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.

Experimental study on dynamic regulation of acetylcholine in striatum of rat Parkinson disease model and behavior observation.

In order to explore the role of acetylcholine in the pathogenesis of Parkinson's disease (PD), the changes in the concentration of acetylcholine (Ach) in the striatum, the apoptosis of substantia nigra cells, the ultrastructure and the changes of Nissl cells in rats during the morbidity of PD, and the corresponding behaviors in rats with PD were observed. Rat PD model was established by using the modified Thomas method. Eighty-one rats were randomly divided into normal control, sham operation and PD groups and their behavior features were observed at post-operative day (POD) 7, 14 and 21 as three subgroups (n=9 each). The concentration of Ach in the striatum was determined by using high-performance liquid chromatography. The apoptosis of substantia nigra cells was assayed by using TUNEL method. The ultrastructural changes in the substantia nigra were observed under the electron microscopy, and the survival of neurons in the substantia nigra area was examined by using Nissl staining. In PD group at POD 7 to 21, the damage in the substantia nigra area was gradually aggravated, the concentration of Ach, apoptosis rate and turns of rotation were gradually increased, and the number of Nissl cells was gradually reduced over the time as compared with the normal control and sham operation groups (all P<0.05). It was concluded that there exist dynamic changes in Ach concentration, ethology and apoptosis of the substantia nigra cells during the morbidity of PD, suggesting the contribution of apoptosis to the morbidity of PD, and critical role of Ach in the pathogenesis of PD.

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.

Dynamics of nutritional status in dying patients with acute cerebral infarction in central China: a preliminary study.

BACKGROUND AND AIMS: Stroke is the number one cause of death in China. Although the effective management has reduced the mortality and lengthened survival, little attention has been paid to nutritional issues in patients with stroke in China. This study aimed to assess the premorbid nutrition status in dying patients with acute cerebral infarction. METHODS: In this study, a total of 185 acute ischemic stroke patients dying within 30 days were recruited from medical records. Characteristics of dying patients were assessed on admission, and serum biochemical parameters including serum total protein, serum albumin, and serum prealbumin were measured within 24 hours after stroke onset and every week routinely. RESULTS: Among 185 ischemic stroke patients, 86 dying patients experienced their first-ever acute cerebral infarction, while 99 dying patients were experiencing a recurrent cerebral infarction. The prevalence of dysphagia, post-stroke pneumonia, and gastrointestinal hemorrhage in recurrent stroke groups were higher than those in the first-ever stroke group (P<0.01). There were gradually declines in serum total protein, serum albumin, and serum prealbumin in dying patients from admission to death, especially in the recurrent ischemic stroke group, as compared to their normal range. The sensitive sequence of serum nutritional index for dying patients with ischemic infarction was: serum prealbumin>serum albumin>serum total protein. CONCLUSIONS: This study showed that hypoproteinemia and undernutrition were serious in dying patients with acute ischemic stroke, especially in patients with recurrent ischemic stroke. This study also confirmed that serum prealbumin is more sensitive than serum albumin to assess nutritional status. The strategies to improve malnutrition in stroke patients are urgently needed in China.

Mild hypothermia reduces ischemic neuron death via altering the expression of p53 and bcl-2.

OBJECTIVE: Studies exploring roles of p53 and bcl-2 in neuroprotection by hypothermia in focal cerebral ischemia have not provided consistent results. In the present study, we determined whether p53 and bcl-2 are involved in the hypothermia-induced neuroprotection. METHODS: Male Sprague-Dawley rats were divided into four groups: normothermic (37-38 degrees C) ischemia, hypothermic (31-32 degrees C) ischemia, hyperthermic (41-42 degrees C) ischemia and sham-operated group. Global cerebral ischemia was established for 20 minutes using the Pulsinelli four-vessel occlusion model and the brain temperature was maintained at defined levels for 60 minutes following the 20 min ischemia. The mortality in rats was evaluated at 72 hour and 168 hour reperfusion. The expression of p53 and bcl-2 proteins was detected at 24, 48 and 72 hours after reperfusion. At the same intervals, neuron necrosis and apoptosis in brain regions was also detected using hematoxylin and eosin (HE) staining and terminal deoxynucleotldyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). RESULTS: The mortalities of rats in normothemia, hypothermia and hyperthermia groups was 33.3, 16.7 and 50% at 72 hour reperfusion. At 168 hours of reperfusion, the mortality in the three groups was 58.3, 25 and 100%, respectively. In light microscopy studies, necrotic neurons and apoptotic neurons were found in the hippocampus after global cerebral ischemia. Surviving neurons in hippocampus was increased in mild hypothermic ischemia group (p<0.05) and decreased in hyperthermia ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. TUNEL-positive neurons in hippocampus decreased in hypothermic ischemia group (p<0.05 or p<0.01) and increased in hyperthermic ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. The expression of p53 and bcl-2 proteins was found in the neurons of cerebral cortex after global cerebral ischemia. P53 decreased and bcl-2 increased in hypothermia group. CONCLUSION: Hypothermia reduces ischemic neuronal necrosis and apoptosis by reducing p53 and increasing bcl-2 expression. Hyperthermia accelerated ischemic neuronal injury by increasing p53 and reducing bcl-2 expression.

Initiation time of post-ischemic hypothermia on the therapeutic effect in cerebral ischemic injury.

OBJECTIVE: To study the efficacy of mild brain hypothermia beginning at different time intervals on cerebral ischemic injury. METHODS: Male Sprague-Dawley rats were divided into sham-operated group, normothermia (37-38 degrees C) and mild hypothermia (31-32 degrees C) ischemia groups. The last group was subdivided into four groups: 240 minute hypothermia, 30 minute normothermia plus 210 minute hypothermia, 60 minute normothermia plus 180 minute hypothermia, and 90 minute normothermia plus 150 minute 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 minute cerebral ischemia and 240 minute reperfusion, and used to measure the levels of malondialdehyde (MDA), lactate, water content and the amounts of electrolytes, such as sodium (Na(+)), potassium (K(+)) and calcium (Ca(++)). RESULTS: Mild hypothermia beginning at 0-60 minutes decreased the levels of malondialdehyde and lactate (p<0.05 or p<0.01), decreased water content, Na(+) and Ca(++), and increased the amount of K(+) (p<0.05 or p<0.01) in ischemic tissue, except the amounts of Na(+), K(+) and Ca(++) in mild hypothermia beginning at 60 minute ischemia group (p>0.05). Mild hypothermia beginning at 90 minutes had little effect on the levels of targeted molecules, water content and amounts of electrolytes of Na(+), K(+) and Ca(++) in ischemic tissue (p>0.05). DISCUSSION: Post-ischemic mild brain hypothermia can decrease the accumulation of lactate and lipid peroxidation in ischemic tissue, and delay the development of brain edema following ischemic reperfusion. The best neuroprotection of mild hypothermia to attenuate ischemic injury was begun within 60 minutes.

Therapeutic effect of post-ischemic hypothermia duration on cerebral ischemic injury.

OBJECTIVE: To study the efficacy of post-ischemic mild brain hypothermia lasting for different time intervals on cerebral ischemic reperfusion injury. METHOD: Male Sprague-Dawley rats were divided into a sham-operated group, normothermia (37-38 degrees C) ischemia group and mild hypothermia (31-32 degrees C) group. The last group was subdivided into four groups: 30 minute hypothermia plus 210 minute normothermia, 60 minute hypothermia plus 180 minute nomothermia,120 minute hypothermia plus 120 minute normothermia, and 240 minute hypothermia (n=8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model. Brain tissue was collected following a 20 minute cerebral ischemia and 240 minute reperfusion, and was used to measure the levels of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin(5-HT) and hydroxyindoleacetic acid (5-HIAA), nitrite (NO(2)), endothelin-1 (ET(1)), tumor necrosis factor alpha(TNFalpha) and interleukin-1beta (IL-1beta). Serum was collected to measure the levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK) and its brain band isoenzyme (CK-BB). RESULTS: Hypothermia lasting for 60-240 minutes delayed the decrease in these amino acids, postponed the decrease in DA, NE and 5-HT and increase in hydroxyindoleacetic acid (5-HIAA), and decreased the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue. Hypothermia also decreased the levels of LDH, AST, CK and CK-BB in serum as compared to normothermia group (p<0.05 or p<0.01). Hypothermia lasting for 30 minutes delayed the decreases in these amino acids and 5-HT and increase in 5-HIAA in brain tissue (p<0.05), but failed to influence the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue and the amounts of LDH, AST, CK and CK-BB in serum as compared to normothermia ischemia group (p>0.05). CONCLUSIONS: Post-ischemic mild brain hypothermia can significantly suppress the excessive release of amino acids, monoamine neurotransmitters and inflammation response in ischemic tissue. It can also stabilize the function of the cell membrane, which is associated with the mechanism of cerebral protection by mild hypothermia. These results suggest that mild hypothermia should be applied immediately after ischemia and last for more than 60 minutes in order to obtain neuroprotective effects.

Synthesis of glutathione C60 derivative and its protective effect on hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells.

Oxidized glutathione C(60) derivative has been synthesized and characterized in our research. As a novel derivative of C(60), the glutathione C(60) derivative is soluble in dimethylsulfoxide, dimethylformamide and dimethylacetamide. Rat pheochromocytoma (PC12) cells are treated with hydrogen peroxide and underwent cytotoxicity; apoptotic death is determined by MTT assay, flow cytometry analysis, PI/Hoechst 33342 staining and glutathione assay. The results suggest that glutathione C(60) derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity.

[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.

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.

Exon deletions of parkin gene in patients with Parkinson disease.

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

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.

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.

Rapid inhibition of the glutamate-induced increase of intracellular free calcium by magnesium in rat hippocampal neurons.

By using Fura-2/AM, the effects of magnesium (Mg2+) on the glutamate-induced increase of intracellular free calcium ([Ca2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1 x 10(-5) mol/L glutamate; Group B receiving 1 x 10(-5) mol/L glutamate and 1 x 10(-5) mol/L Mg2+ simultaneously; Group C receiving 1 x 10(-5) mol/L glutamate again after [Ca2+]i in group B back to the baseline. The results showed that in group A, [Ca2+]i was obviously increased. In group B, the changes in [Ca+] i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the delta[Ca2+]i was slightly decreased. It was suggested that Mg2+ could quickly inhibit the rise of [Ca2+]i induced by glutamate in the cultured hippocampal neurons in rats.

Induction of increased intracellular calcium in astrocytes by glutamate through activating NMDA and AMPA receptors.

To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium signal was investigated by monitoring the fluctuation of intracellular Ca2+ concentration ([Ca2+]i) on the basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamate on the intracellular calcium signal were observed after blockage of NMDA and (or) AMPA receptors. It was found that L-glutamate could induce an increased [Ca2+]i in most of the cells in concentration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selective antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, a selective antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively. The treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

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.

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.

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.

[A novel point mutation in parkin gene was identified in an early-onset case of Parkinson's disease].

OBJECTIVE: To investigate the distribution of possible novel mutation of parkin gene in variant subset patients with Parkinson's disease (PD) in China and to explore the role of parkin gene in the pathogenesis of PD. METHODS: Seventy patients were divided into early onset and late-onset groups, and 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 with standard procedures. Mutations of parkin gene (exons 1-12) in all subjects mentioned above were screened by PCR-SSCP. And in the samples with abnormal SSCP result, further sequencing was performed to confirm the mutation and its location. RESULTS: A new missense mutation (Gly284Arg) on exon 7 was found in a sample, while 4 samples from all subjects exhibited abnormal mobility shift on SSCP electrophoresis. All mentioned DNA variants were sourced from the samples of the patients with early-onset PD. CONCLUSION: Point mutation in parkin gene also contributes partly to the development of early-onset Parkinson's disease in Chinese population.