluation of organ injury. However, larger animals consume more resources associated with the animals and animal husbandry than smaller animal species. Compared to these other species, minipigs share many physiological similarities with humans while also offering several financial, breeding, and handling advantages (Jacobs 2006; Vodicka et al. 2005). In addition, minipigs are an excellent large animal model to study radiation damage to selected organs such as skin, kidneys, gastrointestinal system, and lungs compared to mice, dogs, or NHPs (Williams 2010; Rajkot 2015). The most widely used strains of minipig in radiation research are the Göttingen, and to a lesser extent the Sinclair and Yucatan. In the limited literature on Göttingen and Sinclair minipigs in radiation models, the Göttingen appears to be more sensitive to radiation exposure (Kenchegowda 2018), presenting an interesting physiological difference between the two strains that may be useful in elucidating the mechanisms associated with multi-organ injury of ARS.

In animal models, hematopoietic responses are suggestive of survival rates (Moroni, Lombardini et al. 2011), indicating the importance in studying the manifestation of H-ARS. Furthermore, H-ARS develops after a relatively low dose of radiation and concomitantly with GI-ARS when higher doses are administered (MacVittie et al. 2012). The necessity of a strong immune response has also been demonstrated when bolstered hematopoiesis from bone marrow supplementation, or leukocyte growth factor (LGF) based treatments (Welte et al. 1987; Hercus et al. 2009), lead to increased animal survival after radiation exposure (Terry and Travis 1989; Singh et al. 2015). Due to the vital role of the hematopoietic response, a radiation exposure model constituting lethal injury associated with systemic organ responses is necessary for impactful MCM development.

In general, there is sparse research on ARS in minipigs compared to other species because they have only recently been incorporated into radiation related studies. Moroni et al. have contributed much to the field of minipig H-ARS. They first demonstrated the LD_50/30 of male Göttingens to be between 1.7 and 1.9 Gy with no described medical support (Moroni, Coolbaugh et al. 2011). As seen in other species, it was shown that male Göttingens also experience decreased lymphocytes, platelets, red blood cells, and an initial spike of neutrophils followed by neutropenia. Presentation of these classic symptoms of ARS supports the use of the Göttingen minipig to simulate the human condition. To further illustrate the translational ability of Göttingens in the H-ARS model, Moroni et al. reproduced the ability of Granulocyte-Colony Stimulating Factor (G-CSF) to hasten neutrophil recovery after irradiation as seen in humans (Waselenko et al. 2004; Moroni et al. 2013). It must be acknowledged that there were a relatively limited number of animals used on this study, and further research is required to fully support this claim. A distinct difference between Göttingen and Sinclair minipigs is their sensitivity to radiation exposure under similar study conditions. Sinclair minipigs were shown to have 100% survival 45 days after the highest dose administered (2.3 Gy) while only 25% of Göttingens survived at the same dose (Kenchegowda 2018). To our knowledge, further research has not been conducted to differentiate the mechanistic response from radiation exposure between Göttingen and Sinclair minipigs.

The goal of these studies were to develop models of ARS in both the Göttingen and the Sinclair minipig with a focus on lethality and hematopoietic changes as a tool for advancement of treatments for radiation exposure. Weight matched male Göttingens and Sinclairs received TBI and were evaluated for up to 45 days following exposure for clinical signs of morbidity or mortality. Both animal strains demonstrated similar physiological responses to radiation as seen in humans with minimal clinical pathology and gross necropsy observation differences between strains, despite the noted differences in radiosensitivities.

Materials and methods

Göttingen and Sinclair minipigs

Twenty-one male Göttingen minipigs (Marshall Bioresources, North Rose, NY) and twenty-seven male Sinclair minipigs (Sinclair Bioresources, Columbia, MO) were procured in various cohorts for two separate studies. Upon receipt, animals were placed into a 14-day quarantine. Prior to placement on study, animals were implanted with vascular access ports (VAP) and allowed at least 2 weeks of recovery between surgery and irradiation. When selecting animals from the respective vendors, body weights and sexual maturity (different for the 2 strains) were used to normalize the selected animals. On the day of irradiation, animals had body weights ranging from 7.2 to 13.2 kg with average weights of Göttingen minipigs at 11.1 kg and Sinclair minipigs at 10.2 kg. At time of irradiation, the average age of Göttingen minipigs was 5 months and Sinclair minipigs 3.6 months. Minipigs were uniquely identified by ear tags placed prior to receipt at Lovelace Biomedical and Environmental Research Institute (LBERI). The Study Design is outlined in Table 1. It should be noted that data included herein are a subset of information collected from larger studies containing both male and female animals. Only results from male minipigs are included in the present manuscript; a comparison manuscript between male and female Göttingen minipigs is in preparation. As a result, the statistical power in some groups has been reduced and an uneven distribution can be seen. Throughout, data from the 1.8 Gy dose group in Göttingen minipigs are subject to limited interpretation due to that group only containing one animal.

Table 1. Comparison of animal numbers and irradiation doses per group in Göttingens and Sinclairs.

Göttingen study		Sinclair study
Group (N)	Dose (Gy) [% Received]	Group (N)	Dose (Gy) [% Received]
1 (4)	1.50 [100.2]	1 (3)	0
2 (3)	1.60 [100.0]	2 (6)	1.94 [100.8]
3 (6)	1.70 [100.5]	3 (5)	2.30 [101.5]
4 (1)	1.80 [101.7]	4 (5)	2.45 [99.7]
5 (3)	1.95 [99.7]	5 (5)	2.60 [100.4]
6 (4)	2.10 [99.4]	6 (3)	2.90 [100.2]

Husbandry

Animal studies were approved by the LBERI Institutional Animal Care and Use Committee; conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International; and carried out in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council 2011). Animals were provided ad libitum water and twice daily food (Harlan Teklad, Madison, WI) unless fasted prior to sedation for vascular port implantation, irradiation, and euthanasia.

Irradiation and dosimetry

Animals were exposed at LBERI using a 6-MV X-ray beam generated from a clinical linear accelerator (LINAC) and delivered at an average midline dose rate of 54.3 cGy/min (Varian 600c LINAC; Varian Medical Systems, Inc. Palo Alto, CA). Prior to irradiation, the instrument output was verified using a Lucite block physical phantom with both an NIST-traceable PTW ionization chamber and thermoluminescent dosimeters (TLDs). The acceptance criteria for both verifications was 2%.

Minipigs were sedated with acepromazine (1.1 mg/kg, SC) and anesthetized with isoflurane via facemask. The minipig was positioned entirely inside the light field of the instrument. Animals were irradiated to each animal’s midline bilaterally and uniformly at half of the target dose per side. The irradiation procedures started immediately after the initiation of the daily light-cycle to minimize disturbance to animals’ photoperiods; all irradiations were completed by approximately noon each day.

Health status monitoring

Minipigs were observed for morbidity and mortality twice daily by trained laboratory personnel. Animals were weighed prior to the start of the study for randomization and again on each blood collection day.

Field supportive care

Baytril supportive care treatment (5 mg/kg; PO SID) was initiated when neutrophil counts dropped below 1000/mL in Göttingen following methods used by Farese and MacVittie (Farese et al. 2012; MacVittie et al. 2012). In later studies, Sinclair minipigs received scheduled administration of oral gentamicin (2 mg/kg, SID) and oral amoxicillin (10 mg/kg, BID) from days 3 to 30 in agreement with BARDA minipig model development and harmonization efforts.

Blood collections

Throughout the course of the study, blood was collected from the VAP or a peripheral vein if the VAP did not maintain patency. Blood collections were obtained for hematology and clinical chemistry.

Göttingens had hematology samples collected at baseline, just prior to irradiation, 3 h post irradiation, and 1, 3, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 37, and 45 days post-irradiation. The results were used to determine the onset of daily antibiotic (Baytril) treatment as well as to determine H-ARS progression. Clinical chemistry samples were collected prior to irradiation, 3 h post-irradiation, and 1, 3, 7, 10, 14, 21, 28, 30, 37, and 45 days post-irradiation.

Sinclairs had hematology samples collected before and 3, 5, and 10 h after irradiation on day 0, and on days 1, 3, 7, 14, 21, 28, 35, and 45 days post-irradiation. Clinical chemistry samples were collected prior to irradiation and 1, 7, 14, 21, 28, 35, and 45 days post-irradiation.

Clinical pathology

For hematology analyses, whole blood was collected with syringe and needle and placed into vacutainers containing K₃EDTA as an anticoagulant. Hematology samples were analyzed by automated analysis using an ADVIA^TM 120 Hematology System (Siemens Medical Solutions, Malvern, PA). For clinical chemistry analyses, whole blood was placed into serum separator tubes for centrifugation and separated into cellular and serum fractions. Serum samples were analyzed on a Hitachi Modular Analytics Clinical Chemistry System (Roche Diagnostics, Indianapolis, IN).

Euthanasia

At scheduled necropsy (day 45) or in cases of moribundity, animals were sedated with acepromazine (1.1 mg/kg, IM) and tranquilized by administration of ketamine (20 mg/kg, IM) and xylazine (2 mg/kg, IM). After sedation, animals were euthanized by an overdose of a barbiturate-based sedative (≥1 mL/4.5 kg, IV). Euthanasia was confirmed by cessation of respiration, lack of palpable heartbeat, and bilateral thoracotomy. Detailed gross necropsies were performed at scheduled necropsy or morbundity. Euthanasia criteria included abnormal activity in the form of ambulation difficulties, decreased consumption of food and water, self-mutilation, reluctance to move for over 24 h; clinical conditions consisting of excessive bleeding or hemorrhage, severe dehydration that was unresponsive to fluid therapy, respiratory distress or labored breathing, unresponsive infections of the eyes or upper respiratory tract to antibiotic therapy; a greater than 20% loss of baseline bodyweight for greater than 72 h; or abnormal appearance in the form of rough coat, unwillingness to lift head, tucked abdomen, pallor, and/or exudates around the eyes and/or nose. Multiple, simultaneous instances of these criteria and their severity were evaluated to determine the use of euthanasia.

Statistical analysis

The association between the exposure dosage and mortality was assessed using probit regression (Bliss 1934), and survival data in different dosages were analyzed using Kaplan–Meier estimation. Parametric one-way analysis of variance (ANOVA) was used to test the differences on hematology parameters between exposure groups. For Sinclair minipigs, exposure groups were compared to the shams; and for Göttingen minipigs, higher dosage groups were compared to the lowest exposure dosage 1.50 Gy group. As data frequently exhibit either skewed distribution or variability scaled linearly with mean values, the assumptions of parametric ANOVA were verified using the Shapiro–Wilk test (Shapiro and Wilk 1965) for normality of residuals and Levene’s test (Levene 1960) for homogeneity of variances (p ≤ 0.05). If either of the parametric assumptions was violated, data were log-transformed prior to analysis. Where there was a significant exposure effect in the ANOVA model (p ≤ 0.05), Dunnett’s test (Dunnett 1955, 1980) was used to adjust the multiple comparison. Generalized estimating equation was also used to assess the dose–response effect on hematology parameters for each study date. In this analysis, animals within the same exposure dosage group are nested within their corresponding group, and exposure doses are considered as continuous variable. Statistical calculations were performed using the SAS® software system, Version 9.4 (SAS Inc., Cary, NC), and all reported p-values were based on two-sided tests and assessed at significance level less than or equal to 0.05.

Results

Irradiation and dosimetry

Göttingen minipigs were exposed to a range of doses from 1.50 Gy to 2.10 Gy while Sinclair minipigs received target doses ranging from 1.94 to 2.9 Gy in addition to an untreated control group. On an average, all animals irradiated as part of this study received measured doses within 4% of the targeted dose; 41 of the 45 animals (91%) had measured doses within 3% of the targeted dose; and 34 of the 45 animals (76%) had measured doses within 2% of the targeted dose. The average measured dose was 100.4% of the targeted dose.

Survival of minipigs after total body irradiation

Göttingen and Sinclair minipigs were irradiated with a total body dose ranging from 1.50 Gy to 2.10 Gy and 1.94 Gy to 2.90 Gy, respectively. Animals were monitored through necropsy on day 45. The LD_20/45, LD_50/45, and LD_80/45 and their corresponding 95% confidence intervals calculated by probit analysis for each strain are presented in Table 2. Each study irradiated animals both lower than the calculated LD_20/45 and equal to or higher than the calculated LD_80/45 with lethal dose ranges being higher in Sinclairs than in Göttingens.

Table 2. Comparison of lethal dose between strains with 95% confidence intervals.

Strain	Death probability	Lethal dose (Gy)	95% Confidence Interval
			Lower limit	Upper limit
Göttingen	0.20	1.70	1.30	1.85
	0.50	1.89	1.73	2.32
	0.80	2.10	1.92	3.47
Sinclair	0.20	2.42	1.79	2.52
	0.50	2.53	2.39	2.93
	0.80	2.65	2.54	4.27

Kaplan–Meier curves depicting the proportion of animals surviving following a single dose of total body irradiation are presented by strain in Figure 1. An expected relationship between survival and dose was observed in both strains; as dose increased, survival rates decreased. In Göttingens, the threshold for mortality appeared between 1.60 and 1.70 Gy. The 1.80 Gy dose group only contained one male animal; therefore, this 100% survival was likely due to the small number of subjects. According to probit lethal dose calculations from this study, 1.80 Gy should have resulted in an approximate 35% death probability (per statistical analysis). A significant decrease in survival occurred between the 1.60 Gy and 2.10 Gy dose groups (p < 0.05). Sinclair minipigs had their lethality profile shifted to the right compared to the Göttingen curve and were observed to have a lethality threshold between 2.30 and 2.45 Gy. The 2.90 Gy group had significantly more deaths compared to the 1.94 and 2.30 Gy dose groups (p < 0.05).

Figure 1. Percent survival in Göttingen and Sinclair Minipigs irradiated with a range of doses demonstrated by Kaplan–Meier curves. Göttingens are presented in the left panel with Sinclairs presented on the right. ^a1.60 Gy versus 2.10 Gy (p < 0.05); ^b2.90 Gy versus 1.94 and 2.30 Gy (p < 0.05).

Sigmoidal dose–response curves (shown in Figure 2) illustrate the lethality shift observed between strains. This comparison also demonstrates that the Sinclair curve has a steeper slope than the Göttingen curve, indicating a smaller range between non-lethal doses and LD₁₀₀ doses in Sinclair animals compared to Göttingen animals: $$\text{G}ö\text{ttingen\hspace{0.17em}slope\hspace{0.17em}}-\text{\hspace{0.17em}8}.0\text{5\hspace{0.17em}}\left(\text{probit}\left(\text{mortality}\right)=-\text{5}.0\text{6\hspace{0.17em}}+\text{\hspace{0.17em}8}.0\text{5\hspace{0.17em}}*\text{\hspace{0.17em}log}\left(\text{dosage}\right)\right).$$ $$\text{Sinclair\hspace{0.17em}slope\hspace{0.17em}}-\text{\hspace{0.17em}18}.0\text{2\hspace{0.17em}}\left(\text{probit}\left(\text{mortality}\right)=-\text{16}.\text{75}+\text{18}.0\text{2}*\text{log}\left(\text{dosage}\right)\right).$$

Figure 2. Predicted probabilities of mortality over a range of doses with 95% confidence intervals. Minipigs were exposed to several doses of radiation as depicted by points on the graph above.

Hematology by dose

Hematology response to radiation by dose was evaluated and is presented in Figure 3. Irradiation caused an immediate drop in white blood cell count followed by a small spike and then a gradual increase to day 45 that did not approach baseline in either strain (Figure 3(A)). Sinclair minipigs exposed to radiation showed a significant decrease in white blood cell number compared to sham controls (p < 0.05). Significant dose-dependent decreases were detected in both strains (p < 0.05).

Figure 3. Hematological responses to a single radiation dose in Göttingen and Sinclair minipigs on day 0 by dose. Panels on the left depict Göttingens and panels on the right depict Sinclairs: (A) white blood cell counts, (B) platelet counts, (C) neutrophil counts, (D) lymphocyte counts, (E) hematocrit percentages, and (F) mean cell hemoglobin mass. ^aIrradiated, Sinclair, white blood cells, neutrophils, and lymphocytes were different than shams with dose responses on multiple days (p < 0.05). ^bBoth minipig strains showed dose-dependent changes in white blood cells, lymphocytes, and hematocrit (p < 0.05). ^cOn day 14, Sinclair platelet nadirs were different with dose responses continuing through day 45 (p < 0.05). ^dGöttingen animals had dose-dependent changes in mean cell hemoglobin on days 37 and 45 (p < 0.05).

Irradiation on day 0 caused a steep decline in platelet counts until nadir on approximately day 14 followed by a gradual increase in all dose groups in both minipig strains (Figure 3(B)). Sinclair nadirs on day 14 were different with a significant dose-dependent decrease in platelet count (p < 0.05). Dose dependency continued through day 45. In Göttingens, animals in the 1.70, 1.95, and 2.10 Gy groups did not recover thrombocytopenic levels of platelets. Sinclairs in all groups except the 2.90 Gy group were able to recover their platelet counts above the thrombocytopenia threshold (50,000 cells/µL) by the end of the study. However, none of the animals in either group achieved their pre-radiation baseline counts.

In both strains, blood neutrophil levels decreased gradually from irradiation to nadir on day 21 (Figure 3(C)). This was followed by a gradual increase in levels to day 45. Similar to platelets, Göttingens in the 1.70, 1.90, and 2.10 Gy dose groups never observed neutrophil counts that exceeded neutropenia criteria. Sinclairs in the 2.45 Gy group did not recover from neutropenia by the end of the study and no 2.9 Gy animals survived to the end of study. None of the animals in either strain achieved their pre-radiation baseline counts. Neutrophil changes were similar among radiation groups in Göttingens. A significant decrease in neutrophil concentrations in irradiated groups compared to shams existed in Sinclair minipigs along with a dose-dependent decrease beginning on day 7 (p < 0.05).

Lymphocyte response to radiation exposure in Göttingens and Sinclairs was similar to white blood cell responses. Irradiation resulted in an immediate and sharp drop in lymphocyte counts followed by a slight increase to day 45 in both minipig strains (Figure 3(D)). None of the groups in either strain recovered to baseline counts in the time span of the study. Dose responses were detected in both strains at various time points (p < 0.05). Irradiated Sinclairs showed significant decreases in lymphocyte counts compared to Sham animals (p < 0.05). An unexpected increase in lymphocytes was noted in Shams beginning on day 0, furthering the difference between Shams and irradiated animals.

Hematocrit response to radiation was dose dependent in both minipig strains (Figure 3(E), p < 0.05). All doses in both strains displayed an initial spike and subsequent decrease in hematocrit percent after radiation exposure.

The magnitude of mean cell hemoglobin change in response to irradiation differed between minipig strains (Figure 3(F)). In Göttingen minipigs, a dose-dependent increase was detected on days 37 and 45. A similar, though not as robust, dose response was found in Sinclairs.

Irradiation exposure changed all of the aforementioned hematological parameters. Overall trends were similar between strains in all parameters except hematocrit. Further, dose-dependent changes were detected on at least one day for all parameters in both strains. Other statistically significant aberrations were detected, but proved to be biologically insignificant and are, therefore, not included throughout the manuscript.

Hematology by survival

Hematology parameters were evaluated over time in both strains of animals and are presented by strain (Göttingen versus Sinclair) and by survival (survivors versus decedents) in Figure 4. For both strains, survivor and decedent results were averaged independent of dose group. Sinclair calculations did not include animals in the Sham group.

Figure 4. Comparison of survival and decedent hematology parameters in Göttingen and Sinclair minipigs exposed to a radiation on day 0 regardless of dose. Göttingen parameters are in the left panels and Sinclair parameters are in the right panels: (A) white blood cell counts, (B) platelet counts, (C) neutrophil counts, (D) lymphocyte counts, (E) hematocrit percentages, and (F) mean cell hemoglobin mass.

White blood cell response to irradiation was similar between minipig strains and between survival groups; white blood cell counts dropped acutely post irradiation (Figure 4(A)) and remained at low levels with no major increases for the remainder of the study.

Platelet counts after radiation exposure were also similar in both strains (Figure 4(B)). A decline following radiation occurred until nadir at which point, counts began a gradual increase until day 45. Decedents in both strains failed to show this gradual rise after nadir with the last few time points being significantly lower than their surviving counterparts suggesting that thrombocytopenia is also an important contributor to mortality.

The pattern of neutrophil changes was different in Göttingen and Sinclair minipigs (Figure 4(C)). Göttingen decedents and survivors were largely similar throughout the study. However, nadirs on day 21 were lower in decedents. Deceased animals recovered to levels similar to survivors just prior to death. Due to the lack of bacteriology and histopathology data, it is difficult to further substantiate the exact role neutrophils played in the survival or death within these studies.

Lymphocyte counts responded to radiation exposure in a manner similar to total white blood cells (Figure 4(D)). Both strains exhibited decreases in counts immediately following radiation that were not recovered by day 45. In both minipig strains, survivors and decedents were similar.

Hematocrit percentages displayed similar responses in both strains (Figure 4(E)). Decedents began to diverge from survival groups on approximately day 7 and continued to decrease significantly until death. This suggests that animals unable to maintain higher levels of hematocrit are unable to survive.

Mean cell hemoglobin increased after irradiation until day 45 in both strains (Figure 4(F)). Göttingen decedents had lower mean cell hemoglobin mass throughout all sampling time points than survivors and showed significantly lower mass from day 14 until death. Conversely, Sinclair decedents were similar to survivors until day 6 at which point decedents exhibited a significant increase over survivors immediately before death.

Ultimately, hematology parameter responses were similar between strains with the exception of mean cell hemoglobin and neutrophils. Mean cell hemoglobin mass in decedents was inversely affected in Göttingens compared to Sinclairs. Sinclair decedents showed a difference in neutrophil counts compared to survivors, while Göttingen decedents showed changes from survivors that were less robust than Sinclairs. In both strains, decedents showed significant decreases in platelets and hematocrit compared to survivors. These findings suggest that these parameters play an important role in survival of radiation exposure regardless of dose.

Instances and duration of thrombocytopenia are presented in Table 3. In Göttingens, the mean day of nadir fell on either day 14 or 21 independent of dose with the exception of the 1.95 Gy group which occurred on day 30, the day in which the second decedent in the group was euthanized. The majority of groups had thrombocytopenia onset on day 11. Duration of thrombocytopenia and day of onset and duration of severe thrombocytopenia was largely varied among groups. In Sinclair minipigs an overall dose–response was more clear. The mean day of nadir decreased from days 21 to 14 in all groups above 1.94 Gy. Groups irradiated with 2.3 Gy or more entered thrombocytopenia with groups exposed above 2.45 Gy entering severe thrombocytopenic states. Interpretation of duration is limited due to sampling regimen within the study and the observed survival. Sinclair minipig groups were unlike Göttingens in that groups with decedents were able to show recovery form thrombocytopenia. Overall, the two strains behaved differently in terms of thrombocytopenia. There appeared to be a stronger relationship between thrombocytopenia and survival in Göttingens and thrombocytopenia and dose in Sinclairs.

Table 3. Onset and duration of platelet related thrombocytopenia and severe thrombocytopenia in two minipig strains exposed to a single dose of radiation.

Göttingen Minipigs							Sinclair Minipigs
Dose (Gy)	Mean day of Nadir	<50,000/µl^a		<10,000/µl^b		Mean day of recovered	Dose (Gy)	Mean day of Nadir	<50,000/µl^a		<10,000/µl^b		Mean day of recovered
		Day of onset	Duration (days)	Day of onset	Duration (days)				Day of onset	Duration (days)^e	Day of onset	Duration (days)^e
	12–37	8–12	3–11	10–28	1–7	37–45		7–21	14–21	1–4	14–21	1–2	21–45
1.50	14	11	8	14	1	45	Sham	–	–	–	–	–	–
1.60	21	11	7	–	–	37	1.94	21	–	–	–	–	–
1.70^f	14	11	10	13	4^c	–	2.30	14	14	1	–	–	21
1.80	14	11	3	–	–	37	2.45^i	14	14	3	14	2	35
1.95^g	30	10	10	11	7	–	2.60^j	14	14	2	14	1	28
2.10^h	21	11	10	14	3^c	–	2.90^k	14	14	1	14	1	–

^aThrombocytopenia.

^bSevere thrombocytopenia.

^cDays were not consecutive.

^dFirst sample day.

^eNumber of sample days.

^fTwo animals did not survive to day 45. One was removed on day 38 and the other on day 32.

^gTwo animals did not survive to day 45. One was removed on day 14 and the other on day 30.

^hOnly one animal survived to day 45. Two animals were removed on day 15 and one on day 32.

ⁱTwo animals did not survive. Both animals were removed on day 22.

^jThree animals did not survive. Animals were removed on days 12, 18, and 21.

^kNone of the animals survived. One was removed on day 15 with others being removed on day 16.

Day of onset and duration of neutropenia can be found in Table 4. Göttingen minipigs hit neutrophil nadir and severe neutropenia on day 21 regardless of dose. The day of neutropenic onset varied among dose groups but did not appear to be tightly related to dose. However, duration of neutropenia increased as dose increased with the exception of the lone animal in the 1.80 Gy dose group that experienced neutropenia for less time than the Göttingens in the 1.70 Gy group. All Göttingens had severe neutropenia on day 21, the nadir, with Göttingens in the higher dose group undergoing severe neutropenia for longer durations than their lower dose counterparts.

Table 4. Onset and duration of neutrophil related neutropenia and severe neutropenia in irradiated Göttingen and Sinclair minipigs.

Göttingen Minipigs							Sinclair Minipigs
Dose (Gy)	Mean day of Nadir	<1000/µl^a		<500/µlb		Mean day of recovered	Dose (Gy)	Mean day of Nadir	<1000/µl^a		<500/µl^b		Mean day of recovered
		Day of onset	Duration (days)^e	Day of onset	Duration (days)^e				Day of onset	Duration (days)^e	Day of onset	Duration (days)^e
13–30	1–21	3–10	11–30	1–5	37–45		7–28	1–21	1–6	3–21	1–5	35–45
1.50	21	14	5	21	1	45	Sham	21	–	–	–	–	–
1.60	21	21	4	21	2	45	1.94	21	7	5	21	2	35
1.70^f	21	12	6^c	21	1	–	2.30	21	7	4	21	1	35
1.80	21	0^l	4^c	21	1	37	2.45^i	21	7	6	21	3	–
1.95^g	21	13	7	21	5	–	2.60^j	21	7	6	14	3	45
2.10^h	21	11	10	21	5	–	2.90^k	14	7	2	14	1	–

^aNeutropenia.

^bSevere neutropenia.

^cDays were not consecutive.

^dFirst sample day.

^eNumber of sample days.

^gTwo animals did not survive to day 45. One was removed on day 14 and the other on day 30.

^hOnly one animal survived to day 45. Two animals were removed on day 15 and one on day 32.

ⁱTwo animals did not survive. Both animals were removed on day 22.

^jThree animals did not survive. Animals were removed on days 12, 18, and 21.

^kNone of the animals survived. One was removed on day 15 with others being removed on day 16.

^lThis animal was clinically normal. Cause of the abnormal value was unknown.

Similarly, all irradiated Sinclairs had neutrophil nadir on day 21 with the exception of animals in the 2.1 Gy dose group that reached nadir at day 14. All dose groups had neutropenia onset occur on day 7 with duration increasing with increasing dose. The 2.90 Gy dose groups experienced the shortest duration due to animals dying or reaching moribund criteria shortly after onset. The highest two dose groups began severe neutropenia earlier, on day 14, than the lower three doses that experienced onset on day 21. Duration of severe neutropenia increased with increasing dose until 2.90 Gy. Sinclairs in the 2.90 Gy group only survived one day following onset of severe neutropenia. Sinclairs in the 2.30 Gy groups were the only animals to recover prior to scheduled euthanasia. Ultimately, in Göttingen minipigs, the duration of severe neutropenia is increased by higher radiation doses. As with platelets, Sinclair minipigs experienced neutropenia in response to increasing radiation dose.

Clinical chemistry by dose

Effects of irradiation on several clinical chemistry parameters by dose are presented in Figure 5. Albumin:globulin ratios did not exhibit any changes in response to radiation treatment with group means being similar among all dose groups in both strains (Figure 5(A)). Beginning on day 37, a dose-dependent decrease in Göttingen creatinine occurred and continued through the remainder of the study (Figure 5(B)). Sinclair creatinine levels remained similar in all dose groups at all timepoints. Dose dependent increases in glucose were detected in both strains on day 21 (Figure 5(C)). Globulin concentrations did not respond dose dependently to radiation (Figure 5(D)).

Figure 5. Clinical chemistry responses to a one time irradiation dose on day 0 to Göttingen and Sinclair minipigs by dose. The left panels depict Göttingens while the right panels depict Sinclairs: (A) albumin:globulin ratio, (B) creatinine concentration, (C) glucose concentration, and (D) globulin concentration.

Clinical chemistry by survival

Clinical chemistry parameters collected from minipigs originating from two different strains and exposed to a single dose of radiation are presented in Figure 6 by survival/decedent and strain. Albumin:globulin (AG) ratios in surviving groups were similar between strains (Figure 6(A)). However, Göttingen decedents displayed significantly lower AG ratios from day 21 to death compared to survivors. Interestingly, Sinclair decedents were similar to survivors for the duration of the study.

Figure 6. Comparisons of survival and decedent clinical chemistry responses to a single irradiation dose in Göttingen and Sinclair minipigs. Left panels display Göttingen results and right panels display Sinclair results: (A) albumin:globulin ratio, (B) creatinine concentrations, (C) glucose concentrations, and (D) globulin concentrations.

Göttingen creatinine concentrations spiked around day 20 and returned to normal on day 30 (Figure 6(B)). Sinclair creatinine concentrations were largely unchanged over time and were not altered by radiation exposure. In both strains, decedents and survivors showed similar creatinine concentrations.

Radiation affected blood glucose levels differently in Göttingen and Sinclair minipigs (Figure 6(C)). Glucose concentrations in Göttingen minipigs varied throughout the time of the study as surviving Göttingens displayed large peaks that were followed by decreases in glucose on various days of the study. Deceased Göttingens did not show these drastic increases and decreases. Sinclair survivors exhibited one gradual increase beginning on day 7 followed by a gradual decrease beginning on day 21. Deceased Sinclairs responded to irradiation with an immediate and marked increase in blood glucose that resulted in a significant difference from day 0 to death.

Globulin concentrations in Göttingen and Sinclair survivors were largely unaffected by radiation exposure (Figure 6(D)). Deceased Göttingen minipigs exhibited increases in globulin beginning on day 14 that far exceeded levels in surviving animals. Conversely, deceased animals originating from the Sinclair strain started with lower globulin levels and maintained this difference until mortality.

Clinical chemistry parameters were mostly similar between survivors in each strain. However, decedents from each strain showed different responses of AG ratio, glucose, and globulin. These appear to be strain specific changes indicating a difference in response between strains and may suggest a difference leading to a lack of survival.

Moribund euthanasia

Table 5 shows a detailed euthanasia criteria comparison to the gross findings between strains. The calculated death probability was added to this table to assist in correlation of the study results. Göttingen minipigs in the lowest two dose groups had 100% survival, but were then euthanized as a part of scheduled study endpoints. The most common findings for animals at these dose levels was discoloration of the lungs, indicating possible hemorrhagic events. As expected, as irradiation dose increased there was an increased incidence of animals that met moribund criteria and an increased number of gross abnormal findings. Death dates did vary among groups, but the 1.95 and 2.10 Gy groups had the earliest euthanasia dates. Beginning at a dose of 1.70 Gy, various organs were discolored at the time of necropsy. In the lone 1.80 Gy animal, lungs were the only tissue with gross abnormalities. Beginning at 1.95 Gy, the majority of animals showed potential hemorrhaging on several of the major organs including the heart, lungs, stomach, skin, intestine, pancreas, eyes, and bladder with increased severity.

Table 5. Euthanasia criteria comparison. Minipigs from the Göttingen and Sinclair strains were irradiated with a range of sublethal to lethal doses and followed through to scheduled euthanasia 45 days post-irradiation.

Göttingen Minipigs			Sinclair Minipigs
Dose (calculated death probability)	Euthanasia criteria (number animals/total)	Gross findings (number animals/total)	Dose (Gy)	Euthanasia criteria (number animals/total)	Gross findings (number animals/total)
1.50 (0.03)	Scheduled Study Endpoint (4/4)	Study Day: 45 (4/4) Gross Findings: All animals showed minimal to mild signs of hemorrhaging on the lungs. One animal also had additional discoloration of the skin and jejunum.	Sham (0)	Scheduled Study Endpoint (3/3)	Study Day: 45 (3/3) Gross Findings: Two animals had no visible lesions or abnormalities at necropsy. One animal showed potential hemorrhaging in the right lung lobe and jejunum.
1.60 (0.09)	Scheduled Study Endpoint (3/3)	Study Day: 45 (3/3) Gross Findings: All animals had minimal to mild sign of hemorrhaging on the lung lobes. One animal also had an irregular and calculus bladder	1.94 (0)	Scheduled Study Endpoint (6/6)	Study Day: 45 (6/6) Gross Findings: Four animals had no visible lesions or abnormalities at necropsy. Two animals had minimal to mild signs of hemorrhage on the lung surfaces. One animal possessed a mass on the left side of the thoracic region
1.70 (0.20)	Scheduled Study Endpoint (4/6) Abnormal Activity and Infection (1/6) Respiratory Distress (1/6)	Study Day: 45 (4/6); 32 (1/6); 38 (1/6) Gross Findings: One animal had no visible lesions or abnormal findings. All others had minimal to marked discoloration indicative of hemorrhaging on various organs	2.30 (0.04)	Scheduled Study Endpoint (5/5)	Study Day: 45 (5/5) Gross Findings: Four animals had no visible lesions or abnormalities at necropsy. One animal presented with minimal signs of hemorrhage on the lung surfaces
1.80 (0.35)	Scheduled Study Endpoint (1/1)	Study Day: 45 (1/1) Gross Findings: The animal displayed minimal sign of hemorrhage on the left caudal lung lobe	2.45 (0.27)	Scheduled Study Endpoint (3/5) Abnormal Activity and Respiratory Distress (1/5) Abnormal Activity and Appearance (1/5)	Study Day: 45 (3/5); 22 (2/5) Gross Findings: One scheduled animal had no visible lesions or abnormal findings. Two animals had blood clots with one of these also having an unusually small thymus. Two moribund animals had minimal to marked indications of hemorrhaging on the majority of organs
1.95 (0.60)	Scheduled Study Endpoint (1/3) Abnormal Activity (1/3) Hemorrhage (1/3)	Study Day: 45 (1/1); 14 (1/1); 30 (1/1) Gross Findings: One animal presented no visible lesions or abnormalities. Two animals displayed minimal to mild sign of hemorrhaging on various organs. One of these animals also had prominent mediastinal hemorrhaging	2.60 (0.68)	Scheduled Study Endpoint (2/5) Abnormal Activity and Appearance (1/5) Infection and Abnormal Appearance (1/5) Found Dead (1/5)	Study Day: 45 (2/5); 12 (1/5); 18 (1/5); 21 (1/5) Gross Findings: All animals showed mild to moderate potential hemorrhaging of various organs. One of the moribund animals also had lung adhesion and eye conjunctivitis
2.10 (0.80)	Scheduled Study Endpoint (1/4) Abnormal Appearance (2/4) Hemorrhage (1/4)	Study Day: 45 (1/4); 15 (2/4); 32 (1/4) Gross Findings: Two animals had no visible lesions or abnormalities. The other two animals had minimal to moderate indications of hemorrhaging on various organs	2.90 (1.00)	Found dead (2/3) Abnormal Activity and Respiratory Distress (1/3)	Study Day: 15 (1/3); 16 (2/3) Gross Findings: All animals showed minimal to marked sign of hemorrhage on various organs. One animal also had eye discoloration and conjunctivitis with another also having a depressed right caudal lung lobe

Sinclair minipigs in the lowest three dose groups all survived to scheduled euthanasia, but had gross necropsy findings that included discoloration in the lungs in at least one animal from each group. Beginning with the 2.45 Gy dose group, animals began showing gross signs of hemorrhaging in several major organs, which included the thymus, brain, heart, kidneys, lungs, mediastinal lymph nodes, pericardium, skin, spleen, stomach, testes, muscle, intestine, pancreas, and bladder. Potential hemorrhaging increased in severity and frequency as dose increased. Overall, both strains had increasing frequency and severity of discoloration indicating hemorrhagic events with increasing irradiation doses. This was expected since doses were targeted to cause hematopoietic dysfunction that resulted in significant decreases in platelets, which likely resulted in bleeding and ultimately possibly triggering multi-organ failure.

Gross findings and clinical observations

Common gross findings by dose and severity are presented in Figure 7. As expected, moribund animals had more clinical abnormalities than those that underwent scheduled euthanasia at day 45 post-irradiation. Additionally, moribund animals died earlier than scheduled animals, thus linking a time dependence to gross findings at necropsy. Common findings at necropsy included purple to dark red discoloration of the lungs, skin, heart, and GI tract. This discoloration appears to be indicative of organ hemorrhaging and can be used as a clinical sign of bleeding. In Göttingens, dose-dependent patterns were difficult to establish as animals in the lower dose groups generally displayed abnormalities as frequently and with comparable severity as animals in higher dose groups. This is confounded by the 1.80 Gy group only containing one animal. Additionally, animals in the 1.70 Gy dose group presented the most severe findings in three out of four organs.

Figure 7. Percent of animals exposed to a range of radiation doses experiencing gross abnormalities (hemorrhage specifically) by severity at necropsy. Severity ranged from no visible lesion (NVL) to marked: (A) lung, (B) skin, (C) heart, and (D) GI.

Sinclair minipigs differed from Göttingens in gross findings where a clear dose-dependency was observed. Generally, instances and severity of abnormal organ discoloration increased with dose. Shams did display lung and GI discoloration; however, these findings were in the same animal and were minimal.

In both strains of minipigs, the most common clinical observations included petechia and bruising (which may have been caused by ecchymosis). Red skin was common only in Göttingens which was observed to have the majority of animals in the 1.50–1.70 Gy dose groups with abnormally red skin appearing around day 14 and continued for the majority of the remainder of the study. Red skin in minipigs is similar to clinical symptoms of ARS seen in humans, showing a clear translation from the minipig model to humans. In addition to red skin, Göttingens also displayed petechia and bruising. Petechia occurred in almost all of the animals regardless of dose. Generally, petechia appeared early and persisted for the majority of the study. Bruising in Göttingens was also found in almost all animals, but failed to establish a clear dose-dependent pattern. All but one decedent animal had petechia up to the day of euthanasia and all animals displayed bruising for numerous days prior to euthanasia.

Sinclair clinical observations were more dose-dependent than Göttingen observations. At least two Sinclairs from all groups, including Sham, were observed to have bruising for a span of time throughout the course of the study. As irradiation dose increased, severity of bruising increased from slight in one sham animal to extreme in the 2.10 Gy animals. Like bruising, severity of petechia increased to extreme on more than three sites of the body in the higher radiation groups. Additionally, extreme bruising and petechia appeared to precede moribund euthanasia. Sinclair animals did not display skin reddening similar to Göttingens, which is likely due to all Göttingens having light pink skin that shows abnormalities easily. Sinclairs have darker colored skin that is variable, making it difficult to call observations presenting on their skin. Handling and procedures may also cause bruising, as seen in the Sham animals. However, the increased severity is likely a response to increasing radiation doses, an expected result of external irradiations.

It is difficult to draw patterns between Göttingen clinical observations and gross findings as dose dependence was not observed in either parameter. Conversely, high dose Sinclairs exhibited more severe organ hemorrhaging at a higher frequency along with an increase in extreme bruising and petechia seen during clinical observations. This indicates that high doses of radiation, such as 2.90 Gy, cause hemorrhagic emergencies that manifest both internally and externally and ultimately precede death as all Sinclairs in the 2.90 Gy group underwent moribund euthanasia. Unlike other large animal models or external irradiation, there were little to no findings of inappetence or lethargy in either minipig strain.

Discussion

Increasing threats of nuclear incidents and the everlasting danger of working in nuclear energy facilities have demonstrated the need for MCMs for radiation exposure. Currently, there are no completely effective MCMs or combination of MCMs for individuals exposed to potentially lethal amounts of radiation. The only current treatments approved by the FDA for irradiated individuals are LGF-based pharmaceuticals such as Neupogen®, Neulasta®, and Leukine® (Clark et al. 2005; Singh et al. 2018; US FDA 2018) which stimulates leukopoiesis after radiation exposure, increasing the likelihood of survival (MacVittie et al. 2012). Additional pharmaceuticals as effective as, or more effective than Neuopogen®, along with pharmaceuticals capable of addressing the thrombocytopenia and other vascular and coagulation dysfunctions contributing injury observed in radiation victims are needed to increase the survival rate of individuals exposed to lethal doses of radiation.

Because of the lethal nature of radiation exposure, animal models that represent the human condition are optimal for MCM development. Minipigs are regarded as an equivalent species for drug validation due to their physiological similarities to humans as compared to other large animal species such as NHPs or dogs (Augustine et al. 2005; Jacobs 2006). In addition, the cost of purchasing, housing, feeding, and training personnel to work with minipigs is much less than for NHPs making them possibly a more accessible species for testing. For facilities capable of breeding and holding animal colonies, minipigs are also advantageous when compared to NHPs as Göttingens reach sexual maturity at no later than 5 months of age (Ellegaard 2008), whereas NHPs reach sexual maturity between 3 and 7 years of age depending on the species (Kessler 1986). With their decreased cost, and equivalent or greater translational potential, minipigs present a strong candidate for animal models of radiation exposures. The goal of this study was to develop models of H-ARS in Göttingen and Sinclair minipigs as tools for development of effective MCMs.

In the Göttingen minipig, indicators of H-ARS have been shown to include an LD_50/45 of 1.7–1.9 Gy without medical management and decreases in platelets, lymphocytes, and neutrophils (Moroni, Coolbaugh et al. 2011; Kenchegowda 2018). The Göttingens typically experience thrombocytopenia and neutropenia, with nadirs between 14 and 20 days post-irradiation (Moroni, Lombardini et al. 2011). In addition, similar to what was observed in this study, the animals typically experience widespread hemorrhaging, particularly in the heart, lungs, brain, and intestines (Moroni, Coolbaugh et al. 2011). To our knowledge, an LD₅₀ has not been previously determined for Sinclairs. Sinclairs have been found to have similar neutrophil and platelet response profiles to the Göttingens, although their profiles had not been fully defined (Kenchegowda 2018). In this study, the LD_50/45 was determined to be 1.89 and 2.53 Gy with supportive care (denoted as “field” supportive care) constituting prophylactic antibiotics in Göttingens and Sinclairs, respectively. This LD₅₀ was consistent with the lethal dose demonstrated by Moroni et al. despite differences in study variables. It should be noted that minipigs were implanted with VAPs, which are known to increase susceptibility to immune system complications (Douard et al. 1999); however, no complications were noted during this study. In all Göttingen dose groups, thrombocytopenia nadirs occurred on days 11–14, and on day 14 for Sinclairs dosed with 2.30–2.90 Gy. Neutropenia developed inconsistently in the Göttingens, ranging from starting on the same day of irradiation to starting 21 days post-irradiation, with no dose dependent correlation. More frequent blood collections, rather than once weekly procedure followed in this study after day 14, may have resulted in more consistency for this parameter. The neutrophil count nadirs were consistent, however, occurring on day 21 in all animals. The neutrophil count nadir may have occurred later in the Göttingens in our study as compared to other studies due to supplying antibiotics once neutropenia developed. In contrast, neutropenia occurred on day 7 in all Sinclairs dosed with 1.94–2.90 Gy and nadirs occurred on day 21 for 1.94–2.45 Gy animals and day 14 for 2.60–2.90 Gy animals. While there were no time points between days 14 and 21, it is noteworthy that the higher dose groups consistently experienced neutrophil nadirs in the earlier time point. Consistent with the literature, Göttingens experienced hemorrhage in the lungs, heart, skin, and GI (Moroni, Coolbaugh et al. 2011); however, hemorrhaging did not occur in the hearts and GI of all irradiated animals, as has been reported, but in select animals seemingly independent of dose. In lethally irradiated animals, the lungs were the most commonly effected organ (found in 73% of all animals), followed by the heart (47%) and the GI (40%). It was noted that all moribund euthanized animals, with the exception of one 1.50 Gy-dosed Göttingen, were found to have both heart and GI lesions. Göttingens exposed to 2.10 Gy were the only Göttingens that were consistently found with hemorrhaging in the lungs, skin, heart, and GI. Conversely, Sinclairs experienced hemorrhaging in a clear dose-dependent manner in the same four organs, with lesions being found in the 2.45–2.90 Gy animals. No clear trend could be observed between the occurrence of gross lesions and changes in neutrophil or platelet counts. Unlike previous research, the lungs were the most affected organ in both strains from TBI; independent of dose in the Göttingens and dependent of dose in the Sinclairs. It is important to note that both minipig strains experienced little to no inappetence or lethargy after irradiation in these studies. This likely also contributed to no change in animal body weight over the course of the study (data not shown). This is one feature of the minipig that diverges from other animal models and differs with the human condition.

Predicting survival, or rather detecting critical physiological changes that consistently precede mortality, is a vital component of medical management for irradiated victims. Current methods of predicting survival include monitoring hematopoietic parameters such as white blood cells, neutrophils, lymphocytes, and platelets (Goans et al. 1997; Fliedner et al. 2007; Azizova et al. 2008). Observing changes in these parameters is particularly important for individuals with H-ARS, where bone marrow damage is the largest contributor to injury and, ultimately, death. As a result, animal models of ARS need to clearly reproduce these trends as predictors of mortality in order to provide the best platform for MCM development. In our study, we demonstrated that both Göttingens and Sinclairs exhibit hematological profiles seen in humans exposed to radiation. Decreases in platelets and neutrophils were observed in all animals exposed to radiation. For both parameters, counts decreased more drastically in the decedents than in the survivors. The neutrophils began to recover in the Göttingen decedents after reaching their nadirs, while there was no recovery of neutrophils in the Sinclairs. However, this may be the result of a few late moribund animals from non-hematological reasons. Platelet counts did not recover in either strain of decedents; however, the recovery in the Sinclair survivors was much faster and more pronounced than in the Göttingen survivors. Another parameter that strongly predicted survival was hematocrit levels, which plummeted without recovery in decedents Göttingens and Sinclairs. This, along with thrombocytopenia, likely played a role in the increased hemorrhaging observed in decedents from both strains.

Despite differences in radiosensitivity, both strains exhibited similar physiological predictors of survival. However, there were some instances where the two strains differed. One example is mean cell hemoglobin, where it decreased in Göttingen decedents but spiked just before death in Sinclair decedents as compared to respective strain survivors. In correlation, the lungs of Göttingens were more universally damaged but with less severity, whereas Sinclairs less frequently had damage to the lungs but with higher severity. These two phenotypes may be linked due to the damage of the lungs causing decreased respiration capacity, leading to changes in blood oxygen content, and potentially mean cell hemoglobin counts. Changes in mean cell hemoglobin counts may also have been the result of increased internal bleeding and vascular injury. More research is necessary to determine if there is a connection in the lung damage of these two strains and their respective variable mean cell hemoglobin levels. Bruising and petechiae also coincided with the increased gross lesions observed in the animals exposed to higher doses of radiation. Like gross observations at necropsy, clinical observations in-life were seen in a more dose dependent manner in Sinclairs than in Göttingens. Another example of differences between the two strains is their changes in glucose levels after radiation exposures. In Göttingens, glucose was largely unchanged after exposure, with no difference between survivors and decedents. In contrast, glucose levels in decedent Sinclairs rose drastically until death. Interestingly, this phenotype occurred in Sinclairs independent of radiation dose. There are many factors that influence blood glucose levels. This increase in Sinclairs may simply be the result of increased stress to their bodies, decreased energy demand of those approaching mortality, or the death of cells and thus the decreased demand for glucose. One study noted that rats exposed to 54 Gy to the brain had decreased glucose utilization, as assessed by deoxyglucose-6-phosphate phosphorylation for 4 days and further decreased utilization for 4 weeks, following exposure (Ito et al. 1986). Again, this may be explained by decreased glucose demand in the cells. However, this demonstrates that glucose may be a biomarker for mortality prediction that is already easy to measure due to advancements in diabetes management. More investigation on the effect of radiation on blood glucose levels may prove valuable in predicting survival after radiation exposures. There is limited research focusing on clinical chemistry profile changes after externally irradiated Göttingens and none to our knowledge on Sinclairs. The difference in dose tolerance between the two strains may be attributed to the in-bred nature of Göttingens as compared to the out-bred nature of Sinclairs. Göttingen minipig breeding is strictly regulated, maintaining less than 10% inbreeding, to sustain genetic uniformity and prevent genetic drift (Simianer 2010). The most striking genetic difference between the two strains is the “pituitary dwarfism” exhibited by Göttingens, which is a genetically fixed trait and not considered a genetic defect. Sinclair breeding is maintained through a least-related breeding program, mitigating any potential in-breeding (Sinclair Bio-Resources – Sinclair™ Miniature Swine n.d.). This breeding program may make Sinclairs an animal model more genetically diverse like humans compared to Göttingens or in-bred rodents. In contrast, the in-breading of Göttingens may make them a more reliable model between studies as each animal is as close to a clone to itself as possible, while Sinclairs may show variation between studies with their more heterogeneous genetic background. While these differences in genetic background may contribute to their differences in radiosensitivies, there are factors that may contribute to the differences we observed between Göttingen and Sinclair minipigs that were not observed in these studies.

A limiting factor for this study is the limited blood collections following day 14 for both minipigs strains. More frequent collections may result in a more precise understanding of the neutrophil and thrombocyte profiles for Göttingens and Sinclairs. A second limitation was the lack of a control group for the Göttingens. A third limitation was the age of the animals, as they were matched by weight, rather than age, leading to the Göttingens being approximately a month and a half older than the Sinclairs. A fourth and significant difference between the studies was the antibiotic schedule. Initially, Göttingens were administered Baytril when their neutrophil counts fell below 1000/mL as a prophylactic treatment to prevent infection from occurring. Later, Sinclairs were given gentamicin and amoxicillin on days 3–30, regardless of neutrophil count. The differences in antibiotics delivered and when they were given may have had an impact on observed responses to radiation.

In this study, we developed a TBI model of ARS in Göttingen and Sinclair minipigs to create a platform for radiation MCM development. The LD_50/45 of both strains was determined in context with field supportive care of antibiotics. Göttingens and Sinclairs presented similar hematological profiles to those observed in humans and other animal species, with decreases in neutrophils, lymphocytes, platelets, and hematocrit. In addition, clinical chemistry parameters albumin:globulin ratio, creatinine, glucose, and globulin were assessed in minipigs after radiation to add to the work of Moroni, Coolbaugh et al. (2011). The animals were also examined upon gross necropsy and found to experience hemorrhaging in the lungs, skin, heart, and GI. This work will provide a template for future studies to evaluate potential MCMs that could be used to treat civilians, medical responders, and military personnel who are all at risk of radiation exposure.

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

This work was supported in part or wholly by funds from the Biomedical Advanced Research and Development Authority, Department of Health and Human Services, under Contract no. HHSO100200100004I and Task order no. HHSO10033001T. The authors acknowledge the continuous discussion, insight, and constructive critique of colleagues at BARDA.

Notes on contributors

Melanie Doyle-Eisele

Melanie Doyle-Eisele, PhD, is the Director of Life Sciences and Senior Scientist at Lovelace Biomedical, Albuquerque, NM.

Jeremy Brower

Jeremy Brower, PhD, is an Associate Research Scientist and Study Director at Lovelace Biomedical, Albuquerque, NM.

Kenneth Aiello

Kenneth Aiello is a Research Associate at Lovelace Biomedical, Albuquerque, NM.

Emily Ferranti

Emily Ferranti is a Research Associate at Lovelace Biomedical, Albuquerque, NM.

Michael Yaeger

Michael Yaeger is a Research Associate at Lovelace Biomedical, Albuquerque, NM.

Guodong Wu

Guodong Wu is an Associate Research Scientist and Statistician at Lovelace Biomedical, Albuquerque, NM.

Waylon Weber

Waylon Weber PhD is a Senior Scientist and Study Director at Lovelace Biomedical, Albuquerque, NM.