Polyvinyl alcohol/chitosan wound dressings loaded with antiseptics.

Wound care remains a challenge in healthcare. This work aimed to develop a new polyvinyl alcohol (PVA)/chitosan (Ch) based wound dressing able to ensure protection, hydration and a controlled release of antiseptics, as alternative to actual treatments. Two distinct formulations (1:1 and 3:1, w/w) were prepared, sterilized by autoclaving and characterized concerning surface morphology, degradation over the time, mechanical properties and hydrophilicity. Both dressings revealed adequate properties for the intended purpose. The dressings were loaded with chlorhexidine (CHX) and polyhexanide (PHMB) and the drug release profiles were determined using Franz diffusion cells. The release of PHMB was more sustained than CHX, lasting for 2 days. As the amounts of drugs released by PVA/Ch 1:1 were greater, the biological tests were done only with this formulation. The drug loaded dressings revealed antibacterial activity against S. aureus and S. epidermidis, but only the ones loaded with PHMB showed adequate properties in terms of cytotoxicity and irritability. The application of this elastic dressing in the treatment of wounds in a dog led to faster recovery than conventional treatment, suggesting that the material can be a promising alternative in wound care.

Controlled release of moxifloxacin from intraocular lenses modified by Ar plasma-assisted grafting with AMPS or SBMA: An in vitro study.

Intraocular lenses (IOLs) present an alternative for extended, local drug delivery in the prevention of post-operative acute endophthalmitis. In the present work, we modified the surface of a hydrophilic acrylic material, used for manufacturing of IOLs, through plasma-assisted grafting copolymerization of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), with the aim of achieving a controlled and effective drug release. The material was loaded with moxifloxacin (MFX), a commonly used antibiotic for endophthalmitis prevention. The characterization of the modified material showed that relevant properties, like swelling capacity, wettability, refractive index and transmittance, were not affected by the surface modification. Concerning the drug release profiles, the most promising result was obtained when AMPS grafting was done in the presence of MFX. This modification led to a higher amount of drug being released for a longer period of time, which is a requirement for the prevention of endophthalmitis. The material was found to be non-cytotoxic for rabbit corneal endothelial cells. In a second step, prototype IOLs were modified with AMPS and loaded with MFX as previously and, after sterilization and storage (30days), they were tested under dynamic conditions, in a microfluidic cell with volume and renovation rate similar to the eye aqueous humour. MFX solutions collected in this assay were tested against Staphylococcus aureus and Staphylococcus epidermidis and the released antibiotic proved to be effective against both bacteria until the 12th day of release.

Controlled drug release from hydrogels for contact lenses: Drug partitioning and diffusion.

Optimization of drug delivery from drug loaded contact lenses assumes understanding the drug transport mechanisms through hydrogels which relies on the knowledge of drug partition and diffusion coefficients. We chose, as model systems, two materials used in contact lens, a poly-hydroxyethylmethacrylate (pHEMA) based hydrogel and a silicone based hydrogel, and three drugs with different sizes and charges: chlorhexidine, levofloxacin and diclofenac. Equilibrium partition coefficients were determined at different ionic strength and pH, using water (pH 5.6) and PBS (pH 7.4). The measured partition coefficients were related with the polymer volume fraction in the hydrogel, through the introduction of an enhancement factor following the approach developed by the group of C. J. Radke (Kotsmar et al., 2012; Liu et al., 2013). This factor may be decomposed in the product of three other factors EHS, Eel and Ead which account for, respectively, hard-sphere size exclusion, electrostatic interactions, and specific solute adsorption. While EHS and Eel are close to 1, Ead>>1 in all cases suggesting strong specific interactions between the drugs and the hydrogels. Adsorption was maximal for chlorhexidine on the silicone based hydrogel, in water, due to strong hydrogen bonding. The effective diffusion coefficients, De, were determined from the drug release profiles. Estimations of diffusion coefficients of the non-adsorbed solutes D=De×Ead allowed comparison with theories for solute diffusion in the absence of specific interaction with the polymeric membrane.

Simulation of the hydrodynamic conditions of the eye to better reproduce the drug release from hydrogel contact lenses: experiments and modeling.

Currently, most in vitro drug release studies for ophthalmic applications are carried out in static sink conditions. Although this procedure is simple and useful to make comparative studies, it does not describe adequately the drug release kinetics in the eye, considering the small tear volume and flow rates found in vivo. In this work, a microfluidic cell was designed and used to mimic the continuous, volumetric flow rate of tear fluid and its low volume. The suitable operation of the cell, in terms of uniformity and symmetry of flux, was proved using a numerical model based in the Navier-Stokes and continuity equations. The release profile of a model system (a hydroxyethyl methacrylate-based hydrogel (HEMA/PVP) for soft contact lenses (SCLs) loaded with diclofenac) obtained with the microfluidic cell was compared with that obtained in static conditions, showing that the kinetics of release in dynamic conditions is slower. The application of the numerical model demonstrated that the designed cell can be used to simulate the drug release in the whole range of the human eye tear film volume and allowed to estimate the drug concentration in the volume of liquid in direct contact with the hydrogel. The knowledge of this concentration, which is significantly different from that measured in the experimental tests during the first hours of release, is critical to predict the toxicity of the drug release system and its in vivo efficacy. In conclusion, the use of the microfluidic cell in conjunction with the numerical model shall be a valuable tool to design and optimize new therapeutic drug-loaded SCLs.

Polymorphic GGC repeat differentially regulates human reelin gene expression levels.

The human gene encoding Reelin (RELN), a pivotal protein in neurodevelopment, includes a polymorphic GGC repeat in its 5' untranslated region (UTR). CHO cells transfected with constructs encompassing the RELN 5'UTR with 4-to-13 GGC repeats upstream of the luciferase reporter gene show declining luciferase activity with increasing GGC repeat number (P < 0.005), as predicted by computer-based simulations. Conversely, RELN 5'UTR sequences boost reporter gene expression above control levels in neuronal SN56 and N2A cell lines, but 12- and 13-repeat alleles still yield 50-60% less luciferase activity compared to the more common 8- and 10-repeat alleles (P < 0.0001). RELN "long" GGC alleles significantly blunt gene expression and may, through this effect, confer vulnerability to human disorders, such as schizophrenia and autism.

Structural study of Cu(2-x)Se alloys produced by mechanical alloying.

The crystalline structures of the superionic high-temperature copper selenides Cu(2-x)Se (0 < x < 0.25) produced using mechanical alloying were investigated using X-ray diffraction (XRD). The measured XRD patterns showed the presence of peaks corresponding to the crystalline superionic high-temperature alpha-Cu(2)Se phase in the as-milled sample, and its structural data were determined by means of a Rietveld refinement procedure. After heat treatment in argon at 473 K for 90 h, this phase transforms to the superionic high-temperature alpha-Cu(1.8)Se phase, whose structural data were also determined by Rietveld refinement. In this phase, a very low occupation of the trigonal 32(f) sites ( approximately 3%) by Cu ions is found. In order to explain the evolution of the phases in the samples, two possible mechanisms are suggested: (i). the high mobility of Cu ions in superionic phases and (ii). the important diffusive processes in the interfacial component of samples produced by mechanical alloying.

Novel proteoglycan epitope expressed in functionally discrete patterns in primate cortical and subcortical regions.

The molecular diversity of neuronal subpopulations was examined with a new monoclonal antibody, 8B3, that recognizes a condroitin sulfate proteoglycan expressed in anatomically discrete domains of central nervous system regions. In the neocortex, interneurons display 8B3 immunoreactivity in a rostrocaudal gradient, with a distinctive staining pattern that distinguishes known cytoarchitectonic and functional boundaries. The distribution pattern of 8B3 immunoreactivity in subcortical structures is very restricted. In the striatum, 8B3 stains spiny stellate neurons clearly defining a compartment that may correspond to the matrix. Gradients of immunoreactivity are detected in the putamen, globus pallidus, and deep cerebellar nuclei, where the most dense areas of 8B3 immunoreactivity corresponds to zones of polysynaptic projections to association prefrontal cortex. In contrast, the sensorimotor domains express lower levels of immunoreactivity. Only the projection neurons of the ventrolateral nucleus and the GABAergic neurons of the reticular nucleus express significant 8B3 immunoreactivity in the thalamus. In the spinal cord, 8B3 immunoreactivity is primarily associated with a subpopulation of motor neurons in the ventral horn and neurons in Clarke's nucleus. The complex distribution pattern reflects novel aspects of the functional organization of cortical and subcortical systems in the CNS of the primate brain and represents a potentially useful tool to assess subpopulations of neurons and brain areas as putative targets in human disease.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

[Pelvic lipomatosis]. / Lipomatosis pélvica.

Pelvian lipomatosis is a proliferative process of fatty tissue in the perivesical and perirectal pelvian space. The etiology is unknown and some authors consider that in reality is a localized form of obesity. Diagnosis can be incidental, or be part of a sequence within a study for unspecific symptoms such as pelvian complains or perineal problems. As part of the presentation of a clinical case report, the authors make a revision of this disease, focusing in several particular aspects, like diagnostic methods, treatment, etc.

Dynamic expression suggests multiple roles of the eph family receptor brain-specific kinase (Bsk) during mouse neurogenesis.

The eph family ligands and receptors have been implicated in mediating topographic neuron-target interactions. We recently isolated Bsk, a new member of the eph family receptors, and showed that it is expressed primarily in the brain. To investigate the role of Bsk in the development of the nervous system, we examined the temporal and spatial patterns of Bsk expression using in situ hybridization. We report here that Bsk expression exhibits dynamic changes during embryogenesis. In early embryos, Bsk is widely transcribed in the nervous system, including the forebrain, midbrain, hindbrain and spinal cord. Bsk expression in the midbrain, hindbrain and spinal cord, however, gradually decreases while in the forebrain increases over time. By embryonic day 18, the most intense Bsk expression was found in the limbic system. High levels of the expression in the limbic system persisted throughout post-natal development and remained stable in the adult up to 24 month. The topography of Bsk expression is in the form of gradients in several regions of the brain, including the lateral septum, spinal cord, as well as the hippocampus. Selective expression was also observed in Purkinje cells. Our findings on the topography of Bsk expression provide support to potential roles of Bsk in topographic projection. Our analyses further suggest that there may be other novel functions of Bsk in early neurogenesis in addition to potential roles in topographic mapping.

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): I. Adult rat brain.

The search for molecular markers common to neural structures that are functionally related has become an attractive strategy for neurobiologists interested in identifying mechanisms involved in the formation of patterned connections. One such molecule is the limbic system-associated membrane protein (LAMP), a 64-68 kDa glycoprotein that is expressed in the soma and dendrites of subpopulations of adult neurons in the brain that are functionally associated with classic limbic structures. Such patterned molecular specificity is established prenatally; LAMP is detected during development on the surface of neurons, axonal membranes and pathfinding growth cones. This molecule has now been cloned (lamp) and has been shown to be highly conserved in rat and human. It is a new immunoglobulin superfamily member that has three Ig domains and a glycosyl-phosphatidylinositol (GPI) anchor to the cell membrane. In this study, the distribution of the lamp transcript in the adult rat brain was determined by using in situ hybridization. Generally, the distribution of lamp corresponds well with that of the LAMP protein. Within the cerebral cortex, the transcript is more abundant in areas that are associated with learning/memory and viscerosensory tasks. It is less abundant in somatic sensory and motor areas. The lamp transcript is also ubiquitous in the basal forebrain, amygdala, and preopticohypothalamic areas. In short, the lamp transcript is expressed heavily in areas of the forebrain and diencephalon that have been classically considered limbic and sparsely or moderately in nonlimbic midbrain and hindbrain regions. Correlative analysis of the connectivity patterns of the regions that express greater amounts of the transcript is consistent with a stronger limbic-associated function relative to the regions expressing less lamp. These quantitative differences may be significant in determining the function of LAMP in the adult brain.

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): II. Fetal rat brain.

The limbic system-associated membrane protein (LAMP) is a 64-68 kDa neuronal surface glycoprotein expressed in cortical and subcortical regions of the limbic system of the adult and developing rat central nervous system (CNS). LAMP is a member of the immunoglobulin superfamily of cell adhesion molecules with three Ig domains and is highly conserved between rat and human. In this study, the temporal and spatial pattern of lamp gene expression during fetal rat development was analyzed by using Northern blot analysis and in situ hybridization. In Northern blot analysis, two lamp mRNA transcripts, 1.6 kb and 8.0 kb, identical in size to those present in the adult rat nervous system, were detected in developing neural tissue. In situ hybridization analysis showed close correlation, though not identity, between the expression of lamp mRNAs and the distribution of LAMP in limbic regions of the developing rat CNS, indicative of a more complex regulation of gene expression than was previously thought to be the case. The expression of lamp mRNAs is first detected on about embryonic day (E) 13. The hybridization signal is not seen in the proliferative ventricular zone at any level of the neuraxis, indicating that lamp is expressed in postmitotic neurons. In the cerebral cortex, lamp mRNAs are expressed in limbic cortical regions, such as the perirhinal cortex, prefrontal cortex, and cingulate cortex. In the hippocampus, the hybridization signal is observed in Ammon's horn by E18. The neostriatum, amygdaloid complex, and most hypothalamic areas express lamp mRNAs from early stages (E13-E14) in a pattern consistent with the onset of neurogenesis. The emerging patterns of lamp expression at the outset are similar to those seen in adult hypothalamus and dorsal thalamus. Although the hybridization signal is observed in some nonlimbic areas, including midbrain and hindbrain structures, intense labeling is evident in more classic limbic regions. The high levels of expression of lamp in limbic regions, beginning in early developmental stages, combined with the results of previous functional in vitro and in vivo studies, support a role for LAMP as a recognition molecule involved in the formation of limbic connections.

cDNA cloning and structural analysis of the human limbic-system-associated membrane protein (LAMP).

The limbic-system-associated membrane protein (LAMP) is a 64-68-kDa neuronal surface glycoprotein distributed in cortical and subcortical regions of the limbic system. The human LAMP gene was cloned by RT-PCR using human cerebral cortex mRNA and oligodeoxyribonucleotide (oligo) primers derived from the rat lamp cDNA sequence. The human and rat LAMP cDNAs showed 94% identity at the nucleotide (nt) level, and the encoded 338-amino-acid (aa) polypeptides shared 99% sequence identity. All the important features of LAMP were conserved: (i) the deduced aa sequence reflecting a glycosyl-phosphatidylinositol (GPI)-anchor, (ii) eight putative N-linked glycosylation sites, and (iii) conserved pairs of Cys forming three internal repeats characteristic of the immunoglobulin superfamily (IgSF). Northern blot analysis indicated the presence of two mRNA transcripts in the human brain of a size identical to those identified in adult rat brain. These data indicate that LAMP is a highly conserved new member of the IgSF which, together with the opioid-binding cell adhesion molecule (OBCAM) and neurotrimin, comprises a new subfamily that has been designated as IgLONs. With a unique distribution in limbic structures, LAMP may play an important role in limbic system development and function, as suggested by previous in vitro and in vivo functional studies.

The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting.

The formation of brain circuits requires molecular recognition between functionally related neurons. We report the cloning of a molecule that participates in these interactions. The limbic system-associated membrane protein (LAMP) is an immunoglobulin (Ig) superfamily member with 3 Ig domains and a glycosyl-phosphatidylinositol anchor. In the developing forebrain, lamp is expressed mostly by neurons comprising limbic-associated cortical and subcortical regions that function in cognition, emotion, memory, and learning. The unique distribution of LAMP reflects its functional specificity. LAMP-transfected cells selectively facilitate neurite outgrowth of primary limbic neurons. Most striking, administration of anti-LAMP in vivo results in abnormal growth of the mossy fiber projection from developing granule neurons in the dentate gyrus of the hippocampal formation, suggesting that LAMP is essential for proper targeting of this pathway. Rather than being a general guidance cue, LAMP likely serves as a recognition molecule for the formation of limbic connections.

Comparison of the release of endogenous and of 3H-acetylcholine from slices of rat cerebral cortex.

A comparison of the release of endogenous and of tritiated acetylcholine (3H-ACh) was performed in eserinized slices of rat cerebral cortex labelled in vitro in the presence of 0.43 microM tityustoxin (TsTX), a scorpion venom known to increase the overflow of endogenous acetylcholine. The endogenous and the radioactive ACh were spontaneously released into the organ bath. At the moment when the spontaneous efflux had levelled off, the endogenous ACh collected during 1 min accounted for 1.03 +/- 0.14% of the tissue content and a similar value (0.80 +/- 0.26%) was found for 3H-ACh. The basal outflow as well as the tissue retention of ACh and of 3H-ACh were not modified by exposure to the scorpion venom. In the presence of 20 mM KCl for 1 min, the overflow of both radioactive and endogenous ACh was increased up to approximately 100% above basal levels and this K+-evoked release was almost doubled under the action of TsTX. The results presented are compatible with the view that no major functional differences exist in the rat cortex between responses of pools storing 3H-ACh synthesized from 3H-choline and those storing ACh formed from endogenous substrate.

Comparison of the release of endogenous and of 3H-acetylcholine from slices of rat cerebral cortex

A comparison of the release of endogenous and of tritiated acetylcholine (3H-ACh) was performed in eserinized slices of rat cerebral cortex labelled in vitro in the presence of 0.43 microM tityustoxin (TsTX), a scorpion venom known to increase the overflow of endogenous acetylcholine. The endogenous and the radioactive ACh were spontaneously released into the organ bath. At the moment when the spontaneous efflux had levelled off, the endogenous ACh collected during 1 min accounted for 1.03 +/- 0.14

of the tissue content and a similar value (0.80 +/- 0.26

) was found for 3H-ACh. The basal outflow as well as the tissue retention of ACh and of 3H-ACh were not modified by exposure to the scorpion venom. In the presence of 20 mM KCl for 1 min, the overflow of both radioactive and endogenous ACh was increased up to approximately 100

above basal levels and this K+-evoked release was almost doubled under the action of TsTX. The results presented are compatible with the view that no major functional differences exist in the rat cortex between responses of pools storing 3H-ACh synthesized from 3H-choline and those storing ACh formed from endogenous substrate.

Comparison of the release of endogenous and of 3H-acetylcholine from slices of rat cerebral cortex.

A comparison of the release of endogenous and of tritiated acetylcholine (3H-ACh) was performed in eserinized slices of rat cerebral cortex labelled in vitro in the presence of 0.43 microM tityustoxin (TsTX), a scorpion venom known to increase the overflow of endogenous acetylcholine. The endogenous and the radioactive ACh were spontaneously released into the organ bath. At the moment when the spontaneous efflux had levelled off, the endogenous ACh collected during 1 min accounted for 1.03 +/- 0.14

of the tissue content and a similar value (0.80 +/- 0.26

) was found for 3H-ACh. The basal outflow as well as the tissue retention of ACh and of 3H-ACh were not modified by exposure to the scorpion venom. In the presence of 20 mM KCl for 1 min, the overflow of both radioactive and endogenous ACh was increased up to approximately 100

above basal levels and this K+-evoked release was almost doubled under the action of TsTX. The results presented are compatible with the view that no major functional differences exist in the rat cortex between responses of pools storing 3H-ACh synthesized from 3H-choline and those storing ACh formed from endogenous substrate.

Sensory nerves and inflammation: evidence for the release of a neurogenic permeability factor by tityustoxin.

Venom of the scorpion Tityus serrulatus and its active principle tityustoxin (TsTX), kept in contact with the peripheral cut end of the sciatic or saphenous nerve of the rat, induced inflammatory reactions in the areas supplied by the nerves. The reactions include increased vascular permeability and oedema formation below the tibio-tarsal articulation, and were similar to those evoked by electrical antidromic stimulation of these nerves. When electrical stimulation of the peripheral nerve ending preceded its contact with the toxin or, conversely, when the application of electrical pulses closely followed contact with TsTX, no marked increase in the vascular permeability and oedematous respones, subsequent to the second stimulation, was observed. Anti-histamine and anti-serotonin drugs, as well as substances capable of blocking synthesis of prostaglandins or activation of the kinin system, and also atropine, were ineffective in reducing the responses to TsTX or electrical stimuli. Since the responses were evoked at a distance, in the areas supplied by the nerves, they must be chemically mediated. It is concluded that TsTX and electrical antidromic stimuli affect sensory nerves inducing the release of a permeability-increasing factor, which is responsible for the observed reactions. This factor can be depleted from storage sites by prolonged stimulation of the nerves, is not inhibited by antagonists of known mediators of inflammatory reactions and most probably originates in sensory fibres.