Darstellung apoptotischer und nekrotischer Rattenhepatozyten im Gewebeschnitt nach verschiedenen Fixierungsverfahren und mittels unterschiedlicher Nachweismethoden
Abstract
1. Detection of apoptotic cells and bodies in rat liver was determined by hematoxylin and eosin (H&E)-staining (standard and alcoholic H&E), terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL method), and eosin fluorescence. We used the apoptotic induction model of BURSCH et al. (1984; 1985; 1986). The tissue samples were fixed over various time periods between 1 and 92 days. Three fixation methods were used: formaldehyde only, one day formaldehyde fixation followed by a 70% ethanol fixation, or fixation in carnoy solution. Necrotic cells or other positive structures which could be mistaken for apoptotic hepatocytes were counted as a control for specifity. 2. Standard H&E- and alcoholic H&E-staining To avoid individual changes in the results, the use of standard light microscopy in combination with standard H&E-staining after formaldehyde fixation only is recommended. Based on morphological characteristics, both, routine (one day) and long term (several weeks) fixation with 4% formaldehyde achieved the best apoptotic indices with the lowest variety.
The data obtained after the fixation method with formaldehyde followed by ethanol are less reliable than after use of pure formaldehyde fixation.
Carnoy fixation followed by alcoholic H&E staining showed only very weak staining and was therefore not useful for rapid and specific determination of apoptotic rates by standard light microscopy. 3. Terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL)
Compared to AI obtained by H&E staining and standard light microscopy the AI measured with the TUNEL method were higher but also had bigger variations. With reduced amount of TUNEL-positive apoptotic cells and bodies accompanied by reduced intensity of the staining the use of TUNEL staining for material fixed over longer time periods seems to result in less reliable data. Non-hepatocytes and 22%-40% of the necrotic cells were TUNEL positive and showed intense staining. In conclusion, the TUNEL method in combination with an image processing and analysis system is only useful in combination with morphologic control of positive signals.
The highest AI were determined with fixation of the material in formaldehyde followed by ethanol. The 95% confidence interval, however, was broader than after formaldehyde fixation only, thus the data less reliable. 4. Eosin fluorescence Using the eosin fluorescence technique for estimating apoptotic indices yielded AI quotients (AI treated animals divided by AI of untreated animals) with lower standard deviations compared to the AI quotients calculated from the data obtained via TUNEL staining. After one day of fixation the data obtained via the eosin fluorescence was similar to the data determined by using the H&E staining. However, our study showed that with the eosin fluorescence a reduction of AI is already happening after a fixation period of 7 days. After longer tissue fixation period the AI was reduced in higher values. Easy and fast recognition of apoptotic bodies with the fluorescence method after a short time fixation is still an advantage of this method (STINCHCOMBE, 1996). Necrotic cells showed fluorescence, too. Additionally, we found fluorescing structures (FS) in the tissue that could not be related to an apoptotic event. A morphologic control with H&E is nearly impossible because of the very weak tissue staining due to the alcoholic H&E staining method. In conclusion it leads to the recommendation to use the fluorescence technique only in tissue fixed for less than 7 days.
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