دانلود رایگان مقاله تهیه برشهای فرمالین ثابت مغز انسان برای تحقیقات میکروسکوپی الکترونی

Abstract

Ultra-structural analysis of human post-mortem brain tissue is important for investigations into the pathomechanism of neuropsychiatric disorders, especially those lacking alternative models of studying human-specific morphological features. For example, Von Economo Neurons (VENs) mainly located in the anterior cingulate cortex and in the anterior part of the insula, which seem to play a role in a variety of neuropsychiatric conditions, including frontotemporal dementia, autism and schizophrenia, can hardly be studied in nonhuman animals. Accordingly, little is known about the ultra-structural alterations of these neurons, though important research using qualitative stereological methods has revealed that protein expression of the VENs assigns them a role in immune function. Formaldehyde, which is the most common fixative in human pathology, interferes with the immunoreactivity of the tissue, possibly leading to unreliable results. Therefore, a method for ultra-structural investigations independent of antigenic properties of the fixated tissue is needed. Here, we propose an approach using electron microscopy to examine cytoskeletal structures, synapses and mitochondria in these cells. We also show that our methodology is able to keep tissue consumption to a minimum, while still allowing for the specimens to be handled with ease by using agar embedded slices in contrast to blocks for the embedding procedure. Accordingly, a stepwise protocol utilising 60 μm thick human post mortem brain sections for electron microscopic ultra-structural investigations is presented.

3. Results and discussion

In human pathology, the post-mortem interval is a relevant factor for tissue preservation. It is known that degenerative alterations in the cytoplasm of glial cells and within the axoplasm can occur within 24 h at 4 ◦C, and especially mitochondria are known to be vulnerable to degeneration. The same degenerative effects already appear after 6 h at a storage temperature of 25 ◦C (Hukkanen and Röyttä, 1987). In our study, the tissue had a mean post-mortem interval of 31.89 ± 9.649 h and showed the typical ultra-structural alterations of network-like splitting of the inner part of the myelin sheath (Fig. 3A), as described by Hukkanen and Röyttä (1987). However, other structures such as microtubules, neurofilaments, synapses, vesicles and mitochondria were well preserved (Fig. 3B–D). In particular, entire neurons such as VENs, which are currently of most interest in investigations of psychiatric disorders, could be depicted clearly in a collage of merged micrographs (Fig. 3E). Themost commonfixative inhumanpathology is formaldehyde. Its tissue penetration speed averages from 0.5 to 1 mm/h and so the post-mortem interval is additively extended until the fixation process is completed. However, formaldehyde stabilises the secondary structure of proteins and makes them thermo-stable against high temperatures (Mason and O’Leary, 1991). Unfortunately, the intraand intermolecular cross-linking that confers this thermo-stability also results inadecreasedimmunoreactivity ofthe tissue (Rait et al., 2004). For human post-mortem brain tissue that has been treated with formaldehyde for several weeks, it can be assumed that the antigenicity is affected, and that any immunohistochemical results may be unreliable. Hence, electron microscopic investigations are an alternative option for conducting detailed morphological ultrastructural examinations.