Intrathecal Injection Of MK-801 Research Articles

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn.

To investigate whether the Chinese massage system, Tuina, exerts analgesic effects in a rat model of chronic constriction injury (CCI) by remodeling the synaptic structure in the spinal cord dorsal horn (SCDH). Sixty-nine male Sprague-Dawley rats were randomly and evenly divided into the normal group, sham group, CCI group, CCI + Tuina group, CCI + MK-801 [an -methyl D-aspartate receptor subtype 2B (NR2B) antagonist] group, and CCI + MK-801 + Tuina group. The neuropathic pain model was established using CCI with right sciatic nerve ligation. Tuina was administered 4 d after CCI surgery, using pressing manipulation for 10 min, once daily. Motor function was observed with the inclined plate test, and pain behaviors were observed by the Von Frey test and acetone spray test. At 19 d after surgery, the L3-L5 spinal cord segments were removed. Glutamate, interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels were detected by enzyme-linked immunosorbent assay. The protein expression levels of NR2B and postsynaptic density protein-95 (PSD-95) were detected by Western blot, and the synaptic structure was observed by transmission electron microscopy (TEM). CCI reduced motor function and caused mechanical and cold allodynia in rats, increased glutamate concentration and TNF-α and IL-1β levels, and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH. TEM revealed that the synaptic structure of SCDH neurons was altered. Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801 ( < 0.05 or < 0.01). For the majority of experiments, no significant differences were found between the CCI + MK-801 and CCI + MK-801 + Tuina groups. Chinese Tuina can alleviate pain by remodeling the synaptic structure, and NR2B and PSD-95 receptors in the SCDH may be among its targets.

Blockade of NMDA receptors decreased spinal microglia activation in bee venom induced acute inflammatory pain in rats

Objectives: Microglial cells in spinal dorsal horn can be activated by nociceptive stimuli and the activated microglial cells release various cytokines enhancing the nociceptive transmission. However, the mechanisms underlying the activation of spinal microglia during nociceptive stimuli have not been well understood. In order to define the role of NMDA receptors in the activation of spinal microglia during nociceptive stimuli, the present study was undertaken to investigate the effect of blockade of NMDA receptors on the spinal microglial activation induced by acute peripheral inflammatory pain in rats.Methods: The acute inflammatory pain was induced by subcutaneous bee venom injection to the plantar surface of hind paw of rats. Spontaneous pain behavior, thermal withdrawal latency and mechanical withdrawal threshold were rated. The expression of specific microglia marker CD11b/c was assayed by immunohistochemistry and western blot.Results: After bee venom treatment, it was found that rats produced a monophasic nociception characterized by constantly lifting and licking the injected hind paws, decreased thermal withdrawal latency and mechanical withdrawal threshold; immunohistochemistry displayed microglia with enlarged cell bodies, thickened, extended cellular processes with few ramifications, small spines, and intensive immunostaining; western blot showed upregulated expression level of CD11b/c within the period of hyperalgesia. Prior intrathecal injection of MK-801, a selective antagonist of NMDA receptors, attenuated the pain behaviors and suppressed up-regulation of CD11b/c induced by bee venom.Conclusion: It can be concluded that NMDA receptors take part in the mediation of spinal microglia activation in bee venom induced peripheral inflammatory pain and hyperalgesia in rats.

EphrinB–EphB signaling regulates spinal pain processing via PKCγ

Spinal ephrinB–EphB signaling is involved in the modulation of pain processing. The aim of the present study was to investigate whether protein kinase C-γ (PKCγ) acts as a downstream effector in regulating spinal pain processing associated with ephrinB–EphB signaling in mice. The intrathecal injection of ephrinB2-Fc, an EphB receptor activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PKCγ. Knockdown of spinal PKCγ prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptor blocker, suppressed formalin-induced inflammatory, chronic constriction injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, in addition to reducing the activation of spinal PKCγ. Finally, the intrathecal injection of MK801, an N-methyl-d-aspartate (NMDA) receptor blocker, prevented the pain behaviors and spinal PKCγ activation induced by ephrinB2-Fc. Overall, the results confirm the important role of PKCγ in the regulation of spinal pain processing associated with ephrinB–EphB signaling.

Roles of spinal N-methyl-D-aspartic acid receptor-extracellular signal-regulated protein kinase 5 signaling pathway in naloxone-induced withdrawal response in morphine-dependent rats

Objective To investigate the roles of spinal N-methyl-D-aspartic acid receptor-extracellular signal-regulated protein kinases 5 signaling pathway in naloxone-induced withdrawal response in morphine-dependent rats. Methods Ninety-six adult male SD rats weighting 230-250 g were randomly divided into 4 groups: control group, withdrawal group, DMSO group and MK801 group. Rats were subcutaneously injected with morphine. On day 6, rats were injected with naloxone (intraperitoneal) to precipitate morphine withdrawal syndromes. To identify the function of NMDAR-ERK5 signaling pathway in morphine withdrawal, morphine withdrawal-like behavior test and western blot technique were used in this research. The scores of morphine withdrawal symptom, morphine withdrawal-induced allodynia and the activation of ERK5 in spinal cord were observed after intrathecal injection of MK801. Results There was no withdrawal symptoms and withdrawal-induced allodynia in group A after intraperitoneal injection of naloxone. Compared with group A, withdrawal score (45.2±7.3), score of withdrawal-induced allodynia (14.4±3.7) of group B, withdrawal score (44.7±6.2), score of withdrawal-induced allodynia (13.2±2.7) of group C and withdrawal score (28.3±1.6), score of withdrawal-induced allodynia (5.9±1.1) of group D were significantly increased (P<0.05). Compared with group C, the total withdrawal score (28.3±1.6), score of withdrawal-induced allodynia (5.9±1.1) of group D were significantly decreased (P<0.05). Compared with group A, the expression of spinal p-ERK5 of group B (12 848±621) and group C (12 579±396) were significantly increased (P<0.05). Compared with group C, the expression of spinal p-ERK5 of group D (5 123±546) was significantly decreased (P<0.05). Conclusion The signaling pathway of spinal N-methyl-D-aspartic acid receptor-extracellular signal-regulated protein kinases 5 contributes to naloxone-induced withdrawal response in morphine-dependent rats. Key words: Morphine dependence; Withdrawal symptoms; Spinal cord; N-methyl-D-aspartic acid receptor; Extracellular signal-regulated kinase 5

Chronological changes of mechanical allodynia and spinal microglia activation by an intrathecal injection of MK-801

The neuropathic pain that occurs after peripheral nerve injury may be related to abnormal central activity. The present experiments investigated the effects of MK-801 [N-methyl-D-aspartate (NMDA) receptor antagonist] on neuropathic pain behaviors and microglial activity in rats. Neuropathic pain was produced by L5 spinal nerve ligation of rats. MK-801 was injected to determine whether spinal microglial activation after nerve injury plays a crucial role in the development and/or maintenance of neuropathy through the NMDA receptor. Mechanical allodynia of the hind paw was examined with von Frey filaments postoperatively. Microglial activity was measured by observing changes in immunoreactivity with a microglia marker, OX-42. The MK-801, at a dose of 3 or 30 µg/5 µl, injection group showed higher neuropathic pain threshold and reduction of microglial activity. These results suggest that neuropathic pain behaviors following L5 spinal nerve ligation may be related to altered activity of the microglia involving the NMDA receptor, and chronological changes of microglial activation by MK-801 are related to maintenance of mechanical allodynia.

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Involvement of Spinal NR2B-Containing NMDA Receptors in Oxaliplatin-Induced Mechanical Allodynia in Rats

BackgroundOxaliplatin is a platinum-based chemotherapy drug characterized by the development of acute and chronic peripheral neuropathies. The chronic neuropathy is a dose-limiting toxicity. We previously reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats. In the present study, we investigated the involvement of NR2B-containing N-methyl-D-aspartate (NMDA) receptors in oxaliplatin-induced mechanical allodynia in rats.ResultsRepeated administration of oxaliplatin (4 mg/kg, i.p., twice a week) caused mechanical allodynia in the fourth week, which was reversed by intrathecal injection of MK-801 (10 nmol) and memantine (1 μmol), NMDA receptor antagonists. Similarly, selective NR2B antagonists Ro25-6981 (300 nmol, i.t.) and ifenprodil (50 mg/kg, p.o.) significantly attenuated the oxaliplatin-induced pain behavior. In addition, the expression of NR2B protein and mRNA in the rat spinal cord was increased by oxaliplatin on Day 25 (late phase) but not on Day 5 (early phase). Moreover, we examined the involvement of nitric oxide synthase (NOS) as a downstream target of NMDA receptor. L-NAME, a non-selective NOS inhibitor, and 7-nitroindazole, a neuronal NOS (nNOS) inhibitor, significantly suppressed the oxaliplatin-induced pain behavior. The intensity of NADPH diaphorase staining, a histochemical marker for NOS, in the superficial layer of spinal dorsal horn was obviously increased by oxaliplatin, and this increased intensity was reversed by intrathecal injection of Ro25-6981.ConclusionThese results indicated that spinal NR2B-containing NMDA receptors are involved in the oxaliplatin-induced mechanical allodynia.

Activation of NMDA Receptor is Associated with Up-regulation of COX-2 Expression in the Spinal Dorsal Horn during Nociceptive Inputs in Rats

Cyclooxygenases-2 (COX-2) in the spinal dorsal horn is up-regulated and plays an important role in pain and hyperalgesia induced by nociceptive stimulation. The mechanisms involved in the up-regulation of spinal COX-2 during nociceptive stimulation are yet not well understood. Because the important role of NMDA and its receptor in transmission of nociceptive information in the spinal cord, activation of the spinal NMDA receptor might contribute to the up-regulation of spinal COX-2 expression. The present study was undertaken to demonstrate the above hypothesis by observing changes of COX-2 expression in the spinal dorsal horn in rats subjected to formalin test and intrathecal administration of NMDA, a selective NMDA receptor agonist, in conditions with or without presence of MK-801, an antagonist of NMDA receptor, using methods of Western blotting, reverse transcription polymerase chain reaction and immunohistochemistry. The results showed that intrathecal injection of MK-801, a noncompetitive antagonist of NMDA receptor, significantly suppressed the up-regulation of the COX-2 expression and characteristic pain behavior responses evoked in formalin test. Whereas, intrathecal injection of NMDA significantly up-regulated the expression of COX-2 in the spinal dorsal horn in a time course corresponding to that of nociceptive behavioral responses elicited by the intrathecal NMDA administration. In addition, the up-regulation of the COX-2 expression induced by the intrathecal NMDA was dose-dependent and blocked by prior administration of MK-801. These findings proved that activation of NMDA receptor is associated with the up-regulation of COX-2 expression in the spinal dorsal horn during nociceptive stimulation in rats.

Effect of intrathecal injection of MK‐801, L‐NNA and morphine on the nociceptive behavioral reaction during inflammatory pain

Effect of intrathecal injection of MK‐801, <i>L</i>‐NNA and morphine on the nociceptive behavioral reaction during inflammatory pain

Spinal Opioid Receptor Like1 Receptor Agonist, but Not N-Methyl-d-Aspartic Acid Antagonist, Reverses the Secondary Mechanical Allodynia Induced by Intradermal Injection of Capsaicin in Rats

Secondary mechanical allodynia induced by intradermal injection of capsaicin has been widely used to search for the underlying mechanisms of tissue injury induced mechanical allodynia. However, the capsaicin concentration dependency of the development of secondary mechanical allodynia and the underlying mechanisms of development and maintenance of capsaicin-induced mechanical allodynia are not fully understood. In the present study, we clarify the capsaicin concentration dependency for development and maintenance of secondary mechanical allodynia and the role of spinal opioid receptor like1 (ORL1) receptor and N-methyl-D-aspartate receptor in the development and maintenance of secondary mechanical allodynia induced by an intradermal capsaicin injection. Capsaicin 50 microL of 0.03% induced the most intense secondary mechanical allodynia. Intrathecal injection of nociceptin, an ORL1 receptor agonist, attenuated the maintenance of secondary mechanical allodynia but had no effect on the development of secondary mechanical allodynia. An intrathecal injection of MK801, an N-methyl-D-aspartate receptor antagonist, had no effect on the development and maintenance of secondary mechanical allodynia. These findings suggest that spinal ORL1 receptor should be the target of study for the treatment of secondary mechanical allodynia induced by tissue injury.

Neurophysiological basis for neurogenic-mediated articular cartilage anabolism alteration.

This study was designed to investigate the pathways involved in neurogenic-mediated articular cartilage damage triggered by a nonsystemic distant subcutaneous or intra-articular inflammation. The cartilage damage was assessed 24 h after subcutaneous or intra-articular complete Freund's adjuvant (CFA) injection measuring patellar proteoglycan (PG) synthesis (ex vivo [Na(2)(35)SO(4)] incorporation) in 96 Wistar rats. Unilateral subcutaneous or intra-articular injection of CFA induced significant decrease (25-29%) in PG synthesis in both patellae. Chronic administration of capsaicin (50 mg. kg(-1). day(-1) during 4 days), which blunted the normal response of C fiber stimulation, prevented the bilateral significant decrease in cartilage synthesis. Similarly, intrathecal injection of MK-801 (10 nmol/day during 5 days), which blocked the glutamatergic synaptic transmission at the dorsal horn of signal originating in primary afferent C fibers, eliminated the CFA-induced PG synthesis decrease in both patellae. Chemical sympathectomy, induced by guanethidine (12.5 mg. kg(-1). day(-1) during 6 wk), also prevented PG synthesis alteration. Finally, compression of the spinal cord at the T3-T5 level had a similar protective effect on the reduction of [Na(2)(35)SO(4)] incorporation. It is concluded that the signal that triggers articular cartilage synthesis damage induced by a distant local inflammation 1) is transmitted through the afferent C fibers, 2) makes glutamatergic synaptic connections with the preganglionic neurons of the sympathetic system, and 3) involves spinal and supraspinal pathways.