Establishment of a standardized rat model of chronic radiation proctitis and preliminary evaluation of its association with tubulin

Abstract

BACKGROUND

Chronic radiation proctitis (CRP), a major complication of pelvic radiotherapy, lacks effective treatments and a standardized animal model that recapitulates its late fibrotic and angiogenic pathologies.

AIM

To establish a model that recapitulates the features of human CRP and to investigate the mechanisms of irreversible tissue damage.

METHODS

Rats were randomized into a control group and single-dose irradiation groups (12.5-37.5 Gy). Rectal injury was assessed via 6-month continuous endoscopic observation, the modified Vienna Rectoscopy Score, and histological analysis. Additionally, rectal tissues were analyzed for cytoskeletal protein alterations.

RESULTS

Acute mucosal injury was reversible at doses below 20 Gy. In the 20-35 Gy groups, persistent radiation proctitis, marked telangiectasia, and severe mucosal fibrosis indicated irreversible injury. Furthermore, cytoskeletal proteins, particularly tubulin, decreased sharply at doses ≥ 20 Gy. Vascular endothelial growth factor was mildly upregulated mainly in the deep tissue layers during the acute phase, whereas it was significantly increased in the mucosa of the highdose groups during the chronic phase.

CONCLUSION

Single-dose radiation (20-35 Gy) induces CRP. Reduced tubulin expression marks the early transition to chronic injury (P < 0.0001).

Key Words: Chronic radiation proctitis; Endoscopic views; Endoscopic scoring; Tubulin; Rat model

Core Tip: Chronic radiation proctitis can be induced by a single dose of 20-35 Gy irradiation, which simplifies the modeling procedure while recapitulating two major human pathological features: Telangiectasia and progressive fibrosis. The formation of chronic injury is associated with the destruction of microtubule protein, the low expression of tubulin at 2 weeks after irradiation can serve as an early indicator for the transition of rectal toward chronic radiation injury. After radiation without intervention, around the eighth week, chronic manifestations such as capillary dilation would appear. The observation period for the therapeutic effect should be extended to at least 8 weeks.

INTRODUCTION

Radiotherapy is frequently used to treat pelvic malignancies, with approximately 300000 patients worldwide undergoing pelvic irradiation annually[1]. Among radiation-induced complications, rectal injury is the most common. Radiation proctitis (RP) can be classified as acute or chronic according to symptom onset and the severity of tissue damage[2]. Acute RP (ARP) typically manifests as tenesmus and diarrhea, occasionally accompanied by mild bleeding[3], and generally resolves spontaneously. In contrast, chronic RP (CRP) has a later onset and tends to persist[4]. The most frequent symptom of CRP is rectal bleeding, followed by fibrotic changes leading to strictures[5]. These symptoms significantly reduce patient quality of life, and currently, there is no effective treatment. Thus, CRP remains an important focus of both clinical and basic research.

Previous studies on RP have predominantly utilized acute radiation injury models[6,7], which do not adequately replicate the pathophysiology of chronic injuries resulting from radiotherapy. Therefore, establishing an animal model that closely mimics human CRP progression is essential. Several modeling approaches have been reported, ranging from high-dose[8] to low-dose[9] irradiation, and from single-dose exposure to multiple fractionated low-dose regimens[10,11]. Although these methods induce varying degrees of rectal injury, a standardized animal model and evaluation system for chronic radiation injury remains lacking. In this study, long-term mucosal changes in the rat rectum were monitored by in vivo endoscopy, combined with acute and chronic symptom scoring, histopathological assessments, and molecular biological analyses for comprehensive evaluation.

MATERIALS AND METHODS

Animals and irradiation procedure

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Eastern Theater Command General Hospital (Approval No. DZ24WYXKT 001). Male Sprague-Dawley rats (8 weeks old, specific pathogen-free) weighing 250 ± 20 g were used. All rats were acclimated under standard laboratory conditions with free access to food and water for one week prior to irradiation.

The Gy is the international system of unit for measuring the absorbed dose of ionizing radiation. The rats were randomly divided into 12 groups (eight rats per group), including radiation dose groups of 12.5 Gy, 15 Gy, 17.5 Gy, 20 Gy, 22.5 Gy, 25 Gy, 27.5 Gy, 30 Gy, 32.5 Gy, 35 Gy, and 37.5 Gy, as well as a control group. Rats were immobilized in transparent plastic holders, and irradiation was simultaneously delivered to five rats placed on a wooden board to enhance efficiency (Figure 1). X-ray irradiation was administered using a linear accelerator with a rectangular collimated field. Except for the rectal region, the pelvic area was shielded using a 3 cm thick lead block to minimize exposure of surrounding tissues.

Figure 1 Irradiation setup showing rat fixation and shielding. A and B: The yellow rectangle indicates the irradiation area, while the red area represents the rectal region after tissue shielding.

Endoscopic observation and scoring

The rats were fasted for 12 hours before colonoscopy but had free access to water to facilitate clear visualization. Anesthesia was induced by inhalation of isoflurane, and the rats were placed prone on a thermostatic surgical platform (37 °C). The tail was gently lifted to straighten the rectum. The anus and endoscope were lubricated with paraffin oil, and a cholangioscope (diameter 2.9 mm) was carefully inserted along the anterior rectal wall. During insertion, sterile saline was infused through the instrument channel to improve visibility and prevent intestinal spasms. If resistance occurred, the scope was withdrawn and repositioned at a different angle to avoid mucosal damage or perforation. During withdrawal, the distal colon and entire rectum were thoroughly examined, and both images and videos were recorded.

Sequential endoscopic examinations were conducted at 2 weeks, 4 weeks, 6 weeks, and 8 weeks and at 3 months, 4 months, 5 months, and 6 months after irradiation. These examinations allowed continuous monitoring of mucosal progression from the acute to chronic phases. Findings were evaluated using an endoscopic scoring system developed by modifying the Vienna Rectoscopy Score[12] specifically for rats (Table 1). The scoring was performed by two independent endoscopists who were blinded to the animal grouping.

Table 1 Modified rectoscopic scoring system for rats.

Score (point)	Endoscopic feature
Telangiectasia
0	None
1	Scattered isolated vessels
2	Multiple telangiectasias
3	Diffuse telangiectatic networks
Congested mucosa
0	None
1	Mild hyperemia or edema
2	Diffuse hyperemia
3	Contact bleeding
4	Spontaneous bleeding
Ulceration
0	None
1	Single small ulcer or superficial ulcer
2	Multiple small ulcers or confluent patchy ulcers
3	Deep ulcer
4	Deep ulceration leads to fistula or perforation
Stricture
0	None
1	> 2/3 of the diameter of the rectum
2	1/3-2/3 of the diameter of the rectum
3	< 1/3 of the diameter of the rectum
4	Entirely shut
Necrosis
0	None
1	Extensive necrosis

Euthanasia and sample collection

Rats in the longitudinal endoscopic observation cohort were euthanized at 6 months post-irradiation. For the supplementary mechanistic study cohort (n = 8 per group), four rats from each group were randomly selected and euthanized at 2 weeks (acute phase), while the remaining four rats were euthanized at 3 months (chronic phase).

All rats were humanely euthanized via isoflurane inhalation. Blood samples were collected, allowed to stand at room temperature for 30 minutes, and centrifuged at 3000 rpm for 10 minutes. Serum supernatant was stored at -80 °C. Rectal tissue located 3 cm above the anus was excised, rinsed, and rapidly frozen in liquid nitrogen for storage at -80 °C. Samples intended for Western blot analysis were preserved in liquid nitrogen, whereas tissues for histopathological analysis were fixed in 4% paraformaldehyde.

Histopathological analysis

Rectal specimens were fixed in 4% paraformaldehyde and stained with hematoxylin-eosin (HE) and Masson’s trichrome. Tissue sections (4 m thick) were prepared using a rotary microtome (Leica, Germany), placed onto glass slides, and stained according to standard protocols. Sections were observed under a light microscope (DM2500, Leica) following established methods. Collagen fibers, nuclei, and cytoplasm were stained blue, dark purple, and red/pink, respectively, by Masson’s trichrome staining. Histological changes were quantified using a Radiation Injury Score (RIS) adapted from Langberg et al[13], which assesses mucosal ulceration, inflammatory cell infiltration, edema, vascular narrowing, and submucosal fibrosis. The scoring was performed by two independent observers who were blinded to the animal grouping.

Immunofluorescence analysis

Rectal tissue sections were fixed in 4% paraformaldehyde at room temperature for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes to prevent nonspecific binding. Sections were incubated overnight at 4 °C in a humidified chamber with anti-vascular endothelial growth factor (VEGF) primary antibody (1:100 dilution). After washing, a Cy3-conjugated anti-rabbit IgG (1:200 dilution) was applied for 1 hour at room temperature in darkness. Finally, nuclei were counterstained with DAPI (1:1000 dilution) for 5 minutes at room temperature, protected from light.

Western blot analysis

Rectal tissues were lysed, and total protein was extracted and quantified. Equal protein amounts were mixed with loading buffer, separated by 10% SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked in 5% skim milk for 1 hour, incubated overnight at 4 °C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 hour. Protein bands were visualized using an ECL detection kit, and images were captured with a Tanon 5200 imaging system.

Statistical analysis

All data are expressed as mean ± SD. Statistical analysis was performed using Prism 10 and the statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple groups, or student t-test for two groups. For survival analysis, survival curves were generated using the Kaplan-Meier method, and differences in survival rates between groups were compared by the log-rank (Mantel-Cox) test. Statistical significance was set at P < 0.05.

RESULTS

Effects of different radiation doses on rats

In this study, mortality was minimal in the 12.5-20 Gy groups. In contrast, mortality increased progressively with higher radiation doses (> 20 Gy), and extensive deaths occurred in the 37.5 Gy group during the acute inflammatory phase. Six-month survival analysis revealed that higher radiation doses resulted in earlier mortality, peaking between 120-160 days post-irradiation (Figure 2A). Necropsy demonstrated varying degrees of rectal obstruction extending into the colon, causing difficulties with defecation. Concurrent complications included urinary retention, bladder distension, and highly concentrated urine.

Figure 2 Effects of different radiation doses on rats. A: Survival curve; B: Endoscopic appearance of rat rectum at 2 weeks post-irradiation across different dose groups; C: Dose-endoscopic score curve; D: Hematoxylin-eosin and Masson staining of rectal tissues at 3 months (scale bar = 200 µm); E: Dose-Radiation Injury Score curve. HE: Hematoxylin-eosin.

Endoscopic findings were classified into three severity levels: (1) Mild (12.5-20 Gy): Mild mucosal hyperemia, edema, and occasional pinpoint bleeding; (2) Moderate (22.5-27.5 Gy): Diffuse mucosal hyperemia and edema, thickened mucosa, swollen folds covered with fibrinous white exudate, and increased mucosal fragility with easy bleeding upon contact; and (3) Severe (30-37.5 Gy): Extensive mucosal hyperemia and edema causing luminal narrowing, widespread active bleeding with minimal contact, and visible ulceration under endoscopy (Figure 2B).

Lesions were graded based on capillary dilation, mucosal hyperemia, ulceration, stenosis, and necrosis, and scores were summed. Previous studies by Northway et al[14] indicated pathological injury scores peak between 7-15 days post-irradiation[15]. Accordingly, a dose vs 2-week endoscopic score curve was generated (Figure 2C). Mucosal injury was mild at doses below 20 Gy and progressively worsened with increasing radiation dose. At three months, the 15 Gy group displayed mild injury, showing atrophic mucosal epithelium and slight submucosal fibrosis thickening by HE staining, allowing mucosal self-repair. Higher doses induced significant submucosal fibrosis, arterial wall fibrosis, glandular disorganization, severe rectal wall rigidity, widespread mucosal ulceration, and complete glandular destruction. Full-thickness necrosis occurred at 35 Gy (Figure 2D). RIS trends were consistent with endoscopic scores. Because groups differing by 2.5 Gy showed similar histopathological changes, representative results are presented at 5 Gy intervals (Figure 2E).

VEGF expression increases during the chronic phase of RP

To investigate the relationship between the spatial dynamics of VEGF and the disease stage of RP, immunofluorescence staining was performed on rat rectal tissues at 2 weeks (acute phase) and 3 months (chronic phase) after irradiation. At 2 weeks post-irradiation, all irradiated groups (15-35 Gy) showed mildtomoderate increases in VEGFpositive signals (red). However, these acutephase signals were mainly distributed as fine linear patterns in the deep tissue layers (submucosa or muscular layer), with no obvious expression observed in the mucosal epithelium. At 3 months post-irradiation, the expression level and spatial localization of VEGF underwent marked spatial redistribution. In the 15 Gy group, VEGF signals were nearly abolished and lower than those at 2 weeks. In striking contrast, in the highdose groups of 20-35 Gy (especially the 25 Gy and 30 Gy groups), VEGF signals were not only highly upregulated but also exhibited a prominent shift in localization: Strong VEGF expression accumulated extensively in the mucosal layer, forming a dense positive band along the luminal surface, and infiltrated widely into the surrounding fibrotic tissue (Figure 3).

Figure 3 Immunofluorescence staining for vascular endothelial growth factor in rat rectum at 2 weeks and 3 months post-irradiation. VEGF: Vascular endothelial growth factor.

Endoscopic time-course changes in rats exposed to different radiation doses

Mild (12.5-17.5 Gy, e.g., 15 Gy): Mild mucosal edema and pinpoint bleeding were noted during the acute phase. Later, inflammation resolved and mucosal healing persisted up to 6 months (Figure 4A).

Figure 4 Endoscopic time-course changes in rats exposed to different radiation doses. A: Sequential endoscopic findings in the 15 Gy group; B: Sequential endoscopic findings in the 25 Gy group; C: Sequential endoscopic findings in the 35 Gy group; D: Time-endoscopic score curves for 15 Gy, 25 Gy, and 35 Gy groups.

Moderate (20-25 Gy, e.g., 25 Gy): After partial resolution of acute inflammation, symptoms slightly improved. However, at approximately 8 weeks, various degrees of capillary dilation emerged due to abnormal tissue remodeling. Endoscopy performed between 3-6 months showed luminal narrowing, pale mucosa, and rigid rectal walls (Figure 4B).

Severe (27.5-37.5 Gy, e.g., 35 Gy): Severe mucosal epithelial shedding caused pronounced edema, inflammatory stenosis, and active mucosal bleeding. At 8 weeks, endoscopy revealed red, spiderweb-like vascular patterns. Progressive fibrosis resulted in severe luminal narrowing (Figure 4C). Unlike the 25 Gy group, rats irradiated with 35 Gy maintained persistently high endoscopic scores (Figure 4D). Continuous endoscopic monitoring at doses above 35 Gy was impractical due to high mortality.

Endoscopic observation revealed that groups exposed to doses ≥ 20 Gy developed extensive telangiectasia at approximately 8 weeks post-irradiation. At 6 months, these groups exhibited persistent mucosal injury, failing to heal, and eventually developed CRP. Conversely, rats irradiated below 20 Gy exhibited resolution of inflammation and mucosal recovery.

Significant loss of cytoskeletal proteins in rat rectal tissue following highdose irradiation

To explore the potential molecular alterations underlying CRP induced by highdose irradiation, we extracted proteins from rectal tissues at 2 weeks post-irradiation for Western blot analysis. To evaluate the structural integrity of the cytoskeleton following high-dose irradiation, we investigated the key cytoskeletal components α-tubulin and β-actin as our target proteins of interest. Because these classical housekeeping proteins were severely degraded by irradiation, they were unsuitable as loading controls. Instead, we utilized histone H3 as a reliable internal control, as its stable expression within the nucleus allows it to serve as an accurate proxy for equivalent cell loading. The expression of both cytoskeletal proteins was significantly decreased in the 20 Gy and higherdose groups compared with the 15 Gy group (Figure 5).

Figure 5 High-dose irradiation induces significant degradation of cytoskeletal proteins in rat rectal tissues. A: Representative Western blot images of the cytoskeletal components α-tubulin and β-actin in rectal tissues at 2 weeks post-irradiation across different dose groups (15-35 Gy). Histone H3 was utilized as an internal loading control to represent total nuclear protein and confirm equal cell equivalent loading per lane, demonstrating stable expression across all doses; B and C: Quantitative densitometric analysis of relative α-tubulin (B) and β-actin (C) protein levels, normalized to histone H3. The data indicate a severe, dose-dependent loss of cytoskeletal integrity starting at 20 Gy. Data are presented as mean ± SD. ^aP < 0.05 compared to the 15 Gy group; ^bP < 0.0001 compared to the 15 Gy group. NS: Not significant.

Notably, the loss of αtubulin was extremely dramatic: It exhibited a sharp decline at 20 Gy (P < 0.0001) and was almost completely depleted in the higher-dose groups (25-35 Gy), with no significant differences among the 20-35 Gy subgroups. The β-actin was also significantly downregulated in the 20 Gy and higherdose groups (P < 0.05), but its reduction was milder than that of αtubulin. Given the stable expression of H3, this specific and dramatic decrease suggests extensive cytoskeletal collapse in intestinal tissues during the acute phase, rather than a mere reduction in total cell number.

DISCUSSION

Acute and CRP are not merely sequential stages over time, but represent fundamentally distinct pathological processes. ARP is mainly characterized by mucosal inflammatory infiltration, while the chronic phase predominantly involves regenerative and fibrotic changes[5]. Acute injury reflects a dynamic balance between the inflammatory response and compensatory mucosal repair, which is generally a reversible process. In contrast, the chronic phase is driven by a vicious cycle of persistent microvascular ischemia and fibrosis[16,17]. In our model, extensive telangiectasia observed at 8 weeks in the highdose groups (≥ 20 Gy) represents maladaptive vascular remodeling rather than physiological repair. Chronic rectal injury in rats usually appears at approximately 2 months[18], which is pathologically equivalent to 4-5 years in humans. These vascular abnormalities at 8 weeks indicate the establishment of an irreversible ischemia-inflammation-fibrosis cycle. This is fundamentally different from the transient, self-limiting inflammation seen in the acute phase.

Analyzing inflammatory markers during the acute edematous resolution phase may lead to inaccurate conclusions regarding the long-term effects of drugs. Therefore, pharmacological studies aimed at therapeutic interventions for RP should extend the experimental endpoint to at least 8 weeks to accurately evaluate the improvement of chronic symptoms, especially the typical pathological features of CRP, including telangiectasia, bleeding, and intestinal wall fibrosis.

Compared with histopathological examination relying solely on terminal necropsy, the inclusion of rectal endoscopic observation in the evaluation system offers significant advantages[14,19]. It not only provides more intuitive and comprehensive information on mucosal changes and allows simultaneous observation of the rectum and distal colon, but more importantly, enables continuous longitudinal monitoring of injury progression and repair without the need for animal sacrifice. During the initial establishment of the endoscopic follow-up protocol, we attempted to use an ultra-thin gastroscope with conventional air insufflation. However, due to severe rectal stenosis and obstruction in the experimental animals, air insufflation caused excessive dilation of the proximal colon, resulting in a high mortality rate. In view of this, this study modified the operational strategy by using a thinner-caliber cholangioscope combined with saline perfusion. The slender shaft of the cholangioscope can safely pass through the stenotic rectal segment, and water perfusion instead of air insufflation not only provides a clear visual field but also effectively eliminates the risk of death caused by excessive intestinal dilation. No animal deaths were caused by this improved protocol throughout the study. We recommend the use of a cholangioscope for in vivo longitudinal monitoring of rat rectal injury models.

Fractionated irradiation was not included as a separate group in this study. Previous reports indicate fractionated radiotherapy aims primarily to reduce radiation damage[20,21]. Clinical patients usually receive fractionated low-dose irradiation, allowing normal tissues to undergo limited repair during treatment intervals. In contrast, rats in the present study were subjected to single high-dose irradiation, which eliminates the window for tissue repair. Consequently, pathological progression is more rapid and severe, effectively compressing the timeline of chronic transformation and accelerating abnormal vascular remodeling and fibrosis. Although fractionated radiotherapy permits DNA damage repair, the cumulative effect of repeated irradiation eventually exhausts tissue repair mechanisms. In this study, single high-dose irradiation successfully established a rat model of CRP, whose pathological features, including intestinal wall fibrosis and telangiectasia, are highly consistent with those of advanced CRP in humans. Therefore, we recommend the singledose modeling strategy, which simplifies experimental procedures and minimizes experimental variability caused by repeated irradiation procedures.

Combined with our consecutive endoscopic observations and pathological findings, chronic progressive injury was established in the ≥ 20 Gy groups. Since rats exposed to 37.5 Gy irradiation exhibited high mortality during the acute phase, radiation doses above 35 Gy are not suitable for establishing a rat model of RP. Therefore, a single dose of 20-35 Gy is recommended for the establishment of a rat model of CRP.

The cytoskeleton is mainly composed of microtubules and microfilaments, which jointly maintain cellular structural integrity and functional homeostasis. Microtubules serve as the core driving force for mitosis and intracellular transport, whereas microfilaments are essential for cell movement, contractility, and morphological plasticity. As a key structural basis for tissue regeneration following mucosal injury, an intact cytoskeleton is a prerequisite for epithelial cells to proliferate, migrate toward damaged areas, and cover mucosal defects[22,23]. The fundamental mechanisms of radiation injury include severe cumulative DNA damage and cell cycle arrest, which rapidly deplete the intestinal crypt stem cell pool[24], and further impeding tissue repair. Rapidly proliferating rectal crypt stem cells and microvascular endothelial cells involved in angiogenesis are the most radiosensitive cell populations, and their cytoskeletons are particularly vulnerable to radiation-induced disruption[25]. Growth and repair of the rectal epithelium are highly dependent on the dynamic regulation of tubulin and actin[26]. In our study, the marked reduction in cytoskeletal proteins observed in the ≥ 20 Gy groups indicates that even surviving cells lose essential mechanical functions. They can neither assemble mitotic spindles for proliferation nor form functional cytoskeletal structures required for epithelial migration, ultimately leading to failed epithelial regeneration. The failure of regeneration leaves the submucosa persistently exposed to inflammatory and hypoxic stress, creating a self-reinforcing vicious cycle. Notably, in the group receiving ≥ 20 Gy irradiation, α-tubulin expression was barely detectable, suggesting that the intrinsic regenerative capacity of the rectal tissue was substantially compromised and unlikely to recover spontaneously. Furthermore, when using cytoskeletal protein levels to exclude animals that did not develop chronic injury, α-tubulin exhibited more pronounced alterations and superior discriminatory power compared to other markers. These characteristics render α-tubulin a highly suitable biomarker for predicting the chronic progression of radiation-induced injury.

Irradiation directly injures endothelial cells, leading to disruption of the microvascular basement membrane. Widespread destruction of microvasculature results in insufficient local tissue perfusion[17], thereby establishing a persistent hypoxic microenvironment. Under hypoxic conditions, activation of the hypoxiaHIF1α axis directly drives sustained high expression of VEGF[27]. The initial purpose of upregulating VEGF is to restore tissue oxygenation via angiogenesis. Ideally, compensatory angiogenesis improves perfusion and relieves hypoxia. However, in CRP, VEGF induces structurally abnormal and immature neovessels characterized by tortuous, thin-walled telangiectasia[28,29]. These abnormal vessels not only fail to effectively alleviate hypoxia but also rupture easily. Meanwhile, by increasing vascular permeability[30], VEGF promotes the deposition of protein-rich exudates and accelerates fibrosis. Fibrosis, in turn compresses blood vessels and further exacerbates hypoxia, resulting in the typical clinical pathological features of telangiectasia, bleeding, and intestinal wall fibrosis. These findings also provide a mechanistic explanation for the clinical efficacy of anti-VEGF therapies (such as submucosal bevacizumab injection) and hypoxia-ameliorating strategies (such as hyperbaric oxygen therapy[31,32]).

Our findings provide critical insights into novel targeted therapeutic strategies and future research directions for CRP. Clinically, advanced CRP is notoriously refractory to treatment, and conventional endoscopic hemostasis only offers limited and transient symptomatic relief. Thus, early intervention is essential for RP. In the acute phase, prompt antioxidant intervention is crucial to block the initial oxidative stress cascade and prevent progression toward chronic fibrotic injury. Furthermore, future treatments for refractory RP can target the cellular level by restoring the integrity of the cytoskeletal structure, for example, through exogenous intestinal stem cell transplantation. In addition, to ensure rigorous preclinical evaluation of these novel therapies, it is essential to distinguish individuals prone to spontaneous mucosal healing from those destined to develop refractory injury. The expression level of αtubulin can serve as a robust and highly sensitive early predictive biomarker. Utilizing this marker in future experimental designs will allow researchers to accurately identify and exclude self-healing subjects.

CONCLUSION

A radiation dose between 20 Gy and 35 Gy can be used to establish a rat model of CRP while ensuring adequate survival, thus successfully establishing a CRP model in rats. When assessing pharmacological interventions for RP, the observation period should be extended to at least eight weeks. The low expression of tubulin at two weeks after irradiation can serve as an early indicator of the transition of rectal tissue toward chronic radiation injury.

ACKNOWLEDGEMENTS

We sincerely thank the reviewers for their time, effort, and insightful feedback during the review process of this manuscript.