Ras activation in Hirudo medicinalis angiogenic process

In some leeches like Hirudo medicinalis, any kind of stimulation (surgical wound or growth factor injection) provokes the botryoidal tissue response. This peculiar tissue, localized in the loose connective tissue between gut and body wall, is formed by granular botryoidal cells and flattened endothelial-like cells. Under stimulation, the botryoidal tissue changes its shape to form new capillaries. In mammals, the molecular regulation of the angiogenic phenotype requires coordinated input from a number of signalling molecules: among them the GTPase Ras is one of the major actor. In our current study, we determine whether Ras activation alone would be sufficient to drive vessels formation from leech botryoidal tissue. Our findings indicate that assembly and disassembly of actin filaments regulated by Ras protein is involved in morphological modification of botryoidal tissue cells during leech angiogenic process

Hirudo medicinalis as alternative model for in vivo and in vitro studies on nanomaterials toxicity.

Due to the steady increase of production and use of engineered nanomaterials (NMs), intentional or unintentional discharges into the environment may occur. Since it is critical to develop new methods to fully understand NMs bioaccumulation and cytotoxicity, a reliable model in which analyze NMs effects both in vivo and in vitro is more than ever necessary. Here we propose the leech, Hirudo medicinalis, as alternative animal model to study multi-walled carbon nanotubes (MWCNTs) effects by means of an extended study that includes in vivo and in vitro treatments. First, MWCNTs were dispersed in leeches\u2019 water to mimic a possible environmental exposure. In a second assay, a MWCNTs-supplemented biomatrix was injected in leeches\u2019 body wall. Thirdly, leeches macrophages were isolated and cultured to analyze their responses after MWCNTs in vitro treatment. Our results show that water dispersed MWCNTs evoke in the leech body wall a strong inflammatory response, involving mainly monocyte-macrophages cells. Ultrastructural analysis of MWCNTs-supplemented biomatrix revealed that in leech macrophages MWCNTs are internalized both in an active (phagocytosis) and a passive manner (membrane piercing). Finally, MWCNTs in vitro treatment cause the decrease of cell proliferation rate and the increase of ROS production and of the apoptotic rate. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm Hirudo medicinalis as a good alternative model that can be successfully used to study, both in vivo and in vitro, the possible harmful effects of any nanomaterial

A hedgehog-like signal is involved in slow muscle differentation in Sepia officinalis (Mollusca)

In the tentacle of Sepia officinalis, smooth-like, helical and cross-striated fibres deriving from different populations of myoblasts are present. Myoblasts appear at different times during the development and express two muscle-specific transcription factors: Myf5-like and MyoD-like factors. Myoblasts expressing Myf5 give rise to slow fibres, whereas fast fibres derive from MyoD+ myoblasts. We found that a Hedgehog (Hh)-like signal was present in the central nerve cord of the tentacle from the early stages of development and in a specific population of myoblasts which are the precursors of slow muscle fibres. The model showed interesting similarities with vertebrates, in which Sonic hedgehog is a protein secreted by axial structures (the notochord and neurotube) and is involved in slow muscle differentiation and in survival of muscle precursors

Amyloid/Melanin distinctive mark in invertebrate immunity

Protostomes and Deuterostomes show the same nexus between melanin production, and amyloid fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is conditioned by the redox status/cytoplasmic pH modification, pro-protein cleavage presence, adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (\u3b1-MSH), and neutral endopeptidase (NEP) overexpressions. These events represent the crucial component of immune response in invertebrates, while in vertebrates these series of occurrences could be interpreted as a modest and very restricted innate immune response. On the whole, it emerges that the mechanisms involving amyloid fibrils/pigment synthesis in phylogenetically distant metazoan (viz, cnidaria, molluscs, annelids, insects, ascidians and vertebrates) are evolutionary conserved. Furthermore, our data show the relationship between immune and neuroendocrine systems in amyloid/melanin synthesis. Indeed the process is closely associated to ACTH-\u3b1-MSH production, and their role in stress responses leading to pigment production reflects and confirms again their ancient phylogeny

Functional amyloid formation in LPS activated cells from invertebrates to vertebrates

LPS stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ROS). We have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. Optical and electron microscopic observations of the following LPS activated cell lines [insect (larval hemocytes, IPLB-LdFB and Drosophila Schneider\u2019s S2 cells); mouse (NIH3T3 embryonic fibroblasts); Human (Human Umbilical Vein Endothelial Cells (HUVEC), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. The compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. LPS activation leads to ROS generation, resulting in pH acidification. Stimulated cells show pink cytoplasm in May-Gr\ufcnwald Giemsa differential staining, giving a gross indication of a lower intracellular pH. Moreover the activation of amyloidogenesis is also linked with an extensive production of ACTH and \u3b1-MSH in all cultured cell types. We suggest that amyloidogenesis is a common, physiological cellular response to weak ROS, starting when other anti-stress cellular systems failed to restore homeostasis. The morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process

Treatment of Cavernous Sinus Aneurysms with Flow Diversion: Results in 44 Patients

ABSTRACT BACKGROUND AND PURPOSE: Aneurysms of the cavernous segment of the ICA are difficult to treat with standard endovascular techniques, and ICA sacrifice achieves a high rate of occlusion but carries an elevated level of surgical complications and risk of de novo aneurysm formation. We report rates of occlusion and treatment-related data in 44 patients with cavernous sinus aneurysms treated with flow diversion

In Vivo Isolation and Characterization of Stem Cells with Diverse Phenotypes Using Growth Factor Impregnated Biomatrices

BACKGROUND: The stimulation to differentiate into specific cell types for somatic stem cells is largely due to a series of internal and external signals coming from the microenvironment that surrounds the stem cell. Even though intensive research has been made, the basic mechanisms of plasticity and/or the molecules regulating stem cells proliferation and differentiation are not completely determined. Potential answers concerning the problems could be derived from the studies of stem cells in culture. METHODOLOGY/PRINCIPLE FINDINGS: We combine a new procedure (using the matrigel biopolymer supplemented with a selected cytokine/growth factor) with classic techniques such as light, confocal and electron microscopy, immunohistochemistry and cell culture, to perform an analysis on stem cells involved in the leech (Hirudo medicinalis) repair tissues. The leech has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Our data demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with a selected cytokine/growth factor in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific cell population in vivo. A comparative analysis of biopolymer in vivo sorted stem cells indicates that VEGF recruited cells of a hematopoietic/endothelial phenotype whereas MCP-1/CCL2 isolated cells that were of an early myeloid lineage. CONCLUSION: Our paper describes, for the first time, a method allowing not only the isolation of a specific cell population in relation to the cytokine utilized but also the possibility to culture a precise cell type whose isolation is otherwise quite difficult. This approach could be broadly applied to isolate stem cells of diverse origins based on the recruitment stimuli employed

Identification, Isolation and Expansion of Myoendothelial Cells Involved in Leech Muscle Regeneration

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

Objective: N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification