Effects Of Central Cholinergic System On Carotid Sinus Baroreceptor Reflex And Its Mechanism Under Stress State

Objective:To determine the roles of the central cholinergic system in the stress-induced carotid sinus baroreceptor reflex (CSR) resetting and its mechanism.Methods:Sprague?Dawley rats were divided into two groups at random: unstressed (n=48) and stressed groups (n=48). According to the site of microinjection of the selective cholinergic receptor antagonists, each group was subdivided into a group of intracerebroventricular injection (i.c.v.) and a group of microinjection into the nucleus tractus solitarius (NTS). Stressed groups were subjected to unavoidable electric foot-shock twice daily for a week, each session of foot-shock lasted 2 hours and unstressed groups followed by the same handling and environment but no electric stimulation for fake stresss. The left and right carotid sinus regions were isolated from the systemic circulation with the undamaged sinus nerve, cutting off the aortic nerve and off the vagus nerve under anesthesia with pentobarbital sodium in all rats. The artificial cerebrospinal fluid (ACSF), the selective muscarinic M1 cholinoceptor antagonist pirenzepine (PRZ), the selective M2 cholinoceptor antagonist methoctramine (MTR) and the selective nicotinic N1 cholinoceptor antagonist hexamethonium (HEX) was respectively administrated into the lateral ventricle or the NTS which is one of the brain regions of cholinergic fibers projection and cholinoceptors distribution in both stressed and unstressed groups. The ACSF into the lateral ventricle or the NTS in the stressed and unstressed groups was respectively served as a positive control (stressed) and a negative control (unstressed) in all experiment.After the isolated carotid sinus preparation wae conducted, the intracarotid sinus pressure (ISP) was altered in a stepwise manner to trigger CSR from 0 to 280 mmHg at every step of 40 mmHg and 4 sec, and then returned to 0 mmHg in similar steps. ISP and mean femoral arterial pressure (MAP) were recorded simultaneously. ISP-MAP and ISP-Gain relationship curves were constructed by fitting to the logistic function with five parameters. The CSR functional characteristic parameters, the ISP-MAP and the ISP-Gain relationship curves were separately examined and compared statistically in order to observe the effects of central cholinergic system on CSR and its mechanism in mammalian under the stress state.Results:1. The effects of stress on CSR functionIn the stressed and unstressed groups, the ACSF into the lateral ventricle or the NTS did not obviously change each CSR base level, which was respectively served as a positive control (stressed groups) and a negative control (unstressed groups) in all experiment. In comparison with the unstressed CSR baseline and the CSR level of the negative control, the stressed CSR baseline and the CSR level of the positive control showed significant changes that the ISP-MAP relationship curves were remarkably shifted upwards (P<0.05) and the middle parts of ISP-Gain relationship curves were moved downwards (P<0.05), and the value of the MAP range and maximum gain (Gmax) were decreased (P<0.05), but the set point, saturation pressure (SP) and ISP at Gmax (ISPGmax) were increased (P<0.05).2. Effects of the microinjection of different cholinergic receptor antagonists into the lateral cerebroventricle on the changes in CSR induced by stress and comparison between these effects and the corresponding control level of CSR in the unstressed group2.1 Effects of microinjection of different cholinergic receptor antagonists into the lateral ventricle on the CSR level in the unstressed groups. Microinjection of the selective cholinergic receptors antagonists, PRZ or MTR or HEX with given dose into the lateral ventricles in the unstressed groups, had no obvious effects on both ISP-MAP and ISP-Gain relationship curves and CSR functional parameters, respectively (P>0.05).2.2 Effects of the microinjection of different cholinergic receptor antagonists into the lateral cerebroventricle on the changes in CSR induced by stressMicroinjection of the selective muscarinic M1 cholinoceptor antagonist PRZ or the selective M2 cholinoceptor antagonist MTR into the lateral cerebroventricle in the stressed groups with the corresponding doses used in the unstressed groups, significantly diminished the above-mentioned changes in CSR performance induced by stress, which remarkably moved the posterior semi-parts of ISP-MAP relationship curves downwards (P<0.05), shifted the middle parts of ISP-Gain relationship curves upwards (P<0.05), and increased parameters such as the MAP range (only for MTR) and Gmax (P<0.05), but decreased parameters like SP and ISPGmax (P<0.05), compared with the positive control. The alleviative effects of MTR were remarkably stronger than those of PRZ in the stressed group, which significantly shifted the middle parts of ISP-Gain relationship curves upwards (P<0.05), and decreased parameters SP and ISPGmax (P<0.05), but increased parameters Gmax and the MAP range (P<0.05), compared with the stressed group of PRZ. The responses of CSR level in stressed groups to M1 or M2 cholinoceptor antagonists generally occurred 20 min after the administration and lasted approximately for 15 min. Microinjection of the selective nicotinic N1 cholinoceptor antagonist HEX into the lateral cerebroventricle in the stressed group, however, displayed no significant effects on the stressed CSR baseline and the CSR level of the positive control (P>0.05) but exhibited significant difference from the CSR levels in the stressed groups of the MTR and PRZ (P<0.05).2.3 Comparison between the CSR levels treated by i.c.v. different cholinoceptor antagonists in stressed groups and the corresponding control level of CSR in the unstressed groupsMicroinjection of PRZ or MTR or HEX into the lateral cerebroventricle in the stressed groups could not completely abolish the stress-induced changes in CSR (P<0.05).3. Effects of the microinjection of different cholinergic receptor antagonists into the NTS on the changes in CSR induced by stress and comparison between these effects and the corresponding control level of CSR in the unstressed group3.1 Effects of microinjection of different cholinergic receptor antagonists into the NTS on the CSR level in the unstressed groups.Microinjection of the selective cholinergic receptors antagonists, PRZ or MTR or HEX with given dose into the NTS in the unstressed groups, had no obvious effects on both ISP-MAP and ISP-Gain relationship curves and CSR functional parameters, respectively (P>0.05).3.2 Effects of the microinjection of different cholinergic receptor antagonists into the NTS on the changes in CSR induced by stressMicroinjection of the selective muscarinic M1 cholinoceptor antagonist PRZ or the selective M2 cholinoceptor antagonist MTR into the NTS in the stressed groups with the corresponding doses used in the unstressed groups, also significantly diminished the above-mentioned changes in CSR performance induced by stress, which remarkably moved the posterior semi-parts of ISP-MAP relationship curves downwards (P<0.05), shifted the middle parts of ISP-Gain relationship curves upwards (P<0.05), and increased parameters such as the MAP range (only for MTR) and Gmax (P<0.05), but decreased parameters like SP, ISPGmax and set point (only for MTR) (P<0.05), compared with the positive control. The alleviative effects of MTR were also remarkably stronger than those of PRZ in the stressed group, which significantly moved the posterior semi-parts of ISP-MAP relationship curves downwards (P<0.05), shifted the middle parts of ISP-Gain relationship curves upwards (P<0.05), and increased parameters Gmax and the MAP range (P<0.05), compared with the stressed group of PRZ. The responses of CSR level in stressed groups to M1 or M2 cholinoceptor antagonists generally occurred 12 min after the administration and lasted approximately for 20 min. Microinjection of the selective nicotinic N1 cholinoceptor antagonist HEX into the NTS in the stressed group, however, displayed no significant effects on the stressed CSR baseline and the CSR level of the positive control (P>0.05) but exhibited significant difference from the CSR levels in the stressed groups of the MTR and PRZ (P<0.05).3.3 Comparison between the CSR levels treated by different cholinoceptor antagonists into the NTS in stressed groups and the corresponding control level of CSR in the unstressed groupsMicroinjection of PRZ or MTR or HEX into the NTS in the stressed groups could not completely abolish the stress-induced changes in CSR (P<0.05).Conclusion:1. Stress results in a resetting of CSR , a decrease in reflex sensitivity.2. Microinjection of selective cholinergic receptor antagonist PRZ or MTR or HEX into the lateral ventricle or the NTS with given respective dose under the unstressed state, has neither effects on the CSR function and nor on the arterial blood pressure basal level.3. Microinjection of the selective M1 cholinoceptor antagonist PRZ or the selective M2 cholinoceptor antagonist MTR into the lateral ventricle with given respective dose could obviously attenuate the stress-induced CSR inhibitory resetting, and the alleviative effects of MTR are more stronger than those of PRZ in the stressed group, but the selective N1 cholinoceptor antagonist, HEX into the lateral ventricle may have no effects on the CSR resetting resulted from the stress, which suggest that the M1 and M2 cholinoceptors, especially M2 cholinoceptors, in brain ventricle peripheral nuclei may participate in mediating stress-induced CSR resetting.4. Microinjection of the selective M1 cholinoceptor antagonist PRZ or the selective M2 cholinoceptor antagonist MTR into the NTS with given respective dose could also remarkably diminish the stress-induced CSR inhibitory resetting, and the alleviative effects of MTR are more stronger than those of PRZ in the stressed group, but the selective N1 cholinoceptor antagonist, HEX into the NTS may also have no effects on the CSR resetting resulted from the stress, which indicate that the M1 and M2 cholinoceptors, especially M2 cholinoceptors, in the NTS may also involve in regulating stress-induced CSR resetting, and the descending cholinergic pathway from the hypothalamus to NTS may be involved in mechanisms of the stress-inhibited CSR function.5. Microinjection of PRZ or MTR or HEX into the lateral ventricle or the NTS with given respective dose in the stressed state can not completely abolish the stress-induced changes in CSR, which denote that other neuromechanisms may also contribute to effects of the stress on CSR besides central cholinergic system.