The Study Of Neuroprotective Effects And Its Mechanisms Of Z-Ligustilide Against Subarachnoid Hemorrhage In Rats

BackgroundSubarachnoid hemorrhage is a severe neurosurgical emergency. Although SAH comprises about 5%–10% of all strokes, it is responsible for 25% of all stroke deaths. Numerous intensive researches on mechanisms and pathophysiology of SAH were performed recently. Although various preventative and therapeutic interventions have been applied in clinic practice to improve the outcome of SAH, its mortality and disability still remain at a relatively high level. A considerable percentage of patients still experience permanent neurologic and/or cognitive impairments. The overall prognosis of patients harboring SAH remains poor. Previous studies have demonstrated that secondary brain damage as a result of SAH is one of the leading causes of poor outcome. Thus, prevention and management of secondary brain damage is critical for improving the overall outcome of patients with SAH.Radix Angelica sinensis is one of most popular medicinal plants in traditional Chinese Medicine, which has been used in clinic for thousands of years. Z-Ligustilide, one of the main active ingredients of Radix Angelica sinensis, has various pharmacological effects. Recent researches have reported that LIG provided significant neuroprotective effects on transient forebrain ischemia in mice, permanent forebrain ischemia in rats and focal cerebral ischemia in rats. Ligustilide induces vasodilatation in murine aorta and superior mesenteric artery via inhibiting Ca²⁺ influx and release. It also inhibits proliferation of smooth muscle cells and improves microcirculation. However, the protective effects of LIG on the secondary brain injury following SAH have been not yet reported.The aim of this study was to evaluate the potential therapeutic use of LIG as a treatment option following a SAH-induced brain injury and explore the mechanisms and targets of the neuroprotection of LIG. The results of the present study may provide new theoretical and experimental data for the management of SAH and development and utilization of Angelica sinensis.PART ONE THE NEUROPROTECTIVE EFFECTS OF Z-LIGUSTILIDE AGAINST SUBARACHNOID HEMORRHAGE IN RATSObjective1 To establish a subarachnoid hemorrhage model with secondary neurological injury and delay cerebral vasospasm.2 To assess neuroprotective effects of Z-ligustilide on subarachnoid hemorrhage in rats.Methods1 The male Sprague-Dawley rats were randomly assigned to ten groups as follows: (1) Sham groups: Sham-1d, 3d, 5d, 10d, 14d groups; (2) SAH groups: SAH-1d, 3d, 5d, 10d, 14d groups.SAH model was established by a modified rat double hemorrhage model. Survival analysis, neurological evaluation, brain water content, histological examination of brains and basilar arteries, and diameter, wall thickness and cross-section area of basilar arteries were investigated to assess this model.2 The male SD rats were randomly assigned to the following four groups: (1) Sham group, (2) SAH+vehicle group, (3) SAH+LIG5 group (5mg/kg LIG treatment), and (4) SAH+LIG20 group (20mg/kg LIG treatment). General observations, survival analysis, neurological evaluation, brain water content, and blood brain barrier permeability were measured, and morphologic changes in the brains were observed by Hematoxylin-Eosin staining and Nissl staining to evaluate protective effects of LIG on SAH model rats.Results1 Compared with the Sham group, the survival time was significantly shortened and neurological scores were reduced in SAH group. The brain water content was increased on 1d and 3d after SAH. The ischemic brain injury was observed in the cortex and hippocampus of SAH group. Edema and typical neural apoptosis were observed and the number of neurons was reduced in the above-mentioned regions. HE staining of basilar artery showed a decrease in diameter and cross-section area, and an increase in wall thickness in SAH group. Some histopathological changes of BA were observed in SAH group, including a corrugated internal elastic lamina, a shrunken endothelium, and contracted smooth muscle cells. The spasm of BA reached its peak on 5d after SAH.2 The administration of LIG (20 mg/kg) improved neurological scores, reduced brain edema and BBB permeability. LIG treatment also alleviated histopathological changes in the cortex and hippocampus. LIG treatment had no significant effect on the survival time of SAH rats.Conclusion1 The rat SAH model established by a modified rat double hemorrhage model could induce obvious secondary brain injury and CSV with a typical time course. It is a reliable model for studying mechanisms of both brain injury and CSV induced by SAH2 LIG treatment provided exact neuroprotective effects for SAH model rats via ameliorate neurological deficits, reduce brain edema and BBB permeability, and decrease the neuronal apoptosis and necrosis. PART TWO THE MECHANISMS OF NEUROPROTECTION OF Z-LIGUSTILIDE AGAINST SUBARACHNOID HEMORRHAGE IN RATSObjectiveTo investigate the possible mechanisms of neuroprotection of LIG against SAH.Methods1 Grouping: The male SD rats were randomly assigned to the following four groups: (1) Sham group, (2) SAH+vehicle group, (3) SAH+LIG5 group (5mg/kg LIG treatment), and (4) SAH+LIG20 group (20mg/kg LIG treatment).2 The effect of LIG treatment on CSV following SAH and the possible mechanism was investigated.(1) Morphologic changes in the basilar arteries were observed using transmission electron microscopy and light microscopy.(2) Morphometric analysis was performed to detect wall thickness and cross-section area of basilar arteries.(3) TUNEL staining was used for assessing apoptosis of the endothelial cells of BA.(4) The levels of specific apoptosis-associated protein in the BA, including p53, Bax, Bcl-2, and caspase-3, were evaluated using immunohistochemistry and Western blot analysis.3 The effect of LIG treatment on hemorrheology in the SAH model rats was evaluated.The variables of hemorrheology were measured using an automatic blood viscometer dynamic analysis apparatus.4 The effects of LIG treatment on free radical clearance and neural apoptosis in the cortex after SAH was evaluated.(1) Superoxide dismutase activity in the cerebral cortex was determined.(2) Malondialdehyde content in the cerebral cortex was determined.(3) TUNEL staining was used for assessing apoptosis of the neurons in the cortex.(4) The levels of specific apoptosis-associated protein in the cortical neurons, including p53,Bax, Bcl-2, and caspase-3, were evaluated using immunohistochemistry and Western blot analysis.Results1 LIG treatment significantly ameliorated pathological changes of BAs.2 LIG enlarged cross-section area, and decreased wall thickness of BAs after SAH.3 It also reduced TUNEL-positive endothelial cells of BAs and cerebral neurons. 4 The levels of pro-apoptotic proteins p53 and cleaved caspase-3 were down-regulated and anti-apoptotic protein Bcl-2 was up-regulated in endothelial cells of BAs and cerebral neurons by LIG treatment, whereas it had no effect on pro-apoptotic protein Bax expression.5 Administration of LIG slightly improved whole blood viscosity, whole blood reduced viscosity, erythrocyte aggregation index, e rythrocyte rigidity index, and erythrocyte deformability index, although these findings were not statistically significant.6 The activity of SOD was enhanced and the level of MDA was reduced by LIG treatment.ConclusionLIG could protect the secondary ischemic brain injury induced by SAH. The underlying mechanisms may be partly related to relieving the delay CSV following SAH by anti-endothelial apoptosis, reducing free radical in the brains, and anti-neural apoptosis.