Histopathology of Acute Radiation Syndrome and Delayed Effects in Rhesus Macaques

Advancing Radiation Research: Insights from Non-Human Primate Models on Delayed Effects of Acute Radiation Exposure

The acute physiological effects of irradiation in multiple organs have been well characterised in small animal models, particularly mice. To-date three medical countermeasures (MCMs) have been FDA-approved under its ‘Animal Rule’ for treatment of the Acute Radiation Syndrome (ARS). However, all are for the haematopoietic syndrome.

Evidence for delayed effects of acute radiation exposure (DEARE) has mainly come from survivors of nuclear accidents or from radiation oncology. To develop MCMs to treat the prolonged or delayed damage it is necessary to first understand the pathology, its causes and timelines in relation to radiation dose. To do this a validated animal model is required in both small and large species.

In US NIH (NIAID) funded studies, Macvittie et al. developed a model where non-human primates (NHPs) were irradiated with 10-12Gy with minimal bone marrow sparing (tibiae, ankles and feet were outside the radiation field) to enable survival through the haematopoietic-ARS, continuing up to 180 days post-irradiation. In a recently published book by George A. Parker histopathologic findings in several organs from these studies were described.

Jejunum and colon

• Depletion of the epithelial mucosa within days of irradiation was observed in all irradiated animals, with complete denudation seen only after 12Gy.
• From 7 days post-irradiation evidence of repair was noted, with an increase in Ki67 labelling in crypt cells and Lgr5 expressing stem cells. Repair was largely complete in most animals by Day 45 but in some mucosal injury persisted up to 180 days.
• Inflammatory cell infiltration was not prominent in the intestine but during the first 45 days there was an increased presence of MxA expressing cells (activated immunocytes) in the submucosa of some animals and an increase in CD163 positive cells (pro-fibrotic macrophages) in the mucosa, submucosa and serosa (although this is likely to be due to phenotype switching of the macrophages rather than infiltration of a new population).

Mesenteric lymph node

• Lymphoid depletion was observed in all animals up to Day 50 and in numerous animals up to Day 180 (although corticosteroids given as supportive care may have influenced the severity of this during the latter stages of the study).
• Up to Day 50 there was an increased presence of MxA positive cells and CD163 and MPO positive macrophages in the outer parts of the lymph nodes, suggesting the cells’ migration from the intestine. There was also an increase in CD38 pro-inflammatory macrophages that was not seen in the intestine.
• Increased numbers of LPS positive macrophages indicated passage of enteric microbes (or fragments) through the intestinal barrier and subsequent translocation to the lymph node throughout the study, suggesting the intestines remained ‘leaky’ over time. Bacterial colonisation was noted in the lymph nodes of a couple of animals.
• There was increased collagen deposition in the cortex of the lymph nodes throughout the study.

Kidney

• Up to 30 days post-irradiation, proteinic material was present in the urinary spaces of the glomeruli, consistent with injury to the capillaries. Beyond 100 days proteinaceous casts were commonly found in the renal tubules, suggestive of microhaemorrhage.
• After 100 days there was also a high incidence of interstitial and glomerular fibrosis, sometimes associated with reduced CD31 (endothelial cells) expression. Glomeruli also had increased mesangial matrix, coincident with increased a-SMA labelling, increased trichrome-positive and Periodic Acid/Schiff positive staining.
• As in the intestine, there was an increased presence of CD163 macrophages.

Lung

• From 85 days post-irradiation interstitial fibrosis was commonly seen, often associated with pleural fibrosis. More pronounced areas of fibrosis had reduced levels of CD31 labelling. Alveolar oedema was often noted, indicative of endothelial injury.
• In the later stages there were increased presence of CD163, CD206 and MPO positive macrophages, suggesting they may be involved in generation or perpetuation of fibrotic processes in the lung. Proliferation of Type II pneumocytes that expressed CTGF in areas of damage suggested that they may also be involved.

Heart

• Areas of myocardial fibrosis and myocardial degeneration were seen in some animals throughout the 180 days. Staining for collagen I, a-SMA and CTGF increased in areas of fibrosis. Galectin-3 levels increased in areas of degeneration.
• Animals irradiated with 12Gy had an unusual pattern of caspase-3 labelling, unlike the whole cell staining typically seen in apoptotic cells.

Taken together this work provides for the first time a comprehensive time map of the tissue changes consistent with the development of DEARE in a well-controlled in vivo study. Whilst this has been known to include the development of fibrosis in many tissues this current study also demonstrates the persistent damage to the endothelial vasculature in some organs and the incomplete recovery of the gut over time.

Epistem Services

At Epistem our key project leaders each have a minimum of 20 years’ experience developing and using gastrointestinal radiation models to evaluate potential countermeasures, including both ARS/DEARE (alongside radiotherapy models). This team are supported by an in-house expert histology/immunohistochemistry lab.

• Histological analytical services include measures of enterocyte loss / recovery (including characterization of the target cell population and response kinetics) and quantitative analysis of histological stains and immunohistochemical labelling of a variety of biomarkers using the Aperio Scanscope® image analysis platform.
• The Histology and Immunohistochemistry team at Epistem have a wealth of experience in developing IHC protocols and troubleshooting working protocols for both automated (Ventana Discovery Ultra) and manual applications. We offer a bespoke service which can be suited to your project. We can work readily with FFPE, frozen tissues, with chromogenic and fluorescent detection systems.
• Epistem also offers gene expression analysis services and RNA‑friendly laser capture microdissection of target cell populations.
• Epistem perform in vitro models of normal and irradiated intestinal epithelium using organoids derived from mouse, rat, canine and human intestine. Multiple Test Articles, such as drug candidates and tool molecules, can be assayed in multi-well format assays using freshly passaged organoids to assess the effect on organoid viability and production of crypt-like structures.

To discuss your projects with our experts, don’t hesitate to contact us here.