Eastwood S L, Harrison P J
University Department of Psychiatry, Warneford Hospital, Oxford, UK.
Neuroscience. 1999;93(1):99-106. doi: 10.1016/s0306-4522(99)00096-2.
Most in situ hybridization histochemistry studies of messenger RNA in human brain have been carried out on frozen tissue. Recently, autoclaving has been reported to enable routinely processsed material to be used for in situ localization of messenger RNA. We have investigated whether autoclaving also permits in situ hybridization histochemistry to be used quantitatively. To do this, we targeted synaptophysin messenger RNA with a 35S-labelled oligonucleotide probe in autoclaved, formalin-fixed, paraffin wax-embedded sections of the hippocampal formation of 11 schizophrenics and 11 controls. We compared the results with those seen on frozen sections from adjacent blocks, which had been used previously to demonstrate a loss of the messenger RNA in schizophrenia. Synaptophysin messenger RNA was readily detected in the autoclaved sections. The hybridization signal correlated strongly with that seen in the frozen sections. We found a similar pattern and magnitude of decreased synaptophysin messenger RNA in schizophrenia in the autoclaved sections as we had in the frozen sections, including the selective preservation of synaptophysin messenger RNA in CA1. The reduction of synaptophysin messenger RNA was replicated when six subjects with schizophrenia not included in the earlier study were considered separately. We conclude that autoclaving renders formalin-fixed, paraffin wax-embedded sections of human brain suitable for quantitative in situ hybridization histochemistry. This has considerable implications, given the wider availability, better morphology and easier handling of fixed than frozen human brain tissue. Using this material, we confirmed the finding of decreased synaptophysin messenger RNA in the hippocampal formation in schizophrenia, furthering the evidence for synaptic pathology in this region in the disorder.
大多数关于人脑中信使核糖核酸的原位杂交组织化学研究都是在冷冻组织上进行的。最近，有报道称高压灭菌能够使常规处理的材料用于信使核糖核酸的原位定位。我们研究了高压灭菌是否也能使原位杂交组织化学用于定量分析。为此，我们用35S标记的寡核苷酸探针靶向突触素信使核糖核酸，该探针应用于11名精神分裂症患者和11名对照者海马结构经高压灭菌、福尔马林固定、石蜡包埋的切片中。我们将结果与相邻组织块冷冻切片的结果进行比较，这些冷冻切片此前已被用于证明精神分裂症中信使核糖核酸的缺失。在经高压灭菌的切片中很容易检测到突触素信使核糖核酸。杂交信号与冷冻切片中的信号高度相关。我们发现在经高压灭菌的切片中，精神分裂症患者突触素信使核糖核酸减少的模式和程度与冷冻切片中相似，包括CA1区突触素信使核糖核酸的选择性保留。当单独考虑早期研究未纳入的6名精神分裂症患者时，突触素信使核糖核酸的减少得以重现。我们得出结论，高压灭菌使人类大脑经福尔马林固定、石蜡包埋的切片适用于定量原位杂交组织化学。鉴于固定的人类脑组织比冷冻的更容易获得、形态更好且处理更方便，这具有相当重要的意义。利用这种材料，我们证实了精神分裂症患者海马结构中突触素信使核糖核酸减少的发现，进一步证明了该疾病中该区域存在突触病理学证据。
