Brain slice viability determined under normoxic and oxidative stress conditions: involvement of slice quantity in the medium

ABSTRACT

Objective: In vitro acute adult brain slice methods are instruments in developing our knowledge of the nervous system. Optimization of this method for obtaining high-quality brain slices is extremely important in terms of consistency and reliability of the experimental results. Although some important topics such as slice thickness, temperature, and composition of the physiological medium have been studied for optimization, involvement of slice quantity in medium on tissue viability has not been investigated yet.

Methods: Different number of slices (1, 3, or 6 slices) were incubated under normoxic or some prooxidant stress conditions induced by oxygen–glucose deprivation (OGD), H₂O₂, FeSO₄+ ascorbic acid, or menadione to evaluate the effect of slice density on tissue viability.

Results:Slice quantity in the normoxic incubation medium caused a significant increase in 2,3,5-triphenyltetrazolium chloride (TTC) staining intensity of the slices. Similarly, increase in the slice quantity in the medium also protected the slices against either OGD, H₂O₂, FeSO_4, or menadione-induced decrease in TTC staining. In addition to TTC staining, lactate dehydrogenase leakage or malondialdehyde and reactive oxygen species production under normoxic or ischemia-like conditions were also attenuated by increasing slice quantity in the medium.

Conclusion: These results show that when using brain slices method for investigating the structural and functional features of brain at the molecular and cellular levels, both slice quantity in the medium and incubation volume should be considered first. Increasing slice quantity or decreasing incubation volume probably causes an increase in the concentration of endogenous substance(s) involved in neuroprotection.

KEYWORDS:

• Brain slices
• incubation volume
• slice quantity
• oxidative stress
• TTC staining

Acknowledgments

The authors would like to thank Gökçe Kızılkaya at School of Foreign Languages, Bahcesehir University for editing the grammar and spelling of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by a grant from the Uludag University Research Foundation [HDP(T)-2014/33].

Notes on contributors

Zulfiye Gul

Zulfiye GUL is assistant professor at Department of Medical Pharmacology, Bahcesehir University. She graduated from Istanbul University Faculty of Pharmacy with a B.Sc degree in Pharmacy in 2008 and completed her Ph.D. degrees on Pharmacology and Clinical Pharmacology at Uludag University Faculty of Medicine in 2017. Her research interests are based on oxidative stress, neurodegenerative diseases and neurotransmitter signaling. Her aim with this article is to present important implications about the slice density and incubation volume for optimizing the brain slice incubation method in addition to frequently researched topics such as slice thickness, temperature, or composition of the physiological medium.

M. Cagatay Buyukuysal

M. Cagatay Buyukuysal is assistant professor in Biostatistics at Zonguldak Bülent Ecevit University. His research and scholarship have mainly focused on biased and non-biased regression models. Buyukuysal is the biostatistics editor of national journals indexed at Ulakbim and local indexis such as Medical Journal of Western Black Sea, Turkish Journal of Radiology and Turkish Journal of Diabetes and Obesity. In addition to these Buyukuysal has published articles in leading journals. His aim with this article is to share personal experiences. By referring to his own previous publications he intends to further contextualise these findings. In this article I, together with Zulfiye Gul, take a broader approach and refer to other research equally relevant for this text. .

R. Levent Buyukuysal

R. Levent Buyukuysal is professor in Medical Pharmacology at Uludag University. His research has mainly focused on neurotransmitter signaling in physiologic and pathologic conditions. Buyukuysal has published over fifty articles and chapters in leading journals and edited book volumes. His aim with this article is to define the new important factor of brain slices methods and share personal experiences. By referring to his own previous publications and other research, he intends to further summarise considerable factors that affect the application of brain slices method.