Qing-Jun Li

An anti-nociceptive role for ceftriaxone in chronic neuropathic pain in rats

&NA; Glial glutamate transporter‐1 (GLT‐1) plays an essential role in the maintenance of glutamate homeostasis and is involved in the development and maintenance of pathological pain. The present study was undertaken (1) to observe the anti‐nociceptive effects of ceftriaxone (Cef) in a chronic neuropathic pain model induced by chronic constrictive nerve injury (CCI) of the sciatic nerve and (2) to identify the role of spinal GLT‐1 in the process. CCI induced significant thermal hyperalgesia and mechanical allodynia, which began from postoperative day 3 and lasted to day 21. This long‐term hyperalgesia was accompanied by significant down‐regulation of GLT‐1 expression in the L4–L6 segments of the spinal dorsal horn, as revealed by immunohistochemistry and Western blot. Intraperitoneal preventive and therapeutic administration of Cef effectively prevented or reversed, respectively, the development of thermal hyperalgesia, mechanical allodynia, and GLT‐1 down‐regulation in the spinal dorsal horn. To further determine whether the above anti‐nociceptive effects of Cef are a result of the up‐regulation of spinal GLT‐1 expression and its function, we further observed the effects of intrathecal administration of Cef in the same model. It was found that intrathecal administration of Cef led to the specific up‐regulation of GLT‐1 expression and glutamate uptake (3H‐glutamate) in the spinal dorsal horn, and similar anti‐nociceptive effects to those of intraperitoneal administration of Cef. The above effects of intrathecal Cef administration were all significantly inhibited by intrathecal administration of GLT‐1 antisense oligodeoxynucleotides (As‐ODNs). These results indicate that Cef plays an anti‐nociceptive role by up‐regulating spinal GLT‐1 expression and its function.

The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats.

Glial glutamate transporter-1 (GLT-1) plays an essential role in removing glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP), the present study was undertaken to observe in vivo changes in the expression of GLT-1 and glial fibrillary acidic protein (GFAP) in the CA1 hippocampus during the induction of BIT, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of BIT in rats. Immunohistochemistry for GFAP showed that the processes of astrocytes were prolonged after a CIP 2 days before the lethal ischemic insult, which could protect pyramidal neurons in the CA1 hippocampus against delayed neuronal death induced normally by lethal ischemic insult. The prolonged processes extended into the area between the pyramidal neurons and tightly surrounded them. These changes made the pyramidal layer look like a ‘shape grid’. Simultaneously, the prolonged and extended processes showed a great deal of GLT-1. Western blotting analysis showed significant upregulation of GLT-1 expression after the CIP, especially when it was administered 2 days before the subsequent lethal ischemic insult. Neuropathological evaluation by thionin staining showed that DHK dose-dependently blocked the protective role of CIP against delayed neuronal death induced normally by lethal brain ischemia. It might be concluded that the surrounding of pyramidal neurons by astrocytes and upregulation of GLT-1 induced by CIP played an important role in the acquisition of the BIT induced by CIP.

A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1 region of rats

Pharmacologically blocking or stimulating studies have showed the crucial role of adenosine receptors in the protective effect of cerebral ischemic preconditioning (CIP). However, little is know about whether the adenosine receptors are up-regulated in the process. In the present study, changes in expression of adenosine receptors in the CA1 hippocampus after a short CIP in a period of 3 min were investigated in rat four-vessel occluding (4VO) brain ischemic model using immunohistochemistry. The experiments were performed on groups of sham, 4 h, 1, 3, and 7 days (n = 6 in each group) after the CIP. The number and immunostaining density of immunoreactive cells for A1 and A2b adenosine receptors in the CA1 hippocampus were significantly increased after the CIP. For A1 adenosine receptor, the increase occurred in CA1 pyramidal neurons. While for A2b adenosine receptor, the increase occurred in the stratum radiatum of the CA1. The immunoreactive cells for A2b receptor showed distinct morphological characteristics of astrocytes. The increases were consistent in time course (1-7 days) with the development of the ischemic tolerance induced by the CIP. It was concluded that up-regulation of adenosine receptors may also play an important role in the protective effect of CIP.

Limb ischemic preconditioning induces brain ischemic tolerance via p38 MAPK.

It has been reported that limb ischemic preconditioning (LIP) could induce brain ischemic tolerance. In the present study, we investigated the role of p38 MAPK in the induction of brain ischemic tolerance by observing expression of phosphorylated p38 (p-p38) MAPK in the hippocampus after LIP and the effect of p38 MAPK inhibitor SB 203580 on the protection of LIP against delayed neuronal death (DND) in the CA1 hippocampus induced normally by brain ischemic insult. The results of Flow cytometry and Western blotting showed that expression of p-p38 MAPK initially increased at 6 h after LIP compared with sham group in the CA1 hippocampus. The increases reached peak at 12 h and lasted to 24 h after LIP. Expression of p-p38 MAPK was also increased in the CA3/dentate gyrus (DG) regions after LIP, but the beginning and peaking times were 1 and 3 days after LIP, which were relatively later than those in the CA1. Histological evaluation showed that LIP protected the CA1 hippocampal pyramidal neurons against DND induced by global brain ischemic insult for 8 min, suggesting the occurrence of brain ischemic tolerance. Pretreatment with SB 203580 at 30 min before LIP effectively blocked the ischemic tolerance induced by LIP. Together, it could be concluded that activation of p38 MAPK played an important role in the brain ischemic tolerance induced by LIP, and that components of the p38 MAPK cascade might be targets to modify neuronal survival in ischemic tolerance.

The role of nitric oxide in the neuroprotection of limb ischemic preconditioning in rats.

Brief limb ischemia was reported to protect neurons against injury induced by subsequent cerebral ischemia-reperfusion, and this phenomenon is known as limb ischemic preconditioning (LIP). To explore the role of nitric oxide (NO) in neuroprotection of LIP in rats, we observed changes in the content of nitric oxide (NO) and activity of NO synthase (NOS) in the serum and CA1 hippocampus of rats after transient limb ischemic preconditioning (LIP), and the influence of NG-nitro-l-arginine methylester (l-NAME), a NOS inhibitor, on the neuroprotection of LIP against cerebral ischemia-reperfusion injury. Results showed that NO content and NOS activity in serum increased significantly after LIP compared with the sham group. The increase showed a double peak pattern, in which the first one appeared at time 0 (immediate time point) and the second one appeared at 48xa0h after the LIP (Pxa0<xa00.01). The NO content and NOS activity in the CA1 hippocampus in LIP group showed similar change pattern with the changes in the serum, except for the first peak of up-regulation of NO content and NOS activity appeared at 6xa0h after LIP. Pretreatment with l-NAME before LIP blocked the neuroprotection of LIP against subsequent cerebral ischemic insult. The blocking effect of l-NAME was abolished with pretreatment of l-Arg. These findings indicated that NO may be associated with the tolerance of pyramidal cells in the CA1 hippocampus to ischemia induced by LIP in rats.

The role of extracellular signal-regulated kinases in the neuroprotection of limb ischemic preconditioning

To clarify the role of phosphorylated extracellular signal-regulated kinases (pERK1/2) in the neuroprotection of limb ischemic preconditioning (LIP) in rats, we investigated the expression of pERK1/2 using Western blot and flow cytometry in the hippocampus after LIP and the effect of pERK1/2 inhibitor PD 98059 on the neuroprotection of LIP against delayed neuronal death (DND) in the CA1 hippocampus normally induced by severe ischemic insult. It demonstrated that pERK1/2 in the hippocampus increased after LIP. In the CA1 hippocampus, ERK1/2 activation began to increase at 6h and reached peak at 12h after LIP, and decreased to sham level at 5d after LIP. On the other hand, in the CA3/DG, pERK1/2 enhanced at 1d, reached peak at 3d, and lasted to 5d after LIP. Pretreatment with PD 98059 before LIP blocked the neuroprotection of LIP in a dose-dependent manner. These findings supported that the upregulation of pERK1/2 in the CA1 hippocampus contributed to the neuroprotection of LIP against DND normally caused by the brain ischemic insult.

Activation of p38 MAPK participates in brain ischemic tolerance induced by limb ischemic preconditioning by up-regulating HSP 70.

This study investigates whether activation of p38 MAPK by the up-regulation of HSP 70 participates in the induction of brain ischemic tolerance by limb ischemic preconditioning (LIP). Western blot and immunohistochemical assays indicated that p38 MAPK activation occurred earlier than HSP 70 induction in the CA1 region of the hippocampus after LIP. P-p38 MAPK expression was up-regulated at 6h and reached its peak 12h after LIP, while HSP 70 expression was not significantly increased until 1 day and peaked 2 days after LIP. Neuropathological evaluation by thionin staining showed that quercetin (4 ml/kg, 50mg/kg, intraperitoneal injection), an inhibitor of HSP 70, blocked the protective effect of LIP against delayed neuronal death that is normally induced by lethal brain ischemic insult, indicating that HSP 70 participates in the induction of brain ischemic tolerance by LIP. Furthermore, SB 203580, an inhibitor of HSP 70, inhibited HSP 70 activation in the CA1 region of the hippocampus induced by LIP either with or without the presence of subsequent brain ischemic insult. Based on the above results, it can be concluded that activation of p38 MAPK participates in the brain ischemic tolerance induced by LIP at least partly by the up-regulation of HSP 70 expression.

The neuroprotective effect of propofol against brain ischemia mediated by the glutamatergic signaling pathway in rats

Several mechanisms are involved in the neuroprotection of propofol against ischemia, but influences of propofol on the binding properties of glutamate receptors and the uptake of glutamate in brain ischemia are not known. The present study was undertaken to investigate these issues in rat global brain ischemic model using methods of neuropathological evaluation, radioligand binding assay with and uptake test for L-3H-glutamate. It was shown that propofol used in anesthetic doses protected pyramidal neurons in the hippocampal CA1 subfield against delayed neuronal death normally induced by global brain ischemia. Simultaneously, the propofol decreased the value of maximal number of binding sites (Bmax), increased the value of equilibrium dissociation constant (Kd), and increased the glutamate uptake in the CA1 subfield. These findings indicate that it is, at least partly, via modulating the binding properties of glutamate receptors and the uptake of glutamate that propofol protects neurons against ischemic injury.

Fluorocitrate, an Inhibitor of Glial Metabolism, Inhibits the Up-Regulation of NOS Expression, Activity and NO Production in the Spinal Cord Induced by Formalin Test in Rats

Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague–Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I–II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord.

Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways

The present study was undertaken to observe in vivo changes of expression and phosphorylation of ERK1/2 proteins during brain ischemic preconditioning and effects of inhibiting generation of nitric oxide (NO) on the changes to determine the role of ERKs in the involvement of NO participating in the acquired tolerance. Fifty-five Wistar rats were used. Brain ischemic preconditioning was performed with four-vessel occlusion for 3xa0min. Total ERK1/2 proteins and phospho-ERK1/2 in the CA1 hippocampus were assayed with Western immunoblot. Total ERK1/2 proteins did not change in period from 5xa0min to 5xa0days of reperfusion after preconditioning stimulus. While the level of phospho-ERK1/2 increased obviously to 223, 237, 300, 385 and 254% of sham level at times of 5xa0min, 2xa0h, 1, 3 and 5xa0days after preconditioning stimulus, respectively (Pxa0<xa00.01). Administration of L-NAME, an inhibitor of NO synthase, 30xa0min prior to preconditioning stimulus failed to induce change in total ERK1/2 proteins (Pxa0>xa00.05). However, phospho-ERK1/2 increased only to 138 and 176% of sham level at 2xa0h and 3xa0days after preconditioning stimulus, respectively, when animals were pretreated with L-NAME. The magnitudes of the increase were obviously low compared with those (237 and 385%) in animals untreated with L-NAME at corresponding time points (Pxa0<xa00.01), which indicated that phosphorylation of ERK1/2 normally induced by preconditioning stimulus was blocked apparently by administration of L-NAME. The results suggested that phosphorylation of ERK1/2, rather than synthesis of ERK1/2 proteins, was promoted in brain ischemic preconditioning, and that the promotion was partly mediated by NO signal pathway.