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The peripheral nervous system has a striking regeneration potential and after damage extensive changes in the differentiation state both of the injured neurons and of the Schwann cells are observed. Schwann cells, in particular, undergo a large scale change in gene expression becoming able to support axonal regeneration. Nerve injury is generally associated to inflammation and activation of the coagulation cascade. Thrombin acts as a polyfunctional signalling molecule exerting its physiological function through soluble target proteins and G-protein-coupled receptors, the protease-activated receptors (PARs) [1]. Recently, we have demonstrated that the activation of the main thrombin receptor, PAR-1, in Schwann cells favours their regenerative potential determining the release of factors which promote axonal regrowth [2]. The pro-regenerative potential of thrombin seems to be exerted in a narrow range of concentrations (pM-nM range). In fact, our preliminary data indicate that high levels of thrombin in the micromolar range slow down Schwann cell proliferation and induce cell death. On the contrary, PAR-1 activating peptides mimic the pro-survival but not the pro-apoptotic effects of thrombin. Controlling thrombin concentration may preserve neuronal health during nerve injury and represent a novel target for pharmacologic therapies., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Background and Aims:The JAK2 V617F mutation is an acquired, somatic mutation present in most patients with myeloproliferative neoplasm (essential thrombocythemia and polycythaemia vera). Those disorders have been associated with arterial and/or venous thrombosis. The role of JAK2 mutations in cerebral ischemia of unknown origin has not been well described.Methods:We report 6 cases of patients hospitalized for ischemic stroke with newly diagnosed chronic myeloproliferative disease from JAK2- V617F mutation. Results:Patients had a variable age between 38-78 years, ischemic stroke in the territory of MCA, PCA, BA, or VA. The findings excluded other frequent causes of ischemic stroke. Of these patients 4 had a normal blood count, only 2 had thrombocytosis or polycythaemia. All patients were treated with oncocarbide. Among these 4 started antiplatelet therapy and 2 anticoagulant therapy.Conclusions:We confirm some data reported in the literature, that JAK2 mutation increases the risk of cerebrovascular thrombosis. Finally, we recommend the detection of JAK 2 mutation in patients with an embolic stroke of an indeterminate source, not only in patients with thrombocytosis or polycythaemia, but also in patients with a normal cell blood count.

Background and Aims:The artery of Percheron (AOP) is a variant of the paramedian arteries with a single trunk stemming off from one posterior cerebral artery to supply both paramedian thalami and the rostral midbrain. Thus, occlusion of this artery results in bilateral thalamic and midbrain infarctions. The symptoms are often characterized by altered mental status, consciousness, and memory impairment, and oculomotor nerve deficit. Usually the etiology is cardioembolic or small vessel disease combined with individual anatomical predisposition. Methods:We report 15 cases of stroke from Percheron artery occlusion, verified at our stroke unit between the years 2007-2018. Results:Patients had varying degrees of altered state of consciousness, cognitive impairment, hemiparesis, dysarthria and oculomotor disturbance. All of these patients have a bilateral thalamic infarction and a midbrain involvement at CT scan or cerebral MRI. The diagnostic assessment revealed: atrial fibrillation in 3 patients, patent foramen ovale with moderate-severe right to left shunt in 2 patients, endocavitary thrombotic apposition in diffuse parietal hypokinesia in 1 patient, severe carotid atheromasy in left ICA (70%) in 1 patient with fetal origin of left PCA, thrombosis of the apex of the basilar in 1 patient. Of all these patients, only two received a thrombolytic therapy.Conclusions:Our data are in line with the data in the literature and therefore confirm:- the great clinical variability of AOP infarction- the importance of neuroimaging, in particular MRI for acute differential diagnosis and therefore acute thrombolytic therapy

Infection, neurodegeneration, and other conditions associated with loss of brain homeostasis, induce changes in microglial morphology, gene expression and function, generally referred to as “activation”. Alzheimer’s disease (AD) is the most common dementia and is characterized by neuroinflammatory changes, including alterations in the morphology and distribution of microglia and astrocytes, and deposition of complement and other inflammatory mediators. Our previous observations show that microglial cells challenged in vitro with amyloid peptides clustered and rounded up, dramatically changing their morphology. Besides, in these cells we observed the early acetylation and then the phosphorylation of STAT3 which is required for the expression of the epsilon isoform of 14-3-3, a marker of Abeta-activated microglia (1, 2). We applied affinity partitioning approach combined with high throughput mass spectrometric analysis in order to identify variation of proteins on plasma membrane of BV2 immortalized microglia upon treatment with amyloid peptides. By this method several proteins up- or down-regulated by amyloid treatment were identified in microglial plasma membrane. Among them annexins (5 and 7), IFITM3 and MARK3. These data have been confirmed in primary microglial cultures., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Dupuytren’s contracture (DC) is a benign fibroproliferative disease causing fibrotic nodules and fascial cords with resultant debilitating flexion contracture deformities. The present study was designed to characterize pro-inflammatory cytokines and growth factors involved in the genesis, progression and recurrence of the disease to optimize therapeutic agents and strategies for controlling Dupuytren’s disease. The expression of pro-inflammatory cytokines and other growth factors was detected by immunohistochemistry and immunofluorescence in the fibrotic nodules and normal palmar fascia resected respectively from patients affected by Dupuytren’s contracture and Carpal Tunnel Syndrome (as negative controls). RT-PCR analysis was performed to quantify the expression of TGF-β1, IL-1β and VEGFa in the myofibroblasts and fibroblasts isolated from Dupuytren’s nodules. Histological analysis showed the high cellularity and rate of proliferation of Dupuytren’s tissue with the presence of myofibroblastic isotypes. Our data showed the strong expression of TGF-β1, IL-1β and VEGFa in Dupuytren’s fibromatosis nodules suggesting a direct role of these markers in the onset, progression and recurrence of the disease. Our observations suggest that TGF-β1, IL-1β and VEGFa may be considered potential therapeutic targets in the treatment of Dupuytren’s disease. Moreover, a new innovative therapy may be represented by the combined use of specific inhibitors of these growth factors., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Protease-activated receptor-1 (PAR-1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR-1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR-1 mRNA increased in response to sciatic nerve transection, both in the proximal and in the distal part of the lesioned nerve (1). Other authors revealed that functional PAR-1 receptor exists specifically in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier in the sciatic nerve (2). Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination (3). The present study was aimed to determine if the activation of PAR-1 affects the neurotrophic properties of Schwann cells. We observed a specific staining for PAR-1 in Schwann cells of rat sciatic nerve and also in primary Schwann cell cultures. To study the role of PAR-1 in Schwann cell cultures, we activated this receptor with a specific activating peptide (PAR-1 AP). Conditioned medium from PAR-1 AP-treated Schwann cells reduced the LDH release of PC12 cells respect to the medium of the untreated cells, suggesting that the stimulation of PAR-1 induces the production of pro-survival molecules. Also an increased neurite outgrowth on PC12 cells was observed using the conditioned medium from Schwann cells treated with PAR-1 AP respect to the control obtained from untreated cells. The synthesis and secretion of several factors produced by Schwann cells treated with PAR-1 AP were investigated by proteomics, western blot and RT-PCR analyses. By these experiments we identified as putative neurotrophic candidates some molecules, such as Macrophage migration inhibitory factor, Syndecan 4 and Annexin A2., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

PAR-1 (Protease-activated receptor–1) is a G-protein-coupled receptor that elicits cellular responses to extracellular proteases such as thrombin. In the peripheral nervous system (PNS), the expression and/or the role of PAR-1 are still poorly investigated. Several authors speculated that many functions in PNS, such as motor, secretory, vascular, nociceptive, inflammatory or regenerative processes, may be regulated by PARs (Vergnolle et al. 2003; Shavit et al., 2008; Wang et al., 2013). The present study was aimed to determine if PAR-1 activation affects neurotrophic properties of Schwann cells. By double immunofluorescence experiments we observed a specific staining for PAR-1 in S100β-positive cells of rat sciatic nerve and sciatic teased fibres. Moreover, PAR-1 was highly expressed in Schwann cell cultures obtained from both neonatal and adult rat sciatic nerves. When PAR-1 specific agonists were added to these cultures a higher proliferation rate was observed. Moreover, conditioned medium from primary Schwann cells treated with PAR-1 agonists increased cell survival and neurite outgrowth of PC12 cells. Therefore synthesis and secretion of several factors by Schwann cells treated with PAR-1 agonist peptides were investigated by RT-PCR, western blot and proteomics analysis. By these experiments some molecules, including extracellular matrix components and adhesion molecules, were identified as putative neurotrophic candidates., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Autophagy is generally considered a degradation pathway involved in many neurodegenerative processes. It can be observed in different stress conditions such as starvation generally improving cell survival. Our previous results described the occurrence of autophagy in neuronal cultures exposed to the toxic compound trimethyltin (TMT) (1). TMT belongs to a family of organotin compounds with wide industrial and agricultural applications, especially as heat stabilizers in PVC production and as biocides. In the nervous system TMT determines the selective destruction of neurons in specific brain regions such as the olfactory bulb and the hippocampus. When this toxic molecule was administered to glial cells we observed in astrocytes a rapid block of the autophagic flux and a consequent increased expression of LC3 and p62 which can be observed both in cultured astrocytes and in the brain of intoxicated animals. Conversely, in microglia autophagy was not impaired in the same conditions and p62 accumulation was not observed neither in vitro primary cultures, nor in brain sections of TMT-treated rats. The protein p62 (also known as SQTM1) is known to be selectively degraded through autophagy and its accumulation activates the transcription factor Nrf2 by sequestering Keap1 (2). To note the block of autophagy has been reported to exert an immunosopressive effect in macrophages (3). Thus, the impairment of autophagy in astrocytes could be related to their limited production of pro-inflammatory cytokines and nitric oxide respect to microglia observed after TMT treatment., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Autophagy is an intracellular degradation process that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including neurodegenerative diseases. Our previous data show that in hippocampal and cortical neurons the neurotoxic compound trimethyltin (TMT) activates the autophagic pathway (Fabrizi et al., 2012). Recently we extended our analysis to astrocytes, the main population of glia of the central nervous system. As already observed in neurons, in astrocytes autophagy is rapidly induced after TMT administration. LC3-II which is a distinctive marker of autophagy rapidly appeared in TMT-treated astrocytes but then it accumulates indicating a precocious block of the autophagic pathway. The inhibition of autophagy by 3-methyladenine at the level of the autophagosome formation partially rescues astrocytes from TMT-induced cell death. Interestingly, an impairment of autophagy was also observed by other authors following intoxication with arsenic and could represent a common feature of different environmental toxins., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

The occurrence of motor neuron death is the milestone of amyotrophic lateral sclerosis (ALS). Therefore, morphological analysis along decades focussed on motor neuron loss as the sole marker to score disease severity. Recently, non autonomous cell death took a prominent role to explain the need for additional cell types to induce motor neuron degeneration in ALS. This concept encompasses a variety of inflammatory cells including resident astroglia and microglia. Despite tremendous clinical and basic research activity the role of inflammation remains controversial. On the other hand very recently non autonomous cell death creped back in considering neuronal cell types differing from motor neurons. In the present communication evidence is provided demonstrating that interneurons and other neuronal cell types participate to degeneration. Remarkably, this multi-neuronal non-autonomous motor neuron degeneration extends to mutiple models of motor neuron disorders. Interneurons degenerate earlier and more than motor neurons, thus suggesting a key role in the pathophysiology of motor neuron disease. Additionally, these findings portray ALS as a whole spinal cord disorder rather than a disease affecting solely motor neurons. Novel concepts of abnormal cell to cell communication are likely to underlie multi-neuronal disease spreading. This work was supported by research grant PRIN 2010-2011 FF, Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Trimethyltin (TMT) is an organotin compound which is considered a useful tool to obtain an animal model of neurodegeneration associated with cognitive impairment (Pompili et al., 2011; Geloso et al., 2011). In the present work, this model was used in order to investigate the animal behaviour in association with the immunohistochemical expression of dopaminergic system (D1- and D2-like receptors and dopamine transporters DAT, VMAT-1 and -2) and cells viability (NEU-N) in the rat hippocampus and facial nucleus regions. TMT-treated group showed impaired spatial reference memory in a Morris water maze task compared to control group whereas the memory consolidation tested 24h after was preserved. In the open field, TMT-treated rats showed a decreased in time spent in rearing episodes reflecting a lower interest to explore a novel environment. In the hippocampal area of TMT-treated group, cell viability was significantly reduced by 45.9% whereas the D1, D2, DAT and VMAT- 2 receptor proteins immunoreactivity was significantly decreased by 57.5, 72.8, 64.1, 72.1%. In the facial nucleus, immunoreactivity reduction was observed only for dopamine transporters (average: 60% about) while the NEU-N reduction was 40%. These data were confirmed by real time RT-PCR analysis. These results suggest a differential involvement of the D1-type and D2-type receptors in the regulation of learning and memory. Besides, alterations on the functional ratio of DAT to VMAT-2 could predispose the cells to injury even at very low doses of TMT. The data obtained in facial nucleus demonstrate a different sensibility to xenobiotic of dopamine receptors and transporters. The TMT model could contribute to elucidate the role of dopaminergic system on two different CNS regions. Supported by PRIN 2008 - prot. 20089MANHH_002 and prot. 20089MANHH_003., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Pituitary adenomas are a diverse group of tumors arising from the pituitary gland. Typically, they are small, slow-growing, hormonally inactive lesions that come to light as incidental findings on radiologic or postmortem examinations, although some small, slow-growing lesions with excessive hormonal activity may manifest with a clinical syndrome. The family of neurotrophins plays a key role in the development and maintenance of the pituitary endocrine cell function and in the regulation of hypothalamo-pituitary-adrenocortical axis activity. The objective of our experimental study is to investigate the localization of the neurotrophins, their relative receptors and to detect the expression level of Ki-67 to determine whether all these factors participate in the transformation and development of human pituitary adenomas. A very strong expression of Neurotrophin-3 (NT-3) and its receptor TrKC was observed in the extracellular matrix (ECM) and vessel endothelium, together with a clear/marked presence of Brain-derived neurotrophic factor (BDNF), and its receptor TrKB, thus confirming their direct involvement in the progression of pituitary adenomas. On the contrary, NGF (Nerve growth factor) and its receptor TrKA and p75NTR were weakly expressed in the epithelial gland cells and the ECM.

Thrombin, a multifunctional serine protease, is a key enzyme in the coagulation cascade. Most of its actions are mediated by a G protein-coupled protease activated receptor (PAR-1) which is highly expressed in glial cells especially after injury (Pompili et al., 2006; Pompili et al., 2011) . In the peripheral nerves thrombin and PAR-1 specific agonist peptides produce changes in nerve conduction compatible with a conduction block. Aim of the present study is to determine if the activation of this receptor affects the neurotrophic properties of Schwann cells. In peripheral nerves PAR-1 was predominantly observed by immunofluorescence on non-compacted Schwann cell microvilli at the node of Ranvier. Moreover, PAR- 1 was highly expressed in Schwann cell cultures obtained from both neonatal and adult rat sciatic nerves. When PAR-1 specific peptides were added to these cultures an increased proliferation rate was observed. The synthesis and secretion of several growth factors by Schwann cells treated with PAR-1 agonist peptides were studied by RT-PCR, western blot and proteomics analyses., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Trimethyltin (TMT) is considered a useful tool to obtain an experimental model of neurodegeneration. TMT is known to cause neurotoxicant effects especially marked in the hippocampus. Despite many studies are published, there are poor literature on the interaction of this xenobiotic with dopaminergic system. In the present work, we investigate in rat brain, after 21 days following TMT intraperitoneal administration, the cells viability (N-NEU) and the animal behaviour in association with the immunohystochemical expression of dopamine receptors (D1- and D2-like) and transporters membrane (DAT) and vesicular monoamine trasporters (VMAT-1 and -2) in rat hippocampus and facial nucleus. The animal behaviour shows a significant reduction of spatial reference memory in a Morris water maze task according with the reduction (70% Vs control) of hippocampus dopaminergic system expression, despite the cell viability is maintained at about 50%. In the facial nucleus, a different reduction of dopamine receptors and trasporters (30% against 60%) was observed while the N-NEU reduction was 40%. These results suggest that the toxic interaction of TMT with the dopaminergic system in rat hippocampus may be responsible for learning and memory deficits. Data obtained in facial nucleus demonstrate different sensitivity of dopamine receptors and dopamine transporters to xenobiotic. Supported by PRIN 2008 - prot. 20089MANHH_002, Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and industrial catalysts in chemical reactions. The mechanism underlying TMT selective tissue-specific pattern of cellular damage is not completely known unless a correlation exists between TMT toxicity and the expression of stannin, a highly-conserved protein mainly localized within mitochondria. Ultrastructural studies performed on samples obtained from the brain of humans and rodents intoxicated with TMT describe increased number of lysosomes and big vacuoles suggestive of altered autophagy. Our results show that autophagy inhibitors added to TMT dramatically increased the toxicity of this compound both in hippocampal and cortical neurons. Conversely, autophagy inducers such as rapamycin and lithium prevent the neurotoxicity of TMT. Due to its diverse targets, the action of lithium may be complex and its neuroprotective property is sometimes controversial. Our data show that in acute treatment experiments hippocampal and cortical neurons behave quite differently when challenged with lithium. The neuroprotective effect of lithium acutely administered against TMT in hippocampal but not in cortical neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. Lithium is also effective in preventing TMT toxicity in astrocyte cell cultures in a range of concentrations of 0.2-4 mM., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011