PHB-HV 3D Scaffold supports osteoblast growth

Bone tissue engineering requires a biocompatible scaffold that supports cell growth and enhances the native repair process. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a biodegradable 3D scaffold with 88.1 â 0.3% porosity and pore size of 163.5 â 0.1 mm. Previous studies demonstrated the potential of PHB-HV as a scaffold in spinal cord repair. The aim of this study was to evaluate PHB-HV as a scaffold for bone regeneration by assessing the cytocompatability of this scaffold.

Galanin-mediated behavioural hyperalgesia from the dorsomedial nucleus of the hypothalamus involves two independent descending pronociceptive pathways

Activation of the dorsomedial nucleus of the hypothalamus (DMH) by galanin (GAL) induces behavioural hyperalgesia. Since DMH neurones do not project directly to the spinal cord, we hypothesized that the medullary dorsal reticular nucleus (DRt), a pronociceptive region projecting to the spinal dorsal horn (SDH) and/or the serotoninergic raphe-spinal pathway acting on the spinal 5-HT3 receptor (5HT3R) could relay descending nociceptive facilitation induced by GAL in the DMH. Heat-evoked paw-withdrawal latency (PWL) and activity of SDH neurones were assessed in monoarthritic (ARTH) and control (SHAM) animals after pharmacological manipulations of the DMH, DRt and spinal cord. The results showed that GAL in the DMH and glutamate in the DRt lead to behavioural hyperalgesia in both SHAM and ARTH animals, which is accompanied particularly by an increase in heat-evoked responses of wide-dynamic range neurons, a group of nociceptive SDH neurones. Facilitation of pain behaviour induced by GAL in the DMH was reversed by lidocaine in the DRt and by ondansetron, a 5HT3R antagonist, in the spinal cord. However, the hyperalgesia induced by glutamate in the DRt was not blocked by spinal ondansetron. In addition, in ARTH but not SHAM animals PWL was increased after lidocaine in the DRt and ondansetron in the spinal cord. Our data demonstrate that GAL in the DMH activates two independent descending facilitatory pathways: (i) one relays in the DRt and (ii) the other one involves 5-HT neurones acting on spinal 5HT3Rs. In experimental ARTH, the tonic pain-facilitatory action is increased in both of these descending pathways.

Alterações fisiológicas do sistema nervoso central de rã pelas toxinas de Cl. perfringens, Cl. oedematiens e Cl. septicum

The authors carried on experiences in order to confirm the neurotoxic theory of gas gangrene explained by Pacheco & Costa, uning preparations of isolated cord-posterior train of Leptodactylus ocellatus as described by OZORIO DE ALMEIDA & Cols. Frogs were intoxicated 3 days before the test with parcially purified toxins of Cl. perfringens, Cl. oedematiens and Cl. septicum. The intoxication produced a shortening of spinal reflexes duration time of such preparations, showing a typical alteration of the reflex activity of the spinal cord.

Alterações do sistema nervoso central pelas toxinas do Clostridium perfringens

The progressive intoxication of dogs with Clostridium perfringens toxin promotes degenerative neural lesions analogous to that found in acute intoxication. In progressive intoxication, the neuroglia is more proliferative and the myeline sheats are altered. The alterations were not circunscribed to spinal cord and medulla but were extensive to other nervous organs.

Sôbre a patogenia da Grangrena gasona

Since von Hibler gas grangrene has been considered a local infection with systemic symptoms. When we consider some of the symptoms of gas gangrene, those of the central nervous system are in evidence beeing similar to those observed in tetanus and botulism. It is likely therefore that gas gangrene intoxication and the disease caused by it are of neurotoxic nature. With Almeida Cardoso and Araujo Costa we were able to demonstrate lesions in the central nervous system of animals wich had been intoxicated during a short period of time as well in those with intoxication of longer duration. In acute intoxication, after intracreneal inoculation, severe alterations were seen within 20 to 30 minutes in the cells of the spinal cord, specially in motor cells and also in some cells of the posterior cord and spinal bulb. The changes consisted in chromatolysis and picnosis and were more marked in animals intoxicated with Clostridium histolyticum and Cl. perfringens toxines. Myelin sheet was unchanged. in delayed intoxication with greater and repeated dosis lesions of the central nervous system (brain, protuberance, medula ablongate and medula spinal) were observed. They consisted in hyperemia, perivascular hemorrages in white and grey substances, oedema, accumulation of glia cells with enlarged and hyperchromatic nuclei, fragmentation of the myelin sheet and balooning degeneration of the described by Spielmeyer. Such changes were found in the swollen and hemorragic zones and were generally similar to those found in the acute type of Spielmeyer 9acute swelling and liquefation). Other changes found sometimes were agglutination of Nissl's bodies, sinous appearence of the dendritic endings, shruncken cells of Spielmeyer and neuronophagy around "ghost" cells. In short the changes...

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.

Cellular schwannomas of the intracranial and intraspinal compartment: morphological and immunological characteristics compared with classical benign schwannomas.

The concept of cellular schwannoma as an unusual benign tumor is well established for peripheral nerves but has never been tested in neurosurgical series. In order to test the validity of this concept in cranial nerves and spinal roots we performed an analysis of the clinical and morphological characteristics of 12 cellular and 166 classical benign schwannomas. Immunohistochemical detection of antigen expression in Schwann cells including proliferating cell nuclear antigen (PCNA) was also performed. This study shows that cellular schwannomas in neurosurgical series manifest at a lower age than the classical benign variant and occur mainly in the spinal roots. Mitotic activity and sinusoidal vessels appear more frequently in cellular schwannomas and constitute with high cellularity, the most valuable criteria separating both entities. The postoperative course in both types of tumors was free of metastases or sarcomatous changes. Immunoexpression of S-100 protein, vimentin, epithelial membrane antigen and glial fibrillary acidic protein is not statistically different between the two variants. In contrast, PCNA is more highly expressed in cellular schwannomas. These These results confirm the concept that cellular schwannomas are a clinico-pathological variant of benign schwannomas and provide significant support for the introduction of this entity in neurosurgical oncology.

Chondromas of the spine: report of two cases and review of the literature.

We report two cases of vertebral chondroma presenting with spinal cord compression. In one case there was ultimate malignant transformation.

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Regulation of the akt signalling in human skeletal muscle atrophy

Abstract : The term "muscle disuse" is often used to refer collectively to reductions in neuromuscular activity as observed with sedentary lifestyles, reduced weight bearing, cancer, chronic obstructive pulmonary disease, chronic heart failure, spinal cord injury, sarcopenia or exposure to microgravity (spaceflight). Muscle disuse atrophy, caused by accelerated proteolysis, is predominantly due to the activation of the ATP-dependent ubiquitin (Ub) proteasome pathway. The current advances in understanding the molecular factors contributing to the Ub-dependent proteolysis process have been made mostly in rodent models of human disease and denervation with few investigations performed directly in humans. Recently, in mice, the genes Atrogin-1 and MuRF1 have been designated as primary candidates in the control of muscle atrophy. Additionally, the decreased activity of the Akt/GSK-3ß and Akt/mTOR pathways has been associated with a reduction in protein synthesis and contributing to skeletal muscle atrophy. Therefore, it is now commonly accepted that skeletal muscle atrophy is the result of a decreased protein synthesis concomitant with an increase in protein degradation (Glass 2003). Atrogin-1 and MuRF1 are genes expressed exclusively in muscle. In mice, their expression has been shown to be directly correlated with the severity of atrophy. KO-mice experiments showed a major protection against atrophy when either of these genes were deleted. Skeletal muscle hypertrophy is an important function in normal postnatal development and in the adaptive response to exercise. It has been shown, in vitro, that the activation of phosphatidylinositol 3-kinase (PI-3K), by insulin growth factor 1 (IGF-1), stimulates myotubes hypertrophy by activating the downstream pathways, Akt/GSK-3ß and Akt/mTOR. It has also been demonstrated in mice, in vivo, that activation of these signalling pathways causes muscle hypertrophy. Moreover, the latter were recently proposed to also reduce muscle atrophy by inhibiting the FKHR mediated transcription of several muscle atrophy genes; Atrogin-1 and MuRF1. Therefore, these targets present new avenues for developing further the understanding of the molecular mechanisms involved in both skeletal muscle atrophy and hypertrophy. The present study proposed to investigate the regulation of the Akt/GSK-3ß and Akt/mTOR signalling pathways, as well as the expression levels of the "atrogenes", Atrogin-1 and MuRF1, in four human models of skeletal muscle atrophy. In the first study, we measured the regulation of the Akt signalling pathway after 8 weeks of both hypertrophy stimulating resistance training and atrophy stimulation de-training. As expected following resistance training, muscle hypertrophy and an increase in the phosphorylation status of the different members of the Akt pathway was observed. This was paralleled by a concomitant decrease in FOXO1 nuclear protein content. Surprisingly, exercise training also induced an increase in the, expression of the atrophy genes and proteins involved in the ATP-dependant ubiquitin-proteasome system. On the opposite, following the de-training period a muscle atrophy, relative to the post-training muscle size, was measured. At the same time, the phosphorylation levels of Akt and GSK-3ß were reduced while the amount of FOXO1 in the nucleus increased. After the atrophy phase, there was also a reduction in Atrogin-1 and MuRF1 contents. In this study, we demonstrate for the first time in healthy human skeletal muscle, that the regulation of Akt and its downstream targets GSK-3ß, mTOR and FOXO1 are associated with both thé skeletal muscle hypertrophy and atrophy processes. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of both upper and lower motor neurons, which leads to severe muscle weakness and atrophy. All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls. ALS patients displayed an increase in Atrogin-1 mRNA and protein content which was associated with a decrease in Akt activity. However there was no difference in the mRNA and phospho-protein content of FOXO1, FOXO3a, p70S6K and GSK-3ß. The transcriptional regulation of human Atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via an other signalling pathway. Chronic complete spinal cord injury (SCI) is associated with severe muscle atrophy which is linked to co-morbidity factors such as diabetes, obesity, lipid disorders and cardiovascular diseases. Molecular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood. The aim of the present study was to determine if there was an increase in catabolic signalling targets such as Atrogin-1, MuRF1, FOXO and myostatin, and decreases in anabolic signalling targets such as IGF, Akt, GSK-3ß, mTOR, 4E-BP1 and p-70S6K in chronic complete SCI patients. All measurements were performed in biopsies taken from 8 complete chronic SCI patients and 7 age matched healthy controls. In SCI patients when compared with controls, there was a significant reduction in mRNA levels of Atrogin1, MuRF1 and Myostatin. Protein levels for Atrogin-1, FOX01 and FOX03a were also reduced. IGF-1 and both phosphorylated GSK-3ß and 4E-BP1 were decreased; the latter two in an Akt and mTOR independent manner, respectively. Reductions in Atrogin-1, MuRF1, FOXO and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signalling proteins regulating anabolism such as IGF, GSK3ß and 4E-BP1 would reduce the ability to increase protein synthesis rates in this chronic state of muscle wasting. The molecular mechanisms controlling age-related skeletal muscle loss in humans are poorly understood. The present study aimed to investigate the regulation of several genes and proteins involved in the activation of key signalling pathways promoting muscle hypertrophy such as GH/STAT5/IGF, IGF/Akt/GSK-3ß/4E-BP1 and muscle atrophy such as TNFα/SOCS3 and Akt/FOXO/Atrogin-1 or MuRF1 in muscle biopsies from 13 young and 16 elderly men. In the older, as compared with the young subjects, TNFα and SOCS-3 were increased while growth hormone receptor protein (GHR) and IGF-1 mRNA were both decreased. Akt protein levels were increased however no change in phosphorylated Akt content was observed. GSK-3ß phosphorylation levels were increased while 4E-BP1 was not changed. Nuclear FKHR and FKHRL1 protein levels were decreased, with no changes in their atrophy target genes, Atrogin-1 and MuRF1. Myostatin mRNA and protein levels were significantly elevated. Human sarcopenia may be linked to a reduction in the activity or sensitivity of anabolic signalling proteins such as GHR, IGF and Akt. TNFα, SOCS-3 and myostatin are potential candidates influencing this anabolic perturbation. In conclusion our results support those obtained in rodent or ín vitro models, and demonstrate Akt plays a pivotal role in the control of muscle mass in humans. However, the Akt phosphorylation status was dependant upon the model of muscle atrophy as Akt phosphorylation was reduced in all atrophy models except for SCI. Additionally, the activity pattern of the downstream targets of Akt appears to be different upon the various human models. It seems that under particular conditions such as spinal cord injury or sarcopenia, .the regulation of GSK-3ß, 4eBP1 and p70S6K might be independent of Akt suggesting alternative signalling pathways in the control of these the anabolic response in human skeletal muscle. The regulation of Atrogin-1 and MuRF1 in some of our studies has been shown to be also independent of the well-described Akt/FOXO signalling pathway suggesting that other transcription factors may regulate human Atrogin-1 and MuRF1. These four different models of skeletal muscle atrophy and hypertrophy have brought a better understanding concerning the molecular mechanisms controlling skeletal muscle mass in humans.

Paper de RIP140 en la fisiopatogenia de la adrenoleucodistròfia lligada a l'X.

La adrenoleucodistrofia ligada al X (X-ALD) es un enfermedad neurometabólica fatal caracterizada por una desmielinización cerebral progresiva infantil (CCALD) o por una neurodegeneración de la médula espinal (adrenomieloneuropatía, AMN), insuficiencia adrenal y acumulación de ácidos grasos de cadena muy larga (AGCML) como el ácido hexacosanoico (C26:0) en tejidos. La enfermedad está causada por mutaciones en el gen ABCD1 el cual codifica para un transportador peroxisomoal que importa AGCML. El ratón knockout para Abcd1 (Abcd1-) desarrolla alteraciones en la médula espinal que mimetizan el modelo de enfermedad AMN con inicio de los síntomas a los 20 meses. Previamente, nuestro grupo evidenció mediante análisis de transcriptómica, una desregulación mitocondrial en el modelo murino Abcd1- . En este trabajo demostramos que tanto en el ratón Abcd1- como en la sustancia blanca afectada de pacientes X-ALD hay una depleción mitocondrial. Para poder explicar esta depleción, estudiamos los niveles de un repressor de la biogenesis mitocondrial, RIP140. En cultivo organotípico de cortes de médula espinal observamos un aumento de los niveles proteicos de RIP140 en el ratón Abcd1- y también un aumento mediado por C26:0. Estos resultados indican que la sobreexpresión de RIP140 puede ser la responsable de la depleción mitocondrial presente en el ratón Abcd1- y una posible nueva diana terapèutica para la X-ALD.

Glioprotective impact of cell-permeable peptides in preclinical models of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by progressive degeneration of upper and lower motor neurons. It is mostly sporadic, but about 2% of cases are associated with mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1). A major constraint to the comprehension of the pathogenesis of ALS has been long represented by the conviction that this disorder selectively affects motor neurons in a cell-autonomous manner. However, the failure to identify the events underlying the neurodegenerative process and the increased knowledge of the complex cellular interactions necessary for the correct functioning of the CNS has recently focused the attention on the contribution to neurodegeneration of glial cells, including astrocytes. Astrocytes can hurt motor neurons directly by secreting neurotoxic factors, but they can also play a deleterious role indirectly by losing functions that are supportive for neurons. Recently, we reported that a subpopulation of astrocytes degenerates in the spinal cord of hSOD1G93A transgenic mouse model of ALS. Mechanistic studies in cultured astrocytes revealed that such effect is mediated by the excitatory amino acid glutamate.On the bsis of these observations, we next used the established cell culture model as a tool to screen the glioprotective effect of innovative drugs, namely cell-permeable therapeutics. These consist of peptidic effector moieties coupled to the selective intracellular peptide transporter TAT protein. We initially validated the usefulness of these molecules demonstrating that a control fluorescent peptide enters astrocytes in culture and is retained within the cells up to 24-48 h, according to the timing of our cytotoxicity experiments. We then tested the impact of specific intracellular peptides with antiapoptotic properties on glutamate-treated hSOD1G93A- expressing astrocytes and we identified one molecule that protects the cells from death. Chronic treatment of ALS mice with this peptide had a positive impact on the outcome of the disease.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Do glial cells control pain?

Management of chronic pain is a real challenge, and current treatments focusing on blocking neurotransmission in the pain pathway have only resulted in limited success. Activation of glia cells has been widely implicated in neuroinflammation in the central nervous system, leading to neruodegeneration in many disease conditions such as Alzheimer's and multiple sclerosis. The inflammatory mediators released by activated glial cells, such as tumor necrosis factor-α and interleukin-1β can not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions. Pain can also be potentiated by growth factors such as BDNF and bFGF that are produced by glia to protect neurons. Thus, glia cells can powerfully control pain when they are activated to produce various pain mediators. We will review accumulating evidence supporting an important role of microglia cells in the spinal cord for pain control under injury conditions (e.g. nerve injury). We will also discuss possible signaling mechanisms in particular MAP kinase pathways that are critical for glia control of pain. Investigating signaling mechanisms in microglia may lead to more effective management of devastating chronic pain.