| Objective: In this study,two types of drug delivery methods,subcutaneous injection approach and simple diffusion delivery(SDD)approach were applied to the administration of the neuroprotective drug,rasagiline,based on the establishment of 6-OHDA parkinson’s disease model.By means of DTI and FLASH techniques,the fractional anisotropy(FA)and transverse relaxation time(T2*)values in the PD related brain zone,substantia nigra(SN)and corpus striatum(CS)were measured dynamically and in vivo to reveal the corresponding pathologic changes during the development of PD,and further to evaluate the neuroprotection effect of rasagiline and the validity of the above two delivery methods.Methods: Animal procedure: thirty-four male Sprague-Dawley rats were randomly divided into 5 groups,Control group: control group receiving 0.9% saline supplemented with 0.1% ascorbic acid(2μl)by intracranial injection,n=6Low dose 6-OHDA group: animals receiving 6-OHDA(10μg),dissolving in 0.9% saline supplemented with 0.1% ascorbic acid(2μl)by intracranial injection,n=6High dose 6-OHDA group: animals receiving 6-OHDA(15μg),dissolving in 0.9% saline supplemented with 0.1% ascorbic acid(2μl)by intracranial injection,n=6SDD group: animals receiving the same dose of 6-OHDA as G2.After the injection,rasagiline,20μg/kg,which was dissolved in 2 μl 0.9% saline,was immediately injected by SDD method at one time,n=8.Subcutaneous injection group: animals receiving the same dose of 6-OHDA as the low dose 6-OHDA group.After the injection,animals received daily i.h.doses of rasagiline(2mg/kg)for 6w,n=8.After the skin preparation and sterilization,the head skin of the rats were incised and the subcutaneous tissue,superficial and deep fascial layer,the periosteum were carefully divided to display the skulls between the fontanelle and posterior fontanelle.The rats were then fixed on the stereotaxic apparatus and 6-OHDA or rasagiline was injected into right SN(AP:-4.8mm;R: +1.9mm and DV:-3.6 relative to Bregma).MRI: MRI scanning was carried out onthe first week,second week,forth week and sixth week after the injection.The coronary T2 WI was collected firstlyand used as a base,the axial T2 WI,DTI and FLASH images were further obtained.The center and orientation of DTI and FLASH were copied from axial T2 WI.The image processing method of DTI: the T2 W images were selected as the structure images and then were fused with the DTI images.The ROI was drawn on the layer which showing the maximum level of SN and CS.The image processing method of FLASH: the images including the SN and CS of different TE value were labeled respectively to get the T2* image.The image showing the clearest anatomical characters(TE=15ms)was chosen as the structure image.The ROI which was 5mm2 was drawn on the layer showing the maximum level of SN and CS.Immunohistochemistry: the rats were sacrificed after the last MR scan(6w after modeling)and the brains were collected for slicing,deparaffinating,tissue repair,incubation,DAB developing and counterstain.The histological sections were observed under the microscope and photographed.Statistical analyses were performed with the SPSS13.0 software.The time effects on FA and T2* values were analyzed using the repeated measurement design.The FA and T2* values on the right side of different time points were compared using one-way ANOVA.The bilateral FA and T2* values in SN and CS were compared using paired t-test.Differences were considered significant at P<0.05.Results: 1 The FA and T2* values of the low and high dose 6-OHDA groups 1.1 The FA value in the right SN of the low and high 6-OHDA groups on the 1st were both obviously lower than that of the control group and the left side of their own group(P<0.05),and the FA value was 22.62% lower than that of the control group for the low dose group and 25.30% lower than that for the high dose group.On the 6th week,the FA values of the two model groups were both higher than those of the control group and the left side of their own,and the FA value was 38.34%,which was higher than that of the control group for the low dose group and 39.87% higher than that for the high dose group.The FA values in the right CS on the 4th week of the high and low dose 6-OHDA groups were obviously lower than that of the control group and the left of their own,and the FA value was 28.91%,which was higher than that of the control group for the low dose group and 24.37% higher than that for the high dose group.The FA values in the right SN of the two different dose 6-OHDA groups both showed a gradually increased tendency.The FA values in the right CS of the two different dose 6-OHDA groups showed a gradually decreased tendency during 1-4w,which was the lowest on the 4th week,and then showed a gradually increased tendency in 4-6w.1.2 On 6w,the T2* values of the two different dose 6-OHDA groups were lower than that of other 3 groups.There were significant differences on the T2* values in the right SN among the different groups(P<0.05),which decreased gradually in the low and high 6-OHDA groups.2 There was no significant difference for the FA value in the right SN among the CS of the SDD,subcutaneous injection group and the group in the observed time(P>0.05).The FA and T2* values in the SDD and subcutaneous injection groups didn’t change much over time,and no correlativity has been shown between them.There were significant differences of the FA and T2* values between the two drug treatment groups(SDD and subcutaneous injection groups)and the low and high dose 6-OHDA groups.The values in the SN decreased on the 1st week and increased on the 6th week and in the CS the value was lowest on the 4th week.However,the FA value in the SDD and subcutaneous injection group were both close to that of the control group.The T2* values in the right SN of the two treatment group were lower than that of the model groups.Conclusions:1 The FA and T2* values in the right CS and SN have some regular changes after the injection of 6-OHDA.The FA values in SN significant decreased at early stage,and gradually increased with time,which has shown a strong relationship with the observation time.There was no obvious change for the FA values of the both side of SN and CS in SDD and subcutaneous injection groups,which were both similar to that of the control group.The FA value in the injection side of the CS reached its lowest level on 4th week,and existed significant differences with the other three groups.The FA values in SDD and subcutaneous injection groups were also similar to that of the control group.These findings provided a strong evidence for the effectiveness of the two methods of administration.2 The T2* values in the right SN of the low dose 6-OHDA and high dose 6-OHDA groups presented a negative trend with the observation time,which indicated that with the progression of PD,the iron deposition aggravated gradually and the rasagiline were effectively against iron deposition.3 There was correlation between the FA and T2* values in the right SN of the low dose and high dose groups,namely the T2* value decreased the FA value,which indicated that there was the possibility of an association between neuron injury and the iron deposition. |
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