Staining of the rat cortical slices with 2,3,5 triphenyltetrazolium chloride (ttc) either in normoxic and ischemic conditions: Effect of incubation conditons on staining of the slices


Thesis Type: Doctorate

Institution Of The Thesis: Bursa Uludağ University, Sağlık Bilimleri Enstitüsü, Turkey

Approval Date: 2017

Thesis Language: Turkish

Student: Zülfiye Gül

Supervisor: RİFAT LEVENT BÜYÜKUYSAL

Abstract:

Objective: The accurate quantitation of tissue damage produced under in vivo or vitro conditions is important to the investigation of injury mechanisms and therapies. Although 2,3,5 triphenyltetrazolium chloride (TTC), a marker of the mitochondrial enzyme activity, is widely used to assess the effects of cerebral ischemia, many controversial results, probably due to experimental models used, are easily seen in the literature. In present study we aimed to demonstrate whether number of the brain slices in the medium alters their staining intensities with TTC and if present, whether changes in the other parameters related to tissue damage also show a close similarity under in vitro conditions. Methods: 1, 3, 6 cortical slices (0.4 mm) prepared from female Sprague Dawley rats were placed into the incubation plates containing 2 ml oxygenated physiological medium. After 60 min of preincubation period, cortical slices were incubated either in normoxic or four different oxidative stress conditions; namely in vitro ischemia or H2O2, (1 mM), FeSO4 (0.1 mM) + ascorbic acid or menadione (1 mM) models. At the end of each oxidative stress models, slices were stained with 0.5% TTC. Tissue MDA and ROS levels and the leakage of LDH into the medium were also studied for determination of the tissue damage occurred under these experimental conditions. Results: When incubated in normoxic medium, staining intensities of the slices were highly correlated with the number of slices in the medium. Similarly, other parameters related to tissue damage were also affected in a positive way by the increasing the slice number in the medium. Whereas all oxidative stress models used here caused significant declines in TTC staining intensities in one slice containing group, neither in vitro ischemia nor other models-induced declines reach to statistically significant level in six slices containing group. Alterations in LDH leakage from the slices and tissue MDA ROS levels, on the other hand, were found to be highly correlated with the changes observed in TTC staining intensities.Conclusion: These results clearly indicate that experimental conditions such as slices quantity or incubation volume significantly alters the results observed under normoxic or ischemia-like conditions. Although the mechanism(s) remained to be determined, increasing the slice quantity or decreasing the incubation volume probably causes an increase in the concentration of endogenous substance(s) in the medium which are probably involved in neuroprotection as seen in present study.