Morpho-Physiological Aspects of the Gastrointestinal Tract in Mammals

La tesi verte sullo studio morfo-fisiologico e/o patologico del tratto gastrointestinale (GI) di mammiferi. La prima ricerca è basata sull’identificazione, attraverso metodiche di next generation sequencing, di geni coinvolti nell’insorgenza di acalasia sporadica nell’uomo. Tale patologia risulta caratterizzata dalla presenza di anomalie a carico di neuroni del plesso mienterico e cellule interstiziali di Cajal (ICC), che esita in una compromissione della peristalsi del tratto esofageo. É emerso che nei pazienti con acalasia vs controlli il gene C-KIT è down-regolato, mentre INPP4B è up-regolato. Questi geni risultano essere importanti per lo sviluppo e differenziamento neuronale e delle ICC. Il secondo argomento verte allo studio dei processi di de- e fosforilazione della proteina Tau in condizioni fisiologiche e neurodegenerative. Nell’Alzheimer si osserva un accumulo irreversibile di phospho-Tau che induce neurotossicità. Nell’encefalo di animali letargici la proteina Tau viene iperfosforilata in ipotermia, ma defosforila al rientro della normotermia. Inducendo torpore sintetico nel ratto si è osservato che nel sistema nervoso centrale e nel tratto gastrointestinale la phospho-Tau si accumula durante lo stato ipotermico, ma defosforila al ripristino delle temperature fisiologiche. Comprendere le modificazioni associate alla Tau può rappresentare un valido supporto per meglio comprendere quali meccanismi sono alterati in condizioni patologiche. L’ultima parte delle ricerche è stata finalizzata allo studio della distribuzione dei recettori per cannabinoidi quali CB1R, CB2R, GPR55, PPARα, PPARγ, TRPA1 e 5-HT1aR, nel tratto GI di gatto attraverso metodiche immunoistochimiche. Molti autori riportano come l’attivazione di questi recettori svolga un ruolo chiave nei processi infiammatorie ed in generale sulla sensibilità viscerale. La conoscenza della presenza e distribuzione di questi recettori può rappresentare un efficace contributo per future applicazioni in campo farmaceutico/terapeutico. La parte finale dell’elaborato contiene progetti ai quali ho partecipato che esulano dalla tematica principale della tesi ma che hanno esitato in pubblicazioni o partecipazioni a Congressi.The thesis is focused on a morpho-physiological and pathological study on the gastrointestinal tract (GT) in mammals. The first research talks about the identification, through next generation sequencing, of new genes involved in human sporadic achalasia. This pathology is characterized by abnormalities in myenteric plexus neurons and interstitial cells of Cajal (ICC), that caused esophageal peristalsis impairment. This genetic analysis revealed that achalasia patients vs controls have a downregulation of C-KIT gene and an upregulation of INPP4B. these genes are really important for neuronal and ICC development and differentiation. The second research talks about the processes of de- and phosphorilation of Tau protein in physiological and neurodegenerative conditions. It has been observed that in Alzheimer disease there is an irreversible accumulation of Phospho-Tau that induces neuro-toxicity. In the brain of letargic animals there is a reversible accumulation of phospho-Tau in hypothermia, that is de-phosphorilated when the animals turns to normothermia. It has been observed in a rat artificial model of syntetic torpor that phospho-Tau is accumulated in brain and GT during the hypothermic state, and it’s completely de-phosphorilated turning into normothermia. Understanding the meaning of these modifications associated with Tau protein could be a good tool to discover the mechanisms alterated in pathological neurodegenerative conditions. The third research talks about the distribution on cannabinoid receptors in the cat GI through immunohistochemistry. The receptors studied are CB1R, CB2R, GPR55, PPARα, PPARγ, TRPA1 e 5HT1-aR. Many authors stated that the activation of these receptors has a key role in inflammatory processes and in visceral sensibility. The knowledge of the distribution of these receptors could be helpful for the application in a phamacological/therapeutical field. The last part of the thesis contains all the other projects where I joined, most of them have been published or presented at Congresses

Neuroprotection and neuroregeneration: roles for the white matter

Efficient strategies for neuroprotection and repair are still an unmet medical need for neurodegenerative diseases and lesions of the central nervous system. Over the last few decades, a great deal of attention has been focused on white matter as a potential therapeutic target, mainly due to the discovery of the oligodendrocyte precursor cells in the adult central nervous system, a cell type able to fully repair myelin damage, and to the development of advanced imaging techniques to visualize and measure white matter lesions. The combination of these two events has greatly increased the body of research into white matter alterations in central nervous system lesions and neurodegenerative diseases and has identified the oligodendrocyte precursor cell as a putative target for white matter lesion repair, thus indirectly contributing to neuroprotection. This review aims to discuss the potential of white matter as a therapeutic target for neuroprotection in lesions and diseases of the central nervous system. Pivot conditions are discussed, specifically multiple sclerosis as a white matter disease; spinal cord injury, the acute lesion of a central nervous system component where white matter prevails over the gray matter, and Alzheimer's disease, where the white matter was considered an ancillary component until recently. We first describe oligodendrocyte precursor cell biology and developmental myelination, and its regulation by thyroid hormones, then briefly describe white matter imaging techniques, which are providing information on white matter involvement in central nervous system lesions and degenerative diseases. Finally, we discuss pathological mechanisms which interfere with myelin repair in adulthood

Molecular mechanisms underlying inherited photoreceptor degeneration as targets for therapeutic intervention

Retinitis pigmentosa (RP) is a form of retinal degeneration characterized by primary degeneration of rod photoreceptors followed by a secondary cone loss that leads to vision impairment and finally blindness. This is a rare disease with mutations in several genes and high genetic heterogeneity. A challenging effort has been the characterization of the molecular mechanisms underlying photoreceptor cell death during the progression of the disease. Some of the cell death pathways have been identified and comprise stress events found in several neurodegenerative diseases such as oxidative stress, inflammation, calcium imbalance and endoplasmic reticulum stress. Other cell death mechanisms appear more relevant to photoreceptor cells, such as high levels of cGMP and metabolic changes. Here we review some of the cell death pathways characterized in the RP mutant retina and discuss preclinical studies of therapeutic approaches targeting the molecular outcomes that lead to photoreceptor cell demise

Effects of Chronic Enteropathies on VIPergic and Nitrergic Immunoreactive Neurons in the Dog Ileum

Introduction: The enteric nervous system (ENS) comprises a huge amount of neurons and nerve fibers interposed between the two muscular layers of the tunica muscularis and in the submucosa. Neuropeptides produced by the ENS neurons act as neurotransmitters/neuromodulators, which control intestinal motility and mucosal functions, and play a crucial role also in the regulation of inflammatory processes via cross talk with the local immune system. A growing body of evidence indicates that the gastrointestinal inflammatory response damages the enteric neurons themselves, thus resulting in deregulations in gut motility and mucosal functions. Keywords: Enteric Nervous System; Ileum; Immunohistochemistry; Neuronal Nitric Oxide Synthase; Vasoactive Intestinal Peptide Objective: The purpose of this study was to evaluate quantitatively enteric neurons immunoreactive for the vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the myenteric (MP) and submucosal (SMP) plexus of the ileum of dogs without (CTRL-dogs) and with spontaneous chronic enteritis (inflamed dogs, INF). In addition, the percentage of nNOS immunoreactive neurons co-expressing VIP immunoreactivity (and vice versa) was evaluated. Methods and Material: Animal tissues were collected from the ileum of six control (CTRL) dogs (none had evident gastrointestinal disorders) and ten INF-dogs with chronic enteritis of the ileum. All the enteric neurons, VIPergic and nitrergic neurons were immunohistochemically identified with the anti-HuC/HuD, anti-VIP, and anti-nNOS antibodies, respectively. VIP- and nNOS-immunoreactive neurons were immunohistochemically quantified as a relative percentages, in consideration of the total number of HuC/HuD neurons. Data were expressed as mean \ub1 standard deviation. Results: In the myenteric plexus of INF-dogs, the percentage of VIPergic neurons (16 \ub1 7%) was significantly greater than that observed in the CTRL-dogs (8 \ub1 3%) (P = 0,022). Conversely, in the submucosal plexus of CTRL- and INF-dogs the percentages of VIPergic neurons were similar (31 \ub1 9% and 30 \ub1 11%, respectively; P = 0,786). In the myenteric plexus of INF-dogs, the percentage of nitrergic neurons (24 \ub1 5%) showed only a tendency to decrease in comparison to that evaluated in the CTRL-dogs (29 \ub1 5%) (P = 0.138); also in the submucosal plexus the percentages of nitrergic neurons of CTRL-dogs (8 \ub1 5%) and INF-dogs (7 \ub1 2%) did not show meaningful differences (P = 0.884). Co-localization studies indicated that also the percentages of nitrergic neurons co-expressing VIP immunoreactivity did not change between CTRL- and INF-dogs in the MP (23 \ub1 12% and 24 \ub1 10%, respectively; P = 0.935) and SMP (26 \ub1 16% and 23 \ub1 15%, respectively; P = 0.810). Conclusion: This is the first quantitative study about the VIPergic and nitrergic neurons harbored in the in MP and SMP of the canine ileum and the first comparison between these subclasses of neurons in dogs with and without chronic enteritis. Our findings showed significan

New Improved cGMP Analogues to Target Rod Photoreceptor Degeneration

Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3′,5′-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its RP-phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer SP-CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP. European Union: MSCA-ITN-2017−765441 (transMed) andEuropean Union’s Horizon 2020 research and innovationprogramme under the EJP RD COFUND-EJP N° 825575,grant # 101 (TreatRP).</p

Localization of cannabinoid and cannabinoid related receptors in the cat gastrointestinal tract

none8noA growing body of literature indicates that activation of cannabinoid receptors may exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The present study aimed to immunohistochemically investigate the distribution of the canonical cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) and the putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), transient receptor potential ankyrin 1 (TRPA1), and serotonin receptor 5-HT1a (5-HT1a) in tissue samples of the gastrointestinal tract of the cat. CB1Rimmunoreactivity (CB1R-IR) was observed in gastric epithelial cells, intestinal enteroendocrine cells (EECs) and goblet cells, lamina propria mast cells (MCs), and enteric neurons. CB2R-IR was expressed by EECs, enterocytes, and macrophages. GPR55-IR was expressed by EECs, macrophages, immunocytes, and MP neurons. PPARα-IR was expressed by immunocytes, smooth muscle cells, and enteroglial cells. TRPA1-IR was expressed by enteric neurons and intestinal goblet cells. 5-HT1a receptor-IR was expressed by gastrointestinal epithelial cells and gastric smooth muscle cells. Cannabinoid receptors showed a wide distribution in the feline GIT layers. Although not yet confirmed/supported by functional evidences, the present research might represent an anatomical substrate potentially useful to support, in feline species, the therapeutic use of cannabinoids during gastrointestinal inflammatory diseases.noneAgnese Stanzani, Giorgia Galiazzo, Fiorella Giancola, Claudio Tagliavia, Margherita De Silva, Marco Pietra, Federico Fracassi, Roberto ChiocchettiAgnese Stanzani, Giorgia Galiazzo, Fiorella Giancola, Claudio Tagliavia, Margherita De Silva, Marco Pietra, Federico Fracassi, Roberto Chiocchett

Localisation of cannabinoid and cannabinoid‐related receptors in the equine dorsal root ganglia

Background Growing evidence recognises cannabinoid receptors as potential therapeutic targets for pain. Consequently, there is increasing interest in developing cannabinoid receptor agonists for treating pain. As a general rule, to better understand the actions of a drug, it would be of extreme importance to know the cellular distribution of its specific receptors. The localisation of cannabinoid receptors in the dorsal root ganglia of the horse has not yet been investigated.Objectives To localise the cellular distribution of canonical and putative cannabinoid receptors in the equine cervical dorsal root ganglia.Study design Qualitative and quantitative immunohistochemical study.Methods Cervical (C6-C8) dorsal root ganglia were collected from six horses (1.5 years of age) at the slaughterhouse. The tissues were fixed and processed to obtain cryosections which were used to investigate the immunoreactivity of canonical cannabinoid receptors 1 (CB1R) and 2 (CB2R), and for three putative cannabinoid-related receptors: nuclear peroxisome proliferator-activated receptor alpha (PPAR alpha), transient receptor potential ankyrin 1 (TRPA1) and serotonin 5-HT1a receptor (5-HT1aR).Results The neurons showed immunoreactivity for CB1R (100%), CB2R (80% +/- 13%), PPAR alpha (100%), TRPA1 (74% +/- 10%) and 5-HT1aR (84% +/- 6%). The neuronal satellite glial cells showed immunoreactivity for CB2R, PPAR alpha, TRPA1 and 5-HT1aR.Main limitations The low number of horses included in the study.Conclusions This study highlighted the expression of cannabinoid receptors in the sensory neurons and glial cells of the dorsal root ganglia. These findings could be of particular relevance for future functional studies assessing the effects of cannabinoids in horses to manage pain

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly

Hibernation and Radioprotection: Gene Expression in the Liver and Testicle of Rats Irradiated under Synthetic Torpor

Hibernation has been proposed as a tool for human space travel. In recent years, a procedure to induce a metabolic state known as "synthetic torpor" in non-hibernating mammals was successfully developed. Synthetic torpor may not only be an efficient method to spare resources and reduce psychological problems in long-term exploratory-class missions, but may also represent a countermeasure against cosmic rays. Here we show the preliminary results from an experiment in rats exposed to ionizing radiation in normothermic conditions or synthetic torpor. Animals were irradiated with 3 Gy X-rays and organs were collected 4 h after exposure. Histological analysis of liver and testicle showed a reduced toxicity in animals irradiated in torpor compared to controls irradiated at normal temperature and metabolic activity. The expression of ataxia telangiectasia mutated (ATM) in the liver was significantly downregulated in the group of animal in synthetic torpor. In the testicle, more genes involved in the DNA damage signaling were downregulated during synthetic torpor. These data show for the first time that synthetic torpor is a radioprotector in non-hibernators, similarly to natural torpor in hibernating animals. Synthetic torpor can be an effective strategy to protect humans during long term space exploration of the solar system