Elsevier

Brain Research Bulletin

Volume 130, April 2017, Pages 90-94
Brain Research Bulletin

Research report
Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons

https://doi.org/10.1016/j.brainresbull.2017.01.004Get rights and content

Highlights

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    Viability of cultured cerebellar neurons decreases under streptozotocin toxicity.

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    Pyruvate and insulin attenuate toxic effect of streptozotocin.

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    Streptozotocin increases intracellular calcium in cultured cerebellar neurons.

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    Streptozotocin decreases neuronal mitochondrial membrane potential.

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    Streptozotocin induces ultrastructural alterations in cultured cerebellar neurons.

Abstract

Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer’s disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1–5 mM) in primary culture for 48 h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.

Introduction

Streptozotocin is a glucosamine-nitrosourea compound, particularly toxic to the insulin-producing beta-cells of the pancreas in mammals, applied in medicine for treating certain cancers of the Islets of Langerhans. Intraperitoneal injections of STZ are used in a medical research to produce Type 1 diabetes in animal model (Szkudelski, 2001). In the field of neuroscience and experimental medicine the intracerebroventricular administration of STZ is applied as experimental simulation of sporadic Alzheimer’s disease in vivo during 20 years by now (Mayer et al., 1990). In this model, deficits in hippocampal synaptic transmission and long-term potentiation (LTP) were observed. The decline in LTP correlated with decreased expression of NMDA-receptor subunits NR2A and NR2B. Similarly, there has been a decrease in the expression of brain derived neurotrophic factor (BDNF) (Shonesy et al., 2012). The STZ-intracerebroventricular treated animals developed insulin resistant brain state associated with memory impairment and progressive cholinergic deficits, glucose hypometabolism, tau-hyperphosphorylation, astrogliosis and amyloid angiopathy, oxidative stress and neurodegeneration that share many features in common with sporadic Alzheimer’s disease in humans (Park, 2011, Salkovic-Petrisic et al., 2013, Ejaz Ahmed et al., 2013). The reduction of glucose utilization occurred owing to the decrease of glycolytic enzymatic activities (Plaschke and Hoyer, 1993).

The most part of researches of STZ toxic action in the brain is carried out on the models in vivo. The STZ researches performed on the cultured central nervous system neurons are absent practically. The present study was designed to test the streptozotocin toxic action in primary neuronal cultures in vitro.

Section snippets

Primary cerebellar neuronal cultures

Primary cultures were prepared from the cerebella of 7–8 days old Wistar rats as described elsewhere (Stelmashook et al., 2009). The cerebella were washed with Ca2+- and Mg2+-free PBS (Dulbecco) and incubated in the same solution containing 0.05% trypsin and 0.02% EDTA (15 min, 37 °C). After incubation, the tissue was washed twice in PBS and dissociated by repeated pipetting in a nutrient medium of the following composition: fetal calf serum (10%), minimum essential medium Eagle (90%), glutamine (2

Streptozotocin toxicity in cerebellar granule neurons

Counting of neurons with normal morphology demonstrated that application of STZ (1–5 mM) in culture medium for 48 h with effect from 2.5 mM induced strong dose-dependent CGNs death (Fig. 1). STZ concentrations which cause death more than 50% of neurons have been chosen for further experiments. Under these conditions, the neurons were partially protected from cell death by supplementation of 1 mM pyruvate or 0.01 mM insulin (Fig. 2).

Intracellular calcium ions level and mitochondrial membrane potential

The exposure to STZ (3–4.5 mM) for 5 h induced significantly strong

Discussion

Following the parenteral injection of high doses more 40 mg/kg STZ selectively destroys insulin producing beta cells in the pancreas, causing diabetes type I in adult animals (Szkudelski, 2001). Intracerebroventricular administration of STZ in subdiabetogenic doses (3 mg/kg) to rats or mice is used as experimental simulation of sporadic Alzheimer’s disease in vivo (Ponce-Lopez et al., 2011, Hamidi et al., 2013). Total brain volume is about 769–881 mm3, and lateral ventricles volume is 2.19–2.39 mm3

Conclusions

STZ exposure of cultured neurons causes significantly disturbance of glucose metabolism and mitochondrial function that, in turn, results in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

This work was done with financial support from the Russian Science Foundation (projects No. 16-15-10108). This work was done with the implementation of equipment received under MSU development program PNR5.13.

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