Enteric dysfunctions in experimental Parkinson's disease: alterations of excitatory cholinergic neurotransmission regulating colonic motility in rats

Parkinson's disease (PD) is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiological assay. Electrically and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, while acetylcholine levels were assayed in their incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors. As compared with control rats, at week 4 6-OHDA-treated animals displayed the following changes: decreased in vivo colonic transit rate; impaired electrically evoked neurogenic cholinergic contractions; enhanced carbachol-induced contractions; decreased basal and electrically stimulated acetylcholine release from colonic tissues; decreased ChAT immunopositivity in the neuromuscular layer; unchanged density of HuC/D immunoreactive myenteric neurons; increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were detected also at week 8 post-6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate

Alteration of colonic excitatory tachykininergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration

Background: Parkinson's disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats. Methods: Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1 beta (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR. Results: In colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1 beta levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype. Conclusions: The results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility

Noninvasive near-infrared live imaging of human adult mesenchymal stem cells transplanted in a rodent model of Parkinson’s disease

Background: We have previously shown that human mesenchymal stem cells (hMSCs) can reduce toxin-induced neurodegeneration in a well characterized rodent model of Parkinson's disease. However, the precise mechanisms, optimal cell concentration required for neuroprotection, and detailed cell tracking need to be defined. We exploited a near-infrared imaging platform to perform noninvasive tracing following transplantation of tagged hMSCs in live parkinsonian rats.Methods: hMSCs were labeled both with a membrane intercalating dye, emitting in the near-infrared 815 nm spectrum, and the nuclear counterstain, Hoechst 33258. Effects of near-infrared dye on cell metabolism and proliferation were extensively evaluated in vitro. Tagged hMSCs were then administered to parkinsonian rats bearing a 6-hydroxydopamine-induced lesion of the nigrostriatal pathway, via two alternative routes, ie, intrastriatal or intranasal, and the cells were tracked in vivo and ex vivo using near-infrared technology.Results: In vitro, NIR815 staining was stable in long-term hMSC cultures and did not interfere with cell metabolism or proliferation. A significant near-infrared signal was detectable in vivo, confined around the injection site for up to 14 days after intrastriatal transplantation. Conversely, following intranasal delivery, a strong near-infrared signal was immediately visible, but rapidly faded and was completely lost within 1 hour. After sacrifice, imaging data were confirmed by presence/absence of the Hoechst signal ex vivo in coronal brain sections. Semiquantitative analysis and precise localization of transplanted hMSCs were further performed ex vivo using near-infrared imaging.Conclusion: Near-infrared technology allowed longitudinal detection of fluorescent-tagged cells in living animals giving immediate information on how different delivery routes affect cell distribution in the brain. Near-infrared imaging represents a valuable tool to evaluate multiple outcomes of transplanted cells, including their survival, localization, and migration over time within the host brain. This procedure considerably reduces the number of animal experiments needed, as well as interindividual variability, and may favor the development of efficient therapeutic strategies promptly applicable to patients

Identification of novel loci for the generation of reporter mice

Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress

Influence of Estrogen Modulation on Glia Activation in a Murine Model of Parkinson's Disease

Epidemiological data suggest a sexual dimorphism in Parkinson disease (PD), with women showing lower risk of developing PD. Vulnerability of the nigrostriatal pathway may be influenced by exposure to estrogenic stimulation throughout fertile life. To further address this issue, we analyzed the progression of nigrostriatal damage, microglia and astrocyte activation and microglia polarization triggered by intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in male, female and ovariectomized (OVX) mice, as well as in OVX mice supplemented with 17\u3b2estradiol (OVX+E). Animals were sacrificed at different time points following 6-OHDA injection and brain sections containing striatum and substantia nigra pars compacta (SNc) underwent immunohistochemistry for tyrosine hydroxylase (TH) (dopaminergic marker), immunofluorescence for IBA1 and GFAP (markers of microglia and astrocyte activation, respectively) and triple immunoflorescent to identify polarization of microglia toward the cytotoxic M1 (DAPI/IBA1/TNF\u3b1) or cytoprotective M2 (DAPI/IBA1/CD206) phenotype. SNc damage induced by 6-OHDA was significantly higher in OVX mice, as compared to all other experimental groups, at 7 and 14 days after surgery. Astrocyte activation was higher in OVX mice with respect the other experimental groups, at all time points. Microglial activation in the SNc was detected at earlier time points in male, female and OVX+E, while in OVX mice was detected at all time-points. Microglia polarization toward the M2, but not the M1, phenotype was detected in female and OVX+E mice, while the M1 phenotype was observed only in male and OVX mice. Our results support the protective effects of estrogens against nigrostriatal degeneration, suggesting that such effects may be mediated by an interaction with microglia, which tend to polarize preferentially toward an M2, cytoprotective phenotype in the presence of intense estrogenic stimulation

Transmural remodelling of colonic wall following dopaminergic nigrostriatal neurodegeneration

Background and Aim. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor and non-motor clinical signs, among which gastrointestinal disturbances represent relevant manifestations [1]. Nevertheless, the morphological alterations associated with intestinal dysfunctions in PD have been barely investigated [2]. The present study was aimed at investigating the remodelling of colonic wall in a rat model of PD with central dopaminergic denervation by intra-nigral injection of the neuro-toxin 6-hydroxydopamine (6-OHDA). Methods. Histopathological analysis of the whole colonic wall was performed 4 and 8 weeks after central 6-OHDA injection. Inflammatory infiltrates, collagen deposition as well as the remodelling of intestinal epithelial barrier and tunica muscularis were examined by microscopic techniques (histochemistry/immunohistochemistry/confocal immunofluorescence). Results. Colonic tissue from 8-week 6-OHDA rats were significantly altered, as compared with controls. The tunica mucosa showed: eosinophil infiltration; altered lining epithelium (reduced claudin-1 and transmembrane 16A protein expression) and goblet cells (increased mucus expression); enhanced glial fibrillar acid protein-positive cells and vimentin-positive fibroblast-like cells. Along with transmural collagen deposition, significant changes were observed also in the tunica muscularis: reduced expression of alpha-smooth muscle actin/desmin and increased proliferation index in smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia. Conclusions. A full-thickness structural remodelling occurs in the colon of PD rats 8 weeks after central dopaminergic denervation; the main changes include an alteration of the colonic epithelial barrier along with the activation of the mucosal defence and fibrotic switch of the colonic wall. Overall, these findings suggest that: a) early histological modifications occur in the colon of rats with experimental PD at both mucosal and muscular level; b) these changes and the fibrotic alterations might contribute to bowel motor dysfunctions associated with PD

Systemic administration of an mGluR5 antagonist, but not unilateral subthalamic lesion, counteracts l-DOPA-induced dyskinesias in a rodent model of Parkinson's disease

Altered glutamatergic neurotransmission is central to the expression of Parkinson's disease (PD) symptoms and may underlie l-DOPA-induced dyskinesias. Drugs acting on glutamate metabotropic receptors (mGluR) of group I can modulate subthalamic nucleus (STN) overactivity, which plays a pivotal role in these phenomena, and may counteract dyskinesias. To address these issues, we investigated the effects of a 3-week treatment with mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), or of a subthalamic lesion, on abnormal involuntary movements (AIMs) and associated striatal expression of transcription factor FosB/Delta FosB caused by chronic l-DOPA administration, in rats with a nigrostriatal lesion. MPEP virtually abolished AIMs and reduced, dramatically, striatal expression of FosB/Delta FosB. Reduced FosB/Delta FosB expression, coupled with nonsignificant reduction of AIMs, was also observed in STN-lesioned rats. Our data confirm the role of glutamatergic neurotransmission in the pathogenesis of dyskinesias and the potential of mGluR5 antagonists in the treatment of l-DOPA-induced dyskinesias. © 2007 Elsevier Inc. All rights reserved

Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells

Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients - with or without heterozygous GBA1 mutations - and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies