Mechanisms of neuroprotection and retain the function of central nervous system during hibernation in Syrian hamsters

采用原代培养的仓鼠海马神经元，研究了冬眠调节物质（HRS）如腺苷（ADO）、阿片类药物、组胺和促甲状腺激素释放激素（TRH）对低温诱导细胞死亡（LTCD）的保护作用。在<22℃条件下培养7 d，可诱导LTCD。ADO （10-100 μM）对培养的LTCD神经元具有剂量依赖性。8-环戊基茶碱（CPT， A_1受体拮抗剂）和3,7-二甲基-1-丙基鸟嘌呤（DMPX， A_2受体拮抗剂）均可逆转ADO的神经保护作用。吗啡（一种非选择性阿片受体激动剂）在10-100 μM的体外剂量范围内也能有效地减弱LTCD。吗啡的神经保护作用被纳洛酮（一种非选择性阿片受体拮抗剂）所拮抗。此外，虽然[D-Ala^2， N-Me-Phe^4， Gly-ol^5]-脑啡肽（DAMGO； μ-阿片受体激动剂），[D-Pen^<25 b>]-脑啡肽（DPDPE； δ-阿片受体激动剂…More st）和U-69593 （k-阿片受体激动剂）也有效，但TRH对培养海马神经元的LTCD没有影响。此外，组胺在叙利亚仓鼠体内产生低温，并在体外100 μM下保护海马神经元。组胺的神经保护作用被吡拉胺（H_1受体拮抗剂）逆转。LTCD可能与细胞凋亡有关。这些结果表明，ADO通过对A_1和A_2受体的激动作用来保护体外海马神经元，而吗啡可能通过对μ-、δ-和k-阿片受体的激动作用来激发其神经保护作用。此外，组胺还通过对H_1受体的激动作用保护海马神经元。因此，hrs样腺苷、阿片样和组胺样的低温作用很可能在体外诱导LTCD的神经保护作用。内源性阿片肽参与叙利亚仓鼠冬眠期间体温（Tb）的中央调节系统。根据Tb的变化将其分为i)进入期、ii)维持期和iii)觉醒期。在之前的研究中，我们证实了阿片中枢系统在维持阶段参与结核病调控系统。采用亲和素-生物素-过氧化物酶法对c-Fos和β-内啡肽进行免疫染色。脑室内注射纳洛唑嗪（μ1-阿片受体拮抗剂）能有效唤醒仓鼠。相比之下，10 nM的纳曲多（δ-阿片受体拮抗剂）和非bni （k-阿片受体拮抗剂）都没有中断麻木。体外注射抗β-内啡肽抗体能有效阻断睡眠，而抗内啡肽-1和内啡肽-2抗体无效。睡后1小时，大鼠弓形核（ARC） c-Fos阳性神经元数量较睡前增加。此外，虽然POMC mRNA的表达水平没有变化，但内源性蛋白酶-2 mRNA在ARC中表达水平升高。在ARC中观察Β-Endorphin-like免疫反应性（β-末端IR）。随着冬眠的进行，β-末端IR随ARC降低，随血管终板器官（OVLT）升高。这些结果表明β-内啡肽通过轴突流从ARC转运到OVLT，并通过μ1-阿片受体调节冬眠维持期体温。少

The neuroprotective effects of hibernation-regulating substances (HRS) such as adenosine (ADO), opioids, histamine and thyrotropin-releasing hormone (TRH) on low temperature-induced cell death (LTCD) were examined using primary cultured hamster hippocampal neurons. LTCD was induced when cultures were maintained at <22℃ for 7 days. ADO (10-100 μM) protected cultured neurons from LTCD in a dose-dependent manner. The neuroprotective effects of ADO were reversed by both 8-cyclopenthyltheophilline (CPT, A_1 receptor antagonist) and 3,7-dimethyl-1-propargybcanthine (DMPX; A_2 receptor antagonist). Morphine (a non-selective opioid receptor agonist) was also effective in attenuating LTCD at an in vitro dose-range of 10-100 μM. The neuroprotective effects of morphine were antagonized by naloxone (a non-selective opioid receptor antagonist). In addition, although [D-Ala^2, N-Me-Phe^4, Gly-ol^5]-enkephalin (DAMGO; μ-opioid receptor agonist), [D-Pen^<25>]-enkephalin (DPDPE; δ-opioid receptor agoni … More st) and U-69593 (k-opioid receptor agonist) were also effective, LTCD of cultured hippocampal neurons was not affected by TRH. Furthermore, histamine produced hypothermia in Syrian hamsters and protected hippocampal neurons in vitro at 100 μM. The neuroprotective effect of histamine was reversed by pyrilamine (H_1 receptor antagonist). Apoptosis was probably involved in LTCD. These results suggest that ADO protected hippocampal neurons in vitro via its agonistic actions on both A_1 and A_2 receptors, while morphine probably elicited its neuroprotective effects via agonistic effects on the μ-, δ- and k-opioid receptors. In addition, histamine also protected hippocampal neurons via its agonistic action on the H_1 receptor. Thus, HRS-like adenosine-, opioid- and histamine-like hypothermic actions would most likely induce neuroprotective effects against LTCD in vitro.Endogenous opioid peptides involved in the central regulatory system of body temperature (Tb) during hibernation in Syrian hamsters. The torpor was classified into i) entrance, ii) maintenance and iii) arousal phases according to Tb changes. In previous study, we demonstrated that central opioid system involved in the Tb regulatory system during maintenance phase. Immunostaining of c-Fos and β-endorphin was performed by using avidin-biotin-peroxidase procedure. Intracerebroventricular (icv) injection of naloxonazine (μ1-opioid receptor antagonist) was effective in arousing hamster from torpor. In contrast, neither 10 nM of naltrindole (δ-opioid receptor antagonist) nor nor-BNI (k-opioid receptor antagonist) interrupted the torpor. Moreover, icv injection of anti-β-endorphin antibody was effective in interrupting the torpor, but anti-endomorphin-1 and-2 antibodies were ineffective. The number of c-Fos positive neuron increased in arcuate nucleus (ARC) at 1 hr after torpor onset compared with before torpor. Furthermore, although the expression level of proopiomelanocortin (POMC) mRNA did not change, the endoprotease-2 mRNA incresed in ARC. Β-Endorphin-like immunoreactivity (β-End IR) was observed in ARC. With progress of hibernation, the β-End IR decreased with ARC and increased with organum vasculosum lamina terminalis (OVLT) . These results suggest that the β-endorphin was transported to OVLT from ARC by axonal flow and then regulates body temperature during maintenance phase of torpor via μ1-opioid receptors. Less