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This neuroanatomical study in four, adult, Sprague-Dawley female rats quantified the number of Urothelial (labeled by intravesical DiI dye administration) and Non-Urothelial (labeled by intraparenchymal injection of Fast blue dye) bladder primary afferent neurons (bPANs) located in the T13, L1, L6 and S1 dorsal root ganglia. Additional immunohistochemical labeling using antibodies to detect either Substance P or CGRP further characterized the bPAN samples as peptidergic or non-peptidergic. Cell counts indicated that Urothelial bPANs were more common at the L6/S1 levels and more likely to be identified as peptidergic when compared with bPANs characterized at T13/L1 levels and with Non-Urothelial bPANs. These studies provide additional evidence that at least two distinct neuronal populations, with differing localization of sensory terminals, differing peptide content, and differing projections to the central nervous system, are responsible for bladder sensation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Bladder pain and hypersensitivity to bladder filling are clinically common, but animal models examining syndromes with these features are limited. A rat model of bladder hypersensitivity produced by neonatal bladder inflammation (NBI) has been reported to have many of the clinical features of bladder pain syndromes. The present study sought to determine whether similar hypersensitivity might be induced by NBI in mice. Female C57BL6/J mice had NBI induced on postnatal days P12-14 by the intravesical administration of zymosan. As adults (12-14 weeks of age), the mice were examined for hypersensitivity of their bladders as: spontaneous voiding and evoked cystometrograms at baseline, and visceromotor responses (VMRs) to urinary bladder distension (UBD) following a secondary insult (either repeated bladder inflammation or acute stress induced by footshock). Mice that experienced NBI demonstrated hypersensitivity, when compared with control mice, manifested as increased spontaneous voiding, increased frequency of evoked voids during intravesical saline infusion, and increased vigor of VMRs to UBD following either acute bladder inflammation or acute stress. This recapitulates the hallmark features of clinical painful bladder disorders and suggest utility of this murine model for the study of these disorders while allowing methodological expansion into well-established genetic and immunological models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clodfelder-Miller, Ness and DeBerry.)

This report describes methodological and exploratory investigations of the zymosan-induced neonatal bladder inflammation (NBI) model of interstitial cystitis/bladder pain syndrome (IC/BPS) in female rats. These results validate and extend the currently employed model by evaluating critical timepoints for obtaining treatment effects and identified that a second insult as an adult including repeat intravesical zymosan, intravesical lipopolysaccharide, acute footshock stress, neuropathic nociception (facial) or somatic inflammation (hindpaw) all resulted in magnified visceromotor responses to urinary bladder distension (UBD) in rats which had experienced NBI when compared with their controls. NBI also resulted in increased tone and reactivity of pelvic floor musculature to UBD, as well as increased responsiveness to intravesical potassium chloride solutions, abnormal anxiety measures (elevated plus maze) and an increased number of submucosal petechial hemorrhages following 30 min of hydrodistension of the bladder. These phenotypic findings have correlates to the clinical features of IC/BPS in humans and so support use of this model system to examine mechanisms of and treatments for IC/BPS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ness, DeWitte, DeBerry, Hart, Clodfelder-Miller, Gu, Ling and Randich.)

Aims: Neuromodulation (nerve stimulation) can produce analgesia. One form, bilateral pudendal nerve stimulation (bPNS), suppresses responses to urinary bladder distension (UBD) in hypersensitive rats. Drugs can modify this effect (eg, benzodiazepines, but not opioids, suppress bPNS effects). Prior to a clinical trial of bPNS effects on bladder pain, we felt it was prudent to survey the effects of medications commonly used in patients with bladder disorders., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Antimuscarinic (oxybutynin), β 3 -adrenoceptor agonist (mirabegron, CL316243), α 1 -adrenoceptor antagonist (tamsulosin), antidepressant (amitriptyline), muscle relaxing (baclofen), and sedative (propofol) agents were administered and effects of bPNS on responses to UBD assessed. bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses) were used as nociceptive endpoints., Results: Many of these drugs directly inhibited the VMRs to UBD, but only mirabegron, at the doses employed, significantly reduced inhibitory effects of bPNS. In the presence of the other drugs, bPNS continued to produce statistically significant inhibition of VMRs to UBD., Conclusions: This study suggests that concurrent therapy with drugs used to treat bladder disorders could affect assessment of the effects of bPNS on bladder hypersensitivity. This study gives guidance to clinical trials using bPNS for the treatment of painful bladder syndromes and suggests potential clinical use of some of these medications in the treatment of these same disorders., (© 2020 Wiley Periodicals, Inc.)

Background: Neuromodulation, as a therapeutic modality for pain treatment, is an alternative to opioid therapies and therefore receiving increased interest and use. Neuromodulation at a peripheral nerve target, in the form of bilateral electrical pudendal nerve stimulation (bPNS), has been shown to reduce bladder hypersensitivity in rats and anecdotally reduces pain in humans with pelvic pain of urological origin. Recent studies have identified a role for spinal γ-aminobutyric acid (GABA) receptors in this effect. Concomitant medication use, such as benzodiazepines, could alter responses to neuromodulation, and so before the development of a clinical trial to confirm translation of this potential therapy, the potential interactions between acute and chronic use of benzodiazepines and bPNS were examined in a preclinical model., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Diazepam (1-5 mg/kg intraperitoneal [i.p.]) or vehicle was administered acutely (with or without bPNS) and chronically (5 mg/kg subcutaneous [s.c.] daily for 2 weeks before the final experiment). bPNS was delivered as bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses to urinary bladder distension [UBD]) were used as nociceptive end points. Due to the profound effects of diazepam, the effect of midazolam (0.5-1.0 mg/kg i.p.) on VMRs and bPNS effects was also studied., Results: Diazepam and midazolam both produced a dose-dependent, flumazenil-reversible inhibition of VMRs to UBD. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that had received vehicle injections. Select doses of diazepam and midazolam suppressed the inhibitory effect of bPNS on VMRs., Conclusions: This study suggests that inhibitory effects of bPNS on bladder pain could be suppressed in subjects receiving benzodiazepine therapy, suggesting that potential clinical testing of pudendal nerve stimulation for the treatment of painful bladder syndromes may be confounded by the use of benzodiazepines. Clinical assessment of other forms of neuromodulation should also be screened for impacts of benzodiazepines.

Background and Objectives: Bilateral electrical pudendal nerve stimulation (bPNS) reduces bladder hypersensitivity in rat models and anecdotally reduces pain in humans with pelvic pain of urologic origin. Concomitant opioids are known to alter responses to neuromodulation in some systems. So prior to the development of a clinical trial for purposes of regulatory approval, the preclinical interaction between opioids and stimulation effectiveness was examined., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Morphine was administered acutely (1-4 mg/kg intraperitoneal) or chronically (5 mg/kg subcutaneously daily for 2 weeks prior to the terminal experiment). bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMR; abdominal muscle contractile responses to urinary bladder distension (UBD)) were used as nociceptive endpoints., Results: Morphine produced a dose-dependent inhibition of VMRs to UBD that was naloxone reversible. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that had received acute or chronic subcutaneous morphine injections., Conclusions: This study suggests that inhibitory effects of bPNS can still be evoked in subjects who are receiving opioid therapy, thus giving guidance to potential clinical trials seeking regulatory approval for the treatment of chronic bladder pain., Competing Interests: Competing interests: XS and DEN were employees of Medtronic which funded this study., (© American Society of Regional Anesthesia & Pain Medicine 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Bilateral electrical pudendal nerve stimulation (bPNS) reduces bladder hypersensitivity in rat models of bladder pain and anecdotally reduces pain in humans with pelvic pain of urologic origin. The spinal neurochemical mechanisms of this antinociception are unknown. In the present study, bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Visceromotor responses (VMRs; abdominal muscle contractions) to urinary bladder distension (UBD) were used as a nociceptive endpoint under urethane-isoflurane anesthesia. bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Following determination of the inhibitory effect of bPNS on VMRs, pharmacological antagonists were administered via an intrathecal catheter onto the lumbosacral spinal cord and bPNS effects on VMRs redetermined. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that was statistically reduced by the intrathecal administration of methysergide, WAY100636, CGP35348 and strychnine but was unaffected by naloxone, bicuculline, phentolamine, ondansetron and normal saline. This study suggests that inhibitory effects of bPNS may include serotonergic, GABA-B-ergic and glycinergic mechanisms suggesting the potential for interaction of the neuromodulatory effect with concommitant drug therapies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low- and high-grade squamous intraepithelial lesion (LGSIL and HGSIL), and adjacent "normal" cervical epithelium from six formalin-fixed paraffin-embedded samples were selected. Tissues from these 18 samples were isolated using laser-capture microdissection, RNA was extracted and sequenced. RNA-sequencing generated 2.4 billion raw reads in 18 samples, of which ~50.1% mapped to known and annotated genes in the human genome. There were 40 genes up-regulated and 3 down-regulated (normal to LGSIL) in at least one-third of the sample pairs (same direction and FDR p < 0.05) including S100A7 and KLK6. Previous studies have shown that S110A7 and KLK7 are up-regulated in several other cancers, whereas CCL18, CFTR, and SLC6A14, also differentially expressed in two samples, are up-regulated specifically in cervical cancer. These differentially expressed genes in normal to LGSIL progression were enriched in pathways related to epithelial cell differentiation, keratinocyte differentiation, peptidase, and extracellular activities. In progression from LGSIL to HGSIL, two genes were up-regulated and five down-regulated in at least two samples. Further investigations using co-existing samples, which account for all internal confounders, will provide insights to better understand progression of cervical pre-cancer.

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow., (Copyright © 2011 Elsevier B.V. All rights reserved.)

GSK3beta is prominent for its role in apoptosis signaling and has been shown to be involved in Parkinson's disease (PD) pathogenesis. The overall effects of GSK3beta activity on cell fate are well-established, but the effects of mitochondrial GSK3beta activity on mitochondrial function and cell fate are unknown. Here we selectively expressed constitutively active GSK3beta within the mitochondria and found that this enhanced the apoptosis signaling activated by the PD-mimetic NADH:ubiquinone oxidoreductase (complex I) inhibitors 1-methyl-4-phenylpyridinium ion (MPP+) and rotenone. Additionally, expression of GSK3beta in the mitochondria itself caused a significant decrease in complex I activity and ATP production. Increased mitochondrial a GSK3beta activity also increased reactive oxygen species production and perturbed the mitochondrial morphology. Conversely, chemical inhibitors of GSK3beta inhibited MPP+- and rotenone-induced apoptosis, and attenuated the mitochondrial GSK3beta-mediated impairment in complex I. These results indicate that unregulated mitochondrial GSK3beta activity can mimic some of the mitochondrial insufficiencies found in PD pathology.

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.