The delayed effects of acute radiation exposure within the mouse intestine

Partial-body irradiation (PBI) models with minimal (2.5% or 5%) Bone Marrow (BM) sparing results in Acute GI Damage (GI-ARS). However, it enables longer-term survival against radiation levels that would normally be lethal due to BM Toxicity.

In this long-term study mice that survived both GI-ARS and Haematological-Acute Radiation Syndrome (H-ARS) following X-irradiation with 2.5% shielding were euthanized at pre-determined time-points up to 250 days post-irradiation along with other animals that were euthanized 'for cause' during this period. Numerous organs were collected for the evaluation of specific changes around epithelial and endothelial damage and recovery as well as evidence of collagen deposition in the jejunum and mid-colon. These tissues can be visualized as a reference to evaluate potential mitigators of the delayed effects of acute radiation exposure (DEARE).

In this study 10-12 week old male C57BI/6 mice were anaesthetized and exposed to varying radiation doses – 10, 12 and 13Gy (300kV X-ray, 10mA, with additional filtration to provide a radiation quality of 2.3mm Cu HVL) at 0.82Gy/min. Sections of the animals' jejunum and mid-colon were stained with haematoxylin and eosin (H&E), Masson's Trichrome or immunohistochemically labelled for Bromodeoxyuridine (BrdU) or CD31 (endothelium).

BrdU was administered prior to euthanasia to label proliferating cells. The crypt numbers and size were also evaluated. Measurement of fibrosis was determined by the deposition of collagen in the submucosa and lamina propria as defined by Masson's Trichrome staining. The thickness of the submucosa was also measured. The results are shown below.

 
Crypt Numbers:

The number of crypts per jejunal cross-section or circumference was reduced out as far as Day 200 post-irradiation, with some recovery at Day 250. However, intestinal circumference length was also reduced in many sections (i.e. the jejunum was narrower).

When crypt numbers were evaluated per mm the crypt loss was less dramatic, although still present, but had recovered by Day 250. In animals euthanized 'for cause' there was no increase in jejunal crypt loss, suggesting crypt loss was not the problem in these mice.

Radiation had less of an effect on colonic circumference length, with a mild reduction from Day 200, but overall there was no sustained reduction in crypt number, compared to the age-matched controls, and again no evidence of increased crypt loss in animals euthanized 'for cause'. See Figure 1.

radres-poster-fig-1

Figure 1: Number of crypts per intestinal circumference and per mm of jejunum and mid-colon 75 and 250 days following partial-body irradiation (PBI/BM2.5). 5-10 circumferences of jejunum and 2-10 circumferences of mid-colon per mouse were scored. The mean per group is plotted with Std Dev.

 
BrdU Analysis:

No difference was seen between the irradiated and age-matched control mice up to Day 150. A small increase in labelling at Day 200 was seen between cell positions 5-10 in the jejunum, the location of the clonogenic cells and early transit amplifying cells. See Figure 2.

The increase in proliferating daughter cells was not reflected higher up the crypt or by crypt length. The proliferation appears to be a small effect controlled by homeostasis.

By day 250, in both jejunum and mid-colon, there was a dose-dependent reduction in labelling in the upper crypt, suggesting a shortening of the proliferative lineage. This was consistent with the reduction in the number of cells along the crypt axis.

Levels of proliferation were lower in the colon in the animals euthanized 'for cause' than in the jejunum.

Small areas of difference in crypt labelling and length within tissue regions were also seen. This could be the result of local vascular impairment.

radres-poster-fig-2

Figure 2: Cell positional distribution of BrdU labelling in jejunum and mid-colon 200 and 250 days following PBI/BM2.5. Up to 50 crypt axes were scored per mouse and the mean labelling index at each cell position plotted.

 
Crypt Size:

There were reductions in jejunal crypt and villus physical length (µm), but no consistent trends in size changes, with any effects being modest. Shorter crypts were also seen in the 'for cause' animals. There was a dose-dependent reduction by Day 250 when crypt length was measured.

In the mid-colon, crypts were wider on Day 75 post-irradiation (presumably linked to ongoing regeneration) and remained shorter post-irradiation, with this effect continuing to Day 250 following 13Gy. See Figure 3.

radres-poster-fig-3

Figure 3: Number of cells along a crypt axis, crypt length, width and villus height 75 and 250 days follwing PBI/BM2.5. The number of cells along up to 50 crypt axes were analyzed. Up to 20 crypt lengths, 15 crypt widths and 15 villus heights were measured. The mean per group is plotted with Std Dev.

 
Masson's Trichrome Analysis:

A clear radiation dose-linked increase in submucosal thickness in the jejunum was indicated on Day 75, which remained prevalent but less pronounced, over time with the dose response not present at Day 200. Measurements from animals euthanized 'for cause' did not differ from those that were scheduled to be euthanized. See Figure 4.

In the colon, radiation did cause an increase in submucosal thickness on Days 75 and 150, but there was not a radiation dose response. By Day 250, there was no increase, with indications of a reduction in thickness at 10Gy. The only indication of a difference between the 'for cause' and scheduled groups was a slight increase in thickness 200 days after 12Gy. See Figure 5.

Therefore, it is possible that earlier increases in thickness may be less related to fibrosis than to bowel shortening in response to injury, followed by gradual recovery.

radres-poster-fig-4

Figure 4: Submucosal thickness and % of the analyzed area of both submucosa and lamina propria that contained collagen in the jejunum 75 and 250 days following PBI/BM2.5. The submucosa of 5 circumferences of Masson's Trichrome stained jejunum were analyzed using a colour deconvolution algorithm on the Aperio ScanScope platform. The mean per group is plotted with Std Dev.

radres-poster-fig-5

Figure 5: Submucosal thickness and % of the analyzed area of both submucosa and lamina propria that contained collagen in the mid-colon 75 and 250 days following PBI/BM2.5. The submucosa of 5 circumferences of Masson's Trichrome stained mid-colon were analyzed using a colour deconvolution algorithm on the Aperio ScanScope platform. The mean per group is plotted with Std Dev.

 
CD31 Analysis:

In the jejunal submucosa there was a dose-dependent reduction on Day 75 in both the number of blood vessels/mm2 and the proportion of the submucosal area occupied by blood vessels, a time at which mice also lost weight. The number of vessels/mm2 had started to recover by Day 150 – the vessel area by Day 200, but neither fully recovered by Day 250. Interestingly, the animals euthanized 'for cause' 150-250 days following 12Gy or 13Gy, had a higher blood vessel density and area than those that were scheduled. See Figure 6.

The mean size of the blood vessels within the submucosa was reduced on Day 75 but then recovered at later times (except 150 days after 13Gy which remained low, however the 'for cause' animals at 13Gy recovered this parameter).

In the mid-colon, there was also a dose-dependent reduction in the number and area of blood vessels on Day 75, but these had recovered by Day 150, with the exception of the 13Gy scheduled group. The 13Gy 'for cause' had again recovered this parameter. See Figure 7.

The mean size of blood vessel within the submucosa was reduced on Days 75 and 150 but the former had then recovered by Day 200. Although recovering, the blood vessel size had not completely recovered by Day 250. The levels were higher in the 'for cause' groups once again.

radres-poster-fig-6

Figure 6: Number and area of blood vessels in the submucosa of the jejunum 75, 150 and 250 days following PBI/BM2.5. The submucosa of 5 circumferences of CD31 labelled jejunum were analyzed. The mean per group is plotted with Std Dev.

radres-poster-fig-7

Figure 7: Number and area of blood vessels in the submucosa of the mid-colon 75, 150 and 250 days following PBI/BM2.5. The submucosa of 5 circumferences of CD31 labelled mid-colon were analyzed. The mean per group is plotted with Std Dev.

 
In summary, a narrowing of the jejunum occurred post-irradiation, with some reduction in crypt number and size, but this was not exacerbated in the 'for cause' euthanized mice. The colon remained the same size and crypt numbers were not reduced but crypts were shorter. However, the effects were inconsistent possibly as a result of endothelial damage. On Day 75, there was a reduction in the GI vasculature, mice had lost weight and had breathing problems, but they began to recover, although recovery was not complete by Day 250, indicative of persistent impaired function. Increased vascularity was seen in the 'for cause' euthanized mice as opposed to the scheduled euthanized mice. There was an early (Day 75) increase in submucosal thickness that then resolved and may be as a result of transient bowel shortening in response to injury. There were increased areas and intensity (density) of collagen at the later times post-irradiation, which were often greater in the animals euthanized 'for cause'.

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