Effect of Buyang Huanwu decoction on amino acid content in cerebrospinal fluid of rats during ischemic/reperfusion injury

Highlights

• Brain tissues of rats with MCAO displayed an infarct lesion and nerve injuries.

• We used brain microdialysis and HPLC-FD to moniter the levels of amino acids.

• The excitatory and inhibitory amino acids Increased in the CSF of ischemic rats.

• Treatment BYHWD improved nerve function and reduced the infarct volume.

• Treatment BYHWD significantly decreased EAAs and increased IAAs in the CSF.

Abstract

The inhibitory effect of Buyang Huanwu decoction (BYHWD) on ischemic injury has been proven, but it is not clear how amino acid levels in cerebrospinal fluid (CSF) are associated with BYHWD treatment, nor the mechanism by which BYHWD protects the brain from ischemia/reperfusion injury. We investigated the effect of BYHWD on the amino acid content of CSF in rats during ischemia-reperfusion injury. Ischemia was imposed by right middle cerebral artery occlusion (MCAO). CSF was continuously collected from the striatum via brain microdialysis before and after ischemia/reperfusion. We used on-line derivatization combined with high-performance liquid chromatography with fluorescence detection (HPLC-FD) to determine levels of glutamate (Glu), aspartate (Asp), glycine (Gly), taurine (Tau), and γ-aminobutyric acid (GABA) in CSF. The MCAO model displayed an infarct lesion in the ipsilateral hemisphere and nerve injuries, as the left upper limb was unable to extend and turn leftward. Significant increases in excitatory and inhibitory amino acids were observed in the CSF of the ischemic rats relative to the sham-operated group (P < 0.01). Treatment with BYHWD reduced the areas of cerebral infarction and improved the neurological behavior scores of rats after MCAO. BYHWD treatment was also associated with a significant decrease in excitatory amino acids and increase in inhibitory amino acids in the CSF. Only the higher dose of BYHWD (20 mg/kg) affected all these levels significantly. Attenuated excitatory toxicity and reduced areas of cerebral infarction associated with BYHWD treatment might be due to a protective mechanism induced by BYHWD against ischemia/reperfusion injury.

Introduction

Stroke is the third leading cause of mortality and disability in the world, and ischemic stroke accounts for 85% of all stroke cases [1], [2]. However, no effective treatment has been found to prevent damage to the brain after stroke. Recent studies have reported the protective properties of traditional Chinese medicines against hemorrhagic and ischemic strokes [3], [4], [5]. Buyang Huanwu decoction (BYHWD) derived from “Correction on Errors in Medical Classics” from the Qing dynasty is a well-known traditional Chinese herbal prescription for treating stroke-induced disability [6], [7], [8]. This prescription has been used extensively to improve deficiencies in energy (Qi) and hematological disorders (blood stasis syndromes), owing to its beneficial therapeutic properties [9], [10]. There is evidence that BYHWD has a neuroprotective effect against ischemic stroke. BYHWD improves nervous function and behavior in experimental animals, reduces the cerebral infarct volume, and decreases the rate of infarction and ischemia/reperfusion damage [11], [12]. In addition, BYHWD stimulates neural proliferation, and modulates vascular endothelial growth factor (VEGF) and fetal liver kinase 1 (Flk1) expressions in rat brains with transient focal cerebral ischemia [6]. It is also reported that BYHWD can promote the growth and differentiation of neural cells and inhibit apoptosis of nerve cells [7], [13]. Nevertheless, little is known about the mechanism by which BYHWD protects the brain from ischemia/reperfusion injury.

Amino acids, acting as neurotransmitters, are involved in multiple neurological diseases. The dynamic characterization of amino acids in cerebrospinal fluid (CSF) is especially useful for understanding the pathogenesis of psychiatric disorders [14]. Amino acids may be excitatory or inhibitory (EAA and IAA, respectively) depending on their effects on neurons [15]. Levels of the EAAs glutamate (Glu) and glycine (Gly) in plasma and CSF are significantly higher in patients with a higher degree of neurological deficit, and their excitotoxic activity in patients with cerebral infarction has been demonstrated [16], [17]. In contrast, increases in the levels of IAAs can suppress neuron excitation and alleviate nerve cell damage that was due to EAAs [18], [19].

BYHWD has been shown to suppress the release of Glu, and reduce the expression of metabotropic Glu receptor-1 (Grm1) RNA significantly [12]. Furthermore, it promotes angiogenesis in rats after focal cerebral ischemia and regulates the differential expression of up- and down-regulated genes after focal brain ischemia/reperfusion in these animals [20], [21]. Nonetheless, the nature of the association between the inhibitory effect of BYHWD and the amino acid content in CSF needs to be elucidated. Therefore, in the present study we investigated the effect of BYHWD on the amino acid content of the rat brain during ischemia/reperfusion injury to elucidate the underlying mechanism of the neuroprotective effect of BYHWD against ischemic stroke. Brain microdialysis coupled with high-performance liquid chromatography with fluorescence detection (HPLC-FD) was used to analyze CSF samples. These results provide a scientific basis for the clinical application of BYHWD.