Ligustrazine alleviates spinal cord injury-induced neuropathic pain by inhibiting the TLR4/NF-κB signaling pathway.

OBJECTIVE: To investigate the effects of ligustrazine on neuropathic pain (NPP) in rats with sciatic nerve injury and to provide new scientific insight for broadening the clinical application of ligustrazine. METHODS: Human spinal cord cell line STR cells were transfected with TLR4-mimic or mimic negative control (mimic-NC). After transfection, the STR cells were treated with different concentrations of ligustrazine (0, 0.25, 0.5, 1, 2 µm) for 24 h or 48 h. Cell proliferation was detected by MTT assay and colony formation assay. A rat model was further constructed to evaluate mechanical and cold pain sensitivity behaviors by fiber mechanical stimulation and freezing spray. The extracellular fluids of medial prefrontal cortex (mPFC) and central amygdala (CeA) were collected by intracranial dual-site simultaneous microdialysis. The contents of glutamic acid (Glu), aspartate (Asp), glycine (Gly), and γ-aminobutyric acid (GABA) in extracellular fluids were detected by HPLC. RESULTS: Compared to the 0 µm group, ligustrazine concentration at 0.5 µm significantly decreased the relative cell viability of STR cells and promoted the cell apoptosis rate. Ligustrazine at 0.25 µm significantly reduced the colony number of STR cells (all P<0.05). Compared to the control group, 1 µM ligustrazine significantly increased the protein expression of Bax and cleaved caspase 3 in STR cells but decreased the protein expression of Bcl-2 (all P<0.001). Compared to the control group, 2 µM ligustrazine treatments significantly reduced the protein levels of TLR4 and p-Akt in STR cells (all P<0.001). However, 2 µM ligustrazine treatments did not change the protein expression of Akt (P>0.05). Compared to the control group, the level of TLR4 in STR cells transfected with TLR4-mimic was significantly increased (P<0.001). Compared to the control group, transfection of TLR4-mimic reversed the anti-proliferative and pro-apoptotic effects of ligustrazine on STR cells (all P<0.001). CONCLUSION: The analgesic effect of Ligustrazine on neuropathic pain caused by spinal cord injury may be related to its inhibition of the release of excitatory amino acid transmitters Glu and Gly through the TLR4/NF-κB pathway, regulation of the dynamic balance of excitatory and inhibitory amino acid neurotransmitters, and alleviation of the central sensitization effect caused by the excitotoxicity of Glu.

Comparison of different concentrations of ropivacaine in epidural anesthesia for percutaneous transforaminal endoscopic discectomy: a randomized controlled trial.

BACKGROUND: This study investigated the optimal concentration of ropivacaine epidural anesthesia for clinical use in percutaneous transforaminal endoscopic discectomy (PTED) by comparing the effects of different concentrations. METHODS: Seventy patients scheduled for their first PTED procedure were enrolled in this randomized controlled trial. Patients were randomized to receive ropivacaine at varying concentrations (0.3% or 0.4%). Primary outcome measures included the numeric rating scale (NRS) and hip extension level (HEL). Secondary outcome measures included intraoperative fentanyl dosage and postoperative complications. RESULTS: One patient withdrew due to severe postoperative complications. The remaining 69 patients were allocated to the 0.3% (n = 34) and 0.4% (n = 35) groups, respectively. Baseline characteristics showed no significant differences between the two groups (P > 0.05). The NRS score was significantly lower in the 0.4% group than in the 0.3% group (P < 0.01), whereas the HEL score was significantly higher (P < 0.001). The average fentanyl dose in the 0.4% group was significantly lower than that in the 0.3% group (P < 0.01). Postoperative complications occurred in five and two patients in the 0.3% and 0.4% groups, respectively. CONCLUSION: Although 0.4% ropivacaine (20 mL) impacts muscle strength, it does not impede PTED surgery. Given its effective analgesic properties and few postoperative complications, 0.4% ropivacaine can be considered a preferred dose for PTED. TRIAL REGISTRATION: This study was registered with the Chinese Clinical Trials Registry (Registration number: ChiCTR2200060364; Registration Date: 29/5/2022) and on chictr.org.cn ( https://www.chictr.org.cn/showproj.html?proj=171002 ).

Blocking Pannexin-1 Channels Alleviates Thalamic Hemorrhage-Induced Pain and Inflammatory Depolarization of Microglia in Mice.

Central post-stroke pain (CPSP) is a neuropathic pain syndrome that frequently occurs following cerebral stroke. The pathogenesis of CPSP is mainly due to thalamic injury caused by ischemia and hemorrhage. However, its underlying mechanism is far from clear. In the present study, a thalamic hemorrhage (TH) model was established in young male mice by microinjection of 0.075 U of type IV collagenase into the unilateral ventral posterior lateral nucleus and ventral posterior medial nucleus of the thalamus. We found that TH led to microglial pannexin (Panx)-1, a large-pore ion channel, opening within the thalamus accompanied with thalamic tissue injury, pain sensitivities, and neurological deficit, which were significantly prevented by either intraperitoneal injection of the Panx1 blocker carbenoxolone or intracerebroventricular perfusion of the inhibitory mimetic peptide 10Panx. However, inhibition of Panx1 has no additive effect on pain sensitivities upon pharmacological depletion of microglia. Mechanistically, we found that carbenoxolone alleviated TH-induced proinflammatory factors transcription, neuronal apoptosis, and neurite disassembly within the thalamus. In summary, we conclude that blocking of microglial Panx1 channels alleviates CPSP and neurological deficit through, at least in part, reducing neural damage mediated by the inflammatory response of thalamic microglia after TH. Targeting Panx1 might be a potential strategy in the treatment of CPSP.

[Effect of electroacupuncture at different time points on postoperative urination function in patients with mixed hemorrhoids surgery].

OBJECTIVE: To observe the effect of preoperative, intraoperative and postoperative electroacupuncture (EA) intervention on postoperative urination function in patients with mixed hemorrhoid surgery. METHODS: A total of 240 patients with mixed hemorrhoid surgery under lumbar anesthesia were randomly divided into an EA preconditioning group (group A, 60 cases, 9 cases dropped off), an intraoperative EA group (group B, 60 cases, 4 cases dropped off), a postoperative EA group (group C, 60 cases, 6 cases dropped off), and a non-acupuncture group (group D, 60 cases, 3 cases dropped off). In the groups A, B and C, EA was exerted at Zhongliao (BL 33) and Huiyang (BL 35) , with disperse-dense wave, 4 Hz/20 Hz in frequency, and lasting 30 min, at 30 min before lumbar anesthesia, immediately after lumbar anesthesia and 6 h after surgery, respectively. No EA intervention was performed in the group D. The postoperative urination smoothness score in each group was observed 24 h after surgery. The first urination time, first urination volume, urine residual volume after first urination were recorded, and incidence of indwelling catheterization, postoperative visual analogue scale (VAS) score, number of remedial analgesia, and the incidence of postoperative nausea and vomiting were observed in each group. RESULTS: In the groups A, B and C, the postoperative urination smoothness scores were superior to the group D (P<0.05), and the time of first urination was earlier than the group D (P<0.05). In the group C, the time of first urination was earlier than the group A and the group B (P<0.05), the first urination volume was higher than the group D (P<0.05), and the urine residual volume after first urination was lower than the group D (P<0.05). There was no significant difference in the incidence of indwelling catheterization and postoperative nausea and vomiting among the 4 groups (P>0.05). The VAS scores of the group A, B and C were lower than that in the group D (P<0.05), and the number of remedial analgesia cases was lower than that in the group D (P<0.05). CONCLUSION: EA intervention could promote the recovery of urination function and relieve postoperative pain in patients with mixed hemorrhoids surgery. Early postoperative EA intervention is more conducive to the recovery of urination function.

Antihypertension effect of astragaloside IV during cerebral ischemia reperfusion in rats.

Stroke is one of the leading causes of death from diseases. When the blood supply to the brain tissue is interrupted, neuronal core death occurs due to the lack of glucose and oxygen in min. Blood pressure lowering after ischemic stroke was proven to be an effective strategy to achieve neurovascular protection and reduce the risk of recurrent stroke. Astragaloside IV is a pure small molecular compound isolated from Radix Astragali, and it is well documented that astragaloside IV has neuroprotective effect on cerebral ischemia reperfusion (CIR) injury through many mechanisms, including antioxidant, anti­inflammatory and anti­apoptotic. The present study adopted mean arterial pressure (MAP) monitoring, neurological scoring, 2,3,5­triphenyltetrazolium chloride staining, enzyme­linked immuno­sorbent assay, western blotting and other experimental methods to investigate the effect of astragaloside IV on systemic blood pressure during CIR in a middle cerebral artery occlusion animal model. It was demonstrated that astragaloside IV pretreatment significantly alleviated CIR injury as previously reported. In addition, the elevation of MAP during CIR was significantly inhibited by astragaloside IV administration. Moreover, it was revealed that the expression of Na+­K+­2Cl­ cotransporter isoform 1 in the hypothalamus was inhibited and the subsequent synthesis of vasopressin was reduced by astragaloside IV pretreatment in the CIR animal model. In conclusion, astragaloside IV may alleviate CIR injury partially by lowering systemic blood pressure.

Activation of the Notch signaling pathway in the anterior cingulate cortex is involved in the pathological process of neuropathic pain.

Plastic changes in the anterior cingulate cortex (ACC) are critical in pain hypersensitivity caused by peripheral nerves injury. The Notch signaling pathway has been shown to regulate synaptic differentiation and transmission. Therefore, this study was to investigate the function of the Notch signaling pathway in the ACC during nociceptive transmission induced by neuropathic pain. We adopted Western blotting, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) microinjections, RNA interference targeting Notch1, Hairy and enhancer of split (Hes) 1 or Hes5, electrophysiological recordings, and behavioral tests to verify the link between Notch signaling in ACC and neuropathic pain with adult male Sprague-Dawley rats. Levels of the Notch intracellular domain were increased in ACC on day 7 after chronic constriction injury surgery or spared nerve injury. Meanwhile, the mRNA level of the downstream effector of Notch signaling Hes1 was increased, whereas the level of Hes5 mRNA did not change. Microinjection of DAPT, a γ-secretase (a key enzyme involved in Notch pathway) inhibitor, into ACC significantly reversed neuropathic pain behaviors. Intra-ACC injection of short hairpin RNA-Notch reduced Notch intracellular domain expression and decreased the potentiation of synaptic transmission in the ACC. Moreover, pain perceptions were also alleviated in rats subjected to chronic constriction injury or spared nerve injury. This process was mainly mediated by the downstream effector Hes1, but not Hes5. Based on these results, the activation of the Notch/Hes1 signaling pathway in the ACC participates in the development of neuropathic pain, indicating that the Notch pathway may be a new therapeutic target for treating chronic pain.

Role of T-type Calcium Channels in Generating Hyperexcitatory Behaviors during Emergence from Sevoflurane Anesthesia in Neonatal Rats.

In the current study, we sought to investigate whether T-type Ca2+ channels (TCCs) in the brain are involved in generating post-anesthetic hyperexcitatory behaviors (PAHBs). We found that younger rat pups (postnatal days 9-11) had a higher incidence of PAHBs and higher PAHB scores than older pups (postnatal days 16-18) during emergence from sevoflurane anesthesia. The power spectrum of the theta oscillations (4 Hz-8 Hz) in the prefrontal cortex was significantly enhanced in younger pups when PAHBs occurred, while there were no significant changes in older pups. Both the power of theta oscillations and the level of PAHBs were significantly reduced by the administration of TCC inhibitors. Moreover, the sensitivity of TCCs in the medial dorsal thalamic nucleus to sevoflurane was found to increase with age by investigating the kinetic properties of TCCs in vitro. TCCs were activated by potentiated GABAergic depolarization with a sub-anesthetic dose of sevoflurane (1%). These data suggest that (1) TCCs in the brain contribute to the generation of PAHBs and the concomitant electroencephalographic changes; (2) the stronger inhibitory effect of sevoflurane contributes to the lack of PAHBs in older rats; and (3) the contribution of TCCs to PAHBs is not mediated by a direct effect of sevoflurane on TCCs.

Dependence of Generation of Hippocampal CA1 Slow Oscillations on Electrical Synapses.

Neuronal oscillations in the hippocampus are critical for many brain functions including learning and memory. The underlying mechanism of oscillation generation has been extensively investigated in terms of chemical synapses and ion channels. Recently, electrical synapses have also been indicated to play important roles, as reported in various brain areas in vivo and in brain slices. However, this issue remains to be further clarified, including in hippocampal networks. Here, using the completely isolated hippocampus, we investigated in vitro the effect of electrical synapses on slow CA1 oscillations (0.5 Hz-1.5 Hz) generated intrinsically by the hippocampus. We found that these oscillations were totally abolished by bath application of a general blocker of gap junctions (carbenoxolone) or a specific blocker of electrical synapses (mefloquine), as determined by whole-cell recordings in both CA1 pyramidal cells and fast-spiking cells. Our findings indicate that electrical synapses are required for the hippocampal generation of slow CA1 oscillations.

Defining the Vulnerability Window of Anesthesia-Induced Neuroapoptosis in Developing Dentate Gyrus Granule Cells - A Transgenic Approach Utilizing POMC-EGFP Mice.

Exposure to commonly used anesthetics is associated with widespread neuroapoptosis in neonatal animals. Vulnerability of developing hippocampal dentate gyrus granule cells to anesthetic neurotoxicity peaks approximately 2 weeks after cell birth, as measured by bromodeoxyuridine birth dating, regardless of the age of the animal. The present study examined whether the vulnerable window can be further characterized by utilizing a transgenic approach. Proopiomelanocortin enhanced green fluorescent protein (POMC-EGFP) mice (postnatal day 21) were exposed to 3% sevoflurane for 6 h. Following exposure, cleaved caspase 3, expression of EGFP and differential maturational markers were quantified and compared with unanesthetized littermates. Electrophysiological properties of EGFP+ and EGFP- cells in the subgranular zone and the inner half of the granule cell layer were recorded by whole-cell patch-clamp. We found that sevoflurane significantly increased apoptosis of POMC-EGFP+ granule cells that accounted for approximate 1/3 of all apoptotic cells in dentate gyrus. Apoptotic EGFP- granule cells more frequently expressed the immature neuronal marker calretinin (75.4% vs 45.0%, P < 0.001) and less frequently the late progenitor marker NeuroD1 (21.9% vs 87.9%, P < 0.001) than EGFP+ granule cells. Although EGFP- granule cells were more mature in immunostaining than EGFP+ granule cells, their electrophysiological properties partially overlapped in terms of input resistance, resting membrane potential and action potential amplitude. Our results revealed the POMC stage, when GABA acts as an excitatory neurotransmitter, only partly captures susceptibility to anesthetic neurotoxicity, suggesting the vulnerable window of anesthesia-induced neuroapoptosis extends from the end of POMC+ stage to the post-POMC+ stage when depolarizing glutamatergic inputs emerge.

Ketamine-induced apoptosis in the mouse cerebral cortex follows similar characteristic of physiological apoptosis and can be regulated by neuronal activity.

The effects of general anesthetics on inducing neuronal apoptosis during early brain development are well-documented. However, since physiological apoptosis also occurs during this developmental window, it is important to determine whether anesthesia-induced apoptosis targets the same cell population as physiological apoptosis or different cell types altogether. To provide an adequate plane of surgery, ketamine was co-administered with dexmedetomidine. The apoptotic neurons in the mouse primary somatosensory cortex (S1) were quantitated by immunohistochemistry. To explore the effect of neural activity on ketamine-induced apoptosis, the approaches of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and an environmental enrichment (EE) were performed. Ketamine-induced apoptosis in S1 is most prominent at postnatal days 5 and 7 (P5 - P7), and becomes insignificant by P12. Physiological and ketamine-induced apoptosis follow similar developmental patterns, mostly comprised of layer V pyramidal neurons at P5 and shifting to mostly layer II to IV GABAergic neurons by P9. Changes in neuronal activity induced by the DREADD system bidirectionally regulated the pattern of ketamine-induced apoptosis, with reduced activity inducing increased apoptosis and shifting the lamination pattern to a more immature form. Importantly, rearing mice in an EE significantly reduced the magnitude of ketamine-induced apoptosis and shifted its developmental pattern to a more mature form. Together, these results demonstrate that lamination pattern and cell-type dependent vulnerability to ketamine-induced apoptosis follow the physiological apoptosis pattern and are age- and activity-dependent. Naturally elevating neuronal activity is a possible method for reducing the adverse effects of general anesthesia.

Potentiation of synaptic transmission in Rat anterior cingulate cortex by chronic itch.

Itch and pain share similar mechanisms. It has been well documented that the anterior cingulate cortex (ACC) is important for pain-related perception. ACC has also been approved to be a potential pruritus-associated brain region. However, the mechanism of sensitization in pruriceptive neurons in the ACC is not clear. In current study, a chronic itch model was established by diphenylcyclopropenone (DCP) application. We found that both the frequency and amplitude of miniature excitatory postsynaptic currents in the ACC were enhanced after the formation of chronic itch. The paired-pulse ratio in ACC neurons recorded from the DCP group were smaller than those recorded in control group at the 50-ms interval. We also observe a significant increase in the AMPA/NMDA ratio in the DCP group. Moreover, an increased inward rectification of AMPARs in ACC pyramidal neurons was observed in the DCP group. Interestingly, the calculated ratio of silent synapses was significantly reduced in the DCP group compared with controls. Taken together, we conclude that a potentiation of synaptic transmission in the ACC can be induced by chronic itch, and unsilencing silent synapses, which probably involved recruitment of AMPARS, contributed to the potentiation of postsynaptic transmission.

A lasting effect of postnatal sevoflurane anesthesia on the composition of NMDA receptor subunits in rat prefrontal cortex.

Sevoflurane is widely used in pediatric anesthesia and studies have shown that it is capable of inducing neurodegeneration and subsequent cognitive disorders in the developing brain. However, the evidence that anesthetics are toxic to the human brain is insufficient. N-Methyl-d-aspartate (NMDA) receptors, critical for learning and memory, display expression changes with age and can be modulated by inhalation anesthetics. Generally, NMDA receptor (NR) type 1 is expressed at birth, peaks around the third postnatal week, and then declines slightly to adult levels. NR2Bs slowly decrease and NR2As gradually increase during postnatal development. These developmental switches of NMDA receptor subunits composition mark the transition from immature to adult neural processing and allow for the final maturation of associative learning abilities. In this study, we aimed to evaluate the effect of repeated sevoflurane anesthesia on NMDA receptor subunits composition in the developing rat brain and related behavioral disorders. Six-day-old male Sprague Dawley rats were randomly allocated into either a control group (group con) or a sevoflurane group (group sevo). Group sevo inhaled 2.1% sevoflurane carried by 70% oxygen for 2h each day from postnatal day (PND) 6 to PND 8. The same procedure, without applying the sevoflurane, was executed in group con. The membrane protein expression of NR1, NR2A and NR2B in the prefrontal cortex (PFC) and hippocampus was assessed at the end of the three days of anesthesia and at PND 21. An open field test was carried out to assess spontaneous locomotion on PNDs 21, 28 and 35. Y maze performance was used to assess attention and working memory on PND 28. Sevoflurane induced upregulation of NR1 and NR2B in the PFC at the end of anesthesia. On PND 21, NR1 and NR2B receptors were significantly increased whereas NR2A receptors were significantly decreased in the PFC in group sevo. Sevoflurane-treated rats showed hyper-locomotion and impairment of working memory in the behavior tests. These results indicate that repeated sevoflurane anesthesia at early stage of life can induce a long lasting effect of interfering with NMDA receptor subunits composition in rat PFC. These changes may contribute to the effects of sevoflurane on neuronal development and subsequent neurobehavioral disorders.

Attenuation of Neuropathic Pain by Inhibiting Electrical Synapses in the Anterior Cingulate Cortex.

BACKGROUND: Synaptic mechanisms and neuronal oscillations have been proposed to be responsible for neuropathic pain formation. Many studies have also highlighted the important role of electrical synapses in synaptic plasticity and in neuronal oscillations. Thus, electrical synapses may contribute to neuropathic pain generation. However, previous studies have primarily focused on the role of chemical synapses, while ignoring the role of electrical synapses, in neuropathic pain generation. METHODS: The authors adopted microinjection, RNA interference techniques, and behavioral tests to verify the link between connexin 36 (Cx36) and neuropathic pain. They also studied the selective Cx36 blocker mefloquine in rat chronic constriction injury and spared nerve injury model of neuropathic pain. Electrophysiologic recordings were used to further confirm the behavioral data. RESULTS: The authors found that Cx36, which constitutes the neuron-neuron electrical synapses, was up-regulated in the anterior cingulate cortex after nerve injury (n = 5). Meanwhile, Cx36-mediated neuronal oscillations in the gamma frequency range (30 to 80 Hz) (n = 7 to 8) and the neuronal synaptic transmission (n = 13 to 19) were also enhanced. Neuropathic pain was relieved by disrupting Cx36 function or expression in the anterior cingulate cortex. They also found that mefloquine, which are clinically used for treating malaria, affected gamma oscillations and synaptic plasticity, leading to a sustained pain relief in chronic constriction injury and spared nerve injury models (n = 7 to 12). CONCLUSION: The electrical synapses blocker mefloquine could affect gamma oscillations and synaptic plasticity in the anterior cingulate cortex and relieve neuropathic pain. Cx36 may be a new therapeutic target for treating chronic pain.

Histamine resets the circadian clock in the suprachiasmatic nucleus through the H1R-CaV 1.3-RyR pathway in the mouse.

Histamine, a neurotransmitter/neuromodulator implicated in the control of arousal state, exerts a potent phase-shifting effect on the circadian clock in the rodent suprachiasmatic nucleus (SCN). In this study, the mechanisms by which histamine resets the circadian clock in the mouse SCN were investigated. As a first step, Ca(2+) -imaging techniques were used to demonstrate that histamine increases intracellular Ca(2+) concentration ([Ca(2+) ]i ) in acutely dissociated SCN neurons and that this increase is blocked by the H1 histamine receptor (H1R) antagonist pyrilamine, the removal of extracellular Ca(2+) and the L-type Ca(2+) channel blocker nimodipine. The histamine-induced Ca(2+) transient is reduced, but not blocked, by application of the ryanodine receptor (RyR) blocker dantrolene. Immunohistochemical techniques indicated that CaV 1.3 L-type Ca(2+) channels are expressed mainly in the somata of SCN cells along with the H1R, whereas CaV 1.2 channels are located primarily in the processes. Finally, extracellular single-unit recordings demonstrated that the histamine-elicited phase delay of the circadian neural activity rhythm recorded from SCN slices is blocked by pyrilamine, nimodipine and the knockout of CaV 1.3 channel. Again, application of dantrolene reduced but did not block the histamine-induced phase delays. Collectively, these results indicate that, to reset the circadian clock, histamine increases [Ca(2+) ]i in SCN neurons by activating CaV 1.3 channels through H1R, and secondarily by causing Ca(2+) -induced Ca(2+) release from RyR-mediated internal stores.

Low-dose sevoflurane promotes hippocampal neurogenesis and facilitates the development of dentate gyrus-dependent learning in neonatal rats.

Huge body of evidences demonstrated that volatile anesthetics affect the hippocampal neurogenesis and neurocognitive functions, and most of them showed impairment at anesthetic dose. Here, we investigated the effect of low dose (1.8%) sevoflurane on hippocampal neurogenesis and dentate gyrus-dependent learning. Neonatal rats at postnatal day 4 to 6 (P4-6) were treated with 1.8% sevoflurane for 6 hours. Neurogenesis was quantified by bromodeoxyuridine labeling and electrophysiology recording. Four and seven weeks after treatment, the Morris water maze and contextual-fear discrimination learning tests were performed to determine the influence on spatial learning and pattern separation. A 6-hour treatment with 1.8% sevoflurane promoted hippocampal neurogenesis and increased the survival of newborn cells and the proportion of immature granular cells in the dentate gyrus of neonatal rats. Sevoflurane-treated rats performed better during the training days of the Morris water maze test and in contextual-fear discrimination learning test. These results suggest that a subanesthetic dose of sevoflurane promotes hippocampal neurogenesis in neonatal rats and facilitates their performance in dentate gyrus-dependent learning tasks.

Alleviation of neuropathic pain by regulating T-type calcium channels in rat anterior cingulate cortex.

BACKGROUND: It has been demonstrated that administration of T-type calcium channel (TCC) inhibitors could relieve the neuropathic pain by intraperitoneally or intrathecally. TCCs are not only expressed in dorsal root ganglia or dorsal horn, but also in some of the pain associated brain regions. In the present study, we sought to investigate whether modulating TCCs in the anterior cingulate cortex (ACC) could alleviate the neuropathic pain. RESULTS: (1) Cav3.2 was up regulated in rat ACC after chronic constriction injury (CCI). (2) T-type calcium current intensity was increased in CCI animal model. (3) TCC inhibitor reduced miniature excitatory postsynaptic currents frequency of ACC neurons in CCI animal model. (4) TCC inhibitor suppressed the firing rate of ACC neurons in CCI animal model. (5) Both mechanical and thermal allodynia were partially relieved by ACC microinjection with TCC inhibitor. CONCLUSIONS: TCCs in the ACC may be contributing to the maintenance of neuropathic pain, and the neuropathic pain can be alleviated by inhibiting the neuronal activity of ACC through modulating the TCCs.

Possible role of GABAergic depolarization in neocortical neurons in generating hyperexcitatory behaviors during emergence from sevoflurane anesthesia in the rat.

Hyperexcitatory behaviors occurring after sevoflurane anesthesia are of serious clinical concern, but the underlying mechanism is unknown. These behaviors may result from the potentiation by sevoflurane of GABAergic depolarization/excitation in neocortical neurons, cells implicated in the genesis of consciousness and arousal. The current study sought to provide evidence for this hypothesis with rats, the neocortical neurons of which are known to respond to GABA (γ-aminobutyric acid) with depolarization/excitation at early stages of development (i.e., until the second postnatal week) and with hyperpolarization/inhibition during adulthood. Employing behavioral tests and electrophysiological recordings in neocortical slice preparations, we found: (1) sevoflurane produced PAHBs (post-anesthetic hyperexcitatory behaviors) in postnatal day (P)1-15 rats, whereas it failed to elicit PAHBs in P16 or older rats; (2) GABAergic PSPs (postsynaptic potentials) were depolarizing/excitatory in the neocortical neurons of P5 and P10 rats, whereas mostly hyperpolarizing/inhibitory in the cells of adult rats; (3) at P14-15, <50% of rats had PAHBs and, in general, the cells of the animals with PAHBs exhibited strongly depolarizing GABAergic PSPs, whereas those without PAHBs showed hyperpolarizing or weakly depolarizing GABAergic PSPs; (4) bumetanide [inhibitor of the Cl- importer NKCC (Na+-K+-2Cl- cotransporter)] treatment at P5 suppressed PAHBs and depolarizing GABAergic responses; and (5) sevoflurane at 1% (i.e., concentration<1 minimum alveolar concentration) potentiated depolarizing GABAergic PSPs in the neurons of P5 and P10 rats and of P14-15 animals with PAHBs, evoking action potentials in ≥50% of these cells. On the basis of these results, we conclude that sevoflurane may produce PAHBs by potentiating GABAergic depolarization/excitation in neocortical neurons.

GABAergic excitation of vasopressin neurons: possible mechanism underlying sodium-dependent hypertension.

RATIONALE: Increased arginine-vasopressin (AVP) secretion is a key physiological response to hyperosmotic stress and may be part of the mechanism by which high-salt diets induce or exacerbate hypertension. OBJECTIVE: Using deoxycorticosterone acetate-salt hypertension model rats, we sought to test the hypothesis that changes in GABA(A) receptor-mediated inhibition in AVP-secreting magnocellular neurons contribute to the generation of Na(+)-dependent hypertension. METHODS AND RESULTS: In vitro gramicidin-perforated recordings in the paraventricular and supraoptic nuclei revealed that the GABAergic inhibition in AVP-secreting neurons was converted into excitation in this model, because of the depolarization of GABA equilibrium potential. Meanwhile, in vivo extracellular recordings in the supraoptic nuclei showed that the GABAergic baroreflexive inhibition of magnocellular neurons was transformed to excitation, so that baroreceptor activation may increase AVP release. The depolarizing GABA equilibrium potential shift in AVP-secreting neurons occurred progressively over weeks of deoxycorticosterone acetate-salt treatment along with gradual increases in plasma AVP and blood pressure. Furthermore, the shift was associated with changes in chloride transporter expression and partially reversed by bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor). Intracerebroventricular bumetanide administration during deoxycorticosterone acetate-salt treatment hindered the development of hypertension and rise in plasma AVP level. Muscimol (GABA(A) agonist) microinjection into the supraoptic nuclei in hypertensive rats increased blood pressure, which was prevented by previous intravenous V1a AVP antagonist injection. CONCLUSIONS: We conclude that the inhibitory-to-excitatory switch of GABAA receptor-mediated transmission in AVP neurons contributes to the generation of Na(+)-dependent hypertension by increasing AVP release. We speculate that normalizing the GABA equilibrium potential may have some utility in treating Na(+)-dependent hypertension.

TAK1 activates AMPK-dependent cell death pathway in hydrogen peroxide-treated cardiomyocytes, inhibited by heat shock protein-70.

The aim of this current study is to investigate the potential role of AMP-activated protein kinase (AMPK) in hydrogen peroxide (H2O2)-induced cardiomyocyte death, and focused on the signaling mechanisms of AMPK activation by H2O2. We observed a significant AMPK activation in H2O2-treated cardiomyocytes (both primary cells and H9c2 line). Inhibition of AMPK by its inhibitor or RNAi-reduced H2O2-induced cardiomyocyte death. We here proposed that transforming growth factor-ß-activating kinase 1 (TAK1) might be the upstream kinase for AMPK activation by H2O2. H2O2-induced TAK1 activation, which recruited and activated AMPK. TAK1 inhibitor significantly suppressed H2O2-induced AMPK activation and following cardiomyocyte death, while over-expression of TAK1-facilitated AMPK activation and aggregated cardiomyocyte death. Importantly, heat shock protein-70 (HSP-70)-reduced H2O2-induced reactive oxygen species (ROS) accumulation, the TAK1/AMPK activation and cardiomyocyte death. In conclusion, we here suggest that TAK1 activates AMPK-dependent cell death pathway in H2O2-treated cardiomyocytes, and HSP-70 inhibits the signaling pathway by reducing ROS content.

Chronic hyperosmotic stress converts GABAergic inhibition into excitation in vasopressin and oxytocin neurons in the rat.

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.