Hematological changes as prognostic indicators of survival: similarities between Gottingen minipigs, humans, and other large animal models

Abstract

Background: The animal efficacy rule addressing development of drugs for selected disease categories has pointed out the need to develop alternative large animal models. Based on this rule, the pathophysiology of the disease in the animal model must be well characterized and must reflect that in humans. So far, manifestations of the acute radiation syndrome (ARS) have been extensively studied only in two large animal models, the non-human primate (NHP) and the canine. We are evaluating the suitability of the minipig as an additional large animal model for development of radiation countermeasures. We have previously shown that the Gottingen minipig manifests hematopoietic ARS phases and symptoms similar to those observed in canines, NHPs, and humans.

Principal findings: We establish here the LD50/30 dose (radiation dose at which 50% of the animals succumb within 30 days), and show that at this dose the time of nadir and the duration of cytopenia resemble those observed for NHP and canines, and mimic closely the kinetics of blood cell depletion and recovery in human patients with reversible hematopoietic damage (H3 category, METREPOL approach). No signs of GI damage in terms of diarrhea or shortening of villi were observed at doses up to 1.9 Gy. Platelet counts at days 10 and 14, number of days to reach critical platelet values, duration of thrombocytopenia, neutrophil stress response at 3 hours and count at 14 days, and CRP-to-platelet ratio were correlated with survival. The ratios between neutrophils, lymphocytes and platelets were significantly correlated with exposure to irradiation at different time intervals.

Significance: As a non-rodent animal model, the minipig offers a useful alternative to NHP and canines, with attractive features including ARS resembling human ARS, cost, and regulatory acceptability. Use of the minipig may allow accelerated development of radiation countermeasures.

Figure 1. Dose response curves for bilateral gamma-irradiation of Gottingen minipig.

Six animals per dose were irradiated with Cobalt-60 (0.6 Gy/min). Survival was followed for 30 days after exposure and data were plotted as Kaplan-Meier curves (Panel A). Probit analysis was used to fit survival curves and calculate dose-response (Panel B) and confidence intervals (Panel C).

Figure 2. Ranges (min, max) of blood cell loss and recovery in irradiated minipigs.

Graphs represent the minimum and maximum blood cell values observed over time from 30 animals irradiated with doses ranging from 1.6 to 2.0 Gy at 0.1 Gy dose intervals and pooled together (6 animals/dose).

Figure 3. Histological examination of crypts and villi in the duodenum of irradiated minipigs (1.6 to 2.0 Gy).

Samples were collected and fixed at necropsy. Panel A (1.6 Gy, H&E 100×). Villar length is consistent throughout the section, and is of normal length, lined by a single layer of columnar epithelium with basilar nuclei and abundant eosinophilic apical cytoplasm. Underlying crypts are densely packed. Panel B (1.9 Gy, H&E 100×). Villar length remains consisted in length and in morphology of the overlying epithelium as well as the abundance and morphology of the underlying villar crypts. Panel C (2.0 Gy, H&E 200×). Multifocally throughout the section, villi are blunted and fused (asterix) with multiple villi recovering by denudation by being covered by a single layer of columnar epithelium. Underlying crypts (arrows) have piled epithelium and occasionally are tortuous; however there is no apparent loss of crypts.

Figure 4. Ratio of hematological parameters.

Neutrophil-to-lymphocyte (A), neutrophil-to-platelet (B), and platelet-to-lymphocyte (C) ratios were calculated for sham-irradiated animals (diamonds, n = 4) and for animals irradiated with 1.6–2.0 Gy (squares, n = 30). Ratios were calculated for each individual animal and results were pooled together. Asterisks (*) indicate statistically significant difference between groups (Student T-test); bars represent standard errors.