Muscle stem cells and fibro-adipogenic progenitors in female pelvic floor muscle regeneration following birth injury.

Pelvic floor muscle (PFM) injury during childbirth is a key risk factor for pelvic floor disorders that affect millions of women worldwide. Muscle stem cells (MuSCs), supported by the fibro-adipogenic progenitors (FAPs) and immune cells, are indispensable for the regeneration of injured appendicular skeletal muscles. However, almost nothing is known about their role in PFM regeneration following birth injury. To elucidate the role of MuSCs, FAPs, and immune infiltrate in this context, we used radiation to perturb cell function and followed PFM recovery in a validated simulated birth injury (SBI) rat model. Non-irradiated and irradiated rats were euthanized at 3,7,10, and 28 days post-SBI (dpi). Twenty-eight dpi, PFM fiber cross-sectional area (CSA) was significantly lower and the extracellular space occupied by immune infiltrate was larger in irradiated relative to nonirradiated injured animals. Following SBI in non-irradiated animals, MuSCs and FAPs expanded significantly at 7 and 3 dpi, respectively; this expansion did not occur in irradiated animals at the same time points. At 7 and 10 dpi, we observed persistent immune response in PFMs subjected to irradiation compared to non-irradiated injured PFMs. CSA of newly regenerated fibers was also significantly smaller following SBI in irradiated compared to non-irradiated injured PFMs. Our results demonstrate that the loss of function and decreased expansion of MuSCs and FAPs after birth injury lead to impaired PFM recovery. These findings form the basis for further studies focused on the identification of novel therapeutic targets to counteract postpartum PFM dysfunction and the associated pelvic floor disorders.

Mechanisms governing protective pregnancy-induced adaptations of the pelvic floor muscles in the rat preclinical model.

BACKGROUND: The intrinsic properties of pelvic soft tissues in women who do and do not sustain birth injuries are likely divergent. However, little is known about this. Rat pelvic floor muscles undergo protective pregnancy-induced structural adaptations-sarcomerogenesis and increase in intramuscular collagen content-that protect against birth injury. OBJECTIVE: We aimed to test the following hypotheses: (1) the increased mechanical load of a gravid uterus drives antepartum adaptations; (2) load-induced changes are sufficient to protect pelvic muscles from birth injury. STUDY DESIGN: The independent effects of load uncoupled from the hormonal milieu of pregnancy were tested in 3- to 4-month-old Sprague-Dawley rats randomly divided into the following 4 groups, with N of 5 to 14 per group: (1) load-/pregnancy hormones- (controls), (2) load+/pregnancy hormones-, (3) reduced load/pregnancy hormones+, and (4) load+/pregnancy hormones+. Mechanical load of a gravid uterus was simulated by weighing uterine horns with beads similar to fetal rat size and weight. A reduced load was achieved by unilateral pregnancy after unilateral uterine horn ligation. To assess the acute and chronic phases required for sarcomerogenesis, the rats were sacrificed at 4 hours or 21 days after bead loading. The coccygeus, iliocaudalis, pubocaudalis, and nonpelvic tibialis anterior musles were harvested for myofiber and sarcomere length measurements. The intramuscular collagen content was assessed using a hydroxyproline assay. An additional 20 load+/pregnancy hormones- rats underwent vaginal distention to determine whether the load-induced changes are sufficient to protect from mechanical muscle injury in response to parturition-associated strains of various magnitude. The data, compared using 2-way repeated measures analysis of variance followed by pairwise comparisons, are presented as mean±standard error of mean. RESULTS: An acute increase in load resulted in significant pelvic floor muscle stretch, accompanied by an acute increase in sarcomere length compared with nonloaded control muscles (coccygeus: 2.69±0.03 vs 2.30±0.06 µm, respectively, P<.001; pubocaudalis: 2.71±0.04 vs 2.25±0.03 µm, respectively, P<.0001; and iliocaudalis: 2.80±0.06 vs 2.35±0.04 µm, respectively, P<.0001). After 21 days of sustained load, the sarcomeres returned to operational length in all pelvic muscles (P>.05). However, the myofibers remained significantly longer in the load+/pregnancy hormones- than the load-/pregnancy hormones- in coccygeus (13.33±0.94 vs 9.97±0.26 mm, respectively, P<.0001) and pubocaudalis (21.20±0.52 vs 19.52±0.34 mm, respectively, P<.04) and not different from load+/pregnancy hormones+ (12.82±0.30 and 22.53±0.32 mm, respectively, P>.1), indicating that sustained load-induced sarcomerogenesis in these muscles. The intramuscular collagen content in the load+/pregnancy hormones- group was significantly greater relative to the controls in coccygeus (6.55±0.85 vs 3.11±0.47 µg/mg, respectively, P<.001) and pubocaudalis (5.93±0.79 vs 3.46±0.52 µg/mg, respectively, P<.05) and not different from load+/pregnancy hormones+ (7.45±0.65 and 6.05±0.62 µg/mg, respectively, P>.5). The iliocaudalis required both mechanical and endocrine cues for sarcomerogenesis. The tibialis anterior was not affected by mechanical or endocrine alterations. Despite an equivalent extent of adaptations, load-induced changes were only partially protective against sarcomere hyperelongation. CONCLUSION: Load induces plasticity of the intrinsic pelvic floor muscle components, which renders protection against mechanical birth injury. The protective effect, which varies between the individual muscles and strain magnitudes, is further augmented by the presence of pregnancy hormones. Maximizing the impact of mechanical load on the pelvic floor muscles during pregnancy, such as with specialized pelvic floor muscle stretching regimens, is a potentially actionable target for augmenting pregnancy-induced adaptations to decrease birth injury in women who may otherwise have incomplete antepartum muscle adaptations.

Central Amygdala Circuits Mediate Hyperalgesia in Alcohol-Dependent Rats.

Alcohol withdrawal symptoms contribute to excessive alcohol drinking and relapse in alcohol-dependent individuals. Among these symptoms, alcohol withdrawal promotes hyperalgesia, but the neurological underpinnings of this phenomenon are not known. Chronic alcohol exposure alters cell signaling in the central nucleus of the amygdala (CeA), and the CeA is implicated in mediating alcohol dependence-related behaviors. The CeA projects to the periaqueductal gray (PAG), a region critical for descending pain modulation, and may have a role in alcohol withdrawal hyperalgesia. Here, we tested the roles of (1) CeA projections to PAG, (2) CeA melanocortin signaling, and (3) PAG µ-opioid receptor signaling in mediating thermal nociception and alcohol withdrawal hyperalgesia in male Wistar rats. Our results demonstrate that alcohol dependence reduces GABAergic signaling from CeA terminals onto PAG neurons and alters the CeA melanocortin system, that CeA-PAG projections and CeA melanocortin signaling mediate alcohol withdrawal hyperalgesia, and that µ-opioid receptors in PAG filter CeA effects on thermal nociception.SIGNIFICANCE STATEMENT Hyperalgesia is commonly seen in individuals with alcohol use disorder during periods of withdrawal, but the neurological underpinnings behind this phenomenon are not completely understood. Here, we tested whether alcohol dependence exerts its influence on pain modulation via effects on the limbic system. Using behavioral, optogenetic, electrophysiological, and molecular biological approaches, we demonstrate that central nucleus of the amygdala (CeA) projections to periaqueductal gray mediate thermal hyperalgesia in alcohol-dependent and alcohol-naive rats. Using pharmacological approaches, we show that melanocortin receptor-4 signaling in CeA alters alcohol withdrawal hyperalgesia, but this effect is not mediated directly at synaptic inputs onto periaqueductal gray-projecting CeA neurons. Overall, our findings support a role for limbic influence over the descending pain pathway and identify a potential therapeutic target for treating hyperalgesia in individuals with alcohol use disorder .

Corticotropin-releasing factor in ventromedial prefrontal cortex mediates avoidance of a traumatic stress-paired context.

Post-traumatic stress disorder (PTSD) affects 7.7 million Americans. One diagnostic criterion for PTSD is avoidance of stimuli that are related to the traumatic stress. Using a predator odor stress conditioned place aversion (CPA) model, rats can be divided into groups based on stress reactivity, as measured by avoidance of the odor-paired context. Avoider rats, which show high stress reactivity, exhibit persistent avoidance of stress-paired context and escalated alcohol drinking. Here, we examined the potential role of corticotropin-releasing factor (CRF), a neuropeptide that promotes anxiety-like behavior in mediating avoidance and escalated alcohol drinking after stress. CRF is expressed in the medial prefrontal cortex (mPFC). The dorsal and ventral sub-regions of the mPFC (dmPFC and vmPFC) have opposing roles in stress reactivity and alcohol drinking. We hypothesized that vmPFC CRF-CRFR1 signaling contributes functionally to stress-induced avoidance and escalated alcohol self-administration. In Experiment 1, adult male Wistar rats were exposed to predator odor stress in a CPA paradigm, indexed for avoidance of odor-paired context, and brains processed for CRF-immunoreactive cell density in vmPFC and dmPFC. Post-stress, Avoiders exhibited higher CRF cell density in vmPFC, but not the dmPFC. In Experiment 2, rats were tested for avoidance of a context repeatedly paired with intra-vmPFC CRF infusions. In Experiment 3, rats were stressed and indexed, then tested for the effects of intra-vmPFC CRFR1 antagonism on avoidance and alcohol self-administration. Intra-vmPFC CRF infusion produced avoidance of a paired context, and intra-vmPFC CRFR1 antagonism reversed avoidance of a stress-paired context, but did not alter post-stress alcohol self-administration. These findings suggest that vmPFC CRF-CRFR1 signaling mediates avoidance of stimuli paired with traumatic stress.

Traumatic Stress Promotes Hyperalgesia via Corticotropin-Releasing Factor-1 Receptor (CRFR1) Signaling in Central Amygdala.

Hyperalgesia is an exaggerated response to noxious stimuli produced by peripheral or central plasticity. Stress modifies nociception, and humans with post-traumatic stress disorder (PTSD) exhibit co-morbid chronic pain and amygdala dysregulation. Predator odor stress produces hyperalgesia in rodents. Systemic blockade of corticotropin-releasing factor (CRF) type 1 receptors (CRFR1s) reduces stress-induced thermal hyperalgesia. We hypothesized that CRF-CRFR1 signaling in central amygdala (CeA) mediates stress-induced hyperalgesia in rats with high stress reactivity. Adult male Wistar rats were exposed to predator odor stress in a conditioned place avoidance paradigm and indexed for high (Avoiders) and low (Non-Avoiders) avoidance of predator odor-paired context, or were unstressed Controls. Rats were tested for the latency to withdraw hindpaws from thermal stimuli (Hargreaves test). We used pharmacological, molecular, and immunohistochemical techniques to assess the role of CRF-CRFR1 signaling in CeA in stress-induced hyperalgesia. Avoiders exhibited higher CRF peptide levels in CeA that did not appear to be locally synthesized. Intra-CeA CRF infusion mimicked stress-induced hyperalgesia. Avoiders exhibited thermal hyperalgesia that was reversed by systemic or intra-CeA injection of a CRFR1 antagonist. Finally, intra-CeA infusion of tetrodotoxin produced thermal hyperalgesia in unstressed rats and blocked the anti-hyperalgesic effect of systemic CRFR1 antagonist in stressed rats. These data suggest that rats with high stress reactivity exhibit hyperalgesia that is mediated by CRF-CRFR1 signaling in CeA.

Predator odor stress alters corticotropin-releasing factor-1 receptor (CRF1R)-dependent behaviors in rats.

Humans with stress-related anxiety disorders exhibit increases in arousal and alcohol drinking, as well as altered pain processing. Our lab has developed a predator odor stress model that produces reliable and lasting increases in alcohol drinking. Here, we utilize this predator odor stress model to examine stress-induced increases in arousal, nociceptive processing, and alcohol self-administration by rats, and also to determine the effects of corticotropin-releasing factor-1 receptors (CRF1Rs) in mediating these behavioral changes. In a series of separate experiments, rats were exposed to predator odor stress, then tested over subsequent days for thermal nociception in the Hargreaves test, acoustic startle reactivity, or operant alcohol self-administration. In each experiment, rats were systemically injected with R121919, a CRF1R antagonist, and/or vehicle. Predator odor stress increased thermal nociception (i.e., hyperalgesia) and acoustic startle reactivity. Systemic administration of R121919 reduced thermal nociception and hyperarousal in stressed rats but not unstressed controls, and reduced operant alcohol responding over days. Stressed rats exhibited increased sensitivity to the behavioral effects of R121919 in all three tests, suggesting up-regulation of brain CRF1Rs number and/or function in stressed rats. These results suggest that post-stress alcohol drinking may be driven by a high-nociception high-arousal state, and that brain CRF1R signaling mediates these stress effects.

Nicotine dependence produces hyperalgesia: role of corticotropin-releasing factor-1 receptors (CRF1Rs) in the central amygdala (CeA).

Because tobacco use has a large negative health and financial impact on society, it is critical to identify the factors that drive excessive use. These factors include the aversive withdrawal symptoms that manifest upon cessation of tobacco use, and may include increases in nociceptive processing. Corticotropin-releasing factor (CRF) signalling in the central amygdala (CeA) has been attributed an important role in: (1) central processing of pain, (2) excessive nicotine use that results in nicotine dependence, and (3) in mediating the aversive symptoms that manifest following cessation of tobacco exposure. Here, we describe three experiments in which the main hypothesis was that CRF/CRF1 receptor (CRF1R) signalling in the CeA mediates nicotine withdrawal-induced increases in nociceptive sensitivity in rats that are dependent on nicotine. In Experiment 1, nicotine-dependent rats withdrawn from chronic intermittent (14-h/day) nicotine vapor exhibited decreased hind paw withdrawal latencies in response to a painful thermal stimulus in the Hargreaves test, and this effect was attenuated by systemic administration of the CRF1R antagonist, R121919. In Experiment 2, nicotine-dependent rats withdrawn from nicotine vapor exhibited robust increases in mRNA for CRF and CRF1Rs in CeA. In Experiment 3, intra-CeA administration of R121919 reduced thermal nociception only in nicotine-dependent rats. Collectively, these results suggest that nicotine dependence increases CRF/CRF1R signalling in the CeA that mediates withdrawal-induced increases in sensitivity to a painful stimulus. Future studies will build on these findings by exploring the hypothesis that nicotine withdrawal-induced reduction in pain thresholds drive excessive nicotine use via CRF/CRF1R signalling pathways.