The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Mouse urinary behavior is quantifiable and is used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent, and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying nonconcentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.

Laboratory mice are used to identify causes of urinary dysfunction including prostate-related mechanisms of lower urinary tract symptoms. Effective use of mice for this purpose requires a clear understanding of molecular, cellular, anatomic, and endocrine contributions to voiding function. Whether the prostate influences baseline voiding function has not been specifically evaluated, in part because most methods that alter prostate mass also change circulating testosterone concentrations. We performed void spot assay and cystometry to establish a multiparameter “baseline” of voiding function in intact male and female 9-wk-old (adult) C57BL/6J mice. We then compared voiding function in intact male mice to that of castrated male mice, male (and female) mice treated with the steroid 5α-reductase inhibitor finasteride, or male mice harboring alleles ( Pbsn4cre/+; R26RDta/+) that significantly reduce prostate lobe mass by depleting prostatic luminal epithelial cells. We evaluated aging-related changes in male urinary voiding. We also treated intact male, castrate male, and female mice with exogenous testosterone to determine the influence of androgen on voiding function. The three methods used to reduce prostate mass (castration, finasteride, and Pbsn4cre/+; R26RDta/+) changed voiding function from baseline but in a nonuniform manner. Castration feminized some aspects of male urinary physiology (making them more like intact female mice) while exogenous testosterone masculinized some aspects of female urinary physiology (making them more like intact male mice). Our results provide evidence that circulating testosterone is responsible in part for baseline sex differences in C57BL/6J mouse voiding function while prostate lobe mass in young, healthy adult mice has a lesser influence.

Metastatic estrogen receptor alpha positive (ERα+) cancers account for most breast cancer mortality. Cancer stem cells (CSCs) and dense/stiff extracellular matrices are implicated in aggression and therapy resistance. We examined this interplay and response to mTOR inhibition using ERα+ adenocarcinomas from NRL-PRL females in combination with Col1a1 (mCol1a1) mice, which accumulate collagen-I around growing tumors. Orthotopic transplantation of tumor cells to mCol1a1 but not wildtype hosts resulted in striking desmoplasia. Mammary tumors in mCol1a1 recipients displayed higher CSC activity and enhanced AKT-mTOR and YAP activation, and these animals developed more and larger lung metastases. Treatment with the mTOR inhibitor, rapamycin, with or without the anti-estrogen, ICI182780, rapidly diminished mammary tumors, which rapidly reversed when treatment ceased. In contrast, lung metastases, which exhibited lower proliferation and pS6RP, indicating lower mTOR activity, were unresponsive, and mCol1a1 hosts continued to sustain greater metastatic burdens. These findings shed light on the influence of desmoplastic tumor microenvironments on the CSC niche and metastatic behavior in ERα+ breast cancer. The differential mTOR dependence of local mammary tumors and pulmonary lesions has implications for success of mTOR inhibitors in advanced ERα+ disease.

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available.

Collagen architecture in dog appears similar to that in humans when cross sections are compared side-by-side. Canine collagen organization is affected by both age and androgen status, suggesting these factors may contribute to collagen accumulation in some males.

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation utilizing transurethral instillations of E. coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributes exclusively to bladder, 100 and 200 µL volumes distributes to bladder and prostate, and a 500 µL volume distributes to bladder, prostate and ureter. A threshold optical density (OD) of 0.4 E. coli UTI89 in the instillation fluid is necessary for significant (p < 0.05) prostate colonization. E. coli UTI89 infection results in a low frequency, high volume spontaneous voiding pattern. This phenotype is due to exposure to E. coli UTI89, not catheterization alone, and is minimally altered by a 50 µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation is isolated to the dorsal prostate and is accompanied by increased collagen density. This is partnered with increased density of PTPRC+, ProCOL1A1+ co-positive cells and decreased density of ACTA2+, ProCOL1A1+ co-positive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

A subset of patients receiving radiation therapy for pelvic cancer develop radiation cystitis, a complication characterized by mucosal cell death, inflammation, hematuria, and bladder fibrosis. Radiation cystitis can reduce bladder capacity, cause incontinence, and impair voiding function so severely that patients require surgical intervention. Factors influencing onset and severity of radiation cystitis are not fully known. We tested the hypothesis that genetic background is a contributing factor. We irradiated bladders of female C57BL/6, C3H, and BALB/c mice and evaluated urinary voiding function, bladder shape, histology, collagen composition, and distribution of collagen‐producing cells. We found that the genetic background profoundly affects the severity of radiation‐induced bladder fibrosis and urinary voiding dysfunction. C57BL/6 mice are most susceptible and C3H mice are most resistant. Irradiated C57BL/6 mouse bladders are misshapen and express more abundant collagen I and III proteins than irradiated C3H and BALB/c bladders. We localized Col1a1 and Col3a1 mRNAs to FSP1‐negative stromal cells in the bladder lamina propria and detrusor. The number of collagen I and collagen III‐producing cells can predict the average voided volume of a mouse. Collectively, we show that genetic factors confer sensitivity to radiation cystitis, establish C57BL/6 mice as a sensitive preclinical model, and identify a potential role for FSP1‐negative stromal cells in radiation‐induced bladder fibrosis.