Quanhong Zhou

Propofol prevents cerebral ischemia-triggered autophagy activation and cell death in the rat hippocampus through the NF-κB/p53 signaling pathway

Propofol (2,6-diisopropylphenol) has been shown to attenuate neuronal injury under a number of experimental conditions; however, the mechanisms involved in its neuroprotective effects remain unclear. We therefore investigated whether inhibition of p53 induction by propofol contributes to the neuroprotection of cerebral ischemic cell death through both autophagic and apoptotic mechanisms. A transient global cerebral ischemia-reperfusion (I/R) model was produced with a 10-min, 2-vessel occlusion. The change in target genes including damage-regulated autophagy modulator (DRAM), microtubule-associated protein 1 light chain 3 (LC3), Beclin 1, cathepsin D, cathepsin B, p53-upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 upon p53 inhibition was assessed with the co-administration of the intravenous anesthetic propofol and 3-methyladenine (3-MA), Pifithrin-alpha (PFT-α) or SN50. The I/R-induced increases of protein levels of p53 and LC3-II were significantly inhibited by treatment with propofol, 3-MA or PFT-α. The I/R-induced increases of protein levels of DRAM, Beclin 1, active cathepsin D and cathepsin B were significantly inhibited by treatment with propofol, PFT-α or SN50. The negative effects of the I/R-induced up-regulation of PUMA and Bax and the down-regulation of Bcl-2 in the rat hippocampus were all blocked by treatment with propofol, PFT-α or SN50. Our results suggest that cerebral I/R can induce nuclear factor-kappa B-dependent expression of p53. The autophagic and apoptotic mechanisms participate in programed cell death by regulating the p53-mediated pathway. Our results are the first to show that propofol, at clinically relevant concentrations, attenuated cell death through both autophagic and apoptotic mechanisms in the rat hippocampus after a cerebral I/R insult.

The effect of intrathecal administration of glial activation inhibitors on dorsal horn BDNF overexpression and hind paw mechanical allodynia in spinal nerve ligated rats.

Recent studies have suggested that activated glia in the spinal cord may play a vital role at different times during spinal nerve ligation (SNL)-induced neuropathic pain; therefore, glial activation inhibitors have been used as effective painkillers. Brain-derived neurotrophic factor (BDNF) is also known to be a powerful pain modulator, but it remains unclear how it contributes to the glial activation inhibitor-based treatment. This study revealed the following results: (1) intrathecal administration of minocycline (a microglial activation inhibitor) could prevent mechanical allodynia during the initiation of SNL-induced neuropathic pain, and its action was associated with the elimination of BDNF overexpression in the dorsal horn; (2) the spinal injection of fluorocitrate (an astrocytic activation inhibitor) but not minocycline could reverse mechanical allodynia during the maintenance phase of SNL-induced pain, and its action was also related to a decrease in BDNF overexpression in the dorsal horn; and (3) treatment with TrkB/Fc (a BDNF-sequestering protein) had a similar effect during both the early development and maintenance periods. These results led to the following conclusions: (1) elevated BDNF expression in the dorsal horn was required to develop and maintain neuropathic pain; (2) minocycline could only prevent mechanical allodynia in the early stages, possibly by inhibiting BDNF release from microglia; and (3) fluorocitrate could reverse existing mechanical allodynia, and its action was associated with the inhibition of BDNF upregulation induced by astrocytic activation.

Brain-derived neurotrophic factor–activated astrocytes produce mechanical allodynia in neuropathic pain

Neuropathic pain management is challenging for physicians and a vexing problem for basic researchers. Recent studies reveal that activated spinal astrocytes may play a vital role in nerve injury-induced neuropathic pain, although the mechanisms are not fully understood. We have found increased glial fibrillary acidic protein (GFAP) expression, a hallmark of reactive gliosis, and elevated brain-derived neurotrophic factor (BDNF) expression in the dorsal horn in a rat model of allodynia induced by spinal nerve ligation (SNL). The high GFAP expression and mechanical allodynia that SNL induces were prevented by the intrathecal injection of the BDNF-sequestering fusion protein TrkB/Fc. Additionally, mechanical allodynia and GFAP overexpression was induced by the spinal administration of exogenous BDNF to naive rats, and exogenous BDNF given together with fluorocitrate, an astrocytic metabolism inhibitor, inhibited allodynia and GFAP upregulation. Exogenous BDNF also activated the astrocytes directly when tested in vitro. Furthermore, intrathecal administration of BDNF-stimulated astrocytes also induced mechanical allodynia in naive rats. All of these results indicate that astrocytes activated by BDNF might contribute to mechanical allodynia development in neuropathic pain in rats.

TTX-R Na+ current-reduction by celecoxib correlates with changes in PGE2 and CGRP within rat DRG neurons during acute incisional pain

The present study was undertaken to investigate whether celecoxib could regulate the tetrodotoxin-resistant (TTX-R) sodium channel current in rat dorsal root ganglia (DRG) and whether prostaglandin E2 (PGE2) and calcitonin gene-related protein (CGRP) were involved in celecoxibs analgesia during acute incisional pain. Seventy-five rats were randomly allocated into three groups. Group A was the control group receiving a placebo (sugar pill) 1 h before and 12 h after surgery (right hind paw incisional pain). Group B was the test group receiving celecoxib 30 mg/kg orally 1 h before and 12 h after surgery. Group C was the naive group receiving a sham operation. The changes in the mechanical withdrawal thresholds, PGE2 and CGRP concentration in incisional paw tissue and DRG, and total TTX-R sodium channel current density in small DRG neurons were investigated 1 h before the operation and 2 h, 6 h, 12 h, 24 h, 48 h and 96 h after the operation. The results showed both of a decrease in mechanical withdrawal thresholds and an increase of TTX-R sodium channel current density in DRG neurons in group B were significantly lower than those of group A at 24 h and 48 h after the operation (P<0.05). The increase in PGE2 and CGRP concentrations at incisional paw tissue and DRG neurons in group B were lower than those of groups A at 24 h and 48 h after the operation (P<0.05). This study indicates that: 1) celecoxib can inhibit TTX-R sodium channel current density in rat DRG neurons; 2) PGE2 and CGRP participate in celecoxibs analgesic effect on acute incisional pain.

Effect of metabotropic glutamate 5 receptor antagonists on morphine efficacy and tolerance in rats with neuropathic pain

The metabotropic glutamate 5 (mGlu5) receptor is involved in both pain processing and modulation of µ-opioid induced antinociception and antihyperalgesia. Systemic mGlu5 receptor antagonists 2-methyl-6-phenylethynylpyridine (MPEP) or 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine (MTEP) provide antihyperalgesic effects in various pain models, but few studies have shown their interaction with morphine in neuropathic pain models. The aim of this study is to compare the effects of systemic and intrathecal MPEP/MTEP on morphine efficacy and tolerance in rats with chronic neuropathic pain. L5-6 spinal nerve ligation (SNL) was used to establish neuropathic pain model in rats. The Von Frey test and the hot water tail flick test were employed as behavior tests. Low, medium and high doses of MPEP/MTEP were tested for their effect on both acute morphine efficacy and chronic morphine tolerance. SNL provides sustained neuropathic pain on the ipsilateral hind paw of rats. Both systemic and intrathecal MPEP/MTEP had antiallodynia effects and boosted morphines efficacy in a dose-dependent manner in the Von Frey tests but not in the tail flick tests. In fact, high doses of MTEP and MPEP attenuated morphines antinociceptive effect in the latter test. After intrathecal chronic co-administration with morphine, low-doses of MTEP/MPEP attenuated morphine tolerance in both tests. Systemically, only MTEP attenuated morphine tolerance, and only in the Von Frey tests, not in the tail flick tests, whereas MPEP had no effect on morphine tolerance in either tests. The therapeutic use of mGlu5 receptor antagonists may have distinct effects in different pain models.

Inhibition of MPEP on the development of morphine antinociceptive tolerance and the biosynthesis of neuronal nitric oxide synthase in rat spinal cord

We evaluated the ability of spinally administered 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of the metabotropic glutamate receptor subtype 5 (mGluR5), and 2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, to modulate the antinociceptive action and tolerance of intrathecal (i.t.) morphine infusion in rats, and assessed the expression of spinal nitric oxide synthase (NOS). MPEP co-infused with morphine not only preserved the analgesia and retarded the development of antinociceptive tolerance, but also partially inhibited the up-regulation of spinal nNOS protein. However, the loss of morphine antinociceptive effect and tolerance were accelerated when CHPG and morphine were co-infused, while spinal nNOS activity was significantly up-regulated. We hypothesize that activation of mGluR5 and NMDA receptors occurs after the appearance of antinociceptive tolerance to morphine. The activation of these receptors might stimulate an increased concentration of intracellular calcium and activation of PKC, which both play a vital role in the development of morphine antinociceptive tolerance and expression of spinal NOS. The synergistic effect which seems to exist between mGluRs and iGluRs may also contribute to this phenomenon.

Protein Kinase C Isoforms Distinctly Regulate Propofol-induced Endothelium-dependent and Endothelium-independent Vasodilation.

Abstract: Protein kinase C (PKC) isoforms improve endothelial nitric oxide synthase activity and contractile Ca2+ sensitivity in blood vessels. These actions may have opposite effects on propofol-induced vasodilation. This study examines the hypothesis that propofol induces relaxation by enhancing the PKC-mediated nitric oxide synthesis in endothelium and/or inhibiting the PKC-regulated Ca2+ sensitivity in vascular smooth muscle (VSM). Propofol (1–100 &mgr;M) induced greater relaxation in endothelium-intact rings compared with denuded rings, and this effect was antagonized by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In contrast, treatment with the general PKC inhibitor GF-109203X augmented both the endothelium-dependent and endothelium-independent relaxation induced by propofol, and this enhancement was more profound in the intact rings at lower propofol concentrations. The enhancement was unaffected by L-NAME. Interestingly, calphostin C (an inhibitor of conventional and novel PKCs) and Gö-6976 (an inhibitor of conventional PKCs) had similar effects in augmenting propofol-induced relaxation in endothelium-denuded rings. Downregulation of novel isoforms not only reduced the norepinephrine-elicited contraction but also decreased the magnitude of propofol-induced relaxation. In vascular smooth muscle cells, propofol prevented norepinephrine-elicited phosphorylation of myosin light chain. Propofol can increase the PKC-mediated availability of nitric oxide but inhibit the novel PKC-regulated Ca2+-sensitization, which provides a novel explanation for the mechanism of propofol-induced vasodilation.

Gabapentin attenuates morphine tolerance through interleukin-10.

In this study, we examined the anti-inflammatory mechanism by which gabapentin attenuates morphine tolerance in rats. Gabapentin enhanced the antinociceptive effect of morphine and attenuated chronic morphine tolerance when administered intrathecally with morphine in naive rats. We found that a 7-day chronic intrathecal injection of morphine increased the expression of proinflammatory cytokines and decreased interleukin-10 (IL-10) levels in the rat spinal cord. These changes were minimized when gabapentin was combined with morphine. In addition, the effects of gabapentin were reversed by coadministration of the anti-IL-10 antibody. Our findings indicate that enhancement of the antinociceptive effect of morphine by gabapentin may occur through upregulation of the anti-inflammatory cytokine IL-10 and inhibition of proinflammatory cytokines in the rat spinal cord.

Lipid metabolism disturbances and AMPK activation in prolonged propofol-sedated rabbits under mechanical ventilation

Aim:To explore the mechanisms underlying the propofol infusion syndrome (PRIS), a potentially fatal complication during prolonged propofol infusion.Methods:Male rabbits under mechanical ventilation through endotracheal intubation were divided into 3 groups (n=6 for each) that were sedated with 1% propofol (Group P), isoflurane (Group I) or isoflurane while receiving 10% intralipid (Group II), respectively. Blood biochemical parameters were collected at 0, 6, 12, 18, 24 and 30–36 h after the initiation of treatments. The hearts were removed out immediately after the experiments, and the level of tumor necrosis factor (TNF)-α in the hearts were studied using immunohistochemistry. AMP-activated protein kinase (AMPK) and phospho-AMPK in the hearts were assessed using Western blotting.Results:The mortality rate was 50% in Group P, and 0% in Groups I and II. The serum lipids and liver function indices in Group P were significantly increased, but moderately increased in Group II. Significant decreases in these indices were found in Groups I. All the groups showed dramatically increased release of creatine kinase (CK). Intense positive staining of TNF-α was found in all the heart samples in Group P, but only weak and neglectful staining was found in the hearts from Group II and Group I, respectively. AMPK phosphorylation was significantly increased in the hearts of Group P.Conclusion:Continuous infusion of large dose of propofol in rabbits undergoing prolonged mechanical ventilation causes hyperlipidemia, liver dysfunction, increased CK levels, AMPK activation and myocardial injury. The imbalance between energy demand and utilization may contribute to PRIS.

Optimal interval for hot water immersion tail-flick test in rats.

Background The hot water tail-flick test is widely used to measure the degree of nociception experienced by laboratory animals. This study was carried out to optimise interval times for the hot water immersion tail-flick tests in rats. Method Ten different intervals from 10 s to 1 h were tested in 60 Sprague–Dawley male rats. At least eight rats were tested for each interval in three consecutive hot water tail-flick tests. Dixons up-and-down method was also used to find the optimal intervals. The same rats were then divided into two groups. In Group N, naloxone was injected to reverse the prolonged latency times, whereas saline was used in the control Group S. Results Intervals of 10 s, 20 s, 30 min and 1 h did not significantly impact latencies, yielding similar results in three consecutive tests (p > 0.05). However, interval times of between 30 s and 20 min, inclusively, caused significantly prolonged latencies in the second and third tests (p < 0.001). Dixons up-and-down method showed that 95% of the rats had prolonged latencies in hot water tail-flick tests at intervals longer than 32 s. Naloxone reversed prolonged latencies in Group N, whereas the latencies in Group S were further prolonged in 5 min interval tests. Conclusion The optimal intervals for hot water tail-flick tests are either shorter than 20 s or longer than 20 min. The prolonged latencies after repetitive tests were attributable to an endocrine opioid.