Decoding bladder state from pudendal intraneural signals in pigs.

Neuroprosthetic devices used for the treatment of lower urinary tract dysfunction, such as incontinence or urinary retention, apply a pre-set continuous, open-loop stimulation paradigm, which can cause voiding dysfunctions due to neural adaptation. In the literature, conditional, closed-loop stimulation paradigms have been shown to increase bladder capacity and voiding efficacy compared to continuous stimulation. Current limitations to the implementation of the closed-loop stimulation paradigm include the lack of robust and real-time decoding strategies for the bladder fullness state. We recorded intraneural pudendal nerve signals in five anesthetized pigs. Three bladder-filling states, corresponding to empty, full, and micturition, were decoded using the Random Forest classifier. The decoding algorithm showed a mean balanced accuracy above 86.67% among the three classes for all five animals. Our approach could represent an important step toward the implementation of an adaptive real-time closed-loop stimulation protocol for pudendal nerve modulation, paving the way for the design of an assisted-as-needed neuroprosthesis.

Combined immunohistochemical protocols to differentiate macrophages within the mononuclear-phagocyte system.

BACKGROUND: The "mononuclear phagocyte system" (MPS) refers to dispersed mononuclear monocytes and macrophages and is used to distinguish them from polymorphonuclear cells. The term "histiocyte" indicates large cells with voluminous granulated cytoplasm, sometimes containing engulfed particles, recognized as fully differentiated end cells of the MPS. Dendritic cells (DC) represent another diversified population whose inclusion in the MPS is still debated. The diverse cells of the MPS cannot all be characterized by single antigen markers or unique functions expressed at all stages of cell differentiation or activation. Nevertheless, in a diagnostic setting, their reliable identification plays a major role when a specific therapy must be established. Understanding the heterogeneity among MPS cell populations is indeed relevant to define different therapeutic approaches that can range from the use of antibiotics to immunomodulatory agents. For this reason, we attempted to establish a protocol to reliably identify the proportion of macrophages within the mononuclear phagocyte system in a tissue and/or in a given inflammatory population. METHODS: the Tafuri method was used in different double immunofluorescence protocols using an anti-Iba-1, anti-MAC387, and anti-CD11b-CD68-CD163-CD14-CD16 antibody. RESULTS AND DISCUSSION: in normal canine skin the anti-Iba-1 antibody stained an epidermal cell population (i.e. Langerhans cells) and scattered cells within the dermal compartment. MAC387 was unable to stain cells containing Leishmania amastigotes in leishmaniasis-diagnosed samples as the anti-CD11b-CD68-CD163-CD14-CD16 antibody did. By using a combination of staining protocols to differentiate macrophages within the whole histiocytic infiltrate we validated the use of a cocktail of rabbit monoclonal antibodies raised against CD11b, CD68, CD163, CD14, CD16 to stain skin macrophages.

The laminar organization of the motor cortex in monodactylous mammals: a comparative assessment based on horse, chimpanzee, and macaque.

The architecture of the neocortex classically consists of six layers, based on cytological criteria and on the layout of intra/interlaminar connections. Yet, the comparison of cortical cytoarchitectonic features across different species proves overwhelmingly difficult, due to the lack of a reliable model to analyze the connection patterns of neuronal ensembles forming the different layers. We first defined a set of suitable morphometric cell features, obtained in digitized Nissl-stained sections of the motor cortex of the horse, chimpanzee, and crab-eating macaque. We then modeled them using a quite general non-parametric data representation model, showing that the assessment of neuronal cell complexity (i.e., how a given cell differs from its neighbors) can be performed using a suitable measure of statistical dispersion such as the mean absolute deviation-mean absolute deviation (MAD). Along with the non-parametric combination and permutation methodology, application of MAD allowed not only to estimate, but also to compare and rank the motor cortical complexity across different species. As to the instances presented in this paper, we show that the pyramidal layers of the motor cortex of the horse are far more irregular than those of primates. This feature could be related to the different organizations of the motor system in monodactylous mammals.

Osteopontin expression in healing wounds of horses and in human keloids.

REASONS FOR PERFORMING STUDY: Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN: Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS: Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS: Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS: Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man.

HisTOOLogy: an open-source tool for quantitative analysis of histological sections.

HisTOOLogy is an open-source software for the quantification of digital colour images of histological sections. The simple graphical user interface enables both expert and non-expert users to rapidly extract useful information from stained tissue sections. The software's main feature is a generalizable colour separation algorithm based on k-means clustering which accurately and reproducibly returns the amount of colour per unit area for any stain, thus allowing the quantification of tissue components. Here we describe HisTOOLogy's algorithms and graphical user interface structure, showing how it can be used to separate different dye colours in several classical stains. In addition, to demonstrate how the tool can be employed to obtain quantitative information on biological tissues, the effect of different hepatic tissue decellularization protocols on cell removal and matrix preservation was assessed through image analysis using HisTOOLogy and compared with conventional DNA and total protein content assays. HisTOOLogy's performance was also compared with ImageJ's colour deconvolution plug-in, demonstrating its advantages in terms of ease of use and speed of colour separation.

Adenomatous polyposis coli protein deletion leads to cognitive and autism-like disabilities.

Intellectual disabilities (IDs) and autism spectrum disorders link to human APC inactivating gene mutations. However, little is known about adenomatous polyposis coli's (APC's) role in the mammalian brain. This study is the first direct test of the impact of APC loss on central synapses, cognition and behavior. Using our newly generated APC conditional knock-out (cKO) mouse, we show that deletion of this single gene in forebrain neurons leads to a multisyndromic neurodevelopmental disorder. APC cKO mice, compared with wild-type littermates, exhibit learning and memory impairments, and autistic-like behaviors (increased repetitive behaviors, reduced social interest). To begin to elucidate neuronal changes caused by APC loss, we focused on the hippocampus, a key brain region for cognitive function. APC cKO mice display increased synaptic spine density, and altered synaptic function (increased frequency of miniature excitatory synaptic currents, modestly enhanced long-term potentiation). In addition, we found excessive ß-catenin levels and associated changes in canonical Wnt target gene expression and N-cadherin synaptic adhesion complexes, including reduced levels of presenilin1. Our findings identify some novel functional and molecular changes not observed previously in other genetic mutant mouse models of co-morbid cognitive and autistic-like disabilities. This work thereby has important implications for potential therapeutic targets and the impact of their modulation. We provide new insights into molecular perturbations and cell types that are relevant to human ID and autism. In addition, our data elucidate a novel role for APC in the mammalian brain as a hub that links to and regulates synaptic adhesion and signal transduction pathways critical for normal cognition and behavior.

The small intestine of the adult New Hampshire chicken: an immunohistochemical study.

The presence and distribution of glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP), gastric-releasing peptide (GRP) and glucagon immunoreactivity were studied in the small intestine of the New Hampshire chicken using immunohistochemistry. This is the first report of the presence of GIP-immunoreactive (ir) cells in avian small intestine. GIP, GRP and glucagon immunoreactivity was localized in the epithelium of the villi and crypts of the duodenum, jejunum and ileum. In particular, both in the duodenum and in the jejunum immunoreactive endocrine cells to GIP, GRP and glucagon were observed. In the ileum, we noticed GIP-ir and glucagon-ir cells. GRP-ir was found in nerve fibres of all three segments of the small intestine. The distribution of these bioactive agents in the intestinal tract of the chicken suggests that GIP and glucagon may play a role in the enteropancreatic axis in which intestinal peptides modulate pancreas secretion.

Neuropeptide Y in the brain and retina of the adult teleost gilthead seabream (Sparus aurata L.).

The presence of neuropeptide Y (NPY) in the brain and retina of gilthead seabream (Sparus aurata L.) was investigated for the first time. For this investigation we employed an immunoperoxidase technique and the western immunoblot analysis using an antiserum raised against porcine NPY. The results showed that NPY-immunoreactivity was widely distributed in the brain of S. aurata. In particular, we have found NPY-immunoreactive (ir) neurons in the area ventralis telencephali pars centralis and pars lateralis, in the area dorsali telencephali pars centralis subdivision two and in nucleus intermedius thalami. An intense NPY-ir was detected in the telencephalon, in the optic tectum, in the thalamus, hypothalamus and in the vagal lobes. Scarce positive fibres were seen in the olfactory bulbs. NPY-ir amacrine cells were observed in the retina. The western immunoblot analysis revealed a protein band with a mobility corresponding to that of synthetic NPY. Our findings are, in general, in agreement with those obtained in other teleosts. The extensive distribution of NPY indicates for this peptide a key role in basic physiological actions, including visual and gustatory inputs processing.

Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas.

UNLABELLED: Side population (SP) cells defined by their ability to efflux Hoechst dye 33342 (Hst), demonstrate functional stem cell capabilities in adult murine tissues and may represent organ-specific stem cells. We examined adult human (Hu) and rhesus macaque (Rh) pancreatic tissue for the presence of SP cells. METHODS: Hu cadaver (n = 4) and Rh donor (n = 5) pancreata were dispersed with collagenase and separated by density gradient centrifugation to relatively enrich fractions for islet, ductal, and acinar tissue in human and islet and nonislet tissue in Rh. Single cell suspensions were incubated with varying Hst concentrations to determine optimal conditions for SP cell analysis. Cellular heterogeneity was assessed using a panel of monoclonal antibodies positive for hematopoietic and/or endothelial cells. RESULTS: Hu SP cells comprised approximately 0.12%, 0.08%, and 0.45% of the gated populations for Hu islet, ductal, and acinar fractions respectively. In Rh, 5.5% and 3.7% of the islet and nonislet fractions were identified as SP cells. FACS analysis of Hu pancreas-derived SP cells indicated that greater than 95% were CD45(-), and only 6% were CD34(+)CD45(-). A similar phenotype was detected in Rh pancreas-derived SP cell populations: greater than 70% were CD45(-) and less than 2% were endothelial lineage positive. CONCLUSIONS: SP cells are found in both islet- and nonislet-enriched fractions of the adult Hu and Rh pancreas. The majority of pancreatic SPs are CD45(-) and CD34(-), suggesting nonhematopoietic lineage. Further preclinical study is needed to establish the phenotype and functional role of adult tissue-specific versus tissue-resident stem cells.

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis.

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.

Treatment of malunions and mal-nonunions of the femur and tibia by detailed preoperative planning and the Ilizarov techniques.

Internal fixation has been the mainstay of treatment for post-traumatic deformities. External fixation has been used for correction of deformity in malunions and mal-nonunions. Treatment goals of achieving complete deformity correction with restoration or improvement of function were successful in this very complex group of malunions despite the numerous problems, obstacles, and complications of treatment.

Comparative assessment of gait after limb-salvage procedures.

We performed metabolic studies of gait in eighteen patients who had had above-the-knee amputation, block resection and arthrodesis of the knee, or block resection and rotationplasty for a malignant tumor of the distal end of the femur or the proximal end of the tibia. According to the measurement of consumption of oxygen, the patients who had had rotationplasty walked most efficiently. Those who had had arthrodesis used more oxygen and walked at a slower rate.

Management of displaced extension-type supracondylar fractures of the humerus in children.

The cases of 230 patients who had a displaced extension-type supracondylar fracture of the humerus were reviewed retrospectively. The results of treatment by four different methods were assessed clinically and compared. The mean length of follow-up was 4.6 years (range, one to nine years). The highest percentages of excellent results were achieved by percutaneous Kirschner-wire fixation (78 per cent), skeletal traction (67 per cent), and open reduction with internal fixation (67 per cent). Closed reduction and application of a cast was associated with a significantly lower percentage of early and late complications, including Volkmann ischemic contracture and cubitus varus. It is recommended that treatment with a cast be reserved for undisplaced fractures only. Percutaneous Kirschner-wire fixation is advocated as the method of choice for the majority of displaced fractures.