A Yorkshire swine (Sus scrofa domesticus) model for nerve regeneration and ischemia based on the sciatic nerve and femoral artery.

Animal studies are essential to biomedical research and the cornerstone is a reproducible animal model. While there are many reports on rodent peripheral nerve injury models, a large animal model is essential to confirm the effects of nerve regeneration over the longer distances of regeneration required in humans. Swine have long been used as a large animal model for other surgical and biomedical studies. This paper represents a novel neurovascular injury model in the Sus scrofa domesticus swine (American Yorkshire pig). This paper will describe our experience and recommendations with pre-operative, operative and post-operative protocols and our refinements to produce an effective model.

Comparison of the effects of osmotic pump implantation with subcutaneous injection for administration of drugs after total body irradiation in mice.

The increasing potential for radiation exposure from nuclear accidents or terrorist activities has intensified the need to develop pharmacologic countermeasures against injury from total body irradiation (TBI). Many initial experiments to develop and test these countermeasures utilize murine irradiation models. Yet, the route of drug administration can alter the response to irradiation injury. Studies have demonstrated that cutaneous injuries can exacerbate damage from radiation, and thus surgical implantation of osmotic pumps for drug delivery could adversely affect the survival of mice following TBI. However, daily handling and injections to administer drugs could also have negative consequences. This study compared the effects of subcutaneous needlesticks with surgical implantation of osmotic pumps on morbidity and mortality in a murine model of hematopoietic acute radiation syndrome (H-ARS). C57BL/6 mice were sham irradiated or exposed to a single dose of 7.7 Gy 60Co TBI. Mice were implanted with osmotic pumps containing sterile saline seven days prior to irradiation or received needlesticks for 14 days following irradiation or received no treatment. All irradiated groups exhibited weight loss. Fewer mice with osmotic pumps survived to 30 days post irradiation (37.5%) than mice receiving needlesticks or no treatment (70% and 80%, respectively), although this difference was not statistically significant. However, mice implanted with the pump lost significantly more weight than mice that received needlesticks or no treatment. These data suggest that surgical implantation of a drug-delivery device can adversely affect the outcome in a murine model of H-ARS.

Establishment of Early Endpoints in Mouse Total-Body Irradiation Model.

Acute radiation sickness (ARS) following exposure to ionizing irradiation is characterized by radiation-induced multiorgan dysfunction/failure that refers to progressive dysfunction of two or more organ systems, the etiological agent being radiation damage to cells and tissues over time. Radiation sensitivity data on humans and animals has made it possible to describe the signs associated with ARS. A mouse model of total-body irradiation (TBI) has previously been developed that represents the likely scenario of exposure in the human population. Herein, we present the Mouse Intervention Scoring System (MISS) developed at the Veterinary Sciences Department (VSD) of the Armed Forces Radiobiology Research Institute (AFRRI) to identify moribund mice and decrease the numbers of mice found dead, which is therefore a more humane refinement to death as the endpoint. Survival rates were compared to changes in body weights and temperatures in the mouse (CD2F1 male) TBI model (6-14 Gy, 60Co γ-rays at 0.6 Gy min-1), which informed improvements to the Scoring System. Individual tracking of animals via implanted microchips allowed for assessment of criteria based on individuals rather than by group averages. From a total of 132 mice (92 irradiated), 51 mice were euthanized versus only four mice that were found dead (7% of non-survivors). In this case, all four mice were found dead after overnight periods between observations. Weight loss alone was indicative of imminent succumbing to radiation injury, however mice did not always become moribund within 24 hours while having weight loss >30%. Only one survivor had a weight loss of greater than 30%. Temperature significantly dropped only 2-4 days before death/euthanasia in 10 and 14 Gy animals. The score system demonstrates a significant refinement as compared to using subjective assessment of morbidity or death as the endpoint for these survival studies.

Non-human Primate Total-body Irradiation Model with Limited and Full Medical Supportive Care Including Filgrastim for Biodosimetry and Injury Assessment.

An assessment of multiple biomarkers from radiation casualties undergoing limited- or full-supportive care including treatment with filgrastim is critical to develop rapid and effective diagnostic triage strategies. The efficacy of filgrastim with full-supportive care was compared with results with limited-supportive care by analyzing survival, necropsy, histopathology and serial blood samples for hematological, serum chemistry and protein profiles in a non-human primate (Macaca mulatta, male and female) model during 60-d post-monitoring period following sham- and total-body irradiation with 6.5 Gy 60Co gamma-rays at 0.6 Gy min-1 Filgrastim (10 µg kg-1) was administered beginning on Day 1 post-exposure and continued daily until neutrophil counts were ≥2,000 µL-1 for two consecutive days. Filgrastim and full-supportive care significantly decreased the pancytopenia duration and resulted in improved animal survival and recovery compared to animals with a limited-supportive care. These findings also identified and validated a multiparametric biomarker panel to support radiation diagnostic device development.

Cercopithecine Herpesvirus 9 (Simian Varicella Virus) Infection after Total-Body Irradiation in a Rhesus Macaque (Macaca mulatta).

This case report describes a rhesus macaque (Macaca mulatta; male; age, 5 y; weight, 6.7 kg) with anorexia, dehydration, lethargy, ataxia, and generalized skin rashes that occurred 30 d after total-body irradiation at 6.5 Gy ((60)Co γ-rays). Physical examination revealed pale mucus membranes, a capillary refill time of 4 s, heart rate of 180 bpm. and respirations at 50 breaths per minute. Diffuse multifocal maculopapulovesicular rashes were present on the body, including mucocutaneous junctions. The CBC analysis revealed a Hct of 48%, RBC count of 6.2 × 10(6)/µL, platelet count of 44 × 10(3)/µL, and WBC count of 25 × 10(3)/µL of WBC. The macaque was euthanized in light of a grave prognosis. Gross examination revealed white foci on the liver, multifocal generalized petechiation on serosal and mucosal surfaces of the gastrointestinal tract, hemorrhagic lymph nodes, and hemorrhagic fluid in the thoracic cavity. Microscopic examination revealed cutaneous vesicular lesions with intranuclear eosinophilic viral inclusions within the epithelial cells, consistent with herpesvirus. Immunohistochemistry was positive for herpesvirus. The serum sample was negative for antibodies against Macacine herpesvirus 1 and Cercopithecine herpesvirus 9 (simian varicella virus, SVV). Samples submitted for PCR-based identification of the etiologic agent confirmed the presence of SVV DNA. PCR analysis, immunohistochemistry, and histology confirmed that lesions were attributed to an active SVV infection in this macaque. This case illustrates the importance of screening for SVV in rhesus macaques, especially those used in studies that involve immunosuppressive procedures.

Gastrointestinal acute radiation syndrome in Göttingen minipigs (Sus scrofa domestica).

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; (60)Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.

Significance of bioindicators to predict survival in irradiated minipigs.

The minipig is emerging as a potential alternative non-rodent animal model. Several biological markers (e.g., blood counts, laboratory parameters, and clinical signs) have been proposed for rapid triage of radiation victims. Here, the authors focus on the significance of bio-indicators for prediction of survivors after irradiation and compare it with human data; the relationship between these biomarkers and radiation dose is not part of this study. Male Göttingen minipigs (age 4-5 mo, weight 9-10 kg) were irradiated (or sham-irradiated) bilaterally with gamma-photons (6°Co, 0.5-0.6 Gy min⁻¹) in the dose range of 1.6-12 Gy. Peripheral blood cell counts, laboratory parameters, and clinical symptoms were collected up to 10 d after irradiation and analyzed using logistic regression analysis and calculating ROC curves. In moribund pigs, parameters such as decreased lymphocyte/granulocyte counts, increased C-reactive protein, alkaline phosphatase values, as well as increased citrulline values and body temperature, significantly (p < 0.002 up to p < 0.0001) discriminated non-survivors from survivors with high precision (ROC > 0.8). However, most predictive within the first 3 d after exposure was a combination of decreased lymphocyte counts and increased body temperature observed as early as 3 h after radiation exposure (ROC: 0.93-0.96, p < 0.0001). Sham-irradiated animals (corresponding to "worried wells") could be easily discriminated from dying pigs, thus pointing to the diagnostic significance of this analysis. These data corroborate with earlier findings performed on human radiation victims suffering from severe hematological syndrome and provide further evidence for the suitability of the minipig model as a potential alternative non-rodent animal model.