Effects of Gamma-Tocotrienol on Intestinal Injury in a GI-Specific Acute Radiation Syndrome Model in Nonhuman Primate

Abstract

The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.

Keywords: gamma-tocotrienol; intestine; nonhuman primates; radiation; radiation countermeasure.

Figure 1

Effects of GT3 on irradiation-induced intestinal damage at day 4 and day 7. (A) Representative images showing the cross sections of proximal jejunum treated with vehicle or GT3 at day 4 and 7 post-irradiation. Histogram showing the measurements of intestinal injury such as (B) mucosal surface length, (C) villous height, and (D) crypt depth. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7).

Figure 2

Effects of irradiation on crypt survival. (A) Representative images showing the transverse sections of proximal jejunum from NHP treated with vehicle or GT3 at day 4 and 7 post-irradiation. (B) Surviving crypts. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7). ns = not significant.

Figure 3

Effects of GT3 on TBI-induced intestinal damage. (A) Representative photomicrograph of TUNEL-positive cells (green) in the intestinal villi. (B) Frequency of TUNEL-positive cells in villi at day 4 and 7 post-TBI in vehicle and GT3-treated groups. (C) Representative photomicrograph of TUNEL-positive cells (green) in the crypt. (D) Frequency of TUNEL-positive cells in the crypts at day 4 and 7 post-TBI in vehicle and GT3-treated groups. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7). ns = not significant.

Figure 4

Effects of GT3 on intestinal stem proliferation as assessed by Ki-67. (A) Representative photomicrograph of Ki-67-positive cells (red) in the crypts. (B) Mean fluorescence intensity of Ki-67 at day 4 and 7 post-TBI in vehicle and GT3-treated groups. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7).

Figure 5

Effects of GT3 on PCNA expression in proximal jejunum post-irradiation. Representative images showing the expression of PCNA in cross sections of proximal jejunum treated with vehicle or GT3 prior to exposure to 12 Gy TBI at day 4 and day 7 post-irradiation.

Figure 6

. Effects of GT3 on claudin-2 expression in intestine. (A) Representative photomicrograph of claudin-2 expression (red) in the cross sections of the intestine at day 4 and day 7 post-irradiation. (B) Quantitation of claudin-2 expression levels in intestinal crypts at indicated time points post-12 Gy TBI. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7). ns = not significant.

Figure 7

Effects of GT3 on ZO-1 expression in intestine. (A) Representative photomicrograph of ZO-1 expression (green) in the cross sections of the intestine at day 4 and day 7 post-irradiation. (B) Quantitation of ZO-1 expression levels in intestine at indicated time points post-12 Gy TBI. The data are presented as average ± SEM; N = 3 (day 4) and N = 5 (day 7).