Expression Of Sonic Hedgehog In The Substantia Nigra In A Rat Model Of Parkinson Disease

BackgroundParkinson disease(PD) refers to a kind of neurodegenerative disease which is based on the deficiencies of dopaminergic neurons in substantia nigra and striatum. Dopamine mediates the signal transduction from nigrostriatal circuit loop to dorsal caudate putamen of surcingle.Dopamine deficiency will cause symptoms like malfunction of extracorticospinal tract,generating static tremor,myotone and bradykinesia.Sonic hedgehog(Shh) protein plays an essential role in differentiating dopaminergic neuron of midbrain during embryonic development.It promotes the survival of embryonic dopaminergic neuron when developing and differentiating,and protects them.Studies showed that when tissues are damaged,some embryonic protein will have stronger expression ability and participate in tissue repair.However no report is on about Shh changes after dopaminergic neuron damage of midbrain. Thus,in the first stage of our experiment,we established PD rats using 6-hydroxydopamine(6-OHDA),then to see the expression of Shh by immunofluorescence histochemical stain.It could be said that this experiment is of high value in the study of reconstruction of nigrostriatal circuit loop. Along with average life span lengthening and aging population,Parkinson disease incidence is apparently increasing.Present treatments include drug treatment and neurosurgical approaches.L-Dopamine and some other drugs may be curative,but after long term use,the curative effect decreases along with some side effect.Neurosurgery on the other hand,is risky and may bring trauma.Until now,we haven't found a simple and efficient treatment of drug to cure PD patients.And developing new treatment and investigating mechanisms of PD is hot in research.Citicoline(CC) is now considered one of the most promising neural protective agents.Maybe through investigation of the effectiveness of this drug in improving neuron function,we can reveal the function of Shh in the self-reparative process of dopaminergic neuron. Citicoline is the precurosor of lecithin synthesis,which is essential in cytolemma, providing bilineurin and vitamin B complex for acetylcholine synthesis.And citicoline can motivate the activation of tyrosine hydroxylase and dopamine releasing, good for safety.It can be used clinically for cerebrovascular disease,especially stroke. It is helpful in treating recognition or behavioral disturbance.Recent reports supported that it was helpful in treating PD.In the second step of our study,we treated PD rat with citicoline by peritoneal injection,to observe the therapeutic effect. And then,we discussed the protection of citicoline to dopaminergic neuron as well as the changes of Shh expression.Part One Establishment of a rat model of Parkinson diseaseObjectiveTo establish a qualified rat model of Parkinson diseaseMethods1.Experimental animals:healthy adult SD rats were evenly randomized into normal control group,sham operated group and PD model group.It doesn't matter if the rat is female or male. 2.By stereotaxis of the brain,we localized four points at the right side of corpora striata within two pin holes,then injected 6-OHDA.3.Behavioral test:Peritoneal injection of apomorphine.Calculation of circles the rat sidespin to uninjured side at 1^st,2^nd,4^th,6^th week(spin circles＜7 r/min were considered successful PD models).4.Data analysis:Data were analyzed by SPSS13.0 statistical package,and were presented as mean±standard deviation.Statistical significance was defined as P＜0.05. Repeated Measures was used to compare the differences in 4 test results.Results1.Left spin circles＞7 r/min in PD rats models induced by apomorphine.Normal group rats or sham operated group rats didn't present rotational behavior,while model group rats all showed rotational behavior.And there were 6 rats had Left spin circles＞7 r/min,and＞210 r/30mins.They were regarded as successful PD rats.Mauchly's Test showed W=0359,P=0.588,which did not deny Mauchly's hypothesis,and F=399.319,P＜0.001.There was significant difference between the results of 4 times behavioral tests(P＜0.001),except the results between the 4^th week and the 6^th week (P=0.057).2.There are other abnormal behaviors in PD rats such as sniffing,foraging, standing up,titillating and face-washing.Part two The up-regulated expression of sonic hedgehog in PD rats' substantia nigraObjectiveTo detecte Sonic Hedgehog's expression in substantia nigra of PD ratsMethods1.Perfusion and fixation of nomal rats,sham operation group and PD rats;2.Produce frozen brain sections; 3.Immunofluorescence double staining of frozen brain section for the the detection of expression of Shh and TH;4.Observation of immunofluorescence double staining of Shh and TH with Laser confocal microscopy;5.Obtained fluorescent value of Shh was quantitatively analyzed by Leica Confocal Software;6.Data analysis:Data were analyzed by SPSS13.0 statistical package.And one-way ANOVA was used to compare the differences of the fluorescent value of 3 groups. Statistics were presented as mean±standard deviation,with statistical significance defined as P＜0.05.Results1.In normal control group,there are large amount of TH positive cells and fibril. The cell body is satiation with much evection.In sham operation group,the condition is the same as in normal control group.And there is no sign of specific staining of Shh;in PD model group,TH positive cells and neural fibril significantly decrease. And the cells are small and undersize,with apparent Shh staining.Also,the two kinds of fluorescence co-localized.2.The fluorescent value of 3 groups was heterogeneity of variance,F＝353.528, P＜0.001.So Welch Measures was used and showed that F＝180.575,P＜0.001.The differences between groups were significant.And there was no significant difference between normal group and sham operated group(P＝0.998,P＞0.05).But,contracted with the normal group and the sham operated group,model group's fluorescent value showed significantly higher than the former two groups(P＜0.001).It was proved Shh's up-regulatedly expresses in PD rat's substantia nigra.ConclusionsShh expression in substantia nigra of PD rats was up-regulated,indicating effectiveness of Shh in dopaminergic neuron of midbrain.Part three Relationship of Protection of Citicoline to Dopaminergic neuron and Shh ExpressionObjectiveTo discuss the protection of citicoline to dopaminergic neuron to changes in Shh expressionMethods1.Thirty PD rat models were evenly randomly divided into sham operation group(with 4μl sodium containing 0.2g/L antiscorbic acid injected at localized four points at the right side of corpora striata within two pin holes,and 20 days after the operation,were peritoneal injected 5%glucose solution once per day,for a continuous 20 days),model group(with 4μl sodium containing 0.2g/L antiscorbic acid injected at localized four points at the right side of corpora striata within two pin holes,and 20 days after the operation,were peritoneal injected 5%glucose solution once per day,for a continuous 20 days),and citicoline treatment group(with 6-OHDA injected at localized four points at the right side of corpora striata within two pin holes,and 20 days after the operation,were peritoneal injected citicoline solution once per day,for a continuous 20 days).2.Three groups were raised in the same environment,and all the rats' hearts were perfused with 4%paraform.And then,took out the brains.3.Frozen brain slices were made with thickness of 35μm,preserved with dishes in 0.02 M PBS solution.4.Brain slices were divided into groups A,B.Group A slices were conducted Sonic Hedgehog-TH immunofluorescence double staining;Group B slices were conducted Shh antibody-DAB staining.5.Data analysis:Data were analyzed by SPSS13.0 statistical package and One-way ANOVA to compare the differences of the rate of both sides of TH-positive cells between 3 groups,presented as meant standard deviation.Statistical significance was defined as P＜0.05.Independent-Samples t Test was performed to compare the differences of Shh-positive cell count in uninjured side between the model group and the treatment group.Results1.Shh-TH double immunofluorescence staining results showed that:contracted with the model group,the treatment group had more TH-positive neurons on the injured side of substantia nigra.The data of 3 groups was heterogeneity of variance, F＝5.058,P＝0.014,P＜0.05.So Welch Measures was used.And it showed that F＝180.575,P＜0.001,which mean the differences between groups were significant. What's more,the data of 3 groups had significant differences from each other (P＜0.001).And Shh-positive expression can be seen in the injured sides of the two groups.2.Shh antibody-DAB staining:both in the injured side,there are some Shh-positive cells in the substantia nigra of model group and treatment group.And Shh positive cells appear bigger and with larger amount in the treatment group.The data of Shh-positive cell count in the injured side was population variance(F＝3.276, P＝0.087,P＞0.05).In this case,it turned out that t＝10.534,P＜0.001,which mean there were significant differences in the two groups.And there wasn't such kind of cells in both sides of substantia nigra of negative control group.Conclusion1.In the PD rat models,citicoline can protect TH-positive cells. The neuroprotective effect of citicoline possibly has relationship with Shh protein. Citicoline might initiates its neuroprotective effect by up-regulating the expression of Shh.