Marco Angelo Cocchi

Epithelial expression of vanilloid and cannabinoid receptors: a potential role in burning mouth syndrome pathogenesis.

Burning mouth syndrome (BMS) is an intra-oral burning sensation for which presently no medical or dental causes have been found, and in which the oral mucosa appears normal. It remains an unknown disease for which there is still no long-term treatment. The aim of this study was to assess the epithelial alteration of transient receptor potential vanilloid channel type 1 (TRPV1) and cannabinoid receptors type 1 (CB1) and type 2 (CB2) in the human tongue. The study was performed on eight healthy controls and eight BMS patients. All patients underwent a 3-mm punch biopsy at the anterolateral aspect of the tongue close to the tip. TRPV1, CB1 and CB2 immuno-histochemistry was carried out showing an altered expression of all receptors. In BMS patients there was increased TRPV1, decreased CB1 and increased CB2 expression in tongue epithelial cells also associated with a change in their distribution. It would appear that these receptors are related to BMS. These data could be useful for future characterization of BMS epithelial markers and therapy.

Endothelial Nitric Oxide Synthase in Dorsal Root Ganglia during Chronic Inflammatory Nociception

Nitric oxide (NO) is a gaseous molecule implicated both in vascular tone and nociceptive transmission. The capillary blood supply to the dorsal root ganglia (DRG) is unique because it is highly permeable to several low and high molecular-weight compounds. This anatomical situation leads to a potential role of endothelial nitric oxide synthase (eNOS) in inflammatory nociception, which is not well established. Therefore, we examined the role of eNOS in DRG in a murine chronic inflammatory pain model induced by complete Freund’s adjuvant using L-N5-(1-iminoethyl)ornithine (L-NIO), a potent inhibitor of eNOS activity. Pain state was examined using a behavioral test. The expression of eNOS, platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF) was examined by immunofluorescence. In control animals, CD31 was detected in vessels; VEGF was localized both in vessels and neurons while a weak eNOS immunopositivity was detected in both vessels and in neurons. Under inflammatory pain conditions, eNOS, CD31 and VEGF immunopositivity increased. Administration of L-NIO significantly attenuated thermal hyperalgesia by 24 h and decreased eNOS activity and CD31 immunopositivity by 7 days. VEGF was unaffected. Our results show that eNOS plays a nociceptive role in the early phases of inflammation while in the later phases it may be involved in neurotrophic support.

Abdominal aortic aneurysm and histological, clinical, radiological correlation.

To date, the pathogenesis of abdominal aortic aneurism (AAA) still remains unclear. As such, the aim of this study was to evaluate changes of the aortic structure during AAA. We analysed the microscopic frame of vessels sections, starting from the primum movens leading to abnormal dilatation. AAA samples were collected and processed through various staining methods (Verhoeff-Van Gieson, Masson Goldner, Sirius Red). Subsequently, the vessel morphology and collagenic web of the tunica media and adventitia were determined and the amount of type I and type III collagen was measured. We also applied immune-histochemistry markers for CD34 and PGP 9.5 in order to identify vascular and nerve structures in the aorta. Immune-positivity quantification was used to calculate the percentage of the stained area. We found increasing deposition of type I collagen and reduced type III collagen in both tunica media and adventitia of AAA. The total amount of vasa vasorum, marked with CD34, and nerva vasorum, marked with PGP 9.5, was also higher in AAA samples. Cardiovascular risk factors (blood pressure, dyslipidemia, cigarette smoking) and radiological data (maximum aneurism diameter, intra-luminal thrombus, aortic wall calcification) increased these changes. These results suggest that the tunica adventitia may have a central role in the pathogenesis of AAA as clearly there are major changes characterized by rooted inflammatory infiltration. The presence of immune components could explain these modifications within the framework of the aorta.

Biological Characterization and In Vitro Effects of Human ConcentratedGrowth Factor Preparation: An Innovative Approach to Tissue Regeneration

Scientific background: Platelet concentrates are nowadays widely applied in different clinical fields to improve soft tissue and bone regeneration. “Concentrated Growth Factors” (CGF) is a new generation of platelet concentrate products, which exhibits an interesting clinical and biotechnological application potential. Aim of the study: The aim of this study is to assess the biological rationale for the use of CGF, by evaluating blood cell localization, the in vitro cumulative release of seven growth factors (PDGF-AB, VEGF, TNF-α, TGF-β1, BDNF, BMP- 2 and IGF-1), its in vitro effects on cell proliferation and its mechanical behavior. Methods: CGFs were obtained from volunteer donors. Blood cell localization was evaluated after properly morphological staining and immunohistochemistry. The amount of growth factors release was measured at 5 hours, 1, 3, 6, 7 and 8 days, using ELISA assay. Cells were cultured with and without CGF and their proliferation were evaluated after 72 hours, performing the quantification of Ki-67, using flow cytometry (FACS). The mechanical response of CGF under compression was also attempted. Results: The results showed that platelets and leukocytes were found in a very thin space called “buffy coat”, localized between the white and red part of CGF. Each growth factor evaluated, had a specific kinetic release with a great variability among subjects. The in vitro cell proliferation was stimulated. CGF showed an “apparent plasticity” and its mechanical response was influenced by fibrin network structure. Conclusion: These findings support the CGF’s clinical use and will allow us to better understand and improve the clinical outcomes.

The myloglossus in a human cadaver study: common or uncommon anatomical structure?

BACKGROUND Additional extrinsic muscles of the tongue are reported in literature and one of them is the myloglossus muscle (MGM). Since MGM is nowadays considered as anatomical variant, the aim of this study is to clarify some open questions by evaluating and describing the myloglossal anatomy (including both MGM and its ligamentous counterpart) during human cadaver dissections. MATERIALS AND METHODS Twenty-one regions (including masticator space, sublingual space and adjacent areas) were dissected and the presence and appearance of myloglossus were considered, together with its proximal and distal insertions, vascularisation and innervation. RESULTS The myloglossus was present in 61.9% of cases with muscular, ligamentous or mixed appearance and either bony or muscular insertion. Facial artery provided myloglossal vascularisation in the 84.62% and lingual artery in the 15.38%; innervation was granted by the trigeminal system (buccal nerve and mylohyoid nerve), sometimes (46.15%) with hypoglossal component. CONCLUSIONS These data suggest us to not consider myloglossus as a rare anatomical variant.

Platelet Rich Plasma stimulates human hair growth in vitro

Several factors are involved in hair growth and cycling (Buffoli et al., 2013). Platelet concentrates have a new important role in regenerative medicine and thus in dermatology, oral, plastic and orthopaedic surgery and hair growth (Franco et al., 2012). In this study we evaluated in vitro the effects of Platelet-Rich Plasma (PRP), an autologous platelet preparation, on hair growth. In particular, we compared four different culture media (Philpott et al., 1990): 1-William’s E culture medium with supplemented factors; 2-William’s E culture medium with supplemented factors and Platelet Rich Plasma; 3-William’s E culture medium without supplemented factors; 4-William’s E culture medium without supplemented factors but with PRP. Hair shaft elongation was measured at 0, 24, 48, 72 and 96 hours: digitally fixed images of slices were analyzed using an image analyzer considering as measurable portion the shaft part between the bulb upper border and the top of the hair end. The values obtained were used to calculate the percentage of elongation for each time. Growth in hair cultured with William’s E medium with supplemented factors and PRP resulted higher with respect to the other media. Moreover, these results suggest that PRP stimulates human hair growth in vitro.

H2O2 stress damage is reversed by melatonin in a spinal cord organotypic model

Spinal cord injury (SCI) is characterized to be a two-step process: the primary lesion consisting of the initial trauma; the secondary damage, characterized by multiple processes including inflammation, oxidative stress and cell death that lead to a significant expansion of the original damage and to an increase of the functional deficit (1). Among the aforementioned processes, the oxidative stress plays a significant role in pathophysiology of SCI. In this study, we evaluated the role of the melatonin, an indoleamine recognized as a potent antioxidant and immunomodulator (2, 3 )Reiter et al., 1995, Favero et al., 2015), on the oxidative stress, the tissue vitality and the neuritic plasticity in an experimental model of organotypic cultures of Sprague Dawley rat spinal cord slice (SPS) treated with hydrogen peroxide (H2O2) and/or melatonin. Five experimental protocols were performed: 1) control; 2) H2O2 exposure (50 μM); 3) melatonin treatment (5-10M for 24 hours); 4) H2O2 exposure and post-treatment with melatonin; 5) H2O2 exposure after pre-treatment with melatonin. Cellular death was investigated by propidium iodide (PI) assay and the vitality by MTT assay. The total thiols (SH) levels, contrasting the oxidative stress, the neuronal specific nuclear protein (NeuN) and the synaptophysin (Syp) immunopositivity were also evaluated. Melatonin significantly decreases the number of dead cells and increases slice vitality, mainly in slices treated before H2O2 exposure. Moreover, melatonin attenuates total thiols decrease and NeuN and Syp immunopositivity reduction. Overall, these findings suggest that melatonin may exert a potential beneficial effect upon the progression of SCI secondary damage, protecting the tissue from a further degeneration.This work was supported by grants from Giorgio Brunelli Foundation for Spinal Cord Injuries Research.

Effect of melatonin on SOD expression in Spinal Cord injury

The pathophysiology of Spinal Cord Injuries (SCI) is characterized by an initial primary injury followed by secondary tissue damage. This involves the production of highly reactive species, such as reactive oxygen species (ROS), reactive nitrogen species (RNS) or free radicals, which cause damage to cell components and lead to oxidative stress. Oxidative stress plays a key role in the pathophysiology of SCI and it can be considered an hallmark for it (1); consequently alleviating oxidative stress could be an effective strategy for the therapeutic intervention of SCI. Melatonin is the major secretory product of the pineal gland and it is recognized as a potent antioxidant and immunomodulator (2). So, the aim of this study was to test the effects of melatonin administration in an organotypic slice culture model of adult Sprague Dawley rat spinal cord. Spinal cord slices were cultured for 7 days in vitro; than they were subjected to mechanical injury by cutter blade for the evaluation of melatonin effects on oxidative stress caused by damage. Four experimental group were used in this study: A) Control Group (CTR) – Organotypic spinal cord slice culture; B) Injury Group (INJURY) – Organotypic spinal cord slice culture mechanically injured by cutter blade; C) Treatment Group (INJURY+MEL) – Organotypic spinal cord slice culture mechanically injured by cutter blade and treated after 24 hours with melatonin for other 24 hours; E) Control Group with melatonin (CTR+MEL) – Organotypic spinal cord slice culture treated with melatonin for 24 hours. After the treatment, adeguated immunohistochemical analysis were performed for superoxide dismutase (SOD). The results obtained show that melatonin administration has a favorable influence on antioxidative processes enhancing SOD immunopositivity in organotypic spinal cord slice cultures. So, these preliminary results indicate a potential therapeutical role for melatonin in preventing secondary tissue damage of SCI.

Combined presence of ophthalmic artery origin from anterior cerebral artery and meningolacrimal artery

In this study we describe a case of an ophthalmic artery (OphA) originating from the pre-communicating segment of the anterior cerebral artery (A1), associated with the presence of a meningolacrimal artery (MLA). The OphA has an anomalous origin in 1-3% of cases and rarely arises from A1, however, the combination of these anatomical variations is unique. Anomalous origins of the OphA are also correlated with a higher incidence of ICA aneurysm (1). Macroscopic and endonasal endoscopic dissections of a cadaver head, which formerly underwent a cone-beam CT scan, were performed. Bilateral samples of the ICA walls were collected and processed for standard hematoxylin-eosin staining and immunofluorescence analysis. The MLA was found on the right side by CT scan and its entrance in the superior orbital fissure was confirmed during head dissection. Hence, performing the endoscopic approach on the same side, the anomalous OphA, originating from the inferior surface of A1 segment and entering the optic canal above the optic nerve, was discovered. This arterial pattern could be explained by the embryological development of the orbital vascular system and it is referred to persistent ventral OphA (2). The histomorphological examination of ICA walls showed a significantly decreased thickness of the tunica media and adventitia on the right side compared to the left one. In addition, fluores- cence microscopy showed that type I and type III collagen were significantly lower in the tunicae media and adventitia of the right side. Since aneurysms of the ICA are related with a low content of collagen in the arterial wall, our results are consistent with current literature.

Aging of brain in hypercholesterolemic mice (ApoE -/-): melatonin receptor distribution

The protective role of melatonin has been investigated [1]. Some studies underlined its significant neuroprotective action with a role in aging processing. In patients with Alzheimer’s Disease, parallel to degenerative tissue changes, there was an overall decrease in the intensity of melatonin receptors in the pineal gland and occipital cortex [2]. Melatonin type 1 (MT1) and type 2 (MT2) receptors disclosed a quite widespread distribution in different brain regions. Recently our group demonstrated that an animal model of hypercholesterolemia, such as ApoE-/- mice, is more susceptible to developing severe liver injury, suggesting that in addition to vascular disease, increased cholesterol products and oxidative stress may also play a role in accelerating aging in the liver [3]. On the basis of this consideration, the aim of our work is to characterize the distribution of MT1 and MT2 in brain of ApoE -/- mice at different age (6 weeks, 16 weeks and 60 weeks) together with senescence markers using immunohistochemical technique to verify the role of these receptors in aging process. The results show an altered distribution of melatonin receptors and synaptic connectivity, indicating a process of aging in ApoE -/- mice and suggesting that melatonin treatment may represent a new approach to reduce brain aging and degeneration.