Mechanisms Involved in the Development of the Chronic Gastrointestinal Syndrome in Nonhuman Primates after Total-Body Irradiation with Bone Marrow Shielding

Abstract

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

FIG. 1

Survival of 10–14 Gy TBI NHPs. Panel A: Average day of survival in each group is indicated by the line. Panel B: Timed necropsies of the TBI-BM5 cohorts exposed to 9–12.5 Gy. Studies are focused on the threshold dose of 10 Gy (hatched box).

FIG. 2

Representative photomicrographs of jejunum after 10 Gy TBI-BM5 in control (panel A), 43 days (panel B), 119 days (panel C) and 181 days (panel D) postirradiation. Arrows show submucosal edema, and arrow heads show increased thickness of smooth muscle. All photos are at 20× magnification.

FIG. 3

Representative photomicrographs of the proximal colon after 10 Gy TBI-BM5 in control (panel A) and 181 days (panel B) postirradiation. All photos are at 20× magnification.

FIG. 4

Changes in serum chemistries over time after 10 Gy TBI-BM5. All values were taken from NHPs that survived more than 100 days. Panels A–F show total protein, albumin, AST, ALT, BUN and creatinine, respectively. P < 0.05 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 5

Dose-related changes in transepithelial electrical resistance (TEER) in muscle-free sections of jejunum (panels A and C) and proximal colon (panels B and D) after TBI (panels A and B) or TBI-BM5 (panels C and D). Total-body irradiated NHPs were euthanized ≤15 days after exposure (panels A and B). The TBI-BM5 animals were euthanized at set intervals between 30 and 190 days postirradiation. *P < 0.05 and **P < 0.01 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 6

Time-dependent changes in transepithelial electrical resistance (TEER) in muscle-free sections of jejunum (panel A) and proximal colon (panel B) after 10 Gy TBI-BM5. Resistance was measured in Ussing chambers in muscle-free sections of jejunum (panel C) and proximal colon (panel D) after 10 Gy TBI-BM5. Resistance is calculated using V = IR where V is set at 1 and I is the current generated by the tissue in response to this challenge. *P < 0.05 and **P < 0.01 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 7

Change in weight over time in surviving NHPs after 10 Gy TBI-BM5 (panel A). Glucose absorption was determined in muscle-free sections of jejunum in response to 20 mM glucose added to the mucosal side of the tissue (panel B). Fold change in gene expression of SGLT1 in jejunal mucosa. *P < 0.05 and **P < 0.01 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 8

Fold changes in mucosal gene expression of pro-inflammatory mediators. IL-17A in mucosa of jejunum (panel A) and proximal colon (panel D); IFN-γ in jejunum (panel B) and proximal colon (panel E); and NOS-2 in jejunum (panel C) and proximal colon (panel F). *P < 0.05 and **P < 0.01 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 9

Fold changes in mucosal Lgr5 gene expression (panel A) and protein (panel B) over time in jejunum. Fold change in mucosal expression of the absorptive cell lineage marker, Hes-1 (panel C), and the enteroendocrine cell lineage marker, NGN-3 (panel D), in LCM samples of intestinal crypt cells. *P < 0.05 and **P < 0.01 vs. 0 (control, solid bars). Values are means ± SEM.

FIG. 10

Schematic of interactions among mucosal barrier function, inflammation and mucosal restitution that contribute to chronic GI syndrome in NHPs after 10 Gy TBI-BM5.