Effects Of Salvia Injection On Synaptic Regeneration Of Neonatal Rats With Hypoxic-ischemic Brain Damage

Objective:(1) To observe the effects of Salvia injection on the brain pathology, neuroethology, expression of synaptophysin and the synaptic ultrastructure at different time points in neonatal rats after hypoxic-ischemic brain damage(HIBD).(2) To compare the different effcts among the groups of Salvia injection of high and low dose and ganglioside injection.(3)To probe the mechanism of traditional Chinese medicine with the function of promoting blood circulation and removing blood stasis in improving the neonates with hypoxic-ischemic brain damage, and to provide the experimental evidence for early application of the Chinese medicine of promoting blood circulation.Methods:(1)135 healthy newborn SD rats of 7-day-old were randomly divided into six groups-- normal group(A), sham group(B), Salvia high dose group(C), Salvia low dose group(D), ganglioside group(E) and the model group(F).(2) The hypoxic-ischemic brain damage model was prepared by ligation of the left carotid artery combined with hypoxic environment according to the method of Rice-Vannucci.(3)The rats were started to receive intraperitoneal injection one day after modeling. The rats(group A,B and F)were injected with sterile saline(9 ml/kg·d), the rats in group C and D were injected with Salvia injection(9 ml/kg·d and 4.5 ml/kg·d partly), and the rats in group E were injected with monosialotetrahexosy l ganglioside injection(6 mg/kg·d). The intraperitoneal injection were last for 7 and 14 days at different time points. The general observation and the neuroethology score were made after HI modeling, the pups’ brain were taken at one day, seven days and fourteen days after the modeling respectively, and the brain pathology was observed by HE staining, the neuronal and synaptic ultrastructure in the frontal cortex were observed by transmission electron microscope, and the expression of synaptophysin was determined by Immunohistochemistry method.Results:(1)General observation: After HI modeling, the pups were mostly in theleft-lateral position, with difficulties in turning over, balance abnormalities, limb shaking and other abnormal behaviors, and the delay of eyes open with the left upper eyelid ptosis.(2) Neuroethology: On the neuroethological score, there was no significant difference between the normal group and the sham group at each time point(P>0.05); the model group and the normal control group were significantly different(P<0.05); there was no significant difference among the intervention groups(P>0.05); At 22-day-old, the scores of Salvia high-dose group and ganglioside group were higher than the model group(P<0.05).(3)Pathology: At 8-day-old in model group, the neurons of the left cerebral cortex and hippocampus were swelled, and the typical shapes were lost; at 15-day-old, the nerve cells were disordered and deep stained, the cell bodies were shrank and deformed, and the neurons were degenerated and necrotic; at 22-day-old, the reduced number of neurons and gliosis were observed in the left cerebral cortex and hippocampus.(4)Expression of synaptophysin: Mean density of SYP in the hippocampus CA1 region rose with age increase gradually in the normal group and sham group, and there was no statistical difference between the two groups at each time point(P>0.05); the expression of SYP in the model group was significantly lower than the control group at each time point(P<0.05); the SYP expression in the drug intervention groups increased compared with the model group(P<0.05); there was no significant difference between the groups of high-dose Salvia and ganglioside(P>0.05). At 15-day-old, there was no significant difference among the high-dose Salvia group, the ganglioside group and the normal group(P>0.05); while there was significant difference between the Salvia low-dose group and the control group(P<0.05). At 22-day-old, the SYP expression of the Salvia high-dose group and the ganglioside group had basically reached level of the normal group and sham group, and there was no statistical significance(P>0.05), while the Salvia low-dose group had not yet reached the level of the normal group(P<0.05)and there was significant difference compared with the Salvia high dose group and the ganglioside group(P<0.05).(5)Synaptic ultrastructure: 8-day-old in model group, the incomplete structure of the frontal cortex neurons, the vague nuclear membrane, the swelling organelle, the less number of synapses and less clear synaptic vesicles were observed in the model group. 15-day-old in model group, the neuronal pyknosis, nuclear membrane depression, the blurred mitochondria structure were observed, the number of synapse and synaptic vesicles was reduced, the synaptic cleft was blurred and postsynaptic density was thinning. 22-day-old in model group, the loose cortexstructure and vacuolar structure were showed, the incomplete neuronal structure, the disappearance of partial nuclear membrane, disintegrated nucleolus, and the cytoplasm ingredients depigmentation were observed. There was no significant mitochondrial cristae structure, the number of synapse and synaptic vesicles was reduced, the synaptic cleft was widened, and the postsynaptic density was uneven thickness. After the intervention treatment of high and low dose Salvia injection and ganglioside injection, the neuronic and synaptic ultrastructure were improved compared with the model group. The damaged neuronal organelle was restored, the number of mitochondria around the nucleus was increased, and the cristaes were visible. There was an increase in the number of synapses, and more clear synaptic vesicles were seen in the presynaptic enlargement. The presynaptic and postsynaptic membrane and the cleft were close to the normal morphology, and the postsynaptic density was thickening.Conclusion:(1)Salvia injection can alleviate the damage of neuronal and synaptic ultrastructure in neonatal rats with hypoxic-ischemic brain damage.(2)The appropriate dose of Salvia injection can promote the hippocampal synaptophysin expression, and improve synaptic plasticity and regeneration in neonatal rats with hypoxic-ischemic brain damage.(3)The mechanism of Salvia injection in treatment of neonatal rats with hypoxic-ischemic brain damage may be associated with the improvement in neuronal ultrastructure and synaptic reorganization.