| Objective:Hypertension is characterized by high level of blood pressure and sympathetic nerve activity, which is closely related to its development and prognosis. It has been proposed that trigeminal neuropathic pain, a chronic nociceptive stimulation to the body, can increase risk of hypertension and its related complications. However, the mechanism by which Trigeminal neuropathic pain increases blood pressure and sympathetic activity is poorly understood. The enhanced glutamatergic input to the rostral ventrolateral medulla (RVLM), which is a key region involved in sympathetic outflow, seems to be an important mechanism of sympathetic hyperactivity and blood pressure elevation in pathophysiolgoical conditions. Therefore, the overall goal of the study is to determine the mechanism and the significance of tonic glutamatergic input to the RVLM in mediating sympathetic overactivity induced by neuropathic pain.Methods:1. Sprague-Dawley (SD) rats with a body weight of 220 g-260 g, were randomly divided into control group (control group), sham operation group (sham group) and model group (trigeminal neuralgia, TN (ION-CCI) group).The TN (ION-CCI) group received bilateral infraorbital nerve chronic constriction surgery, the sham group only underwent exposure of the infraorbital nerve and the control group didn’t receive any surgery or anesthesia. Von-Frey filaments were used to assess the mechanical pain threshold of rats at the same time every five days after the surgery.2. To determine the change of sympathetic activity and blood pressure (BP), the non-invasive rat tail arterial pressure measuring instrument was used to record systemic arterial pressure and heart rate (HR) of conscious rats. The BP and HR under anesthetized condition were measured by femoral arterial catheter. The level of norepinephrine (NE) in 24 h urinary which could define the sympathetic activity was detected by High Performance Liquid Chromatography (HPLC).3. To determine the effect of trigeminal neuropathic pain on glutamate release in the RVLM, RVLM microdialysis and utilizing HPLC were used to measure the concentration of glutamate in dialysate samples.4. The level of NMDAR1 protein expression in RVLM and the level of GLS2, VGLUT2 protein expression in PAG and PVN were determined by western blot.5. C-fos immunostaining was used as a marker of neuron activity. C-fos-positive cells in the periaqueductal gray (PAG), paraventricular nucleus (PVN) and RVLM were plotted by immunohistochemistry.6.Effect of bilateral microinjections of KYN, which is the antagonist of the GluR, into the RVLM on BP, HR and RSNA were observed to determine whether the glutamatergic synaptic transmission played an important role in tonically maintaining the basal cardiovascular activity.Results:1. ION-CCI induced hyperalgesia in rats and had a significant increase on the blood pressure.Compared with control group and sham group, the mechanical pain threshold of rats in TN (ION-CCI) group was significantly decreased. As for the TN (ION-CCI) group rats, MAPs on day 5 (PO5)-20 (PO20) after surgery were significantly higher than that of sham rats and control rats.And increments of MAP in TN (ION-CCI) group rats were most apparent. We also found that the MAP of ION-CCI rats, which was measured through right femoral artery in anesthetic condition, was significantly higher than that of sham rats and control rats 10 days after surgery.2. Effects of ION-CCI on 24 h urinary excretion of NE and RSNAThe 24 h urinary excretion of NE and RSNA were measured on Day 10 after ION-CCI surgery and sham surgery. The 24 h urinary excretion of NE was increased significantly in ION-CCI group compared with the sham group. Meanwhile, the RSNA data was similar to the NE results, and the excitability of ION-CCI group was predominantly higher than the sham group.3. Effects of ION-CCI on the release of glutamate and the expression of NMDAR1 protein in RVLMThe concentration of glutamate in the microdialysis fluid was significantly higher in ION-CCI group compared with sham rats, and started to increase on Day 10 after ION-CCI. Although this increment lasted up to PO30, the value on PO10 reached a peak. However, the concentration of glutamate in sham group kept almost the same level at different time points. Moreover, the level of NMDAR1 on RVLM was also observed, and we found its expression in ION-CCI group was higher than sham group.4. Effect of bilateral microinjections of KYN into the RVLM on BP, HR and RSNAIn sham group, bilateral microinjections of KYN into the RVLM had no effect on resting BP, HR, and RSNA.However, bilateral injection of KYN into the RVLM produced a significant decrease of BP, HR, and RSNA in TN (ION-CCI) group rats.5. ION-CCI had effects on the content of glutamate and the protein expressions of VGLUT2, GLS2 in both PAG and PVNIn the TN (ION-CCI) group, the glutamate concentrations in PAG and PVN are both significantly higher than those of sham group. Moreover, in both PAG and PVN the VGLUT2 levels elevated in ION-CCI group compared with sham group. Levels of GLS2 in the PAG and PVN were also significantly increased in TN (ION-CCI) group.6. ION-CCI had an influence on the activity of neuron in PAG, PVN and RVLM.The TN (ION-CCI) group rats evoked an obvious increase in fos-expression in PAG, PVN and RVLM. The number of fos-positive cells in the PAG was four folds of that in sham group. It should be noted that Fos-positive neurons in TN (ION-CCI) group were much more than that in sham group in PVN area. That is, there were approximately four times the numbers of Fos-positive neurons cells in TN (ION-CCI) group versus the sham group in RVLM.Conclusion:(1) Neuropathic pain effectively increased BP and sympathetic activity in SD rats, and this effect could reach a peak on PO10. (2) Neuropathic pain significantly increased the release of glutamate in the RVLM and enhanced the tonic glutamatergic inputs in the RVLM. (3) The tonic glutamatergic inputs in RVLM mainlyderived from PAG and PVN. |
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