Analysis of NG2-glia in the striatum in 6-OHDA-lesioned Parkinsonian rats treated with L-DOPA by immunoconfocal microscopy
Firstly, we examined the spatial distribution of NG2-glia in the rat striatum of the experimental groups No lesion (control), 6OHDA lesion (6OHDA), 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+L-DOPA(Fig. 2B-E; see methods). The four subdivisions of the striatum (dorsomedial, dorsolateral, ventromedial, ventrolateral) were separately quantified, due to their functional properties, which reflect the diversity of the striatal dopaminergic system connections (Fig. 2F-I).
Parkinsonian rat brains were analyzed 32 days after the neurotoxin microinjection, corresponding to 6OHDA-lesion establishment and L-DOPA/vehicle time therapy. The analysis of the dorsolateral and ventrolateral striatal regions showed a slight increase (15%) in NG2-glia density when compared to the No lesion group. This minor increase was maintained after doxycycline treatment (experimental group6OHDA+DOXY , p<0.05; Fig. 2G and I).
Parkinsonian rats receiving L-DOPA and reporting LID presented a robust NG2-glia density reduction in the dorsolateral, ventrolateral and dorsomedial striatum when compared to the No lesion (control), 6OHDA lesion (6OHDA), 6OHDA+DOXY and 6OHDA+DOXY+L-DOPA groups (6OHDA+Vehicle+L-DOPA , p<0.001; Fig. 2F, G and I). Doxycycline administration before L-DOPA prevented dyskinesia and lead reduced NG2-glia density in the striatum regions (6OHDA+DOXY +L-DOPA ; p<0.001; Fig. 2F, G and I). There was a negative correlation of NG2-glia density with the dyskinesia score (dorsomedial r=-0.83, dorsolateral r=-0.88, and ventrolateral r=-0.64; p<0.0001; Fig. 2J, K and M). There was no NG2-glia immunolabelling alteration in the ventromedial striatum of the animals.
The profile of NG2-glia, analyzed by protein expression immunoblotting in dorsolateral striatum extract, revealed one band of about 26kDa corresponding to NG2-glia in both the control and the lesioned striatum (Fig. 2N). Lesioned rats treated or not with doxycycline showed a slight increase (17%) in NG2-glia expression when compared to No lesionones (6OHDA and 6OHDA+DOXY , p<0.05, Fig. 2N). The NG2-glia expression decreased in the lesioned striatum of L-DOPA-treated parkinsonian rats compared to all other groups (50-60%,6OHDA+Vehicle+L-DOPA , p<0.05, Fig. 2M). Doxycycline treatment induced a significant increase (76%) of NG2-glia immunoreactivity compared to the intensity detected in animals presenting LID (6OHDA+DOXY+L-DOPA ; p<0.05, Fig. 2N).