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EUROPEAN SOCIETY of PATHOLOGY AMSTERDAM 29th CONGRESS Parkinson s disease: myenteric gliosis in an experimental rat model Lina Carvalho 1, Ana Lúcia Silva 2, Sofia Viana 2,3, Inês Pita 2, Cristina Lemos 4, Ana Filipa Ladeirinha 1, Teresa Ferreira 1, Carlos A. Fontes Ribeiro 2,3, Rui Prediger 4,5, Frederico C. Pereira 2,3, Sónia Silva 2,3 (1) Institute of Anatomical & Molecular Pathology, Faculty of Medicine; University of Coimbra, Portugal; (2) Laboratory of Pharmacology and Experimental Therapeutics/Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Portugal; (3) CNC.IBILI, University of Coimbra, Portugal; (4) CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; (5) Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brasil FCT: UID/NEU/04539/2013; COMPETE: POCI-01-0145-FEDER-007440. FCT, Portugal, EXPL-DTP-DES-0104-2013

Introduction Aim Material and Methods Results/Discussion Parkinson s Disease Chronic, progressive neurodegenerative disease characterized by degeneration of dopaminergic neurons in the SNpc coupled with intracytoplasmic inclusions known as LBs MPTP Treated rodents Exhibit time-dependent impairments in olfactory, emotional, cognitive and motor functions Possibly recapitulating those observed during different stages of PD Also, Prior to the motor phase of classical PD there is a prodromal period of several years duration where non motor symptoms may be observed. Autonomic dysfunction (e.g.constipation) Potential clinical biomarker of the premotor phase

Introduction Aim Material and Methods Results/Discussion Evaluate the intestinal function and biochemical alterations before motor impairment occurs by using an intranasal MPTP rat PD model

Introduction Aim Material and Methods Results/Discussion Experimental Procedure in vivo Male Wistar rats (16 weeks old) MPTP i.n. administration MPTP HCl (2 mg/ml) was infused for 4 min (0.1 mg/nostril) MPTP Group N=6 rats Saline Group N=6 rats 12 days Figure 1: Schematic procedures of the intranasal administration of MPTP (adapted from Prediger et al. 2010. Neurotoxicity Research, 17(2), 114 29) Animals were previously anaesthetized and then sacrificed by decapitation Sample Collection i.n. administration of 0.9% NaCl

Introduction Aim Material and Methods Results/Discussion Functional Studies Ileum isolation after animal sacrifice Ileum segments suspended on stainless steel hooks under a basal tension of 29.6 mn into individual organ chambers filled with aerated Krebs Henseleit solution, 37 C 2 hours Contraction with 100 M ACh 30 minutes incubation with antagonist Isometric cumulative concentration-response (CR) curve to DA( 0.01 to 0.9 M) CR curve Figure 2: Example of a cumulative concentration response (CR) curve for DA.

Introduction Aim Material and Methods Results/Discussion Functional Studies Evaluate possible DA modifications on the isolated rat ileum function after a single i.nasal MPTP administration. Study the contractile response of isolated rat ileum to DA in the presence of a D2R antagonist - sulpiride Assess the dopaminergic status in rat ileum, 12 days after i.n. MPTP HPLC Measurement of total DA content Western Blot TH Density Tirosine Hidroxilase rate limiting enzyme for DA biosynthesis Immunohistochemistry D2R (characterize cellular location ) Protein S100β (enteroglial intestinal marker) GFAP (glial fibrillary acidic protein)

Introduction Aim Material and Methods Results/Discussion CONTRACTILE RESPONSE TO DA IN THE WISTAR RAT ILEUM: Figure 3: Contractile response of ileum from control and MPTP treated rats to DA; results are expressed as average values ± S.E.M; vertical bars represent S.E.M. Significance of statistical differences was analyzed by Student's t test. * P <0.05 vs. Saline MPTP imposed a decrease in the maximum contractile effect

Introduction Aim Material and Methods Results/Discussion CONTRACTILE RESPONSE TO DA IN THE WISTAR RAT ILEUM: Effect of D2R antagonist on the CR curve for DA Figure 4: Contractile response of ileum from control rats to DA in the absence and in the presence of 10µM Sulpiride; results are expressed as average values ± S.E.M; vertical bars represent S.E.M. Significance of statistical differences was analyzed by Student's t test. * P <0.05 vs. Saline D2R mediate DA-induced ileum contraction Figure 5: Contractile response of ileum from MPTP-treated rats to DA in the absence and in the presence of 10µM Sulpiride; results are expressed as average values ± S.E.M; vertical bars represent S.E.M. Significance of statistical differences was analyzed by Student's t test. * P <0.05 vs. MPTP. Reduced ileum contraction with Sulpiride control saline group

Introduction Aim Material and Methods Results/Discussion EFFECT OF MPTP TREATMENT IN THE TOTAL CONTENT OF DA IN THE WISTAR RAT ILEUM EFFECT OF MPTP TREATMENT IN Tirosine Hydroxilase ILEAL LEVELS Saline MPTP kda TH 60 Β-actin 42 Figure 6. Impact of MPTP i.n on rat ileum DA total levels - HPLC; the results are expressed as average values ± S.E.M; vertical bars represent S.E.M. (Saline: n= 5; MPTP: n=6). Significance of statistical differences was analyzed by Student's t test. Figure 7: Impact of MPTP i.n. on rat ileum TH levels - Western Blotting. Data are expressed as percentage of control and represent the mean ± SEM (Saline: n= 5; MPTP: n=6). Significance of statistical differences was analyzed bystudent's t test. Dopaminergic dysfunction occurs with preserved DA homeostasis.

Introduction Aim Material and Methods Results/Discussion IHC D2R A 4 2 1 IHC S100β A A 3 B B B 2 1 4 3 Figure 7: Representative D2R immunohistochemical findings on isolated ileum from saline (Panel A) and MPTP (Panel B) treated rats. D2R imunoreactivity appears in brown. D2R positive cells in ganglion cells of both submucous (1) and myenteric (2) plexuses but not on smooth muscle cells of circular (3) and longitudinal layers (4). Figure 8: Representative S100β immunohistochemical findings on isolated ileum from saline (A) and MPTP (B) treated rats. S100β imunoreactivity appears in brown. S100β positive cells in ganglion cells of both submucous (1) and myenteric (2) plexuses, and on smooth muscle cells of circular (3) and longitudinal layers (4).

Introduction Aim Material and Methods Results/Discussion IHC GFAP A A HE A B B B Figure 9: Representative GFAP immunohistochemical findings on isolated ileum from saline (A) and MPTP (B) treated rats. GFAP imunoreactivity appears in brown. GFAP positive cells in ganglion cells of both submucous (1) and myenteric (2) plexuses, but not on smooth muscle cells of circular (3) and longitudinal layers (4). Figure 10: Representative HE findings on isolated ileum from saline (A) and MPTP (B) treated rats.

Introduction Aim Material and Methods Results/Discussion and Future Studies We newly show that this PD model exhibits ileum motile dysfunction; D2R which are located in plexus play a key role on the GI motility; MPTP-treated animals showed a decrease in DA maximum contractile effect (functional studies); MPTP seems to have a detrimental effect in D2R-dependent ileum contractility (functional studies); The small intestine dopaminergic dysfunction was accompanied by preserved DA markers. MPTP group evidenced apparent increase in S100β immunoreactivity and lower GFAP immunoreactivity in myenteric plexus compared to saline group. Investigation of D2R signaling in the small intestine is warranted.

Introduction Aim Material and Methods Results/Discussion Acknowledgments Acknowledgments Pharmacology and Experimental Therapeutics Laboratory, Faculty of Medicine, University of Coimbra IAP-PM Institute of Anatomical & Molecular Pathology, Faculty of Medicine, University of Coimbra