48 ± 0.05) at 3 weeks after surgery (0.95 ± 0.04). In the PD group, FEZ1 protein levels then decreased but at 5 weeks after injury were still PD0325901 cell line higher compared with the sham control group (Figure 2E,F). Along with FEZ1 expression, GFAP expression in striatum and substantia nigra was enhanced at 2 weeks after injury, peaked (0.77 ± 0.04 compared with 0.64 ± 0.03 in striatum, and 0.47 ± 0.05 compared with 0.27 ± 0.04 in substantia nigra), and then decreased (Figure 2G–J). In striatum of PD rats, GFAP expression levels were markedly higher at 2–4 weeks compared with the sham group (Figure 2G,H). However, in substantia nigra, GFAP expression levels were
increased at 2–5 weeks in the PD group compared with the sham group (Figure 2I,J). Because we found increased expression of FEZ1 and GFAP using real-time PCR and Western blot analysis, we chose two time points, 2 and 5 weeks after surgery, to examine brain sections from the PD and sham groups for immunohistochemical staining (Figure 3). This immunohistochemical
analysis at 2 weeks after surgery indicated that FEZ1 protein expression in PD rats Navitoclax was increased compared with the sham group. To determine the cellular localization and the temporal changes of FEZ1 immunoreactivity in brain of PD rats, we performed immunofluorescent staining on transverse cryosections. Because previous data have shown that FEZ1 was expressed in the cytoplasm of astrocytes and neurones and that FEZ1 may play important roles in human astrocytes [28, 29], we investigated whether FEZ1 colocalized with TH (a positive marker for dopamine neurones) or with GFAP (a positive marker for astrocytes). In sham-operated controls, FEZ1 mostly colocalized with TH (Figure 4A) but not with GFAP (Figure 4C). In contrast, at 2 weeks after injury, when FEZ1 had reached peak expression,
we found that FEZ1 was expressed in many TH-negative cells in PD group brain sections (Figure 4A). Double immunofluorescent staining demonstrated that these TH-negative cells were mostly FAD GFAP-positive astrocytes (Figure 4B,C). Cells morphologically looking like TH cells but only stained by FEZ1 might be other types of neurones. Furthermore, triple immunostaining was performed using FEZ1, TH and GFAP to better understand the redistribution of FEZ1 immunostaining in the PD group (Figure 5). Brain tissues from the sham and PD groups (2 weeks after 6-OHDA injection) were transversely sectioned and triple immunolabelled with FEZ1, TH and GFAP (Figure 5A–P). Next, we counted FEZ1-positive cells, FEZ1-positive astrocytes and FEZ1-positive dopamine neurones (Figure 5Q). FEZ1-positive dopaminergic neurones constituted the majority of FEZ1-positive cells in substantia nigra of the sham group. However, FEZ1-positive astrocytes composed the majority of FEZ1-positive cells in the PD group. The proportion of FEZ1-positive in other cell types was unchanged.