In 2009, it was estimated that 80% of the reported 2.6 million new infections of HIV were sexually transmitted.1 Although the worldwide incidence of new infections has decreased approximately 20% from 2001 to 2011, the HIV/AIDS epidemic continues to be a prominent global health concern.2 Recent successful studies using preexposure prophylaxis support the use of microbicides in the vagina and have increased interest in the use of topical drugs both vaginally and rectally.3
The risk of HIV anal transmission is believed to be 10 to 20 times higher than rates of vaginal and oral transmission.4,5 Anal intercourse is practiced by both men and women and is prevalent in up to 35% to 40% of heterosexual relationships; thus, there are several populations at risk that would benefit from the development of an effective rectal microbicide.6,7 Because of potential to cause toxicity that may increase susceptibility, safety studies must be performed.8 Numerous methods to determine vaginal product safety have been used; however, methods must also be developed to ensure that microbicides are safe in the rectum.
Safety studies of topical microbicides for rectal use have used cell cultures,9,10 rectal tissue explants collected retrospectively and prospectively,11 in vivo mouse herpes simplex virus 2 challenge,12 and in vivo macaque studies.13 Caco-2 cell cultures are ideal for initial screening of a large number of products for toxic effects; however, cell cultures are not representative of an in vivo environment, and thus, in vivo models are also necessary.9,10 In a macaque rectal safety model,13 samples from rectal swabs and lavage are evaluated for tissue sloughing, pH, and microflora. During the lavage collection process, tissue injury could occur, potentially limiting specificity of the model. In addition, owing to the expense and limited availability of animals, intermediate large animal models used before testing in NHP are desirable. Human intestinal explants have been harvested from untreated surgical resection specimens or prospectively via colonoscopy in treated volunteers.14 The tissue is cultured and used for evaluation of toxicity using histological techniques, and in the case of pretreated volunteers, the explants allow for susceptibility testing ex vivo after in vivo product use. These explants also have limitations including the need to collect invasive biopsies in humans and architectural deterioration within 24 hours of collection.11 Therefore, there is a critical need for the development and evaluation of suitable large animal models that would provide the sensitivity that is needed to detect potential toxicity of emerging microbicides for rectal application.
High-resolution imaging modalities have the potential to provide sensitive measures of mucosal disruption and can delineate specific locations of injury. Colonoscopy uses white light magnification to visualize gross morphological changes and can be used to direct biopsies or endoscopic-based high-resolution imaging methods. The vaginal epithelial layer is clearly visualized using a high-resolution minimally noninvasive imaging modality, optical coherence tomography (OCT), for vaginal product safety evaluations.15 Optical coherence tomography has also been used extensively in gastrointestinal tract research for evaluation of neoplastic and inflammatory processes.16 Together, these imaging modalities have promised to provide a comprehensive evaluation of the rectum and sigmoid colon.
To further aid in the advancement of the study of microbicides, we established an ovine rectal model for the investigation of topical microbicides in vivo, which is critical for future long-term studies. In addition, we applied the use of novel high-resolution imaging techniques of OCT and colonoscopy to develop a sensitive noninvasive method for evaluating product toxicity of rectal microbicides.
MATERIALS AND METHODS
Eight yearling virginal female sheep were administered a single 8-mL dose of either 0.2% benzalkonium chloride (BZK) solution (n = 4) or an equal volume of phosphate-buffered saline (PBS; n = 4) rectally and then rinsed with saline 30 minutes after treatment. Colonoscopy and OCT images were obtained at baseline and after intrarectal treatment. After rinsing of the colon, OCT imaging was performed at the distal colon, for 2- and 5-cm depths, requiring 5 minutes. Then colonoscopy was performed with OCT imaging simultaneously at the 10-, 20-, and 30-cm depths, with marking of the site by biopsy. The colonoscopy and OCT imaging lasted 10 to 20 minutes. Tissue was then obtained for biopsy after removal of the colon, which occurred approximately 1.5 hours after application of BZK/PBS, and approximately 1 hour after rinsing of the colon; therefore, the timing of OCT imaging and the timing of biopsy collection were separated in time by approximately 1 hour. All procedures were approved by The University of Texas Medical Branch at Galveston Institutional Animal Care and Use Committee and were in accordance with the standards set forth in the Guide for the Care and Use of Laboratory Animals (published by the National Academy of Science, National Academy Press, Washington, DC).
Because a previous study has shown that microbicides can reach distances of 30 cm into the colon,17 imaging was performed up to 30 cm. Colonoscopy images were obtained at 0 to 10, 20, and 30 cm. Optical coherence tomography images were obtained by direct visualization at 2 and 5 cm, and then at 10, 20, and 30 cm through the colonoscope. After posttreatment imaging, biopsies were obtained at all depths at the site of OCT imaging.
For colonoscopy, a dual-channel Pentax EG-3831T gastroscope was introduced rectally after saline flush was performed. A gastroscope was used because the length would allow for use of the flexible OCT probe. The use of this device will be referred to as colonoscopy because the imaging was performed in the colon. Through the colonoscope, the colon was distended with air and debris was flushed from the imaging site with saline. Colonoscopy images were obtained to visualize 4 quadrants at each of the depths into the rectum and colon, including 0 to 10, 20, and 30 cm. Colonoscopy findings were categorized based on erythema, petechiae, and mucosal disruption.
The OCT images were obtained using a time-domain system centered at 1300 nm (Niris OCT system; Imalux, Cleveland, OH) with an imaging probe 2.5 m in length and 2.75 mm in diameter. Optical coherence tomography images at 2 and 5 cm proximal to the anus (inside the rectum) were obtained directly (without the colonoscope) in 4 quadrants at both 2 and 5 cm. The OCT probe was then passed through the colonoscope to obtain images at 10, 20, and 30 cm in 4 quadrants. Imaging progressed from the anus into the colon and for each set of imaging sites corresponding to depths of 2, 5, 10, 20, and 30 cm; OCT images were obtained in 4 quadrants. Optical coherence tomography images were graded by using a previously reported scoring system,18,19 in which category 1 images were normal, category 2 images were partially abnormal with minimal disruption of the mucosal layer, and category 3 images were abnormal with disruption of the mucosal layer across the entire image.
Biopsies were processed and stained with hemotoxylin and eosin. A histology grading system was developed based on the spectrum of pathological observations, and all slides were graded according to the extent of pathological changes (0, absent; 1, mild; 2, moderate; 3, severe). Pathological features used in grading criteria included the following: inflammation of the mucosa and/or submucosa (0–3 based on extent), edema of the lamina propria (0 or 1), necrosis of mucosal crypts or epithelial disruption (0 or 1), epithelial detachment and/or blistering (0 or 1), microabscesses (0 or 1), and hemorrhage (0 or 1). The overall grade was obtained by adding the individual observations scored and ranged from zero (0) to a maximum of 12. Each section was graded based on 5 high-power fields (×10 objective/0.85) per section (4 sections), for a total of 20 measurements per slide. Consistency of duplicate sections was checked on all slides.
Data analysis for the comparison between the baseline, PBS and BZK groups, for the OCT scores data and pathological finding scores was performed using Mann-Whitney nonparametric U test for independent samples. A P value less than 0.05 for a 2-tailed test was considered statistically significant.
Baseline colonoscopy findings were similar to that seen in humans with rugated, smooth pink mucosa through which vascular patterns were visualized.20 Figure 1 shows representative images of the distal colon/rectum before and after treatment with PBS (Fig. 1A, B) or BZK (Fig. 1C, D). Figure 1A shows a representative image of the rectum at 10 cm at baseline, and Figure 1B represents the same site after PBS treatment. Figure 1C is a representative image at 10 cm at baseline, and Figure 1D represents a sheep treated with BZK at the same site after treatment. At baseline, findings of erythema or petechiae were seen in 3 of the 8 sheep. After treatment, erythema was seen in both treatment groups. All BZK-posttreated animals had superficial mucosal disruption, which was primarily noted in the distal colon.
Optical Coherence Tomography
The flexible OCT probe was easily maneuvered through the channel of the colonoscope, allowing images to be taken under direct visualization as seen in Figure 2. Optical coherence tomography images revealed a layered structure (Fig. 3A, B), with mucosa (long arrow), and underlying lamina propria and muscularis in baseline (Fig. 3A) and post-PBS (Fig. 3B) images. The thin superficial columnar epithelium is also visible in Figure 3A and B (short arrow). After treatment with BZK, this layered structure was disrupted, as represented in Figure 3C and denoted by an asterisk (Fig. 3C) showing an area lacking the superficial layer of the mucosa. In addition to the loss of the layered structure, the mucosa and submucosa exhibited a hyporeflective granular appearance, which has been reported to be associated with inflammatory infiltrates.20 A previously established scoring system was used to evaluate the pretreatment and posttreatment images, with higher scores indicating a higher degree of mucosal disruption.18,19 There was no difference in scores between the PBS and BZK groups at baseline (Fig. 4); however, there were significant differences between the baseline and posttreatment scores in the BZK group (P < 0.001) as well as between scores in the post-BZK and post-PBS groups (P < 0.016), with the images from the most distal sites showing a higher degree of toxicity in the BZK treatment group (Fig. 4).
The microanatomy of the sheep rectum is similar to that found in humans. Histology revealed the expected multilayer structure with a mucosa composed of a single layer of columnar epithelium lining the crypts. The submucosa below the single layer of columnar epithelium consisted of a lamina propria with an underlying muscularis. Histology also revealed that the epithelium remained intact after treatment with PBS (Fig. 5A) but after treatment with 0.2% BZK, epithelial and crypt disruption and inflammatory changes were present (Fig. 5B). Histology scores were greater in the BZK-treated group, indicating more inflammation and injury than after treatment with PBS (Table 1). The cumulative pathological scores based on features were significantly different (P < 0.001) between the PBS and BZK groups for the entire rectum at various depths. When the scores were analyzed according to each pathological feature individually, only inflammation and necrosis were statistically significant (P < 0.01) when comparing BZK and PBS treatment groups. When data were analyzed according to distal (2, 5 and 10 cm) or proximal (20 and 30 cm) depth sites, the distal rectum showed statistically significant differences in the pathological features scores (P < 0.001).
Owing to increased risk of acquisition of HIV through rectal compared with vaginal intercourse, safety of microbicides used rectally is a critical part of microbicide development. Mucosal barrier assessment is essential for evaluating the potential for increased risk of HIV and other STI infection. The degree and nature of damage needed to increase susceptibility to infection in the rectum is not fully known but is likely related to the disruption of the epithelial and mucosal barrier and recruitment of inflammatory components targeted by HIV and other STIs. Recent studies have shown that products considered safe in the vagina may be toxic to the rectum21; therefore, preclinical evaluation of chemical agent effects on rectal epithelium is important to the development of rectal microbicides.
In the current study, colonoscopy and OCT with high depth resolution showed that a single 30-minute dose of BZK caused disruption of the mucosal epithelial barrier and inflammation, factors that could lead to increased susceptibility to sexually transmitted infections. In addition, this study introduces a methodology for noninvasive evaluation of human rectal product safety in the sheep model, a large animal model with anatomical and histological similarity to human rectum.
Anatomical and physiological similarities between humans and sheep include the rectum comprising single columnar epithelial cell layer that covers the surface of the mucosa and crypts. Beneath the columnar layer lie the lamina propria and submucosa and then the muscularis. Using endoscopic ultrasound, Huh et al.22 estimated the mean of the human rectal mucosa thickness to be 830 ± 60 μm ranging from 660 to 1130 μm and an average of 495 µm ranging from 260 to 730 μm via histology.23 Similar to findings in the sheep vagina, the sheep colonic mucosa was thinner than humans, measuring 350 ± 20 µm by histology.24 The sheep rectum and colon also have several lymphoid follicles within the lamina propria closely resembling Peyer patches, similar to that seen in human.25 A significant difference noted is that in the sheep rectum the anal verge with stratified squamous epithelium is approximately 0.5 cm in length, whereas the human anal verge measures 2.5 to 4 cm in length.
Furthermore, sheep have been used as animal models for the study of mucosal and innate immunity, vaccine development and delivery, asthma and stem-cell transplantation models, and transgenic and nuclear transfer where cloned lambs were created and used in functional genomics experiments, indicating that immunological responses relevant to the human may be found in the sheep.26 Given the striking similarities, the ovine model is relevant and highly suitable for future in vivo rectal microbicide safety, efficacy, and long-term cumulative exposure studies.
Using high-resolution imaging in a suitable large animal model could provide a means for in vivo testing with improved sensitivity over traditional methods. We have previously shown that white light magnification with colposcopy was less sensitive than OCT imaging in the sheep and human vagina and that the addition of high-resolution methods improved ability to determine subtle toxicity of drugs.18,27 In this current study, we found that distal colonoscopy readily showed acute rectal injury after a single treatment with product. Specifically, sloughing, petechiae, erythema, and bleeding were noted after treatment with BZK, especially in the distal rectum. The mucosal rectal injury observed after application of a single dose of BZK is significantly higher compared with the response in the vagina.18
In the current study, OCT imaging was used to visualize and grade the morphology of the mucosal layer with posttreatment changes quantified by the OCT scoring system. In contrast to colonoscopy, as shown in Figure 3, the depth-resolved OCT images allow for visualization of the temporal response in the deeper layers, such as accumulation of infiltrating cells, thus providing another unique dimension for the diagnostic power of OCT. The hyporeflective, granular layer seen in Figure 3C was typical in images after treatment with BZK and is suggestive of injury to the crypt structure as well as inflammatory infiltrates.20 The current scoring system, initially developed to quantify abnormalities in the superficial vaginal layer,19 also can allow for quantification of change in the rectal mucosal layer caused by inflammatory infiltrates, as it disrupts the normal architecture of this layer.
As discussed above, it is not clear what degree of epithelial or mucosal injury is required to increase susceptibility to pathogens; therefore, being able to delineate the epithelial layer would provide an enabling and sensitive tool for assessing subtle changes in epithelial integrity. The superficial columnar epithelial layer, measuring approximately 20 to 30 µm, was visible in OCT images, suggesting that OCT could be a powerful tool to detect disruption of this thin epithelial barrier. However, this layer is not consistently seen in all baseline images, likely because this thickness is at the level of resolution of our current OCT system (15–20 µm). Improvements in spatial resolution of OCT imaging or the addition of higher-resolution in vivo cellular imaging techniques, such as confocal microendoscopy,28 have the potential to provide a highly sensitive noninvasive tool for quantitative assessment of the integrity of columnar epithelial cell layer and mucosal barrier beyond what can be accomplished using currently available endoscopic-based OCT systems.
Histopathological examination confirmed epithelial injury in the specimens collected after treatment with BZK; namely, disruption of the epithelium and crypt structure was observed, accompanied by inflammatory infiltrates extending to the submucosa. No changes were observed in the specimens collected after treatment with PBS, and a statistically significant difference was observed when comparing scores between the 2 groups.
The combination of these imaging modalities could easily be translated into rectal product toxicity clinical studies or for the evaluation of colorectal disorders. For high-risk patients with multiple comorbidities or in which repetitive biopsies may be harmful, these techniques can provide a noninvasive alternative that is safer than biopsy for tissue evaluation. These imaging techniques could also be used to follow evaluation and progression of anal intraepithelial neoplasia. Further expansion of our model could ultimately be useful in the evaluation of malignant cell transformation and in posttreatment follow-up for patients with cancer who have undergone pelvic radiation therapy and are at risk for radiation proctitis. Ultimately, our model and combined imaging techniques offer methods for long-term evaluation of the rectum in an era where preventative medicine is flourishing.
The methods introduced in this study could provide insight into the degree and time course of damage that results from product use and, coupled with animal models for susceptibility testing, could lead to insight about the link between mucosal disruption in the rectum and risk of infection. It is well known that the detergent and antimicrobial BZK causes tissue injury; therefore, less toxic products can be evaluated in the future to determine the limits of sensitivity of this rectal product safety model. In addition, the 30-minute dosing time schedule in this study only showed acute toxicity. Repeated dosing and imaging allowed by this model could be used to determine long-term effects on the rectal mucosa. Furthermore, in contrast to live imaging with colonoscopy, which provides qualitative assessment of the integrity of the surface of the mucosal barrier, OCT imaging would allow for acquiring large numbers of depth-resolved in vivo “optical” biopsies that can be archived and subsequently graded by a single reader for quantification of injury.
In summary, we have shown that the sheep is a suitable large animal model for testing toxicity of rectal products. With colonoscopy and OCT imaging, morphological abnormalities can be visualized at high resolution. Optical coherence tomography imaging allowed for quantitative scoring of the extent of microbicide-induced injury in colorectal tissue, suggesting that this may be a sensitive tool for noninvasive optical biopsy of colorectal tissue. Based on the current and prior studies, we have shown that the sheep is a suitable model for preclinical assessment of microbicide safety in both the vagina and the rectum.
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