Chuanhan Zhang

Rapid inhibitory effects of corticosterone on calcium influx in rat dorsal root ganglion neurons

Corticosterone may nongenomically affect cell functions in addition to its well-characterized effects on gene expression. The purpose of this study is to examine if corticosterone has a rapid nongenomic effect on excitability of dorsal root ganglion neurons by using patch-clamp and single-cell Ca(2+) microfluometry techniques. The results show that corticosterone has a dose-dependent rapid inhibitory effect on the voltage-dependent calcium currents in dorsal root ganglion neurons. Moreover, corticosterone inhibits [Ca(2+)](i) elevation induced by 50 mM high K(+) within just 3 s. The inhibitory effects of corticosterone on the voltage-dependent calcium current and high K(+)-induced calcium influx diminish after adding protein kinase C inhibitor or pretreatment with pertussis toxin for 24 h. Our results demonstrate an nongenomic effect of corticosterone on the excitability of dorsal root ganglion neurons and the effect is mediated through a putative pertussis toxin-sensitive G-protein-coupled receptor and activation of protein kinase C.

Hesperidin pretreatment protects hypoxia-ischemic brain injury in neonatal rat.

Neonatal hypoxia-ischemic encephalopathy (HIE) remains a major cause of brain damage, leading to high disability and mortality rates in neonates. In vitro studies have shown that hesperidin, a flavanone glycoside found abundantly in citrus fruits, acts as an antioxidant. Although hesperidin has been considered as a potential treatment for HIE, its effects have not been fully evaluated. In this study, the protective effect of hesperidin pretreatment against hypoxia-ischemic (HI) brain injury and possible signal pathways were investigated using in vivo and in vitro models. In vivo HI model employed unilateral carotid ligation in postnatal day 7 rat with exposure to 8% hypoxia for 2.5h, whereas in vitro model employed primary cortical neurons of neonatal rats subjected to oxygen and glucose deprivation for 2.5h. Hesperidin pretreatment significantly reduced HI-induced brain tissue loss and improved neurological outcomes as shown in 2,3,5-triphenyltetrazolium chloride monohydrate staining and foot-fault results. The neuroprotective effects of hesperidin are likely the results of preventing an increase in intracellular reactive oxygen species and lipid peroxide levels. Hesperidin treatment also activated a key survival signaling kinase, Akt, and suppressed the P-FoxO3 level. Hesperidin pretreatment protected neonatal HIE by reducing free radicals and activating phosphorylated Akt.

Cdh1-APC is involved in the differentiation of neural stem cells into neurons.

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that controls cell cycle transition in proliferating cells. Recent studies show that Cdh1–APC is active in postmitotic neurons, which regulates axonal growth and patterning, synaptic development and neuronal survival. However, the role of Cdh1–APC in neural stem cells differentiation remains unknown. Using quantitative reverse transcription-PCR, we observed that Cdh1 was expressed higher in neurons than in neural stem cells in vitro. Cdh1 was upregulated, whereas Id2 (one downstream substrate of Cdh1–APC) was downregulated when primary neural stem cells were induced to differentiate into neurons by all-trans retinoic acid. This observation suggests that Cdh1 is involved in the control of neural stem cells differentiated into neurons.

Role of ATP-sensitive potassium channels in modulating nociception in rat model of bone cancer pain

Bone cancer pain is a major clinical problem and remains difficult to treat. ATP-sensitive potassium (KATP) channels may be involved in regulating nociceptive transmission at the spinal cord level. We determined the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat model of bone cancer pain. The rat model of bone cancer pain was induced by implanting rat mammary gland carcinoma cells (Walker256) into the tibias. KATP modulators (pinacidil and glibenclamide) or the specific Kir6.2-siRNA were injected via an intrathecal catheter. The mechanical withdrawal threshold of rats was tested using von Frey filaments. The Kir6.2 mRNA and protein levels were measured by quantitative PCR and western blots, respectively. Intrathecal injection of pinacidil, a KATP channel opener, significantly increased the tactile withdrawal threshold of cancer cell-injected rats in a dose-dependent manner. In contrast, intrathecal delivery of glibenclamide, a KATP channel blocker, or the specific Kir6.2-siRNA significantly reduced the tactile withdrawal threshold of cancer cell-injected rats. The mRNA and protein levels of Kir6.2 in the spinal cord of cancer cell-injected rats were significantly lower than those in control rats. Our findings suggest that the KATP channel expression level in the spinal cord is reduced in bone cancer pain. Activation of KATP channels at the spinal level reduces pain hypersensitivity associated with bone cancer pain.

The involvement of down-regulation of Cdh1-APC in hippocampal neuronal apoptosis after global cerebral ischemia in rat

Anaphase-promoting complex (APC) and its coactivator Cdh1 are required for maintaining cells in G1 phase of cell cycle in proliferating cells. Recent studies showed that Cdh1-APC was active in post-mitotic neurons, which regulates neuronal survival, differentiation, axonal growth and synaptic development. However, the possible function of Cdh1-APC in ischemic brain injury has not been determined. This study aimed to investigate changes in the activity of Cdh1-APC in hippocampus after global cerebral ischemia in rat. We found that, compared with sham group, the expression of Cdh1 in hippocampus was significantly decreased on 1 and 3 days of reperfusion in ischemia group (P<0.05), while neuronal apoptosis were found in hippocampal CA1 region and the two downstream substrates of Cdh1-APC (SnoN and Skp2) were significantly increased after global cerebral ischemia (P<0.05). This study demonstrates that the down-regulation of Cdh1-APC is associated with neuronal apoptosis in hippocampus following global cerebral ischemia. It brings a prospect to explore the further function of Cdh1-APC in the injured nervous system.

Cdh1 inhibits reactive astrocyte proliferation after oxygen-glucose deprivation and reperfusion.

Anaphase-promoting complex (APC) and its co-activator Cdh1 are required for cell cycle regulation in proliferating cells. Recent studies have defined diverse functions of APC-Cdh1 in nervous system development and injury. Our previous studies have demonstrated the activity of APC-Cdh1 is down-regulated in hippocampus after global cerebral ischemia. But the detailed mechanisms of APC-Cdh1 in ischemic nervous injury are unclear. It is known that astrocyte proliferation is an important pathophysiological process following cerebral ischemia. However, the role of APC-Cdh1 in reactive astrocyte proliferation is not determined yet. In the present study, we cultured primary cerebral astrocytes and set up in vitro oxygen-glucose deprivation and reperfusion model. Our results showed that the expression of Cdh1 was decreased while Skp2 (the downstream substrate of APC-Cdh1) was increased in astrocytes after 1h oxygen-glucose deprivation and reperfusion. The down-regulation of APC-Cdh1 was coupled with reactive astrocyte proliferation. By constructing Cdh1 expressing lentivirus system, we also found exogenous Cdh1 can down-regulate Skp2 and inhibit reactive astrocyte proliferation induced by oxygen-glucose deprivation and reperfusion. Moreover, Western blot showed that other downstream proteins of APC-Cdh1, PFK-1 and SnoN, were decreased in the inhibition of reactive astrocyte proliferation with Cdh1 expressing lentivirus treatment. These results suggest that Cdh1 plays an important role in the regulation of reactive astrocyte proliferation induced by oxygen-glucose deprivation and reperfusion.

Cervical spinal cord compression after thyroidectomy under general anesthesia.

Cervical spinal cord injury is a rare but serious complication after general anesthesia. The risk factors include traumatic cervical injury, cervical spine instability, and difficult airway management. It has also occurred in the absence of cervical instability. Here we report a patient who had a history of intermittent neck pain without numbness. Preoperative radiologic examinations showed degenerative changes in the cervical spine. She developed progressive tingling and numbness in her limbs after thyroidectomy under general anesthesia. Magnetic resonance imaging showed a cervical disc protruding into the canal at C5–C6, which was considered to be induced by surgical positioning. She recovered after anterior cervical decompression and internal fixation surgery.

PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1.

Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

Downregulation of Cdh1 signalling in spinal dorsal horn contributes to the maintenance of mechanical allodynia after nerve injury in rats

Background Anaphase-promoting complex/cyclosome (APC/C) and its co-activator Cdh1 are important ubiquitin-ligases in proliferating cells and terminally differentiated neurons. In recent years, APC/C-Cdh1 has been reported as an important complex contributing to synaptic development and transmission. Interestingly, cortical APC/C-Cdh1 is found to play a critical role in the maintenance of neuropathic pain, but it is not clear whether APC/C-Cdh1 in spinal dorsal cord is involved in molecular mechanisms of neuropathic pain conditions. Results Immunostaining showed that Cdh1 was mainly distributed in dorsal horn neurons of the spinal cord in rats. Its expression was downregulated in the ipsilateral dorsal horn at 14 days after spared nerve injury. Rescued expression of Cdh1 in spinal cord by intrathecal administration of recombinant lentivirus encoding Cdh1 (Lenti-Cdh1-GFP) significantly attenuated spared nerve injury-induced mechanical allodynia. Furthermore, rescued expression of spinal Cdh1 significantly reduced surface membrane expression of GluR1, but increased the expression of GluR1-related erythropoietin-producing human hepatocellular receptor A4 and its ligand EphrinA1 in dorsal horn of spared nerve injury-treated animals. Conclusions This study indicates that a downregulation of Cdh1 expression in spinal dorsal horn is involved in molecular mechanisms underlying the maintenance of neuropathic pain. Upregulation of spinal Cdh1 may be a promising approach to treat neuropathic pain.

SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

Evidence has shown that stromal cell-derived factor (SDF-1/CXCL12) plays an important role in maintaining adult neural progenitor cells (NPCs). SDF-1 is also known to enhance recovery by recruiting NPCs to damaged regions and recent studies have revealed that SDF-1α exhibits pleiotropism, thereby differentially affecting NPC subpopulations. In this study, we investigated the role of SDF-1 in in vitro NPC self-renewal, proliferation and differentiation, following treatment with different concentrations of SDF-1 or a CXCR4 antagonist, AMD3100. We observed that AMD3100 inhibited the formation of primary neurospheres. However, SDF-1 and AMD3100 exhibited no effect on proliferation upon inclusion of growth factors in the media. Following growth factor withdrawal, AMD3100 and SDF-1 treatment resulted in differential effects on NPC proliferation. SDF-1, at a concentration of 500ng/ml, resulted in an increase in the relative proportion of oligodendrocytes following growth factor withdrawal-induced differentiation. Using CXCR4 knockout mice, we observed that SDF-1 affected NPC proliferation in the sub-ventricular zone (SVZ). We also investigated the occurrence of differential CXCR4 expression at different stages during lineage progression. These results clearly indicate that signaling interactions between SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.