Neuroprotective effects of echinacoside in the mouse MPTP model of Parkinson's disease

In the present study, we investigated the neuroprotective effects of echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Cistanches salsa, against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic toxicity. We confirmed that exposure to MPTP in mice leads to permanent behavioral deficits and depletion of dopamine and its metabolites. When administered prior to MPTP, echinacoside reduced behavioral deficits, increased striatal dopamine and dopamine metabolite levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. In addition, pre-treatment with echinacoside significantly reduced caspase-3 and caspase-8 activation in 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in cerebellar granule neurons. Taken together, these findings suggest that echinacoside improves the behavioral and neurochemical outcomes in MPTP mice model of Parkinson's disease and inhibits caspase-3 and caspase-8 activation in cerebellar granule neurons, making the compound an attractive candidate treatment for various neurodegenerative disorders, including Parkinson's disease.     

Behavioural effects of angiotensin II in the mouse following MPTP administration.

1. The effects of the octapeptide angiotensin II (AT II) on some types of behaviour [threshold of seizures induced by timed intravenous infusion of pentylenetetrazol (PTZ), exploratory behaviour: ambulation, rearing and head-dips on a hole-board, and passive avoidance performance--step-through paradigm] were studied following repeated systemic administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to mice (30 mg/kg, i.p., twice daily for 5 days). AT II was administered s.c. (200 micrograms/kg), single and repeated (for 7 days) 7 days after withdrawal of MPTP. 2. PTZ seizure threshold was significantly increased by AT II both in groups with single and repeated administration (stronger than in the group treated only with MPTP and in the group untreated with MPTP). 3. AT II significantly increased the ambulation and rearing as well as the head-dips both on single and repeated administration as compared to groups treated and untreated with MPTP. 4. Administered immediately after the passive training trial AT II, on single and repeated administration, significantly increased latencies, i.e. it facilitated retention, in MPTP-treated mice. 5. These results were discussed in the light of DA and perhaps, GABA receptor supersensitivity developed in susceptible brain structures after withdrawal of repeated MPTP administration. 6. The adaptive changes in the effects of AT II on seizure threshold, exploratory behaviour and retention of passive avoidance performance might be associated with the altered receptor-receptor (AT II-DA-GABA) interactions in the brain.     

大花罗布麻对MPTP型小鼠的多巴胺能神经保护作用研究

目的观察大花罗布麻叶中槲皮素-3-O-槐糖苷和黄酮富集物对MPTP型帕金森症(Parkinson's disease,PD)小鼠模型的多巴胺能(dopaminergic,DA)神经保护作用。方法以C57/bl6小鼠为研究对象,采用MPTP腹腔注射30mg.kg-1连续5d制备PD模型,口服灌胃给予槲皮素-3-O-槐糖苷25mg.kg-1和大花罗布麻叶黄酮富集物250mg.kg-1,造模前7d开始给药。给药结束后进行自主活动和爬杆实验测试动物行为变化,荧光法测定纹状体中多巴胺含量,石蜡切片观察黑质神经细胞病理变化,分析大花罗布麻对多巴胺能神经的保护作用。结果槲皮素-3-O-槐糖苷25mg.kg-1预先给药1wk,能提升MPTP型PD小鼠的运动能力,提高纹状体中多巴胺含量,减轻黑质神经的损伤,增加酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞表达数量。结论槲皮素-3-O-槐糖苷能减轻MPTP对PD小鼠的DA神经损伤。     

KW-6002 protects from MPTP induced dopaminergic toxicity in the mouse

The risk of Parkinson's disease (PD) is associated with a lower intake of caffeine, a non-selective adenosine A2A antagonist. In agreement, genetic or pharmacological inactivation of adenosine A2A receptors in animal models of PD has demonstrated both symptomatic and neuroprotective effects. These findings and the lack of disease modifying therapies have led to intense research on adenosine A2A antagonists as a novel treatment for PD. In the present study the neuroprotective effect of the A2A receptor antagonist KW-6002 was investigated using different models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, which induced dopaminergic terminal and or dopaminergic cell loss and inflammation. Treatment with KW-6002 prevented the loss of dopaminergic striatal terminals and nigral cell bodies and inhibited the nigral microglia activation. Our results confirm previous findings that pharmacological inactivation of A2A receptors inhibits MPTP-induced dopaminergic damage at the level of striatum. In addition, we demonstrate for the first time that, after MPTP treatment in mice, an A2A antagonist is neuroprotective, and has anti-inflammatory effects, at the level of the substantia nigra. Thus, our data further support the use of A2A receptor antagonists as a novel neuroprotective therapy for PD.     

Neuroprotection by tetrahydroxystilbene glucoside in the MPTP mouse model of Parkinson's disease

Our in vitro experiments suggested that tetrahydroxystilbene glucoside (TSG) affords a significant neuroprotective effect against MPP⁺-induced damage and apoptosis in PC12 cells though activation of the PI3K/Akt pathway. This study was aimed to investigate the potential neuroprotective effect of TSG in 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP)-treated mouse model of Parkinson's disease (PD). We found that treatment of TSG protected dopaminergic neurons by preventing MPTP-induced decreases in substantia nigra tyrosine hydroxylase (TH)-positive cells and striatal dopaminergic transporter (DAT) protein levels. Furthermore, it was also associated with increasing striatal Akt and GSK3β phosphorylation, up-regulation of the Bcl-2/BAD ratio, and inhibition of the activation of caspase-9 and caspase-3. These results showed that TSG promoted dopamine neuron survival in vivo, the PI3K/Akt signaling pathway may have mediated the protection of TSG against MPTP, suggesting that TSG treatment might represent a neuroprotective treatment for PD.     

Sex differences in motor behavior in the MPTP mouse model of Parkinson's disease

Sex differences in Parkinson's disease (PD) have been reported in humans and rodent models, with a higher incidence in men and increased severity in male rodents. The current study examined sex differences and the effects of gonadal steroid hormones in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of PD. Male (n = 51) and female (n = 50) mice were gonadectomized and received physiologic replacement with testosterone or estrogen (Experiment 1), or no hormones (Experiment 2). Two weeks later, mice received either MPTP (10 mg/kg per day for 5 days) or saline. Higher doses killed female mice. Mice were tested one week after MPTP for motor performance using rotarod, pole and gait tests. In hormone-treated mice, males significantly outperformed females in all three tests (p < 0.05). Compared with females, males had a greater overall rotarod performance (ORP: 1317.1 ± 98.3 vs. 988.1 ± 95.6), descended a pole faster (7.1 ± 0.6 vs. 9.6 ± 0.7 s), and had longer stride lengths (hindlimb 7.3 ± 0.1 vs. 6.8 ± 0.1 cm). By contrast, ovariectomized female mice receiving saline outperformed castrated males on the rotarod (1296.6 ± 83.3 vs. 811.2 ± 113.7, p < 0.05) and descended a pole faster (9.7 ± 2.0 vs. 15.6 ± 1.9 s, p < 0.05). MPTP significantly impaired ORP (p < 0.05) in hormone-treated males (703.7 ± 65.5) and females (432.8 ± 88.6, p < 0.05). After MPTP, stride length was selectively decreased in males (hindlimb 6.6 ± 0.1 cm, p < 0.05), and pole test performance was unimpaired in either sex. After gonadectomy, MPTP did not decrease motor performance in males (p > 0.05) but significantly reduced ORP in females (975.9 ± 110.3 vs. saline females, p < 0.05). Our results show that small, chronic doses of MPTP produce subtle, sexually-dimorphic impairments in motor performance, but without a loss of tyrosine hydroxylase-positive neurons in the substantia nigra. In gonadectomized mice, this sex difference is reversed.     

MPTP致帕金森模型小鼠海马区NGF基因的表达研究

目的检测帕金森(Parkinson’s disease,PD)模型小鼠海马区(hippocampus)神经生长因子(nerve growth factor,NGF)基因的表达。方法采用腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)建立PD小鼠模型;利用蛋白免疫印迹(Western blot)法和免疫荧光技术(immunofluorescence,IF)检测中脑酪氨酸羟化酶(tyrosine hydroxylase,TH)表达鉴定PD模型建立是否成功;采用足迹分析法和避暗法分别检测小鼠运动能力和认知能力;利用RT-PCR和原位杂交技术(in situ hybridization)检测小鼠海马区NGF基因的表达。结果与对照组相比,模型组的TH、NGF水平均降低,行为学指标偏离正常值,且差异有显著性。结论 NGF可能在PD认知障碍的病理学发生、发展过程中发挥重要作用。     

Protective effects of neuronal nitric oxide synthase inhibitor in mouse brain against MPTP neurotoxicity: an immunohistological study.

We recently reported that neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole, can protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice. It protected against both dopamine depletions and tyrosine hydroxylase (TH) positive neuron decreases in the mouse brain. In the present study, we further examined whether 7-nitroindazole can also protect against the alterations of TH-, microtubule-associated protein 2a,b (MAP2)-, glial fibrillary acidic protein (GFAP)-, parvalbumin (PV)-, dopamine transporter (DAT)-, nNOS- or endothelial NOS (eNOS)-positive cells, in comparison with pargyline as a relatively selective inhibitor of the monoamine oxidase-B (MAO-B). The present study showed that nNOS inhibitor as well as MAO-B inhibitor has a dose-dependent protective effect against MPTP-induced striatal dopamine and DOPAC depletion in mice. Furthermore, the present study revealed that 7-nitroindazole and pargyline can protect the alterations of immunohistochemical changes in the striatum and substantia nigra after MPTP treatment. These protective effects may be, at least in part, produced by the reduction of neuronally derived NO and peroxynitrite caused by MPTP. Our results also demonstrate that MPTP can cause functional damage of interneurons in the substantia nigra. These results suggest the possibility that nNOS inhibitors as well as MAO-B inhibitors may be therapeutically useful in neurodegenerative diseases such as Parkinson's disease. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.     

Kinetics of [3H]MPP+ uptake in dopaminergic neurons of mouse: regional effects of MPTP neurotoxicity

In an effort to determine the specificity of MPTP/MPP+ toxicity with respect to the dopaminergic systems, the effect of prior MPTP treatment on [3H]MPP+ uptake in the striatum and olfactory tubercle of BALB/cBy mice was examined. Kinetic analysis of [3H]MPP+ uptake indicated a reduction of Vmax values in both striatum (49%, P less than 0.05) and olfactory tubercule (26%, P less than 0.05). MPTP treatment did not significantly alter the Km in either region, although MPP+ accumulates in both olfactory tubercle and striatum, these dopaminergic systems show different sensitivity to the neurotoxicity of MPTP/MPP+.     

Galanin: Ca2+-dependent contractile effects on the isolated mouse distal colon

Galanin provoked concentration-dependent contractions of the longitudinal muscle of the mouse distal colon. The responses were not affected by atropine, mepyramine, methysergide, phentolamine, timolol, naloxone or tetrodotoxin. In contrast, the contractile responses were reduced by nifedipine and in Ca2+-free medium. These observations indicate that the contractile effects of galanin on the mouse distal colon result from a direct activation of smooth muscle cells and that these effects are mainly dependent on an increase in Ca2+ inflow.     

Contractile effects of substance P and other tachykinins on the mouse isolated distal colon.

1. Substance P (SP), physalaemin, eledoisin and kassinin induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The responses were not antagonized by atropine (1.5 x 10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), timolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). They were enhanced by tetrodotoxin (TTX, 1.5 x 10(-7) M). These observations indicate that the contractile responses to the tachykinins result from a direct activation of smooth muscle cells. 2. The contractile activity provoked by SP and physalaemin was inhibited by nifedipine (a Ca2+-entry blocker) and was abolished in Ca2+-free EGTA solution. Such data suggest that the myogenic effects of SP and physalaemin are mainly dependent on their ability to promote Ca2+ influx. 3. Eledoisin and kassinin evoked a contractile response in the absence of external Ca2+ and their myogenic activity was, to some extent, resistant to the inhibitory effect of nifedipine. This may indicate that an additional process, probably the release of an intracellularly bound Ca2+ store, participates in the mechanism by which eledoisin and kassinin contract the mouse distal colon. 4. After desensitization of the mouse distal colon to SP, the contractile activity provoked by SP or physalaemin was totally abolished whilst the responses evoked by eledoisin and kassinin were barely affected. These observations and other experimental findings indirectly support the assumption that the mouse distal colon could possess different tachykinin-binding sites.     

Older dopaminergic neurons do not recover from the effects of MPTP

Recent studies have shown that dopaminergic neurons in young mature mice recover fully from toxic injury induced by MPTP. The aim of the present study was to assess whether or not older dopaminergic neurons were capable of similar recovery. We now report that older dopaminergic neurons do not recover from the effects of MPTP. This suggests that with age, neurons in the nigrostriatal system begin to lose their regenerative capacity. This phenomenon could underlie the process of neuronal degeneration known to occur in a number of age-related diseases, including Parkinson's disease.     

Acute electrophysiological and neurochemical effects of administration of MPTP in mice

The changes occurring during the first few hours after subcutaneous administration of the catecholaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated. Injections of MPTP (30-60 mg/kg s.c.) reduced the impulse rate by 12-45% in all dopaminergic neurones tested in the pars compacta of the substantia nigra. Depressions were maximal at 11 min and remained present for more than 2 hr after injection. This effect was completely abolished by prior administration of the catecholamine uptake inhibitor, nomifensine (13-69 mg/kg s.c.), which prevents the toxic metabolite of MPTP 1-methyl-4-phenylpyridine (MPP+) from entering dopaminergic neurones. These results suggest an intraneuronal mechanism underlying the observed depressions in impulse rate. Levels of dopamine (DA) were decreased at 3 hr after administration of MPTP (50 mg/kg s.c.) by 60% and 54% in the striatum and substantia nigra, respectively. Pretreatment with nomifensine (25 mg/kg, intraperitoneally) prevented the decrease in DA only in the striatum. This suggests an acute DA-releasing effect of MPTP in the striatum, mediated by intracellular accumulation of MPP+, while not explaining the depression of activity of DA neurones occurring with a different time course.     

Electrophysiological and neurochemical correlates of the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on central catecholamine neurons in the mouse

Summary 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an agent which produces a parkinsonian syndrome in man. To explore the use of MPTP in a rodent model of parkinsonism, male albino mice (NMRI) were given MPTP (50 mg/kg, s.c.) twice with a 6–8 h interval. Up to 10 weeks after injection, mice were killed and high-pressure liquid chromatography was used to assay dopamine (DA) and noradrenaline (NA) concentrations in various regions of the CNS. At 4 and 10 weeks after injection, DA levels were significantly reduced in occipital cortex (-40%), hippocampus (-30%), and striatum (-60%). NA levels were reduced by 60–80% in frontal and occipital cortex, hippocampus, and cerebellum. Neither DA nor NA concentration was reduced in spinal cord. Dopaminergic denervation was also suggested by electrophysiological data which showed that treatment with MPTP increased the spontaneous discharge rate of caudate neurons and decreased the potency of locally administered phencyclidine, an indirect DA agonist. However, denervation was evidently not complete enough to produce postsynaptic receptor supersensitivity, as MPTP treatment did not increase the potency of locally applied DA, and it did not increase ³H-spiperone binding in striatal membrane preparations. These results suggest that MPTP causes regionally selective and long-term reductions of catecholamine transmission in the CNS of the mouse.This work has been supported by the Swedish MRC (04X-2295, 14X-03185, 14P-5868, 14P-5867); M. Bergvalls Stiftelse, Jeansson's Stiftelse, The Expressen Prenatal Research foundation and The Swedish Council for Planning and Coordination of Research (Project: Chemical Hazards in the Environment).     

Effects of neurotensin on the isolated mouse distal colon

The effects of neurotensin were studied in the isolated mouse distal colon. This peptide had potent stimulatory effects which were of pre- or postjunctional origin according to the concentrations used. At low concentrations (10(-11)-10(-10) M) neurotensin induced neurogenic non-cholinergic contractions which seemed to result from the release of substance P (or substance P-like activity) by enteric excitatory nerves. At higher concentrations (10(-9)-10(-7) M) neurotensin elicited a biphasic effect consisting of transient relaxation rapidly followed by myogenic contraction. The bee venom toxin apamin inhibited the NT-induced relaxation while inhibition of prostaglandin synthesis by indomethacin abolished the contraction phase. All these responses were tightly related to the extracellular Ca2+ concentration. These properties of neurotensin point to a possible role for this peptide as a modulator of colonic motility.     

Behavioural and neurochemical effects of phosphatidylserine in MPTP lesion of the substantia nigra of rats

The present study investigated the effects of intranigral MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) infusion on rats treated with phosphatidylserine and evaluated in two memory tasks and on striatal dopamine levels. The results indicated that MPTP produced a significant decrease in the avoidance number in comparison to sham-operated and non-operated rats submitted to a two-way avoidance task. MPTP-lesioned rats exhibited an increase in the latencies to find the platform in cued version of the water maze in comparison to sham-operated and non-operated animals. The tested toxin reduced striatal dopamine levels in comparison to sham-operated and non-operated groups. A final surprising result was that phosphatidylserine was unable to reverse the cognitive deficits produced by MPTP or the reduction of striatal dopamine levels. In conclusion, the data suggest that MPTP is a good model to study the early impairment associated with Parkinson's disease and phosphatidylserine did not improve the memory impairment induced by MPTP.     

Behavioural effects and supersensitivity in the rat following intranigral MPTP and MPP+ administration

Unilateral intranigral injections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and MPP+ (1-methyl-4-phenylpyridine) were given to young rats and unilateral intranigral injections of MPTP were given to old rats. MPTP in old rats and MPP+ in young rats induced ipsiversive circling for at least one week after injection and contraversive circling after the systemic administration of apomorphine; the number of D-2 receptors (Bmax) in the striatum of the injected hemisphere increased compared with that of control rats. MPTP in young rats induced only short-lasting ipsiversive circling and no contraversive circling after apomorphine; the number of striatal D-2 receptors did not increase. These results suggest that the neurotoxicity of MPTP is age-dependent in the rat, and that MPTP has neurotoxic effects on the nigrostriatal dopaminergic system in old rats and induces dopamine receptor supersensitivity in the denervated striatum.     

钒联合维生素D3对结肠癌荷瘤裸鼠的治疗作用

目的 探讨钒联合维生素D3(VitD3)对结肠癌的治疗作用.方法 100只裸鼠皮下接种HCT116结肠癌细胞,成瘤后原位移植.建立结肠癌裸鼠模型后随机等分为钒(A)组、Vit D3(B)组、钒联合VitD3(C)组和对照(D)组.用药4周后,每组取18只脱颈处死,检测肿瘤体积和转移灶数节,免疫组化检测瘤体增殖细胞核抗原(PCNA)指数,其余裸鼠用于生存试验,流式细胞仪检测肿瘤细胞凋亡和细胞周期.结果 故 C组裸鼠增殖指数显著低于D组(P<0.05),荷瘤裸鼠生存时间显著延长(P<0.05),肿瘤细胞凋亡增加(P<0.05),细胞停留在G0/G1期.结论 钒联合Vit D3具有明显的抗结肠癌细胞增殖、诱导细胞凋亡的作用.     

Oral administration of yessotoxin stabilizes E-cadherin in mouse colon

YTX has been shown to disrupt the E-cadherin-catenin system in cultured epithelial cells, raising some concern that ingestion of seafood contaminated by YTX might favour tumour spreading and metastasis formation in vivo. In order to probe whether YTX might affect cadherin systems in vivo, we have set up a study involving repeated oral dosing of the toxin in mice (1mg/kg/day, for 7 days) and analysis of E-cadherin and N-cadherin in tissue extracts obtained at the end of the dosing scheme, as well as 1 and 3 months after YTX administration. We found that the E-cadherin pools obtained from lung and kidney were not altered by YTX in any of our experimental conditions. Extracts from mouse colon contained intact E-cadherin and an E-cadherin fragment of about 90 kDa (ECRA(90)), displaying a molecular alteration resembling that caused by YTX in cultured cells. We found that the relative proportion of ECRA(90), as compared to intact E-cadherin, was higher in colon extracts from control mice than from YTX-treated animals, indicating that oral administration of YTX to mice stabilizes E-cadherin of mouse colon. No significant difference could be detected in samples prepared from colons obtained 30 or 90 days after termination of YTX treatment. Oral administration of YTX to mice did not lead to a significant increase in the fragments of E-cadherin detectable in serum, neither it altered the N-cadherin pool of mouse heart. Electron microscopy analysis showed no substantial ultrastructural differences between controls and YTX-treated mice. Our findings show that ingestion of food contaminated by YTX poses a low risk of disruption of the E-cadherin system in vivo.     

Mechanisms of the contractile responses to morphine of the mouse colon in vitro

1. Morphine induced a contractile response in the mouse colon which consisted of two phases or components. 2. The first component was dose-related and was inhibited by tetrodotoxin, atropine and naloxone, but was insensible to hexamethonium, propranolol, phentolamine, diphenhydramine and methysergide. 3. The second component of the contractile effect was not modified by atropine, hexamethonium, propranolol, phentolamine, guanethidine or diphenhydramine, but was antagonized by naloxone, tetrodotoxin and serotonin antagonists. 4. Tachyphylaxis was observed only for the first component of the morphine induced contractile response of the mouse colon.