Expression of ASIC3 in the Trigeminal Nucleus Caudalis Plays a Role in a Rat Model of Recurrent Migraine.

Acid-sensing ion channel 3 (ASIC3) is abundant in the trigeminal nervous system and is most sensitive to a slight pH decrease. Recent studies have indicated that ASIC3 in the peripheral trigeminal ganglia is likely involved in the pathogenesis of migraine pain. However, it is unclear whether this receptor plays a role in recurrent migraine, namely, migraine chronicity. Here, we aimed to investigate the role of ASIC3 in an animal model of recurrent migraine (RM). In this study, we established a rat model of RM through repeated administration of inflammatory soup (IS) onto the dura. Then, we tested the mechanical pain thresholds of the face and hindpaws by von Frey filaments. qRT-PCR, Western blot and immunofluorescence labelling were used to detect the expression and localization of ASIC3 in the trigeminal nucleus caudalis (TNC). The protein levels of calcitonin gene-related peptide (CGRP), its receptor component receptor activity modifying protein 1 (RAMP1) and c-Fos were analysed following treatment with the ASIC3 inhibitor APETx2 and activator 2-guanidine-4-methylquinazoline (GMQ). We found decreased pain thresholds after repeated dural inflammatory stimulation, which suggested the establishment of an RM model. Based on this model, we observed elevated expression of ASIC3 in the TNC group compared to that in the Sham group. ASIC3 was primarily expressed in neurons but not in astrocytes of the TNC. Moreover, APETx2 attenuated tactile allodynia and significantly decreased the expression of c-Fos, CGRP and RAMP1, while GMQ aggravated these effects compared to those observed in the IS + vehicle group. These findings indicate a critical role of ASIC3 channels in the pathophysiology of RM, and ASIC3 might represent a potential therapeutic target to prevent the progression of migraine.

Protein Kinase C γ Contributes to Central Sensitization in a Rat Model of Chronic Migraine.

Protein kinase C γ (PKCγ) is a critical regulator of central sensitization and is widely recognized to be involved in the pathogenesis of chronic migraine (CM). However, the function of PKCγ in CM remains unknown. This study investigated the role of PKCγ on pathogenesis of CM. We repeated infusions of inflammatory soup (IS) on the intact dura of conscious rats to model recurrent trigeminovascular or dural nociceptor activation assumed to occur in patients with CM. The von Frey test was then used to detect changes in pain threshold. QT-PCR, western blotting, and double immunofluorescence staining were performed to detect the expression and location of PKCγ in the trigeminal nucleus caudalis (TNC) and the expressions of calcitonin gene-related peptide (CGRP), c-Fos, and phosphorylation level of GluR1 subunit at serine 831. Chelerythrine chloride (CHE) and phorbol 12-myristate 13-acetate (PMA) were administrated to investigate the role of PKCγ in central sensitization. We found that repeated infusions of IS induced mechanical allodynia. PKCγ was significantly increased in TNC after CM. Furthermore, inhibition of PKCγ by CHE relieved allodynia and reduced the expression of CGRP and c-Fos. Activation of PKCγ by PMA aggravated allodynia and increased the expression of CGRP and c-Fos. In addition, inhibition of PKCγ reduced the phosphorylation level of GluR1; in contrast, activation of PKCγ increased the phosphorylation level of GluR1. These results suggest PKCγ-induced GluR1 phosphorylation might participate in central sensitization in a rat model of CM. We suggest that PKCγ is a potential therapeutic target for CM.

PTEN inhibition preserves trigeminal nucleus caudalis neuron activation through tyrosine phosphorylation of the NR2B subunit at Tyr1472 of the NMDA receptor in a rat model of recurrent migraine.

OBJECTIVE: Activation of the trigeminal nucleus caudalis is believed to be involved in the pathomechanism of migraine. Evidence suggests that N-methyl-d-aspartate receptor subtype 2B tyrosine phosphorylation, originating from the trigeminal nucleus caudalis neuron dysfunction, might be a triggering mechanism for recurrent migraine. Phosphatase and tensin homolog is thought to have a neuroprotective effect in various neurologic diseases by regulating N-methyl-d-aspartate receptor subtype 2B or tyrosine phosphorylation. Thus, the aim of this study was to explore whether the recombinant adenovirus AdR-siPTEN attenuates neuron activation in the trigeminal nucleus caudalis in a rat model of recurrent migraine. METHODS: Adenovirus-expressing siPTEN or RFP was independently injected into the spinal trigeminal nucleus of the rat model suffering from recurrent migraine by inflammatory soup stimulation the superior sagittal sinus of rats. Seven days later, tactile sensory testing was performed to detect the tactile threshold. Immunofluorescence, Immunohistochemistry, and western blot assay were done to measure PTEN, NR2B, NR2B-pTyr1472, and c-Fos levels in the trigeminal nucleus caudalis of recurrent migraine rats. RESULTS: A significant increase (p < 0.05) in neuron c-Fos content, an indicator of neuron activation, was detected in the trigeminal nucleus caudalis in a rat model of recurrent migraine. However, neuron activation in the trigeminal nucleus caudalis was attenuated by pretreatment with AdR-siPTEN. Moreover, the attenuated effect was potentially mediated by tyrosine phosphorylation of the NR2B-p1472 tyrosine site in the trigeminal nucleus caudalis, as seen in rat brain slices. CONCLUSION: These results suggest that, phosphatase and tensin homolog might be a novel and promising candidate for future treatment or prophylaxis of recurrent migraine by attenuating neuron activation in the trigeminal nucleus caudalis.

Neuroprotective effects of tetrandrine against vascular dementia.

Tetrandrine is one of the major active ingredients in Menispermaceae Stephania tetrandra S. Moore, and has specific therapeutic effects in ischemic cerebrovascular disease. Its use in vascular dementia has not been studied fully. Here, we investigated whether tetrandrine would improve behavioral and cellular impairments in a two-vessel occlusion rat model of chronic vascular dementia. Eight weeks after model establishment, rats were injected intraperitoneally with 10 or 30 mg/kg tetrandrine every other day for 4 weeks. Behavioral assessment in the Morris water maze showed that model rats had longer escape latencies in training trials, and spent less time swimming in the target quadrant in probe trials, than sham-operated rats. However, rats that had received tetrandrine showed shorter escape latencies and longer target quadrant swimming time than untreated model rats. Hematoxylin-eosin and Nissl staining revealed less neuronal necrosis and pathological damage, and more living cells, in the hippocampus of rats treated with tetrandrine than in untreated model rats. Western blot assay showed that interleukin-1ß expression, and phosphorylation of the N-methyl-D-aspartate 2B receptor at tyrosine 1472, were lower in model rats that received tetrandrine than in those that did not. The present findings suggest that tetrandrine may be neuroprotective in chronic vascular dementia by reducing interleukin-1ß expression, N-methyl-D-aspartate receptor 2B phosphorylation at tyrosine 1472, and neuronal necrosis.

Selection and characterisation of Staphylococcus aureus mutants with reduced susceptibility to the investigational oxazolidinone MRX-I.

MRX-I is a new oxazolidinone antimicrobial under development. In this study, the potential for development of resistance to MRX-I in Staphylococcus aureus was investigated and key mutations were characterised. Determination of spontaneous resistance frequency and the mutant selection window (MSW) were performed with meticillin-susceptible S. aureus (MSSA) ATCC 29213, meticillin-resistant S. aureus (MRSA) ATCC 33591 and two clinical MRSA isolates SA 0016 and SA 0017. Selected resistant mutants were sequenced for 23S rRNA as well as genes encoding the ribosomal proteins L3, L4 and L22. Resistance frequencies for the aforementioned strains were <8.25×10(-12), <6.33×10(-12), <2.96×10(-13) and <4.52×10(-13), respectively, and the MSW of MRX-I was 2-4, 1-4, 1-2 and 1-4 mg/L, respectively. After 30 serial passages, MRX-I minimum inhibitory concentrations (MICs) increased up to 8- to 16-fold both against MSSA and MRSA, whilst linezolid MICs increased 128-fold against MSSA and 16- to 32-fold against MRSA. MRX-I resistance mutations were clustered mainly in 23S rRNA and L3 protein regions. The U2504A transversion in 23S rRNA dominated in MRX-I-resistant mutants. No mutations in L4 and L22 proteins were observed. MRX-I exhibits a low potential to develop resistance in S. aureus, with a reduced resistance propensity compared with linezolid.