High resolution imaging of epithelial injury in the sheep cervicovaginal tract: a promising model for testing safety of candidate microbicides

Abstract

Background: Access to readily available large animal models and sensitive noninvasive techniques that can be used for the evaluation of microbicide-induced changes in tissue could significantly facilitate preclinical evaluations of microbicide safety. The sheep cervicovaginal tract, with stratified squamous epithelium similar to humans, holds promise as a large animal model used before nonhuman primates. In addition, optical coherence tomography (OCT) could enable high resolution visualization of tissue morphology and noninvasive assessment of microbicide-induced epithelial injury.

Methods: We evaluated the dose response of sheep cervicovaginal tract to benzalkonium chloride (BZK). Twenty sheep received treatment with phosphate-buffered saline or BZK solution (2%, 0.2%, or 0.02%). Pre- and posttreatment colposcopy and OCT images were collected and graded based on World Health Organization criteria and a previously reported scoring system, respectively. Biopsies were collected and the degree of epithelial injury and its thickness was assessed based on histology and OCT.

Results: The sheep cervicovagina exhibited anatomic and microscopic features similar to the human. Extensive loss of the epithelium was noted on colposcopy and OCT after application of 2% BZK. Colposcopy detected findings in half of sheep and OCT in all sheep treated with 0.2% BZK. OCT detected differences in the 0.02% BZK-treated group compared with controls, whereas colposcopy failed to detect any changes.

Conclusions: The sheep cervicovagina is similar to humans, and exhibits dose dependent epithelial changes after BZK treatment. These findings suggest that the sheep model and OCT may become valuable tools for the safety evaluation of candidate microbicides, and warrant continued development.

Figure 1

Colposcopy findings in sheep cervix and vagina at baseline and twenty-four hours after a single intravaginal treatment with PBS or 0.02%, 0.2%, or 2% BZK. At baseline, erythema, petechiae, or no findings were detected (a-d). Twenty-four hours after treatment, the PBS (e) and 0.02% treated sheep (f) were similar to baseline. Superficial disruption (peeling) (arrow) is seen after treatment with 0.2% BZK (g). The sheep treated with 2% BZK had global blanching and thickening as well as peeling (arrow) (h).

Figure 2

Representative OCT images of the vagina with corresponding histology. OCT images at baseline and at 24 hours after a single dose treatment with PBS, 0.02% BZK, or 0.2% BZK are shown with the corresponding H&E stained image from biopsy 24 hours after treatment. At baseline, all OCT images are similar, showing the layered structure of an intact epithelium with underlying submucosa (a, b, c). After treatment, the OCT image in the PBS-treated sheep was comparable to baseline (d). The corresponding biopsy confirmed intact epithelium (g). After treatment, with 0.02% BZK treatment the OCT image is also comparable to baseline (e). The corresponding biopsy reveals intact epithelium with evidence of subepithelial inflammation (h). After treatment with 0.2% BZK there was disrupted epithelium, detected both by OCT (f) and histology (i). (OCT image scale is in millimeters. H&E stain 40X magnification. Arrows denote boundary of epithelium and submucosa. ep, epithelium; s, submucosa; de, epithelial disruption).

Figure 3

Representative OCT images of the cervix with corresponding histology. OCT images are shown with corresponding histology from biopsies 24 hours after a single dose treatment with PBS or 0.02% BZK. OCT images and histology images after PBS treatment show intact epithelium (a, c). In contrast, OCT and histology images after 0.02% BZK treatment show epithelium with areas of disruption (b, d). (OCT image scale is in millimeters. H&E stain 40X magnification. Arrows denote boundary of epithelium and submucosa. ep, epithelium; s, submucosa; de, epithelial disruption).

Figure 4

OCT scores in the cervix at baseline and twenty-four hours after a single treatment with PBS (n=8) or 2% (n=2), 0.2% (n=5), or 0.02% (n=5) BZK. At baseline, scores were similar (p=.47). In the sheep treated with 2% and 0.2% BZK, scores were significantly higher after treatment than scores in the PBS-treated sheep (p<0.0001). In the sheep treated with 0.02% BZK, the scores at the cervix were significantly higher than that of PBS (p=0.0004). (Error bars indicate 95% confidence intervals. Asterisks indicate statistical significance.)

Figure 5

OCT scores at baseline and twenty-four hours in the vagina after a single treatment with PBS (n=8) or 2% (n=2), 0.2% (n=5), or 0.02% (n=5) BZK. Baseline scores were similar (p=.25). In the sheep treated with 2% and 0.2% BZK, scores were significantly higher after treatment than scores in the PBS-treated sheep (p<0.0001). In the sheep treated with 0.02% BZK, there were no significant differences in the vagina when compared to PBS treated sheep (p=.96). (Error bars indicate 95% confidence intervals. Asterisks indicate statistical significance.)