Endothelial proliferation and increased blood-brain barrier permeability in the basal ganglia in a rat model of 3,4-dihydroxyphenyl-L-alanine-induced dyskinesia

Abstract

3,4-Dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia is associated with molecular and synaptic plasticity in the basal ganglia, but the occurrence of structural remodeling through cell genesis has not been explored. In this study, rats with 6-hydroxydopamine lesions received injections of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) concomitantly with L-DOPA for 2 weeks. A large number of BrdU-positive cells were found in the striatum and its output structures (globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata) in L-DOPA-treated rats that had developed dyskinesia. The vast majority (60-80%) of the newborn cells stained positively for endothelial markers. This endothelial proliferation was associated with an upregulation of immature endothelial markers (nestin) and a downregulation of endothelial barrier antigen on blood vessel walls. In addition, dyskinetic rats exhibited a significant increase in total blood vessel length and a visible extravasation of serum albumin in the two structures in which endothelial proliferation was most pronounced (substantia nigra pars reticulata and entopeduncular nucleus). The present study provides the first evidence of angiogenesis and blood-brain barrier dysfunction in an experimental model of L-DOPA-induced dyskinesia. These microvascular changes are likely to affect the kinetics of L-DOPA entry into the brain, favoring the occurrence of motor complications.

Figure 1.

Schematic illustration of the experimental design applied in this study. Boxes depict treatment periods with daily injections of saline (white), l-DOPA (gray), and BrdU (hatched). Lines illustrate length of time before and after treatment.

Figure 2.

Epifluorescence photomicrographs of BrdU-positive cells in caudate–putamen (A–C), globus pallidus (D–F), entopeduncular nucleus (G–I), and in the substantia nigra pars reticulata (J–L). Dyskinetic animals (right column) show an increased cell proliferation in all four structures compared with nondyskinetic cases (middle column) and saline-treated animals (left column). Pictures were taken from animals treated with l-DOPA or saline for 14 d (experiment 1). Scale bar, 200 μm.

Figure 3.

Cell proliferation in the basal ganglia after l-DOPA treatment is higher in dyskinetic rats (n = 10) compared with nondyskinetic cases (n = 4) and saline-treated control animals (n = 8). Drawings show coronal sections through the regions examined, and white boxes outline the areas counted from (A–M). Bar histograms show the number of BrdU cells/mm² in each structure and group. ^#p < 0.05 versus nondyskinetic animals; *p < 0.05 versus control animals; ^§p < 0.05 versus intact side within the same group; two-factor ANOVA and post hoc comparisons with Tukey’s HSD test. MCx, Motor cortex.

Figure 4.

The extent of cell proliferation is positively correlated with the severity of dyskinesia. The number of BrdU-labeled cells (average number of cells per square millimeter) counted in the caudate–putamen (A), the globus pallidus (B), the entopeduncular nucleus (C), and the substantia nigra pars reticulata (D) are plotted against the cumulative axial, limb, and orolingual AIM scores (sum of all AIM scores acquired during 5 test sessions) from the l-DOPA-treated animals in experiment 1 (n = 14, except for the EP, in which sections were not available in one of the dyskinetic animals). Dyskinetic rats are represented by white circles and nondyskinetic cases by black circles. The probability value of the regression (p) and the correlation coefficient (R) are given in the bottom right corners of each graphs.

Figure 5.

Confocal three-dimensional reconstructions of BrdU-immunoreactive cells (red) colabeled with GFAP (green; A), NG2 (green; B), or RECA-1 (green; C). Reconstructed orthogonal images are presented as viewed in the x–z and y–z directions (bottom and right panels, respectively). Bottom row (D–F) shows epifluorescence photomicrographs of dual-antigen immunostaining for BrdU (D) and RECA-1 (E; the merged picture is shown in F). Photos were taken in the substantia nigra pars reticulata from a dyskinetic rat. Scale bars: A–C, 20 μm; D–F, 100 μm.

Figure 6.

Microvascular changes associated with l-DOPA treatment. A, Blood vessel lengths were increased in the basal ganglia in dyskinetic animals (n = 8), and the increase reached significance in the EP and SNr. B, In addition, dyskinetic animals showed an increased percentage of blood vessel profiles immunoreactive for the immature endothelial marker nestin in all basal ganglia structures. C, Levels of albumin immunostaining in the neuropil were increased in the EP and SNr in dyskinetic rats. D, The percentage of blood vessel profiles immunoreactive for EBA was reduced in dyskinetic animals in all of the basal ganglia regions examined. In each dataset, measurements taken on the side ipsilateral to the lesion are expressed as percentage of the values from the contralateral intact side (in which no group differences were found). ^#p < 0.05 versus nondyskinetic animals (n = 6); *p < 0.05 versus control animals (n = 8). One-factor ANOVA and post hoc comparisons with Student–Newman–Keuls test. The p value from the one-factor ANOVA is shown in C, panel III, because the ANOVA revealed a significant group effect, but the post hoc test failed to detect significant pairwise differences between specific groups.

Figure 7.

Bright-field photomicrograph sections through the SNr and the EP stained for markers of BBB disruption (EBA and albumin) and angiogenesis (nestin). In both the SNr (A–I) and the EP (J–R), dyskinetic animals (right column) show a loss of EBA expression on blood vessel profiles (C, L), increased albumin staining in the brain parenchyma (F, O), and increased numbers of nestin-positive blood vessels (I, R) compared with both nondyskinetic cases (middle column) and saline-treated control animals (left column). Scale bar, 250 μm.

Figure 8.

Dual-antigen immunostaining of microvessels confirmed the occurrence of BBB dysfunction and angiogenesis in dyskinetic animals. Double immunohistochemistry for EBA and RECA-1 (A–I) or EBA and nestin (J–R) was performed on sections from the SNr. A reduced expression of EBA in dyskinetic animals (A) compared with nondyskinetic (D) and saline-treated control (G) animals was not attributable to a loss of vessels, which were evenly distributed throughout the sections, as shown by RECA-1 staining (B). C, F, and I show merged photomicrographs. Photomicrographs in the three bottom rows show staining for EBA (J, M, P), and nestin (K, N, Q), and the corresponding merged pictures (L, O, R) in the three experimental groups. Dyskinetic animals (K) show an increased number of nestin-positive microvessels compared with nondyskinetic animals (N) and controls (Q). A subset of the nestin-immunoreactive vessels was located in regions with distinct loss of EBA staining (J, K, L). Scale bars, 100 μm.

Figure 9.

Time course of l-DOPA-induced cell proliferation, angiogenesis, and BBB disruption. Rats were treated with l-DOPA for 3 d (n = 4), 6 d (n = 6), 10 d (n = 7), and 14 d (n = 6), and BrdU injections were given during the last 3 d of l-DOPA treatment. Only animals that showed a dyskinetic-like response to l-DOPA were included in the analysis. A, Number of BrdU-positive cells per square millimeter in the different structures examined. B, C, EBA (B) and nestin (C) expression on blood vessel profiles expressed as percentage of immunoreactive vessels within the sample area on the side ipsilateral to the lesion. *p < 0.05 versus control animals (n = 6); p < 0.05 versus 3 d; ^○p < 0.05 versus 6 d; ^♦p < 0.05 versus 10 d. One-factor ANOVA and post hoc comparisons with Student–Newman–Keuls test. The p value from the one-factor ANOVA is shown in B, panel II, because the ANOVA revealed a significant group effect, but the post hoc test failed to detect significant pairwise differences between specific groups.