Mouse model of cervicovaginal toxicity and inflammation for preclinical evaluation of topical vaginal microbicides

Abstract

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.

FIG. 1.

Human endocervical cells are more sensitive to N-9 exposure than human vaginal keratinocytes. Vaginal (Vk2/E6E7, vaginal keratinocytes) and cervical (End1/E6E7, endocervical keratinocytes) cell lines were exposed to N-9 for 10 min (A), 2 h (B), 4 h (C), or 8 h (D). Cells were assayed for viability immediately after N-9 exposure by using an MTS assay. The results are expressed relative to that of mock-exposed cells (viability index). Each graph illustrates the average of at least two independent experiments in which each concentration was examined at least in triplicate for each experiment. Error bars indicate the standard deviations of the calculated mean values.

FIG. 2.

Application of N-9 results in little or no change in mouse vaginal epithelial integrity and immune cell recruitment. Swiss Webster mice were inoculated with 60 μl of 1% N-9 intravaginally. At 10 min, 2 h, 4 h, and 8 h postapplication, mice were sacrificed, and the genital tracts were harvested. Tissue sections were stained with H&E for morphological analyses and anti-CD45 for IHC analyses. Tissue sections for IHC analyses were counterstained with hematoxylin. Two independent experiments were performed with a total of at least five mice at each time point. Since N-9-treated tissues were similar to control tissues in all cases, only representative water- and N-9-treated tissue sections from 2 h postapplication are presented. Arrows indicate examples of CD45-positive cell staining; the inset picture shows higher magnification of the region outlined by box.

FIG. 3.

N-9 causes severe mouse cervical epithelial disruption and immune cell infiltration at 2 h postexposure. Swiss Webster mice were inoculated with 60 μl of water only or 1% N-9 intravaginally. At 10 min and 2 h postapplication, mice were sacrificed, and the genital tracts were harvested. Tissue sections were stained with H&E for morphological analyses and anti-CD45 for IHC analyses. Tissue sections for IHC analyses were counterstained with hematoxylin. Two independent experiments were performed with a total of at least five mice at each time point. Representative tissue sections are presented. Arrowheads indicate regions of epithelial disruption, and arrows indicate regions of intense CD45-positive cell staining. The inset picture shows a higher magnification of CD45-positive cell aggregates surrounding a lymphatic vessel in control tissues. The blue arrowhead indicates an area epithelial disruption was not accompanied by CD45-positive cell infiltration. The red arrows indicate CD45-positive cell infiltration in the absence of epithelial disruption.

FIG. 4.

Mouse cervical epithelial tissues, which are severely disrupted by N-9 at 4 and 8 h postapplication, have undergone regeneration by 24 h postapplication. Swiss Webster mice were inoculated with 60 μl of 1% N-9 intravaginally. At 4, 8, and 24 h postapplication, mice were sacrificed, and the genital tracts were harvested. Tissue sections were stained with H&E for morphological analyses and anti-CD45 for IHC analyses. Two independent experiments were performed with at least two mice at each time point. Representative tissue sections are presented. Arrowheads indicate regions of epithelial disruption; arrows indicate regions of intense CD45-positive cell staining.

FIG. 5.

Intravaginal inoculation of Conceptrol (4% N-9) also results in mouse cervical epithelial disruption and immune cell infiltration. Swiss Webster mice were inoculated with 60 μl of Conceptrol intravaginally. At 10 min and 2, 4, and 8 h postapplication, mice were sacrificed, and the genital tracts were harvested. Tissue sections were stained with H&E for morphological analyses and anti-CD45 for IHC analyses. Tissue sections for IHC analyses were counterstained with hematoxylin. Two independent experiments were performed with a total of at least five mice at each time point. A representative tissue section is presented. Arrowheads indicate regions of epithelial disruption; arrows highlight regions of intense CD45-positive cell staining.

FIG. 6.

Endocervical cells are much less sensitive to PEHMB compared to N-9 after a 2-h exposure. End1 cells were exposed to N-9 or PEHMB for 2 h and then assayed for cellular viability by using an MTS assay. The results are expressed relative to that of mock-exposed cells (viability index). Each graph illustrates the average of two independent experiments in which each concentration was examined at least in triplicate. Error bars indicate the standard deviations of the calculated mean values.

FIG. 7.

Intravaginal inoculation of 1% PEHMB causes minimal mouse cervical epithelial disruption and immune cell infiltration. Swiss Webster mice were inoculated with 60 μl of 1% PEHMB intravaginally. At 10 min and 2, 4, 8, and 24 h postapplication, mice were sacrificed, and the genital tracts were harvested. Tissue sections were stained with H&E for morphological analyses and anti-CD45 for IHC analyses. Tissue sections for IHC analyses were counterstained with hematoxylin. Two independent experiments were performed with a total of at least five mice at each time point. Minimal epithelial disruption and inflammation of the cervical mucosa was observed after the application of 1% PEHMB at all time points evaluated. A representative tissue section from 2 h postapplication is presented. Control (water-treated) and N-9-treated tissue sections from 2 h postapplication are presented for comparison.