To test whether NEP1-40 treatment alone affects the remaining dopaminergic striatal fibers, rats with unilateral striatal 6-hydroxydopamine lesions were infused with NEP1-40 or saline without receiving a transplant. result in more robust effects. Therefore, rats with unilateral striatal 6-hydroxydopamine lesions were grafted with ventral mesencephalic tissue in combination with intraventricular infusions of the Nogo-receptor 1 antagonist NEP1-40. Transplanted rats receiving saline infusions served as controls. To test whether NEP1-40 treatment alone affects the remaining dopaminergic striatal fibers, rats FANCB with unilateral striatal 6-hydroxydopamine lesions were infused with NEP1-40 or saline without receiving a transplant. Motor behavior was assessed prior to the lesion as well as prior and D-(+)-Phenyllactic acid 1, 3, and 5 weeks after the transplantations. At the end of the experimental period the number of graft-derived dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons and graft volume were analyzed. In rats without a transplant, the density of dopaminergic fibers in the striatum was analyzed. We detected that NEP1-40 treatment significantly enhanced graft-derived dopaminergic fiber outgrowth as compared to controls while no effects were detected for graft volume and survival of grafted dopaminergic neurons. Notably, the enhanced dopaminergic fiber outgrowth was not sufficient to improve the functional recovery as compared to controls. Moreover, NEP1-40 infusions in hemi-parkinsonian rats without a transplant did not result in enhanced striatal dopaminergic fiber densities and consequently did not improve behavior. In sum, our findings demonstrate that antagonization of the Nogo-receptor 1 has the capacity to support the engraftment of transplanted mesencephalic tissue in an animal model of Parkinsons disease. and mouse model of PD (Inoue et al., 2007). Moreover, we could show that antagonization of NgR1 by the peptide NEP1-40 D-(+)-Phenyllactic acid significantly improved the survival of dopaminergic neurons and their morphological complexity in fetal main VM cultures (Seiler et al., 2013). Based on these observations, we aimed at investigating whether NgR1 antagonization by NEP1-40 enhances survival and integration of grafted dopaminergic neurons and functional recovery in a hemi-parkinsonian rat model. Materials and Methods Animals Adult female Wistar rats D-(+)-Phenyllactic acid (Janvierlabs, France) were housed at 12 h light dark cycle with food and water = 6; NEP1-40 = 7). Rats from your non-grafted groups (experimental setup 2) experienced the same mini-osmotic pump surgeries (saline = 7; NEP1-40, = 7). Animals were let to recover for 1 week after the surgery. Behavior Test Series The cylinder test is a reliable measure to assess the asymmetry in forelimb use as observed after a unilateral lesion of the dopaminergic nigro-striatal pathway (Brooks and Dunnett, 2009; Schaar et al., 2010) and was evaluated as previously explained (Seiler et al., 2016). In brief the rats were placed in a transparent cylinder (diameter 30 cm and height 41 cm) with mirrors placed around it to allow a 360 degree view on the cylinder walls. The 10 min video recordings of the rats behavior were analyzed by a researched blinded to the treatment groups by counting the number of wall touches with the left, the right and both paws together. To discriminate between a meaningful physiological movement and an accidental touch, only wall contacts by which the rat supported its body weight around the forelimb with extended digits were counted. One animal in the control group had to be excluded from your analysis as it did not touch the wall at all after the lesion. The percentage of left wall touches are calculated according to the formula: [(left + ? of both paw touches)/(left + right + both paw touches)] ? 100 as previously explained (Boix et al., 2015; Seiler et al., 2016). Perfusions Six weeks after the transplantation, rats were anesthetized with Isoflurane (75% N2O, 20% O2, 4.5C5%) followed by an i.p. injection of Narketan (75 mg/kg) and Xylazine (5 mg/kg). Fentanyl (0.005 mg/kg, Janssen-AG, Zug, CH) was i.p. injected just prior to opening the thorax and the rats were perfused with 200 ml ice chilly 0.1 M phosphate buffer saline (PBS, pH 7.4) containing heparin (1000 I. E./100 ml, NOVO Nordisk) followed by 250 ml 4% paraformaldehyde in 0.1 M PBS. The brains were removed from the skull and placed in 4% paraformaldehyde overnight and thereafter cryoprotected in 10% sucrose-PBS answer. Immunohistochemistry Immunohistochemistry was performed as explained previously (Seiler et al., 2016). The brains were cut at 30 m on a cryostat (Leica CM 1900) and mounted on Superfrost slides (Thermo Scientific). Sections were heated in citrate buffer for 30 min and blocked with 10% horse serum in 0.1% Triton-PBS. Main and secondary antibodies were incubated in a 0.1% Triton-PBS answer containing 2.5% horse serum. Slides were incubated with the mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000, Millipore) overnight. After PBS washes, sections were incubated for 2 h with the secondary biotinylated anti-mouse IgG antibody (1:200, Vector Laboratories).
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