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Single-Cell Atlas of a Rat Model of Vaginal Birth Injury induced Stress Urinary Incontinence

ABSTRACT: Stress urinary incontinence (SUI) greatly affects the daily life of numerous women and is closely related to a history of vaginal delivery and aging. We used vaginal balloon dilation to simulate vaginal birth injury in young and middle-aged rats to produce a SUI animal model, and found that young rats restored urethral structure and function well, but not the middle-aged rats. To identify the characteristics of cellular and molecular changes in the urethral microenvironment during the repair process of SUI. We profiled 51,690 individual female rat urethra cells from 24 and 48 weeks old, with or without simulated vaginal birth injury.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE260780 | GEO | 2024/03/31

REPOSITORIES: GEO

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Project description:Periurethral human mesenchymal stem cell (hMSC) injections are associated with functional improvement in animal models of post-partum stress urinary incontinence (SUI). However, limited data exists on the role of hMSCs in modulating gene expression in tissue repair after urethral injury. We quantified temporal gene expression modulation in hMSCs, and injured rat urethral tissue, using RNA seq in an animal model of SUI, over a 3-day time period following urethral injury, and local hMSC therapy. We injected PKH fluorescent-labeled human hMSC into the periurethral space of rats, following a 4h vaginal distention (3 rats per time point). Control rats underwent vaginal distention injury only, and were sacrificed at 12h, 24h, 36h, 72h post injury. Rat urethral and vaginal tissues were frozen and sectioned. Fluorescent labeled hMSCs were distinguished from adjacent, unlabeled rat urethral tissue. RNA was prepared from urethral tissue obtained by laser dissection of frozen tissue sections and sequenced on an Illumina HiSeq 2500. Differentially expressed genes (DEGs) over 72h were evaluated using a 2-group t-test (p<0.05). Our transcriptional analyses identified candidate genes involved in tissue injury, that were broadly sorted by injury and exposure to hMSC, throughout the first 72h of acute phase of injury. DEGs in treated urethra, compared with untreated urethra, were functionally associated with tissue repair, angiogenesis, neurogenesis, and oxidative stress suppression. DEGs included a variety of cytokines, extracellular matrix stabilization and regeneration genes, cytokine signaling modification, cell cycle regulation, muscle differentiation and stabilization. Moreover, our results revealed DEGs changes in the hMSCs (PKH-labelled), which were harvested from injured urethra. The expressions are related to DNA damage repair, transcription activation, stem cell regulation, cell survival, apoptosis, self-renewal, cell proliferation, migration and injury response.

Project description:The rostral ventrolateral medulla (RVLM), a part of the medullary reticular formation, plays a major role in several physiological responses, including cardiovascular and sympathetic nervous system functions. Although aging causes disturbances in the responses of these physiological systems, RVLM involvement in these age-related changes is not clear. Previous work using high-throughput gene expression analysis of the RVLM in aged animals suggested that chemical neurotransmission-related genes might be downregulated with advancing age. Since the RVLM function involves a balance of inhibitory and excitatory inputs, which is largely mediated by gamma-aminobutyric acid (GABA) and excitatory amino acid (EAA) neurotransmission, we hypothesized that aging is associated with altered excitatory and/or inhibitory neurotransmission-related gene expression in the RVLM. To test this hypothesis, we micropunched an RVLM-containing area from young (3–5 months), middle-aged (12–14 months), and aged (22–26 months) Fischer 344 male rats. RNA purified from these micropunches was analyzed using GABA and Glutamate RT2 Profiler PCR arrays (n= 8–10). In addition, the expression of selected genes was validated at the RNA level using TaqMan® based- qPCR and at the protein level using western blotting. All the genes that displayed significant differential expression (1.5-fold, p < .05, FDR < .05) were identified to be downregulated in the RVLM of aged and middle-aged rats compared to young rats. Among the downregulated genes, the percentage of glutamate neurotransmission-related genes was higher than GABA neurotransmission-related genes. Solute carrier family 1 member 6 (Slc1a6) gene showed the highest fold downregulation at the RNA level in the RVLM of aged compared to young rats, and its protein product, Excitatory amino acid transporter 4 (EAAT4), showed a downregulatory trend in the RVLM of aged and middle-aged rats. These results suggest that molecular constituents of both GABA and glutamate neurotransmission might be altered in the RVLM of aged and middle-aged rats, and the changes in glutamate neurotransmission might be more prominent. Investigating age-associated anatomical and functional changes in RVLM GABA and glutamate neurotransmission might provide a foundation for understanding the effects of aging on physiological function.

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Single-Cell Atlas of a Rat Model of Vaginal Birth Injury induced Stress Urinary Incontinence

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