PMC

N-syndecan, a receptor for pleiotrophin, is expressed by nigral dopaminergic neurons. Triple immunolabeling in the substantia nigra pars compacta for TH (A, blue), N-syndecan (B, green) and PTN (C, red) showing co-localization of the three markers in some neurons (solid arrow-heads). The open arrow-head indicates a cell that only contains N-syndecan, the open arrow indicates a cell that contains PTN and TH, and the solid arrows indicate cells immunoreactive for TH that do not contain PTN or N-syndecan. Scale bar: 50 μm.

Pleiotrophin over-expression rescues TH-immunoreactive terminals in the striatum. (A) Tyrosine hydroxylase-immunoreactive (TH-ir) area in the 6-OHDA or vehicle injected striatum of Adβgal and AdPTN treated animals (percent of contralateral side). Sham/Adβgal: 90 ± 1%, Sham/AdPTN: 90 ± 1%, 6-OHDA/Adβgal: 53 ± 3%, 6-OHDA/AdPTN: 63 ± 2%. Student Neuman Keuls post hoc test, after significant interaction in a two-way ANOVA and Logit transformation of the variable [], *p< 0.05. Mean ± SEM. (B) Outlines on photomicrographs illustrate the area used for optical density measurements on a representative coronal section of the striatum (+0.20 mm from bregma). Scale bar: 500 μm.

Inflammatory response after adenoviral infection. (A) Inflammatory infiltrate induced by the adenoviruses as seen in Nissl stained sections in normal rats. The coronal sections correspond to the same Paxinos and Watson stereotaxic atlas plate shown in Figure 3A (right). The infiltrate has a similar extension in Adβgal and AdPTN injected rats and was higher at 7 than 14 days after injection. Scale bars: 500 μm (low magnification images) and 100 μm (high magnification images). (B) Microphotographs showing activated macrophages labeled with antibodies against ED1 (left) or Mac1 (right), in double labeling with PTN (red). Scale bars: 50 μm.

Pleiotrophin over-expression was induced after the lesion to mimic more closely a possible use in patients. Schematic illustration of the experimental design. Rats were injected with Adβgal or AdPTN in the substantia nigra seven days after being injected with 6-OHDA or vehicle in the striatum. The animals were sacrificed 14 days after adenoviral injections (21 days after 6-OHDA injection). Histology and immunohistochemistry were performed on free-floating coronal serial tissue sections of the entire striatum and substantia nigra. Beta-galactosidase activity was detected using X-gal as substrate (A). Pleiotrophin (B) and TH (C and D) were revealed with DAB as chromogen. The microphotographs were taken from sections of 6-OHDA lesioned rats treated with Adβgal (A and D) or AdPTN (B and C).

Pleiotrophin is expressed by dopaminergic neurons but not by astrocytes in the adult substantia nigra pars compacta. (A) Bar graph showing the distribution of cells immunoreactive for PTN and TH in the substantia nigra pars compacta of adult normal rats: 18 ± 4% of TH-immunoreactive neurons expressed PTN (TH-PTN/total TH) and 48 ± 5% of PTN-immunoreactive cells were also TH-immunoreactive neurons (TH-PTN/total PTN). Mean ± SEM, n = 3. (B) Photomicrograph shows the double fluorescence of PTN (red) and TH (green) in adult normal rats. The white arrows indicate double-labeled cells (yellow). (C) The double fluorescence of PTN (red) and GFAP (green) indicates that astrocytes are not PTN-immunoreactive in normal rats. (D) Expression of PTN (red) and TH (green) in normal and 6-OHDA lesioned animals. The photographs were taken from the area indicated in the Paxinos and Watson atlas plate corresponding to bregma -4.80 mm (inset). Scale bars: B and C, 50 μm; D, 200 μm.

Astrocytes over-express pleiotrophin after AdPTN infection. (A) Coronal plates from the Paxinos and Watson atlas illustrating the site of injection of the adenoviruses (left) and the region of the substantia nigra from which the microphotographs have been taken (right). (B) Immunohistochemical detection of PTN on coronal sections at mesencephalic level after AdPTN or Adβgal injection, 7 and 14 days post-injection, in adult normal rats. The strong PTN-immunoreactivity in glial-like cells is only seen in animals treated with AdPTN. The squares in the low magnification images indicate the area shown in the high magnification ones (bottom panel of images). Scale bars: 500 μm (low magnification images) and 100 μm (high magnification images). (C) Double immunohistochemistry showing the co-localization of GFAP and PTN in astrocytes in a control rat 7 days after AdPTN injection. Scale bar: 50 μm.

Pleiotrophin over-expression rescues TH-immunoreactive cell bodies in the substantia nigra pars compacta (SNpc). (A) Values (mean ± SEM) are the percentage of TH-immunoreactive (TH-ir) neurons remaining in the substantia nigra injected with AdPTN or Adβgal compared to the contralateral substantia nigra (100%). Sham/Adβgal: 68 ± 5% (n = 9), Sham/AdPTN: 72 ± 3% (n = 9), 6-OHDA/Adβgal: 50 ± 4% (n = 10), 6-OHDA/AdPTN: 64 ± 3% (n = 11). (B) Photomicrographs show TH immunohistochemistry in a coronal section (-5.3 mm from bregma) from a representative animal of each experimental group, at low (left) and high (right) magnification. Note the higher number of remaining cell bodies in the 6-OHDA rat treated with AdPTN (high magnification image). Consistent with the view that PTN over-expression rescued TH-immunoreactive neurons, post hoc analysis revealed a significant effect of PTN over-expression in 6-OHDA-lesioned animals (*p< 0.05, Student Neuman Keuls). Although it is not indicated in the figure for simplicity, the post hoc comparison between Sham/Adβgal and 6-OHDA/Adβgal is also significant at p < 0.05 level. Scale bars: 500 μm (low magnification images) and 50 μm (high magnification images).