Rusnak M, House S B, Gainer H
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
J Neuroendocrinol. 2003 Oct;15(10):933-9. doi: 10.1046/j.1365-2826.2003.01080.x.
The use of hypothalamic organotypic cultures for the long-term study of mechanisms in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system has been limited by the relatively poor maintenance of the vasopressin MCNs in vitro. Recent studies have shown that addition of ciliary neurotrophic factor (CNTF) to the media significantly reduced the apoptosis of both oxytocin and vasopressin MCNs. Here, we studied various temporal factors in the CNTF treatment that can influence the efficacy of MCN survival. Immunohistochemistry was used to identify and count surviving vasopressin and oxytocin MCNs in the supraoptic nucleus (SON) in hypothalamic slices cultured in the presence of CNTF (10 ng/ml media) for various time intervals, and in situ hybridization for vasopressin mRNA was used to evaluate the vasopressin mRNA gene expression in the SON under the same conditions. The presence of CNTF in the medium for 10 days produced a maximal increase in the survival of vasopressin MCNs (by 11-fold) and in the survival of oxytocin-MCNs (by approximately four-fold) over controls. These effects persisted for an additional 7-10 days even in the absence of CNTF. The ability of CNTF to increase survival of the MCNs or increase vasopressin mRNA levels in the SON required that the CNTF be present during the initial 7-10 days of culture. CNTF failed to rescue vasopressin or oxytocin MCNs when added to the media only for the last 7 days of a total of 14 days in vitro. Similar results were observed when SON vasopressin mRNA levels were measured. These results indicate that the presence of CNTF is required at the outset to rescue the vasopressin and oxytocin MCN from axotomy induced apoptosis, and that, after 10 days in CNTF, the MCNs no longer require the CNTF for survival.
下丘脑器官型培养用于下丘脑 - 神经垂体系统大细胞神经元（MCNs）机制的长期研究，一直受到体外抗利尿激素MCNs维持相对较差的限制。最近的研究表明，向培养基中添加睫状神经营养因子（CNTF）可显著减少催产素和抗利尿激素MCNs的凋亡。在此，我们研究了CNTF处理中可影响MCNs存活效力的各种时间因素。采用免疫组织化学法鉴定并计数在含有CNTF（10 ng/ml培养基）的条件下培养不同时间间隔的下丘脑切片视上核（SON）中存活的抗利尿激素和催产素MCNs，并采用抗利尿激素mRNA原位杂交法评估相同条件下SON中抗利尿激素mRNA基因的表达。培养基中存在CNTF 10天，抗利尿激素MCNs的存活率（提高了11倍）和催产素MCNs的存活率（提高了约4倍）相对于对照组达到最大增加。即使在没有CNTF的情况下，这些效应仍持续另外7 - 10天。CNTF增加MCNs存活率或提高SON中抗利尿激素mRNA水平的能力要求在培养的最初7 - 10天存在CNTF。当仅在总共14天体外培养的最后7天向培养基中添加CNTF时，无法挽救抗利尿激素或催产素MCNs。在测量SON抗利尿激素mRNA水平时观察到类似结果。这些结果表明，一开始就需要CNTF来挽救抗利尿激素和催产素MCNs免受轴突切断诱导的凋亡，并且在CNTF中培养10天后，MCNs不再需要CNTF来维持存活。
