Research reportMethamphetamine causes widespread apoptosis in the mouse brain: evidence from using an improved TUNEL histochemical method
Introduction
Terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) has been generally used to detect cell death in situ. The DNA strand breaks in apoptotic cells can be identified by labeling free 3′-OH termini with modified nucleotides (fluorescein-labeled dNTP) in a TdT enzymatic reaction [9], [28]. However, this enzymatic reaction is often difficult to conduct on slide-mounted sections. Crosslinked proteins due to fixation and precipitating fixatives may block the exposure of free 3′-OH and enzyme activity [20]. Some of the pretreatments, including detergent (Triton X-100), microwave and proteinase K, have been reported to circumvent some of these obstacles although the resulting observations are not always satisfactory [21], [32], [24]. For example, Triton X-100 was found to be inefficient in improving TUNEL sensitivity at 37°C [21]. It has also been reported that microwave pretreatment can improve sensitivity of the technique without noticeable loss of specificity [21], [32]. Nevertheless, although proteinase K pretreatment has been the most popular method of improving TUNEL sensitivity [24], this approach seems to increase the percentage of false positive signals [8], [30]. Thus, in order to better evaluate the apoptosis-triggering potentials of some neurotoxic agents, we thought it necessary to further improve the sensitivity of the TUNEL method.
Herein, we present evidence that the TUNEL method can indeed be improved. Because brain ischemia is a model known to be associated with apoptotic changes in the brain [15], [26], we used it to test the reliability of the improved method. Having established that the method was reliable, we then used it to assess the distribution of apoptotic changes caused by the illicit drug, methamphetamine (METH). METH can induce degeneration not only in monoaminergic terminals but also cell death in rodent brains [4], [5], [11], [12], [13]. However, the TUNEL technique was not used by other laboratories in order to assess the mode of cell death. Herein, a TUNEL technique with an improved pretreatment was used to display the spatial distribution of METH-induced cell death in the mouse brain.
Section snippets
In vivo neuronal death models
All animal use procedures described below were according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local NIDA Animal Care Committee.
Comparison of TUNEL results on adjacent sections
Two sets of adjacent sections were used in order to test the reliability of TUNEL staining. TUNEL-labeled cells were similarly distributed in adjacent sections after 80°C/Triton X-100 pretreatment (Compare Fig. 1A with B; C with D). This suggests that the method can be reliably replicated.
Comparison of TUNEL results after 80°C/Triton X-100, PBS or microwave pretreatment
No TUNEL-labeled cells were detectable in the brains of sham-operated animals pretreated with 80°C plus Triton X-100 (Fig. 2A), indicating no increase in false positivity. Fig. 2B shows that there were
Discussion
Triton X-100, a stable detergent, has been widely used to increase the permeability of antibody in immunohistochemical studies because it can perforate cellular membrane by dissolving its lipids [4]. However, 37°C/Triton X-100 pretreatment was found to be inefficient in improving TUNEL sensitivity [21]. Hypercondensed DNA and proteins around DNA may explain the need of stronger treatment to expose the broken ends of DNA [20].
The results showed that the 80°C/0.5% Triton X-100 pretreatment
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