Changes of c-fos, malondialdehyde and lactate in brain tissue after global cerebral ischemia under different brain temperatures.

Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic (37-38°C) ischemia, mild hypothermic (31-32°C) ischemia, hyperthermic (41-42°C) ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20-min ischemia. The expression of c-fos protein and the levels of malondialdehyde (MDA) and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-fos protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion following 20-min ischemia as compared with the sham-operated group (P<0.01). The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group (P<0.01). It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.

[Changes of inducible nitric oxide synthase in lipopolysaccharide-mediated degeneration of dopaminergic neurons].

OBJECTIVE: To explore whether the upregulation of inducible nitric oxide synthase (iNOS) is involved in lipopolysaccharide (LPS)-induced neurodegeneration. METHODS: 108 SD rats were randomly divided into 2 groups: experimental group and normal control group, and each group was sub-divided into 5 subgroups of 18 rats to undergo examination at different time points (6 h, 12 h, 1 d, 3 d, and 7 d). LPS was stereotaxically infused into the substantia nigra (SN) of left side of the experimental rats and PBS was used instead for the control rats. At different time points different numbers of rats from each subgroup were killed to take out the SN. Biochemical method was used to test the activity of NO and iNOS in 6 rats from each subgroup, iNOS mRNA expression was tested by RT-PCR in 3 rats from each subgroup, and iNOS protein expression was tested by Western blotting in 4 rats from each subgroup. Immunohistochemistry was used to detect the iNOS positive cells. RESULTS: iNOS positive cells were found since 6h after the intranigral injection of LPS, peaked 1d after, began to decrease 3d after, and basically disappeared 7d after; and were not found in the control group and the SN at the opposite side of the experimental rats. The percentage of iNOS-positive neurons 1d after the injection was 45.30 +/- 4.63, significantly higher than that of the control group (0.11 +/- 0.04, P < 0.01). RT-PCR and Western blotting showed that expression of iNOS mRNA and expression of iNOS protein at all time points were all higher than those of the normal controls and PBS controls (all P < 0.01). iNOS activity and NO amount in the LPS-injected SN began to increase 6 h after the injection, significantly higher then that of the control group (P < 0.05), peaked 1d after, (P < 0.01), began to decrease 3d after, and basically returned to normal level. CONCLUSION: Up-regulation of iNOS may be one of the crucial mechanisms in LPS-induced degeneration of DA neurons.

Protective effects of Ginkgo biloba extract on rats during cerebral ischemia/reperfusion.

OBJECTIVE: To study the effect of Ginkgo biloba extract on rats during ischemia/reperfusion and its influence on intracellular calcium in hippocampal neurons. METHODS: Model of intraluminal occlusion of the middle cerebral artery (MCAO) was used to prepare the ischemia/reperfusion cortex tissue. Concentration of MDA was determined by measuring thiobarbituric acid-reactive substance. GSH-PX was quantified using the thiobarbituric acid (TBA) technique. SOD was assayed througha xanthine method. Endogenous amino acids were quantified by high performance liquid chromatographic (HPLC) analysis. Primary culturs of hippocampal neurons were prepared for a free intracellular calcium ([Ca(2+)]I) assay by Fura-2 based single cell microfluoremetric technique. RESULTS: Comparing control and treatment groups, the concentration of SOD and GSH-PX were higher, whereas that of MDA was much lower; the concentration of glutamate and aspartate decreased and that of GABA increased markedly at all time point (P < 0.01), Gly also decreased at some time points (P < 0.05). The differences were significant between the groups of 10 mg/kg, 15 mg/kg and the groups of 5 mg/kg. When 1 x 10(-5) mol/L glutamate was applied with 25 micro g/ml ginkgo biloba extract to cultured neurons, the increase in [Ca(2+)]I was lower than that caused by applying glutamate alone. Its peak value was much lower and increased phase was longer, its declining phase was shorter. After returning to baseline, the application of 1 x 10(-5) mol/L glutamate could induce the reaction to recover. CONCLUSIONS: Ginkgo biloba extract could protect damaged neurons by keeping the balance of inhibitory/excitatory aminoacids, enhancing the free radical scavengers system, and inhibiting the effect of glutamate on [Ca(2+)]I.

Effect of thrombin on blood brain barrier permeability and its mechanism.

BACKGROUND: Previous studies have indicated that thrombin (TM) may play a major role in brain edema after intracerebral hemorrhages (ICHs). However, the mechanism of TM-induced brain edema is poorly understood. In this study, we explored the effect of TM on the permeability of the blood brain barrier (BBB) and investigated its possible mechanism, aiming at providing a potential target for brain edema therapy after ICHs. METHODS: TM or TM + cathepsin G (CATG) was stereotaxically injected into the right caudate nucleus of Sprague-Dawley rats in vivo. BBB permeability was measured by Evans-Blue extravasation. Brain water content was determined by the dry-wet weight method. Brain microvascular endothelial cells were then cultured in vitro. After TM or TM + CATG was added to the endothelial cell medium, changes in the morphology of cells were dynamically observed by phase-contrast light microscopy, and the expression of matrix metalloproteinase-2 (MMP-2) protein was measured by immunohistochemical method. RESULTS: BBB permeability increased at 6 hours after a TM injection into the ipsilateral caudate nucleus (P < 0.05), peaked between 24 hours (P < 0.01) and 48 hours (P < 0.05) after the injection, and then declined. Brain water content changed in parallel with the changes in BBB permeability. However, at all time points, BBB permeability and brain water content after a TM + CATG injection were not significantly different from the respective parameters in the control group (P > 0.05). TM induced endothelial cell contraction in vitro in a time-dependent manner and enhanced the expression of MMP-2 protein. After incubation with TM + CATG, cell morphology and MMP-2 expression did not change significantly as compared to the control group (P > 0.05). CONCLUSIONS: Increased BBB permeability may be one of the mechanisms behind TM-induced cerebral edema. TM induces endothelial cell contraction and promotes MMP-2 expression by activating protease activated receptor-1 (PAR-1), possibly leading to the opening of the BBB.

Experimental study on heterograft of glomus cells of carotid body for hemiparkinsonian rats.

To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopaminergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 min at various time points after grafting. The striata were cut and examined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH+) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were significantly elevated after grafting (P < 0.01), and TH+ cells survived better. The present study demonstrates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH+ cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.

Experimental study on the protective effect of puerarin to Parkinson disease.

The protective effect of puerarin on the Parkinson disease (PD) mice with decreased estrogen level was investigated in order to develop a new potential medicine as a substitute for estrogen for preventing and treating PD. By using immunohistochemical method of avidinbiotin peroxidase complex (ABC), the distribution of the cells positive for tyrosine hydroxylase (TH) and fibres in the substantia nigra of the mouse were observed. These mice were divided into three groups randomly: group A, normal-female-mouse models; group B containing three subgroups, B1 (normal saline), B2 (estrogen), B3 (puerarin); group C containing three sub groups, C1 (normal saline), C2 (estrogen), C3 (puerarin). By using TUNEL the numbers of apoptosis cells in every visual field was counted and the difference between the experimental group and control group was compared. The results showed the numbers of the cells positive for TH were more and the numbers of apoptosis cells were less in the normal-female-mouse models group than in the group of model made after ovariosteresis and the group of model made before ovariosteresis (P < 0.05), respectively. However, there was no significant difference, between the group given estrogen/puerarin and the controls, and between the group given estrogen and given puerarin. (P > 0.05). It was suggested that puerarin may have protective effect on the nigral neurons to PD. Moreover, the protective effect might serve as a surrogate of estrogen and be associated with the apoptosis.

Experimental study on inhibition of neuronal toxical effect of levodopa by ginkgo biloba extract on Parkinson disease in rats.

In order to observe neuronal toxical effect of Levodopa and investigate if using Levodopa together with Ginkgo Bilobar Extract (EGb) would be an workable method to treat Parkinson disease, rat models of Parkinson disease (PD) were made by injecting 6-OHDA stereotaxically to right side of the mesencephic ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Rotational behavioral observation, TUNEL, immunocytochemistry, Nissl's body staining were performed to measure the difference between group treated by Levodopa (50 mg/kg every day for 3 days, 5 days, 7 days, L-dopa group) and group treated by Levodopa combined with EGb (100 mg/kg every day, E-D group). The results showed that in the L-dopa group, the numbers of apoptosis of substantial nigra, rings of rotational behavior were more than those in the E-D group (P < 0.05). The numbers of Nissl's cells in L-dopa group were fewer than in E-D group (P < 0.05). The results suggested that Levodopa had neur toxic effect and EGb may decrease the toxicity of levodopa. The combined use of EGb with Levodopa may be a workable method to treat PD and may be better than using Levodopa alone.

Dynamic expression of bFGF and TGFbeta2 in glomus cell grafts of carotid body in rat model of Parkinson disease.

To investigate the changes in the expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in glomus cell grafts of carotid body in the rat model of 6-hydroxydopamine-induced Parkinson disease, immunohistochemical staining of bFGF and TGFbeta2 in the sections of striate body was done on the 2nd, 4th and 12th week after transplantation. The results showed that on the 2nd week after transplantation, bFGF and TGFbeta2 were not detectable in the glumous cell grafts. On the 4th week after graft, bFGF and TGFbeta2 immunoreactivity was increased within the grafts and at the graft-host interface but was restricted only to astrocytes. In the striatum surrounding the graft, bFGF was expressed persistently, while TGFbeta2 showed transient expression. It was suggested that the transient expression of TGFbeta2 was likely due more to the trauma imposed by the graft procedure than to an intrinsic. The deficiency in astrocytic bFGF early after graft may be responsible for the poor survival of grafted glomus cells of carotid body.

LPS-induced degeneration of dopaminergic neurons of substantia nigra in rats.

In order to investigate the neurotoxicity of lipopolysaccharide (LPS) on the dopaminergic neurons of substantia nigra and the pathogenesis of Parkinson disease, LPS was stereotaxically infused into substantia nigra (SN). At different dosages and different time points with 5 microg LPS, the damage of the dopaminergic neurons in SN was observed by using tyrosine-hydroxylase (TH) immunohistochemical staining. The results showed that 14 days after injection of 0.1 microg to 10 microg LPS into the rat SN, TH-positive (TH+) neurons in the SN were decreased by 5%, 15%, 20%, 45 %, 96% and 99% respectively. After injection of 5 microg LPS, as compared with the control groups, TH+ neurons began to decrease at 3rd day and obviously decrease at 14th day, only 5% of total cells, and almost disappeared 30 days later. The results suggested that LPS could induce the degeneration of dopaminergic neurons in the SN in a dose- and time-dependent manner.

Point mutation in the parkin gene on patients with Parkinson's disease.

To investigate the distribution of possible novel mutations from parkin gene in variant subset of patients with Parkinson's disease (PD) in China and explore whether parkin gene plays an important role in the pathogenesis of PD, 70 patients were divided into early-onset group and late-onset group; 70 healthy subjects were included as controls. Genomic DNA from 70 normal controls and from those of PD patients were extracted from peripheral blood leukocytes by using standard procedures. Mutations of parkin gene (exon 1-12) in all the subjects were screened by PCR-single strand conformation polymorphism (SSCP), and further sequencing was performed in the samples with abnormal SSCP results, in order to confirm the mutation and its location. A new missense mutation Gly284Arg in a patient and 3 abnormal bands in SSCP electrophoresis from samples of another 3 patients were found. All the DNA variants were sourced from the samples of the patients with early-onset PD. It was concluded that Parkin point mutation also partially contributes to the development of early-onset Parkinson's disease in Chinese.

Effects of immediate and delayed mild hypothermia on endogenous antioxidant enzymes and energy metabolites following global cerebral ischemia.

BACKGROUND: The optimal time window for the administration of hypothermia following cerebral ischemia has been studied for decades, with disparity outcomes. In this study, the efficacy of mild brain hypothermia beginning at different time intervals on brain endogenous antioxidant enzyme and energy metabolites was investigated in a model of global cerebral ischemia. METHODS: Forty-eight male Sprague-Dawley rats were divided into a sham-operated group, a normothermia (37°C - 38°C) ischemic group and a mild hypothermic (31°C - 32°C) ischemia groups. Rats in the last group were subdivided into four groups: 240 minutes of hypothermia, 30 minutes of normothermia plus 210 minutes of hypothermia, 60 minutes of normothermia plus 180 minutes of hypothermia and 90 minutes of normothermia plus 150 minutes of hypothermia (n = 8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model for 20 minutes and mild hypothermia was applied after 20 minutes of ischemia. Brain tissue was collected following 20 minutes of cerebral ischemia and 240 minutes of reperfusion, and used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and adenosine triphosphate (ATP). RESULTS: Mild hypothermia that was started within 0 to 60 minutes delayed the consumption of SOD, GSH-Px, GSH, and ATP (P < 0.05 or P < 0.01) in ischemic tissue, as compared to a normothermic ischemia group. In contrast, mild hypothermia beginning at 90 minutes had little effect on the levels of SOD, GSH-Px, GSH, and ATP (P > 0.05). CONCLUSIONS: Postischemic mild brain hypothermia can significantly delay the consumption of endogenous antioxidant enzymes and energy metabolites, which are critical to the process of cerebral protection by mild hypothermia. These results show that mild hypothermia limits ischemic injury if started within 60 minutes, but loses its protective effects when delayed until 90 minutes following cerebral ischemia.

NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes.

AIM: To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in the procedure. METHODS: The fluorescence of calcium was measured by Fura-2/AM (F(345)/F(380)). RESULTS: L-Glutamate induced [Ca(2+)](i) increase in most of the cells in concentration- and time-dependent manner. NMDA 50 mmol/L induced the fluorescence increase by almost three to four times, while the effect of AMPA 50 mmol/L was just half of that of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5; a selective antagonist of the NMDA receptor). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor) abolished the effects of NMDA and AMPA, respectively. D-AP-5 and CNQX simultaneously or respectively attenuated the effect of L-glutamate at different degrees, but could not abolish it entirely. CONCLUSION: Glutamate modulated intracellular Ca(2+) of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.