In vitro effects of hydrogen peroxide on rat uterine contraction before and during pregnancy

Aim To assess the in vitro effect of hydrogen peroxide (H2O2) on uterine contractions in pregnant and non-pregnant rats. Methods The study was performed at the Department of Physiology, College of Medicine, King Saud University from December 2016 to October 2017. Intact uterine samples were obtained from non-pregnant (n = 7-8) and termpregnant (n = 6-7) rats. Small longitudinal uterine strips were dissected and mounted in an organ bath. Isometric force measurements were used to assess the effect of 400, 800, and 1000 μM H2O2 on spontaneous uterine contractions and contractions induced by oxytocin (5 nM), high calcium (Ca+2) solution (6 mmol/L), and high potassium chloride (KCl) solution (60 mmol/L).Results In both term-pregnant and non-pregnant uterine strips, H2O2 elicited a biphasic response, consisting of a transient contraction followed by a persistent decrease in spontaneously generated contractions, contractions induced by oxytocin, and contractions induced by high Ca+2 (all P < 0.01, compared with controls) in a concentration-dependent manner. The effect of H2O2 was more pronounced in non-pregnant than in pregnant rats (P < 0.05). In both groups, H2O2 failed to relax uterine strips pre-contracted with high-KCl solution (P > 0.05 compared with controls). Conclusion H2O2 was shown to be a potent uterine relaxant in pregnant and non-pregnant states. The pregnant uterus better withstood the inhibitory effect of H2O2 than non-pregnant uterus

Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes c-fiber nociceptors with broad action potentials and high nav1.9 expression

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4+) and small dark neurons had similar size distributions. We examined IB4-binding levels in >120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4+ neurons were classified as strongly or weakly IB4+. All strongly IB4+ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, ~50% were strongly IB4+, ~20% were weakly IB4+ and ~30% were IB4–. A{delta} low-threshold mechanoreceptive (LTM) neurons were weakly IB4+ or IB4–. All 33 A-fiber nociceptors and all 44 A{alpha}/beta-LTM neurons examined were IB4–. IB4+ compared with IB4– C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, ~35% were trkA+/IB4+ but with negatively correlated immunointensities; 26% were IB4+/trkA–, and 35% were IB4–/trkA+. We conclude that strongly IB4+ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties

Cutaneous Aβ-Non-nociceptive, but Not C-Nociceptive, Dorsal Root Ganglion Neurons Exhibit Spontaneous Activity in the Streptozotocin Rat Model of Painful Diabetic Neuropathy .

Diabetic peripheral neuropathic pain (DPNP) is the most devastating complication of diabetes mellitus. Unfortunately, successful therapy for DPNP remains a challenge because its pathogenesis is still elusive. However, DPNP is believed to be due partly to abnormal hyperexcitability of dorsal root ganglion (DRG) neurons, but the relative contributions of specific functional subtypes remain largely unknown. Here, using the strepotozotocin (STZ) rat model of DPNP induced by a STZ injection (60 mg/kg, i.p), and intracellular recordings of action potentials (APs) from DRG neurons in anesthetized rats, we examined electrophysiological changes in C-and Aβ-nociceptive and Aβ-low threshold mechanoreceptive (LTM) neurons that may contribute to DPNP. Compared with control, we found in STZ-rats with established pain hypersensitivity (5 weeks post-STZ) several significant changes including: (a) A 23% increase in the incidence of spontaneous activity (SA) in Aβ-LTMs (but not C-mechanosensitive nociceptors) that may cause dysesthesias/paresthesia suffered by DPNP patients, (b) membrane hyperpolarization and a ∼85% reduction in SA rate in Aβ-LTMs by K7 channel activation with retigabine (6 mg/kg, i.v.) suggesting that K7/M channels may be involved in mechanisms of SA generation in Aβ-LTMs, (c) decreases in AP duration and in duration and amplitude of afterhyperpolarization (AHP) in C-and/or Aβ-nociceptors. These faster AP and AHP kinetics may lead to repetitive firing and an increase in afferent input to the CNS and thereby contribute to DPNP development, and (d) a decrease in the electrical thresholds of Aβ-nociceptors that may contribute to their sensitization, and thus to the resulting hypersensitivity associated with DPNP.This research work was supported by a Medical Research Council grant (G0700420) and a Ph.D. studentship from Biotechnology and Biological Sciences Research Council to LD

Changes in expression of Kv7.5 and Kv7.2 channels in dorsal root ganglion neurons in the streptozotocin rat model of painful diabetic neuropathy

Diabetic peripheral neuropathic pain (DPNP), the most debilitating complication of diabetes mellitus, is resistant to current therapy. The pathogenesis of DPNP is still elusive, but several mechanisms have been proposed including abnormal hyperexcitability of dorsal root ganglion (DRG) neurons. The underlying molecular mechanisms of such aberrant hyperexcitability are incompletely understood. Using the streptozotocin (STZ) rat model of DPNP, we have recently provided evidence implicating neuronal Kv7 channels that normally exert a powerful stabilizing influence on neuronal excitability, in the abnormal hyperexcitability of DRG neurons and in pain hypersensitivity associated with DPNP. In the present immunohistochemical study, we sought to determine whether Kv7.2 and/or Kv7.5 channel expression is altered in DRG neurons in STZ rats. We found 35 days post-STZ: (1) a significant decrease in Kv7.5-immunoreactivity in small (<30 μm) DRG neurons (both IB4 positive and IB4 negative) and medium-sized (30−40 μm) neurons, and (2) a significant increase in Kv7.2-immunoreactivity in small (<30 μm) neurons, and a non-significant increase in medium/large neurons. The decrease in Kv7.5 channel expression in small and medium-sized DRG neurons in STZ rats is likely to contribute to the mechanisms of hyperexcitability of these neurons and thereby to the resulting pain hypersensitivity associated with DPNP. The upregulation of Kv7.2 subunit in small DRG neurons may be an activity dependent compensatory mechanism to limit STZ-induced hyperexcitability of DRG neurons and the associated pain hypersensitivity. The findings support the notion that Kv7 channels may represent a novel target for DPNP treatment

Spontaneous Pain, Both Neuropathic and Inflammatory, Is Related to Frequency of Spontaneous Firing in Intact C-Fiber Nociceptors

Spontaneous pain, a poorly understood aspect of human neuropathic pain, is indicated in animals by spontaneous foot lifting (SFL). To determine whether SFL is caused by spontaneous firing in nociceptive neurons, we studied the following groups of rats: (1) untreated; (2) spinal nerve axotomy (SNA), L5 SNA 1 week earlier; (3) mSNA (modified SNA), SNA plus loose ligation of the adjacent L4 spinal nerve with inflammation-inducing chromic gut; and (4) CFA (complete Freund’s adjuvant), intradermal complete Freund’s adjuvant-induced hindlimb inflammation 1 and 4 d earlier. In all groups, recordings of SFL and of spontaneous activity (SA) in ipsilateral dorsal root ganglion (DRG) neurons (intracellularly) were made. Evoked pain behaviors were measured in nerve injury (SNA/mSNA) groups. Percentages of nociceptive-type C-fiber neurons (C-nociceptors) with SA increased in intact L4 but not axotomized L5 DRGs in SNA and mSNA (to 35%), and in L4/L5 DRGs 1–4 d after CFA (to 38–25%). SFL occurred in mSNA but not SNA rats. It was not correlated with mechanical allodynia, extent of L4 fiber damage [ATF3 (activation transcription factor 3) immunostaining], or percentage of L4 C-nociceptors with SA. However, L4 C-nociceptors with SA fired faster after mSNA (1.8 Hz) than SNA (0.02 Hz); estimated L4 total firing rates were ?5.0 and ?0.6 kHz, respectively. Similarly, after CFA, faster L4 C-nociceptor SA after 1 d was associated with SFL, whereas slower SA after 4 d was not. Thus, inflammation causes L4 C-nociceptor SA and SFL. Overall, SFL was related to SA rate in intact C-nociceptors. Both L5 degeneration and chromic gut cause inflammation. Therefore, both SA and SFL/spontaneous pain after nerve injury (mSNA) may result from cumulative neuroinflammation

Activation of K(v)7 channels with the anticonvulsant retigabine alleviates neuropathic pain behaviour in the streptozotocin rat model of diabetic neuropathy

Diabetic peripheral neuropathy (DPN) is the most incapacitating complication of diabetes mellitus. Up to 50% of patients with DPN develop peripheral neuropathic pain (PNP). The underlying ionic and molecular mechanisms of diabetic PNP (DPNP) are poorly understood. However, voltage gated potassium (K7) channels which have been implicated in the pathogenesis of other types of PNP are likely to be involved. Here we examined, in the streptozotocin (STZ) rat model of DPNP, whether activating the Kv7 channels with a potent activator retigabine (ezogabine) would reverse/attenuate behavioural signs of DPNP. STZ rats exhibited behavioural indices of mechanical and heat hypersensitivity, but not cold hypersensitivity or spontaneous pain, 35 days after STZ injection. Retigabine given at a dose of 15 mg/kg (but not at 7.5 mg/kg, i.p.) significantly attenuated mechanical, but not heat hypersensitivity in DPNP rats, and was as effective as the positive control gabapentin. This analgesic effect of retigabine was completely reversed by the K7/M channel blocker XE991 (3 mg/kg, i.p.) indicating that the anti-allodynic effects of retigabine were mediated by K7 channels. In conclusion, the findings suggest that Kv7 channels are involved in DPNP pathogenesis, and that strategies that target their activation may prove to be effective in treating DPNP

L5 Spinal Nerve Axotomy Induces Distinct Electrophysiological Changes in Axotomized L5- and Adjacent L4-Dorsal Root Ganglion Neurons in Rats .

Peripheral neuropathic pain (PNP) is a major health problem for which effective drug treatment is lacking. Its underlying neuronal mechanisms are still illusive, but pre-clinical studies using animal models of PNP including the L5-spinal nerve axotomy (L5-SNA) model, suggest that it is partly caused by excitability changes in dorsal root ganglion (DRG) neurons. L5-SNA results in two DRG neuronal groups: (1) axotomized/damaged neurons in L5- plus some in L4-DRGs, and (2) ipsilateral L4-neurons with intact/uninjured fibers intermingling with degenerating L5-fibers. The axotomized neurons are deprived of peripherally derived trophic factors and degenerate causing neuroinflammation, whereas the uninjured L4-neuorns are subject to increased trophic factors and neuroinflammation associated with Wallerian degeneration of axotomized L5-nerve fibers. Whether these two groups of DRG neurons exhibit similar or distinct electrophysiological changes after L5-SNA remains unresolved. Conflicting evidence for this may result from some studies assuming that all L4-fibers are undamaged. Here, we recorded somatic action potentials (APs) intracellularly from C- and A-fiber L4/L5 DRG neurons to examine our hypothesis that L5-SNA would induce distinct electrophysiological changes in the two populations of DRG neurons. Consistent with this hypothesis, we found (7 days post-SNA), in SNA rats with established pain hypersensitivity, slower AP kinetics in axotomized L5-neurons and faster AP kinetics in L4-nociceptive neurons including decreased rise time in Aδ-and Aβ-fiber nociceptors, and after-hyperpolarization duration in Aβ-fiber nociceptors. We also found several changes in axotomized L5-neurons but not in L4-nociceptive neurons, and some changes in L4-nociceptive but not L5-neurons. The faster AP kinetics (decreased refractory period) in L4-nociceptive neurons that are consistent with their reported hyperexcitability may lead to repetitive firing and thus provide enhanced afferent input necessary for initiating and/or maintaining PNP development. The changes in axotomized L5-neurons may contribute to the central mechanisms of PNP via enhanced neurotransmitter release in the central nervous system (CNS)

Molecular Mechanisms of Adiponectin-Induced Attenuation of Mechanical Stretch-Mediated Vascular Remodeling

Hypertension induces vascular hypertrophy, which changes blood vessels structurally and functionally, leading to reduced tissue perfusion and further hypertension. It is also associated with dysregulated levels of the circulating adipokines leptin and adiponectin (APN). Leptin is an obesity-associated hormone that promotes vascular smooth muscle cell (VSMC) hypertrophy. APN is a cardioprotective hormone that has been shown to attenuate hypertrophic cardiomyopathy. In this study, we investigated the molecular mechanisms of hypertension-induced VSMC remodeling and the involvement of leptin and APN in this process. To mimic hypertension, the rat portal vein (RPV) was mechanically stretched, and the protective effects of APN on mechanical stretch-induced vascular remodeling and the molecular mechanisms involved were examined by using 10μg/ml APN. Mechanically stretching the RPV significantly decreased APN protein expression after 24 hours and APN mRNA expression in a time-dependent manner in VSMCs. The mRNA expression of the APN receptors AdipoR1, AdipoR2, and Tcadherin significantly increased after 15 hours of stretch. The ratio of APN/leptin expression in VSMCs significantly decreased after 24 hours of mechanical stretch. Stretching the RPV for 3 days increased the weight and [3H]-leucine incorporation significantly, whereas APN significantly reduced hypertrophy in mechanically stretched vessels. Stretching the RPV for 10 minutes significantly decreased phosphorylation of LKB1, AMPK, and eNOS, while APN significantly increased p-LKB1, pAMPK, and p-eNOS in stretched vessels. Mechanical stretch significantly increased p-ERK1/2 after 10 minutes, whereas APN significantly reduced stretch-induced ERK1/2 phosphorylation. Stretching the RPV also significantly increased ROS generation after 1 hour, whereas APN significantly decreased mechanical stretch-induced ROS production. Exogenous leptin (3.1nM) markedly increased GATA-4 nuclear translocation in VSMCs, whereas APN significantly attenuated leptin-induced GATA-4 nuclear translocation. Our results decipher molecular mechanisms of APN-induced attenuation of mechanical stretch-mediated vascular hypertrophy, with the promising potential of ultimately translating this protective hormone into the clinic.This work was supported by the Medical Practice Plan (MPP), Faculty of Medicine at AUB to AZ, the National Council for Scientific Research of Lebanon (CNRS-L) to CMG, and the graduate teaching/research assistantship support from Qatar University to SA. The publication of this article was funded by the Qatar National Library

Umbelliferone inhibits spermatogenic defects and testicular injury in lead-intoxicated rats by suppressing oxidative stress and inflammation, and improving Nrf2/HO-1 signaling

© 2020 Alotaibi et al. Introduction: Lead (Pb) is an environmental toxic metal that threatens human health. Umbelliferone (UMB) is a coumarin with known medicinal and protective properties against cytotoxicity. This study explored the ameliorative effect of UMB against Pb-induced testicular toxicity in rats, focusing on steroidogenesis, oxidative stress and inflammation. Materials and Methods: Rats received lead acetate (50 mg/kg) and UMB (25, 50 or 100 mg/kg) via oral gavage for 4 weeks. Results: Pb-intoxicated rats exhibited testicular tissue injury and decreased serum levels of LH, FSH and testosterone. The count, viability, motility and normal morphology of the sperms were decreased accompanied with downregulated steroidogenesis markers in Pbinduced group. UMB prevented testicular injury, increased serum levels of LH, FSH and testosterone, upregulated steroidogenesis markers and improved the semen quality. In addition, UMB attenuated oxidative stress and oxidative DNA damage, downregulated the expression of pro-inflammatory mediators and Bax, boosted antioxidant defenses and Bcl2, and upregulated Nrf2/HO-1 signaling in Pb-intoxicated rats. Conclusion: UMB prevents Pb-induced testicular injury by suppressing oxidative damage, inflammation and cell death, and boosting antioxidant defenses, Nrf2/HO-1 signaling and pituitary-gonadal axis. Thus, UMB may represent a protective and cost-effective agent against Pb testicular toxicity, pending further investigations to elucidate other underlying mechanisms.Open Access funding provided by the Qatar National Library