The Study Of Neurological Injury Following Symptomatic Subarachnoid Hemorrhage In Rabbits And The Effect Of Ecdysterone Therapy

Background: Delayed ischemic neurological deficit(DIND)following subarachnoid hemorrhage (SAH) remains a sever complication, frequently leading to disability or death. Despite adequate research, its pathogenesis is not well understood and lack effective therapeutic strategies. Absence of ideal animal model is the main reason for impeding its further studies. In 2004, Zhang et al introduce the conception of early brain injury (EBI). They said that permeability change of blood brain barrier (BBB), brain edema, elevated intracranial pressure (ICP) and microcirculation compromise all contribute to early brain injury. However, the study that is focus on DIND is still limited. Ecdysterone (EDS), one of the main constituents of Chinese medicine, is a steroid hormone. Some studies have described its protective effect of vascular endothelial cells. Our previous studies showed that EDS could alleviate vascular endothelial cells injury, smooth muscle cells and adventitial fibroblast proliferation which was cause by bloody cerebral spinal fluid (CSF). However, there is no study about the effect of EDS on neurological injury following SAH in vivo.Object:Our aims are to establish a symptomatic SAH model in rabbits, observe its pathological and physiological process, investigate the role of PI3K and apoptosis in neurological injury following SAH, and study the effect of EDS in the treatment of neurological injury on this model.Methods:1. Autologous arterial blood was injected into cisterna magna of rabbit twice 48hr apart after 7 days ligation of the right common carotid artery to establish the symptomatic SAH model. 2. Male rabbits were divided into groups randomly. They were N group (normal control group), L group (rabbits underwent the right common carotid artery ligation), SAH group (twice blood injection was performed ), L-SAH group (twice blood injection was performed after ligation), Nim/SAH group (Nimodipine therapy after model establishment), EDS/SAH 1mg group (EDS therapy 1mg/kg, 4/d, after model establishment), EDS/SAH 3mg group (EDS therapy 3mg/kg, 4/d, after model establishment).3. Ethological changes were evaluated by Strong's score.4. Regional cerebral blood flow (rCBF) was measured by laser doppler flowmetry (LDF).5. Discharge of hippocampal neuron was recorded by extracellular recording technique .6. Glucose, lactate, pyruvate, glutamate concentration and L/P ratio in hippocampus were analysed by microdialysis techni que.7. Brain water content (BWC)was measured to reflect the degree of brain edema.8. Pathological changes of hippocampus and basilar arteries were observed by light microscope and transmission electron microscope.9. Bax and Bcl-2 expression in hippocampus were detected by immunohistochemistry, and TUNEL stain was also studied.10. PI3K expression was assessed by RT-PCR and Western blot.Results:1. The two-hemorrhage model established by injecting autologous arterial blood into cisterna magna 48 hours apart after ligation of the right common carotid artery was a successful symptomatic SAH rabbit model, and its mortality rate was 25%.2. There was no ethological abnormal in L group, and all scores were 0. The ethological scores in 1-7d after SAH of SAH group were 4.17±1.17, 5.33±1.63, 4.67±1.37,4.00±0.89, 2.50±0.55, 1.83±0.41, 1.50±0.55, respectively. It increased in 1-4d significantly (P<0.01), started to decrease in 5d (P<0.05), and returned to normal in 7d when compared with L group. The ethological scores in 1-7d after SAH of L-SAH group were 6.00±1.79,8.00±1.90, 8.00±2.37, 6.50±1.97, 4.33±1.63, 3.00±1.10, 2.17±0.75. It was significant higher than that of L group in 1-7d, and was higher than that of SAH group in 2d, 3d, and 5-7d, especially in 3d, 5d and 6d (P<0.01). The ethological score was low in rabbits with nimodipine or EDS therapy. The ethological scores in 1-7d after SAH of Nim/SAH group were 4.67±1.21, 5.83±1.17, 4.83±0.98, 4.50±1.05, 2.50±0.55, 2.33±0.52, 2.00±0.00. It was lower than that of L-SAH group significantly in 2-5d (P<0.01). The ethological scores in 1-7d after SAH of EDS/SAH 1mg group were 4.67±1.21, 5.50±0.84, 4.83±0.98, 4.33±0.82, 2.33±0.52, 1.67±0.52, 1.50±0.55. It was lower than that of L-SAH group significantly in 2-6d (P<0.01). The ethological scores in 1-7d after SAH of EDS/SAH 3mg group were 4.16±0.75, 5.17±0.75, 4.83±1.17, 4.17±0.75, 2.50±0.55, 1.67±0.52, 1.33±0.52. It was lower than that of L-SAH group in 2-7d (P<0.01) and lower than that of Nim/SAH group in 7d (P<0.05).3. There was no statistical difference of rCBF before and after ligation of the right common carotid artery, and no statistical difference between left and right brain in all groups. Two sides relative rCBF of SAH group in 30min, 1d, 3d and 7d after SAH were 60.63±7.76%(L), 62.33±7.88%(R), 83.92±10.07%(L), 82.73±7.55%(R), 95.07±5.82%(L), 91.83±6.39%(R), 98.28±7.95%(L), 92.07±4.39%(R). It decreased significantly within 1d (P<0.01) and increased in 3d. Two sides relative rCBF of L-SAH group in 30min, 1d, 3d and 7d after SAH were 57.30±6.90%(L), 58.22±10.69%(R), 83.53±5.00%(L), 83.20±6.46%(R), 92.57±4.36%(L), 93.60±13.52%(R), 91.57±5.13%(L), 97.30±5.63%(R). It was lower than that of L group within 1d (P<0.01) significantly and the left relative rCBF was lower than that of SAH group in 3d and 7d (P<0.05). Two sides relative rCBF of Nim/SAH group in 30min, 1d, 3d and 7d after SAH were 58.97±10.69%, 60.47±7.84%, 91.98±5.55%, 94.57±6.06%, 98.82±7.70%, 99.50±9.63%, 101.68±12.79%, 102.80±9.85%. That of EDS/SAH 1mg group were 61.58±8.39%, 57.93±7.87%, 91.73±4.35%, 92.27±4.99%, 94.15±5.82%, 95.07±5.82%, 99.82±10.31%, 97.55±10.71%. That of EDS/SAH 3mg group were 62.82±7.35%, 58.38±7.50%, 91.88±4.50%, 92.30±4.77%, 94.32±6.22%, 95.63±5.54%, 100.30±10.08%, 97.62±11.28%,and relative rCBF of three therapy groups were higher than that of L-SAH group significantly in 1d (P<0.01).4. There was no statistical difference of BWC in 3d after SAH in N group and L group. In SAH group, BWC of two sides of cortex were higher than N group and L group, and that of left hippocampus was higher than N group (P<0.05). BWC in L-SAH group increased in 3d after SAH. They were 80.45±0.47%, 80.37±0.50%, 81.93±0.44%, 81.93±0.65%, 79.32±0.88%, 72.65±0.87% in two sides of cortex and hippocampus, cerebellum and brain stem, respectively. Moreover, BWC of two sides of cortex and hippocampus were higher than that of N, L and SAH groups significantly (P<0.01). The whole brain BWC in L, L-SAH, Nim/SAH, EDS/SAH 1mg and EDS/SAH 3mg group were 77.7±0.35%, 78.56±0.52%, 78.56±0.53%, 78.29±0.40%, 78.06±0.52%, resoectively. BWC of L-SAH and Nim/SAH group were significant higher than that of L group (P<0.01), and BWC of EDS/SAH 3mg group was lower than that of L-SAH group.5. Morphological structural changes of hippocampus and the basilar arterial wall in L-SAH group, including Virchow-Robin space increasion, axon edema, vessel stenosis, vessel wall incrassation, endothelial cell lesions, disorder of VSMC structure, artery corrugation and lumen reduction were detected by light microscope and transmission electron microscope observation, while these changes were not conspicuous in SAH and therapy groups.6. There was no statistical difference of microdialysis analysis of hippocampal interstitial fluid in two sides of brain of all groups (P>0.05). There were no statistic difference of glucose concentrations in all groups and all time points. Lactate concentrations in left hippocampus were 3.48±1.63mmol/l, 7.00±2.76 mmol/l, 7.11±2.17 mmol/l and 4.52±1.58 mmol/l before SAH, 1d, 3d and 7d after SAH, respectively, while in right hippocampus were 3.55±2.4 mmol/l, 6.66±2.90 mmol/l, 7.18±2.33 mmol/l and 4.59±1.91 mmol/l. Pyruvate concentrations in left hippocampus were 122.47±18.32μmol/l, 94.32±19.23μmol/l, 84.85±16.59μmol/l and 84.93±30.46μmol/l before SAH, 1d, 3d and 7d after SAH, respectively, while in right hippocampus were 125.23±16.62μmol/l, 97.07±38.94μmol/l, 92.35±17.79μmol/l and 80.87±25.90μmol/l. L/P ratio in left hippocampus were 29.31±15.24, 72.74±21.36, 83.42±16.81 and 55.79±17.95 before SAH, 1d, 3d and 7d after SAH, respectively, while in right hippocampus were 27.97±14.16, 70.57±24.40, 8.58±23.18 and 58.72±20.58. Glutamate concentrations in left hippocampus were 9.61±7.27 mmol/l, 106.07±42.61 mmol/l, 52.97±26.10 mmol/l and 45.02±57.47 mmol/l before SAH, 1d, 3d and 7d after SAH, respectively, while in right hippocampus were 12.00±8.82 mmol/l, 110.56±31.68 mmol/l, 57.30±24.15 mmol/l, and 34.43±30.46 mmol/l. L/P ratio in right hippocampus of EDS/SAH 3mg group in 3d after SAH was lower than that of other groups. Glutamate concentration in right hippocampus of L-SAH group was higher than that of Nim/SAH group and EDS/SAH 1mg group (P<0.05). Glutamate concentration in hippocampus in EDS/SAH 3mg group was lower than L-SAH group and Nim/SAH group significantly (P<0.01).7. Immunohistochemical stain showed that Bax expression increased in L-SAH group after SAH, it decreased with the use of Nimodipine and EDS; while Bcl-2 stain was strongest in L group and slight positive stain in L-SAH group. TUNEL stain positive cells were more in L-SAH group than that of other groups.8. Expression of PI3K mRNA and protein in hippocampus in L-SAH group decreased 3d after SAH and increased in therapy groups, especially in EDS/SAH 3mg group.Conclusion:1. The two-hemorrhage model established by twice injecting autologous arterial blood into cisterna magna 48 hours apart 7 days after ligation of the right common carotid artery was a symptomatic SAH model with notable neurological deficits;2. After SAH, rabbits experienced a series physiological and pathological process, including brain edema which is more obviously in cortex and hippocampus, rCBF decrease, metabolism disorder, glutamate accumulation in interstitial fluid, apoptosis increase in hippocampus. PI3K and apoptosis might involve in the mechanism of neurological injury after SAH.3. There were ischemic phenomenon such as Virchow-Robin space increasion, gliocyte edema in hippocampus, and vasospasm phenomenon such as vessel stenosis, incrassation of vessel wall, endothelial cell lesions and disorder of VSMC structure in basilar artery of rabbit following twice injecting autologous arterial blood into cisterna magna 48 hours apart 7 days after ligation of the right common carotid artery.4. EDS might have some protective effects on endothelial cells, smooth muscle cells and hippocampus neurons after SAH. It could alleviate neurological deficit and brain edema, raise rCBF, improve metabolism, reduce glutamate accumulation and decrease apoptosis in hippocampus after SAH in rabbits.