EFFECTS OF TRANSIENT FOREBRAIN ISCHEMIA AND RADIX SALVIAE MILTIORRHIZAE (RSM) ON EXTRACELLULAR LEVELS OF MONOAMINE NEUROTRANSMITTERS AND METABOLITES IN THE GERBIL STRIATUM-An in vivo Microdialysis Study

The aim of this study was to investigate the effect of 30 min forebrain ischemia,followed by120 min reperfusion on extracellular fluid(ECF)levels of dopamine(DA),norepinephrine(NE),serotonin(5-HT)and their metabolites,homovanillic acid(HVA)and 5-hydroxyindoleacetic acid(5-HIAA)in the striatum of gerbils,so as to obtain furtherinformation on the mechanism of Radix Salviae Miltiorrhizae(RSM)-inducedneuroprotection.Microdialysis was used to sample the extracellular space.Dialysate wasmeasured by high performance liquid chromatography with electrochemical detector(HPLC-ED).ECF DA,NE levels increased from basal levels by 282,227 and 221 folds,by9.14,8.51 and 8.25 folds,respectively for the three ischemic duration(0-10;11-20;21-30min).ECF DA,NE,5-HT levels in the RSM-treated group were significantly decreased ascompared with those in the control group during ischemia(P【0.01).The results suggestedthat monoamine neurotransmitters were involved in ischemic neuron damage directly orindirectly;and that RSM plays a protective role during cerebral ischemia by attenuating thedysfunctions of monoamine neurotransmitters.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON EAA AND IAA DURING CEREBRAL ISCHEMIA IN GERBILS: A MICRODIALYSIS STUDY

The concentrations of amino acids (AA), stroke index and infarct area were determined in 26 gerbils which were divided into 3 groups: RSM-treated (n = 8), Saline-treated (n = 10) and sham-operated (n = 8). The levels of AA were measured with microdialysis technique in cerebral cortex. The concentrations of neurotransmitter AA, as Glu and GABA and Asp, were significantly increased during the first 60 min after CCA ligation, while the concentra-tions of non-neurotransmitter AA, as Thr and Ser, had no significant changes. In RSM-treated gerbils, the level of Glu was significantly lower than that of the saline-treated, but the GABA in RSM-treated was significantly higher than that of the sa-line-treated. The ratio of Glu/ GABA was significantly decreased after ischemia. The RSM could improve the reduction of ratio of Glu / GABA during 0-30 min and 91-120 min after cerebral ischemia. There were statistically significant decrease in terms of stroke index in RSM-treated group when compared with saline-treated group at 24 h and 16 h after CCA ligation respectively. The RSM has a tendency to decrease the size of infarct area, but no statistical difference. The results suggest that the neurotransmitter AA involve in the pathophysiological procedures of cerebral ischemia and the RSM can attenuate dysfunctions of EAA and IAA. Furthermore, the results also imply that there may be an alternate way to treat cerebral ischemia by inhibiting the presynaptic releasing of Glu and stimulating the releasing of GABA.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON NITRIC OXIDE IN CEREBRAL ISCHEMIC-REPERFUSION INJURY

It has been postulated that nitric oxide (NO) is produced in significant amount on reperfusion of ischemic brain and could cause brain edema and cell death. This study was undertaken in an attempt to determine whether Radix Salviae Miltiorrhizae (RSM) could reduce cerebral NO contents in 4 -vessel occlusion rat model after 30 min of global ischemia and 15 min of reperfusion. There was a significant increase in cerebral NO level in untreated animals as compared with sham-operated ones. By pretreatment with RSM the cerebral NO concentration was reduced to normal level. The possibilities of neuroprotective role of RSM in cerebral ischemic-reperfusion injury were discussed.

THE EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON VASOACTIVE INTESTINAL PEPTIDE IN CEREBRAL ISCHEMIA:AN ANIMAL EXPERIMENT

The levels of vasoactive intestinal peptide(VIP)in three regions of rat brain were assayed in 62 rats.Bilateral common carotid artery ligation was donein 50 rats.Half an hour before ligation 26 rats weregiven 10 g/kg of Radix Salviae Miltiorrhizae(RSM);24 rats were given same volume of normalsaline as controls.A sham operation wasdone in 12 rats.Half an hour(n=30)and 3hours(n=32)after operation,the rats were quicklydecapitated.VIP levels were assayed in cerebralcortex,hippocampus and caudate nucleus,in salin-treated animals,VIP levels of cerebral cortex andcaudate nucleus at 3 hour group were significantlydecreased compared with the sham-operated group.No significant difference was found between RSM-treated and sham-operated groups.The preliminaryresults suggest that VIP may be involved in thepathophysiological procedures of cerebral ischemiaand RSM may attenuate the dysfunction of VIPduring cerebral ischemia.

PROTECTIVE EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON APOPTOSIS OF NEURONSDUREVG FOCAL CEREBRAL ISCHEMIA AND REPERFUSION INJURY

We have found that Radix Salviae Miltiorrhizae (RSM) plays a protective role in ischemic brain injury,which attracted us to investigate the effect of RSM on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic brains at different reperfusion intervals were tested with the method of TdT-mediated dUTP-DIG nick end labeling (TUNEL), and the effect of RSM on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion in rat models (n=18). The results showed that few scattered apoptotic cells were observed in right cerebral hemisphere after LMCA occlusion and reperfusion, and that a lot of apoptotic cells were found in left ischemic cerebral cortex and caudoputamen at 12 h reperfusion, and they reached peak at 24-48h reperfusion. However, in rats pretreated with RSM, the number of apoptotic cells in left cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24h reperfusion as compared with those of saline-treated rats. From this study, we conclude that administration of RSM can reduce the apoptosis of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.

Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Effect of Batroxobin on Neuronal Apoptosis During Focal Cerebral Ischemia and Reperfusion in Rats

We have found that Batroxobin plays a protactive role in ischemic brain injury, which attracted us to investigate the effect of Batroxobin on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic rat brains at different reperfusion intervals were tested with method of TdT-mediated dUTP-DIG nick end labeling (TUNEL) and the effect of Batroxobin on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion and reperfusion in rat models (n=18). The results showed that few scattered apoptosis cells were observed in right cerebral hemispheres after LMCA occlusion and reperfusion, and that a lot of apoptosis cells were found in left ischemic cortex and caudoputamen at 12h reperfusion, and they reached peak at 24h^48h reperfusion. However, in the rats pretreated with Batroxobin, the number of apoptosis cells in left cerebral cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24h reperfusion than that of saline-treated rats. The results indicate that administration of Batroxobin may reduce the apoptosis of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.

GAP-43 Expression and Pathological Changes of Temporal Infarction in R.ats and Effects of Batroxobin

To study the changes of the expression of growth-associated protein-43 (GAP-43) and pathology in temporal infarction of rats photochemically induced and the effects of batroxobin. Methods: immunohistochemical technique and hematoxylin-eosin stain was used to show the changes of the expression of GAP-43 and pathology. Results: In infarction group, GAP-43 expression was markedly increased on the infarction and surronding tissues at 24h cerebral infarction. The expression reached peak level at 72h after cerebral infarction and was decreased at 7d after cerebral infarction. However, in batroxobin-treated group, GAP-43 expression was increased and the pathological changes were much slight as compared with infarction group. Conclusion: The expression of GAP-43 increases in infarction of temporal neocortex and batroxobin promotes the expression of GAP-43 and ameliorates the pathological changes in infarction of temporal neocortex.

Effect of Batroxobin on Expression of Neural Cell Adhesion Molecule in Temporal Infarction Rats and Spatial Learning and Memory Disorder

The effect of Batroxobin expression of neural cell adhesion molecule (NCAM) in left temporal ischemic rats with spatial memory disorder was investigated by means of Morri's water maze and immunohistochemical methods. The results showed that the mean reaction time and distance of temporal ischemic rats for searching a goal were significantly longer than those of sham-operated rats and at the same time NCAM expression of left temporal ischemic region was significantly increased. However, the mean reaction time and distance of Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of NCAM immune reactive cells of Batroxobin-treated rats was more than that of ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats and the regulation of the expression of NCAM is probably related to the neuroprotective mechanism.

THE EFFECT OF RADIX SALVIAE MILTIORRHIZAE(RSM)ON SUBSTANCEP IN CEREBRAL ISCHEMIA—ANIMAL EXPERIMENT

The levels of substance P(SP)in rat brains were assayed in 64 rats.Bilateral common carotid ar-tery ligation was done in 49 rats.Half an hour before ligation,25 rats were given 10 g/kg ofRSM;24 rats were given the same volume of normal saline as controls.Sham operation was donein 15 rats.Half an hour and 3 hours after cerebral ischemia,the rats were quickly decapitated.SP concentration was assayed in the cerebral cortex,caudate nucleus and brain stem.Insaline-treated animals,the SP level of caudate nucleus at 3-hour group was significantly de-creased as compared with the 0.5-hour group and sham-operated group respectively.No signifi-cances were found among RSM-treated groups and sham-operated groups.The SP levels wereshown:brain stem【caudate nucleus】cerebral cortex.The preliminary results suggest that SPmay be involved in the pathophysiologic procedures of cerebral ischemia and RSM mayattenuate the dysfunction of SP during cerebral ischemia.

STUDIES OF QINGYANGSHEN (Ⅱ): MODULATORY EFFECT OF CO-TREATMENT WITH QINGYANGSHEN AND DIPHENYLHYDANTOIN SODIUM ON RAT HIPPOCAMPAL c-fos EXPRESSION DURING SEIZURES

We have reported that, after KA treatment in rats, there was first a significant increase hi hippocampal c-fos expression daring acute seizures and then a long-term inhibition in hippocampal c-fos expression duing chronic seizures. In this experiment, we examined the modulatory effect of Qingyangshen (QYS), a traditional Chinese medicine with antiepileptic property, and diphenylhydantoin sodium (DPH) on hippocampal c-fos expression during seizures. We found that after intra peritonea I injection of QYS+DPH (15 mg/ kg and 50 mg/ kg respectively, q.o.d. x 6), the peak level of hippocampal c-fos mRNA induction during KA-in-duced acute seizures was reduced by 56.27%; while the inhibition of hippocampal c-fos expression during KA-induced chronic seizures was disinhibited to control level. It is suggested that the inhibitory effect of QYS+DPH on KA-induced seizures, chronic seizures in particular, may have much to do with its modulatory effect on hippocampal c-fos expression.

EFFECTS OF ELECTRO-ACUPUNCTURE ON SOMATOSTATIN AND PANCREATIC POLYPEPTIDE IN ISCHEMIC CEREBROVASCULAR DISEASES

The levels of somatostatin(SS)in CSF and blood and pancreatic polypeptide(PP)inplasma were measured by radioimmunoassay in 64 patients with acute ischemiccerebrovascular diseases(ICVD),randomly divided into two groups:group 1(n=31,bothelectro-acupuncture and routine treatments given)and group 2(n=33,routine treatment)and 26 non-ICVD patients were used as controls.The points of electro-acupuncture wereQuchi(LI 12),Waiguan(SJ 5)and Huantiao(GB 30)and Zusanli(St 36).After a courseof treatment,the SS levels in plasma and CSF were significantly increased in the patientsof group 1 with good result and their plasma PP level had no significant change.In thepatients with poor result,however,the PP level was significantly decreased.The resultssuggested that electro-acupuncture might play an active role in alleviating the SSmetabolic disturbance in CNS of ICVD patients.

EVIDENCE FOR AMELIORATION OF CELLULAR DAMAGE IN ISCHEMIC RAT BRAIN BY RADIX SALVIAE MILTIORRHIZAE TREATMENT——IMMUNOCYTOCHEMISTRY AND HISTOPATHOLOGY STUDIES

The changes of somatostatin (SS)—like immunoreactive cells and morphology were observed inthe models of ligation of right MCA and bilateral ligation of CCA respectively.The resultsshowed:(1) a decline of SS immunoreactive nerve cell profiles in right cerebral cortex after 3hligation of MCA as compared with left cerebral cortex and sham operated group,while no sig-nificant difference was found between RSM-treated and saline-treated groups;(2) selectiveischemic cell changes in cerebral cortex,hippocampus and caudate nucleus after 3h ligation ofCCA.The changes included shrinkage and condensation of perikaryon,nuclear pyknosis,cytoplasmic eosinophilia in some neurons.The ischemic changes were more severe insaline-treated group than RSM-treated group.The experiments indicated that the selectiveischemic cell changes could be improved by RSM,suggesting that treatment to modulate SSmetabolism may be indicated after ischemic insults.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON EXTRACELLULAR ADENOSINE AND EVALUATION OF ITS PROTECTIVE EFFICACY IN ISCHEMIC REPERFUSION RAT——Microdialysis, HPLC and Histopathologic Studies

The effects of Radix Salviae Miltiorrhizae (RSM) on extracellular adenosine (Ade) and its metabolites, Le. inosine, hypoxanthine and xanthine, were studied with microdialysis and HPLC techniques during cerebral ischemia-reperfusion induced by 4-vessel occlusion in rat brain. Histological examination of hippocampus was performed 6h after reperfusion. ECF (extracellular fluid) adenosine and its metabolites were compared, between the controls (n= 6) and RSM-treated rats (n= 6). Basal level of Ade and its metabolites release were not greatly affected by pretreatment with RSM, and no significant difference as compared with the sham-operated (n= 6). Ade and its metabolites were dramatically increased after ischemia, and decreased near basal-level and its metabolites remained high at the end of reperfusion. In the RSM-treated animals, the tendency of changes of Ade and its metabolites was just the same as in the controls, but the magnitudes of changes were significantly lower at some different tune points. In sham-operated animals, no changes were observed at different time points both during ischemia (30 min.) and reperfusion (60 min.). Histopathological findings demonstrated that RSM pretreatment results in better histologic preservation of the pyramidal cells in the postischemic reperfusion CA1 sector both qualitatively and quantitatively. These results indicated that RSM protects against cerebral ischemia reperfusion injury.

PROTECTIVE EFFECT OF RADIX SALVIAE MILTIORRHIZAE COMPOSITA ON CEREBRAL ISCHEMIA

The protective effects of Radix Salviae Miltiorrhizae Composita (RSMC) on cerebral ischemia in gerbils produced by unilateral ligation of left common carotid artery and on the development of ischemic cerebral edema were studied. It was found that the administration of RSMC resulted in a very significant decrease in neurological deficits and the time for ex-hibiting first signs of neurological deficit delayed compared with untreated animals. Al-though the values of specific gravity of fresh cerebral tissue on the ligated side of common carotid artery in untreated animals (1.0406± 0.0039 SD) and in RSMC-treated animals (1.0453 ± 0.0009) were decreased significantly compared to that (1.0468± 0.0003) of sham-operated animals, the specific gravity of cerebral tissue in RSMC-treated animals was significantly heavier than that in untreated animals (t= 5.5022, P【 0.001), i.e. the ad-ministration of RSMC decreased the severity of cerebral edema. A significant negative cor-relation was found between stroke index and specific gravity, and it suggests that one of the protective mechanisms of RSMC might be related to its ameliorating cerebral edema.

RADIX SALVIAE MILTIORRHIZAE TREATMENT RESULTS IN DECREASED LIPID PEROXIDATION IN REPERFUSION INJURY

In our pervious investigations we have found that Radix Salviae Miltiorrhizae (RSM) can reduce neurological deficits and ameliorate cellular damages, and its protective actions are associated with its effects on cerebral edema, monoamines, neuropeptides, neurotransmitter aminoacids, thromboxane A2, peroxidation and down-regulation of c-fos gene expression in ischemic animals. The purpose of present study was to investigate the protective effect of RSM against lipid peroxidation in 4-vessel occlusion and reperfusion rat model. We found that in RSM-treated rats the cerebral SOD activity is significantly increased, while the cerebral MDA level is significantly decreased when compared with untreated animals. The results suggested that RSM can reduce the lipid peroxidation and afford cerebroprotection against reperfusion injury.

EFFECT OF QINGYANGSHEN ON HIPPOCAMPAL α-AND β-TUBULIN GENE EXPRESSION DURING KAINIC ACID INDUCED EPILEPTOGENESIS

Using α-and β-tubulin cDNA Probes and Northern blot hybridization technique,we ana-lyzed the effect of intraperitoneally injected Qingyangshen (QYS),a traditional Chinesemedicine with antiepileptic property,and diphenylhydantoin sodium (DPH) on hippocampalα-and β-tubulin gene expression during kainic acid (KA) induced chronic seizures.It wasfound that:1) thirty days after intraperitoneal injection of KA,α-and β-tubulin mRNAs inanimals showing chronic seizures increased 3.02±1.05 and 4.07±1.32 times respectivelycompared with control;2) neither QYS (15 mg/kg,q.o.d.×6) nor DPH (50 mg/kg,q.o.d.×6),when used separately,could inhibit the above mentioned effect of KA;3) whenQYS (15 mg/kg,q.o.d.×6) and DPH (50 mg/kg,q.o.d.×6) were given in combination,the long-term increase in tubuliu gene expression induced by KA was significantly reduced,with α-and β-tubulin mRNAs being decreased to 0.44±0.08 and 0.50×0.10 times of corresponding values in animals treated with KA alone.The results indicate that the mechanismof antiepileptic effect of QYS is at least partially related to the inhibition of tubulin synthe-sis and subsequent reduction in mossy fiber sprouting and neosynaptogensis.

STUDIES OF QINGYANGSHEN (I): DIFFERENTIAL EXPRESSION OF HIPPOCAMPAL c-fos PROTO-ONCOGENE DURING KAINIC ACID INDUCED ACUTE AND CHRONIC SEIZURES

We previously reported that Qingyangshen (QYS), a traditional Chinese medicine with antiepileptic property, had therapeutic effect on kainic acid (KA) induced experimental sei-zures (see JTCM 13 (4): 281-286, 1993 for reference). To investigate the mechanisms underlying the anticonvulsant action of QYS, we analyzed the modulatory effect of QYS on rat hippocampal c-fos proto-oncogene expression during KA-induced epileptogenesis in this and the following paper. The expression of hippocampal c-fos gene during KA-induced seizures were examined first. Rats were intraperitoneally injected with kainic acid (KA, 12mg/kg) and hippocampal c-fos mRNA level was determined by Northern blot analysis during both acute (within one day after KA injection) and chronic (15 days after KA treat-ment) seizures. A mild increase in hippocampal c-fos mRNA level was observed 30 min after KA injection (being 1.98 ± 0.70 times of control level), which corresponded temporally to the occurence of thr first limbic seizures. There was an 11.02 ± 3.33 fold maximal induction of c-fos mRNA at 2h after KA administration, which remained relatively stable even when behavioral seizures continued to aggravate 4h after KA treatment. Twelve hours after KA administration, c-fos mRNA in the hippocampus returned to control level when acute sei-zures began to gradually disappear. In contrast, the induction of hippocampal c-fos mRNA during chronic seizures was inhibited significantly, being reduced by 55.40% compared to control. This suggested that c-fos is in different functional states at acute and chronic stages of epileptogenesis induced by KA.

THE EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON THE CHANGES OF ULTRASTRUCTURE IN RAT BRAIN AFTER CEREBRAL ISCHEMIA

The effect of RSM on ultrastructural alterations of the cortical,hippocampal and caudateneucleus areas brought about by forebrain jschemia in rats were studied.In bothRSM-treated and saline-treated groups the ischemic damage was detected in nearly all an-imals three hours after bilateral common carotid artery ligation,while it was much moremild in RSM-treated animals.The ultrastructural changes consisted of swollenmitochondria,partial loss of cristae,dilatation of rough endoplasmic reticulum and Goigi’scomplex.In addition,some dark neurons

Efficacy and Effect of SI17 Therapy on Pancreatic Polypeptide in Vascular and Tension-Type Headache

Fisher's exact test: P=0.