Elsevier

Brain Research

Volume 904, Issue 1, 15 June 2001, Pages 67-75
Brain Research

Research report
Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats

https://doi.org/10.1016/S0006-8993(01)02450-7Get rights and content

Abstract

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D3 (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague–Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H2O2 alone caused significant cell death. Pretreatment with D3 (10−10 M) protected VM neurons against 6-OHDA- or H2O2-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H2O2 and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.

Introduction

Numerous studies have indicated that glial cell line derived neurotrophic factor (GDNF), in addition to its trophic effects during development [1], [2], [3], has neuroprotective effects against various types of neuronal insults. Administration of GDNF reduces the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP) and 6-hydroxydopamine (6-OHDA) in animal models of Parkinsonism [4] and reduces ischemia-induced expression of nitric oxide, caspase-3, and TUNEL (+) cells as well as the extent of infarction in cerebral cortex [5], [6]. The expression of GDNF is up-regulated after neuronal injury. GDNF mRNA is increased in striatum after 1-methyl-4-phenylpyridinium (MPP+) lesioning [7] and in cortex after ischemia [8], [9]. Moreover, mRNA for GDNF and for the GDNF receptor α-1 (GFR α1) are upregulated in the sciatic nerve and in the CNS after injury in mice [10], and in hippocampus after systemic administration of kainate acid in rats [11]. The induction of GDNF expression during neuronal damage suggests that this protein maybe an endogenous neuroprotective agent. However, there is still little documentation demonstrating that the upregulation of endogenous GDNF can reduce neurotoxin-induced injury.

Recent studies have indicated that endogenous neurotrophic factors can be regulated by different pharmacological treatments. For example, 1,25-dihydroxyvitamin D3 (D3), an active metabolite of vitamin D, is a potent inducer of GDNF. D3 augments GDNF expression in C6 glioma cells [12] and GDNF release in human U-87 MG glioblastoma cells [13]. D3 also increases nerve growth factor (NGF) [14] and transforming growth factor (TGF)-β2 [15] expression in neuroblastoma cells, and elevates NT3/NT4 mRNA levels in astrocytes [16]. We recently reported that pretreatment with D3 increases GDNF levels in rat brain [17]. Taken together, these data suggest that D3 upregulates GDNF and other trophic factors in vivo and in vitro. Moreover, since D3, unlike GDNF, is able to cross the blood–brain barrier, it is possible that systemic administration of this compound could protect against neuronal injury indirectly via an elevation of endogenous trophic factors in brain.

In the present study, we tested this hypothesis by studying effects of D3 against 6-OHDA toxicity for midbrain dopamine (DA) neurons in vivo and in vitro. It has been shown that 6-OHDA is taken into dopaminergic neurons via high affinity reuptake mechanisms where it is oxidized to produce hydrogen peroxide, superoxide and hydroxyl radicals [18]. This is thought to account for the cytotoxicity of 6-OHDA in these neurons. Given this hypothetical mechanism, we further examined whether D3 inhibits H2O2 and 6-OHDA-induced cell toxicity in vitro.

Section snippets

Animals and chemicals

A total of 32 adult male Sprague–Dawley rats (weight >300 g) were used for in vivo study. These animals were either received daily D3 (n=16, 1 μg/ml×1 ml/kg per day) or saline (n=16, 154 mM NaCl, 1 ml/kg per day) injections intraperitoneally for eight consecutive days. On the seventh day of injection, the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.) and unilaterally injected with 6-OHDA (8 μg/3 μl in normal saline containing 0.2 mg/ml ascorbic acid, RBI, Matick, MA, USA.)

Locomotor activity

Sixteen animals were pretreated with saline and 16 additional rats were pretreated with D3. Animals were individually placed in the behavioral monitors. We found that the average peak locomotor activity, the mean from two peak activity periods, was significantly higher in animals which received D3 treatment, as compared to animals that received saline (Fig. 1, P<0.05, t-test). Peak horizontal activity (P=0.0033, t-test), total distance traveled (P=0.0029, t-test), movement time (P=0.0010, t

Discussion

Previous studies have indicated that 6-OHDA-induced DA bundle lesions produce a very rapid and complete DA axotomy, followed by loss of the DA phenotype and nigral cell death, which can be reversed by GDNF administration [27], [28]. We previously found that exogeneous GDNF restores nigral DA and metabolite levels in 6-OHDA-lesioned rats [29], [30]. These data suggest that GDNF is neuroprotective against 6-OHDA lesioning. In this study, we found that D3 pretreatment enhanced peak locomotor

Acknowledgements

This study was supported by the National Institute on Drug Abuse, USPHS and the National Science Council, Taiwan (NSC89-2320-B016-070; 89-2320-B016-041).

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