Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

The TGR5 receptor mediates bile acid-induced itch and analgesia

Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.

High sensitivity recording of afferent nerve activity using ultra-compliant microchannel electrodes: an acutein vivovalidation

Neuroprostheses interfaced with transected peripheral nerves are technological routes to control robotic limbs as well as convey sensory feedback to patients suffering from traumatic neural injuries or degenerative diseases. To maximize the wealth of data obtained in recordings, interfacing devices are required to have intrafascicular resolution and provide high signal-to-noise ratio (SNR) recordings. In this paper, we focus on a possible building block of a three-dimensional regenerative implant: a polydimethylsiloxane (PDMS) microchannel electrode capable of highly sensitive recordings in vivo. The PDMS 'micro-cuff' consists of a 3.5 mm long (100 µm × 70 µm cross section) microfluidic channel equipped with five evaporated Ti/Au/Ti electrodes of sub-100 nm thickness. Individual electrodes have average impedance of 640 ± 30 kΩ with a phase angle of −58 ± 1 degrees at 1 kHz and survive demanding mechanical handling such as twisting and bending. In proof-of-principle acute implantation experiments in rats, surgically teased afferent nerve strands from the L5 dorsal root were threaded through the microchannel. Tactile stimulation of the skin was reliably monitored with the three inner electrodes in the device, simultaneously recording signal amplitudes of up to 50 µV under saline immersion. The overall SNR was approximately 4. A small but consistent time lag between the signals arriving at the three electrodes was observed and yields a fibre conduction velocity of 30 m s−1. The fidelity of the recordings was verified by placing the same nerve strand in oil and recording activity with hook electrodes. Our results show that PDMS microchannel electrodes open a promising technological path to 3D regenerative interfaces.

Compliant Multi-electrode Micro-channel Array for Recording Afferent Nerve Activity

We have fabricated a compliant neural interface to record afferent nerve activity. Stretchable gold electrodes were evaporated on a polydimethylsiloxane (PDMS) substrate and were encapsulated using photo-patternable PDMS. The built-in microstructure of the gold film on PDMS allows the electrodes to twist and flex repeatedly, without loss of electrical conductivity. PDMS microchannels (5mm long, 100μm wide, 100μm deep) were then plasma bonded irreversibly on top of the electrode array to define five parallel-conduit implants. The soft gold microelectrodes have a low impedance of ~200kΩ at the 1kHz frequency range. Teased nerves from the L6 dorsal root of an anaesthetized Sprague Dawley rat were threaded through the microchannels. Acute tripolar recordings of cutaneous activity are demonstrated, from multiple nerve rootlets simultaneously. Confinement of the axons within narrow microchannels allows for reliable recordings of low amplitude afferents. This electrode technology promises exciting applications in neuroprosthetic devices including bladder fullness monitors and peripheral nervous system implants.

Schwann cells can be reprogrammed to multipotency by culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Translational research and therapeutic applications of neural crest-derived stem cells in regenerative periodontology

Regeneration of periodontal tissues aims to utilize tissue engineering techniques to restore lost periodontal tissues including the cementum, periodontal ligament and alveolar bone. Regenerative dentistry and its special field regenerative periodontology represent relatively new and emerging branches of translational stem cell biology and regenerative medicine focusing on replacing and regenerating dental tissues to restore or re-establish their normal function lost during degenerative diseases or acute lesions. The regeneration itself can be achieved through transplantation of autologous or allogenic stem cells, or by improving the tissue self-repair mechanisms (e.g. by application of growth factors). In addition, a combination of stem cells or stem cell-containing tissue with bone implants can be used to improve tissue integration and the clinical outcome. As the oral cavity represents a complex system consisting of teeth, bone, soft tissues and sensory nerves, regenerative periodontology relies on the use of stem cells with relatively high developmental potential. Notably, the potential use of pluripotent stem cell types such as human embryonic stem cells or induced pluripotent stem cells is still aggravated by ethical and practical problems. Thus, other cellular sources such as those readily available in the postnatal craniofacial area and particularly in oral structures offer a much better and realistic alternative as cellular regenerative sources. In this review, we summarize current knowledge on the oral neural crest-derived stem cell populations (oNCSCs) and discuss their potential in regenerative periodontology.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle.

Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9-26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9-20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17-18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 mu m(2) to 805.89 mu m(2). Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers. (C) 2008 Elsevier B.V. All rights reserved.

A light and scanning electron microscopy study of bone healing following inferior alveolar nerve lateralization: An experimental study in rabbits

Purpose: The purpose of this study was to evaluate the bone healing kinetics around commercially pure titanium implants following inferior alveolar nerve (IAN) lateralization in a rabbit model. Materials and Methods: Inferior alveolar nerve lateralization was performed in 16 adult female rabbits (Oryctolagus cuniculus). During the nerve lateralization procedure, 1 implant was placed through the mandibular canal, and the IAN was replaced in direct contact with the implant. During the 8-week healing period, various bone labels were administered for fluorescent microscopy analysis. The animals were euthanized by anesthesia overdose, and the mandibular blocks were exposed by sharp dissection. Nondecalcified samples were prepared for optical light and scanning electron microscopy (SEM) evaluation. Results: SEM evaluation showed bone modeling/remodeling between the IAN and implant surface. Fluorochrome area fraction labeling at different times during the healing period showed that bone apposition mainly occurred during the first 2 weeks after implantation. Conclusions: The results obtained showed that bone healing/deposition occurred between the alveolar nerves in contact with a commercially pure titanium implant. No interaction between the nerve and the implant was detected after the 8-week healing period. Appositional bone healing occurred around the nerve bundle structure, restoring the mandibular canal integrity and morphology.

SEM and Neurohistological Observations of Nerve Endings in the Middle Region of the Tongue of the Collared Peccary (Tayassu tajacu): A Silver Impregnation Method

The presence of lingual papillae and the nerve endings in the middle region of the tongue mucosa of collared peccary (Tayassu tajacu) were studied using scanning electron microscopy and light microscopy, based upon the silver impregnation method. The middle region of tongue mucosa revealed numerous filiform and fungiform papillae. The thick epithelial layer showed epithelial cells and a dense connective tissue layer containing nerve fibre bundles and capillaries. The sensory nerve endings, intensely stained by silver impregnation, were usually non-encapsulated and extended into the connective tissue of the filiform and fungiform papillae very close to the epithelial cells. In some regions, the sensory nerves fibres formed a dense and complex network of fine fibrils. The presence of these nerve fibrils may characterize the mechanisms of transmission of sensitive impulses to the tongue mucosa.

Multiple Pathways Analysis of Brain Functional Networks from EEG Signals: An Application to Real Data

In the present study, we propose a theoretical graph procedure to investigate multiple pathways in brain functional networks. By taking into account all the possible paths consisting of h links between the nodes pairs of the network, we measured the global network redundancy R (h) as the number of parallel paths and the global network permeability P (h) as the probability to get connected. We used this procedure to investigate the structural and dynamical changes in the cortical networks estimated from a dataset of high-resolution EEG signals in a group of spinal cord injured (SCI) patients during the attempt of foot movement. In the light of a statistical contrast with a healthy population, the permeability index P (h) of the SCI networks increased significantly (P < 0.01) in the Theta frequency band (3-6 Hz) for distances h ranging from 2 to 4. On the contrary, no significant differences were found between the two populations for the redundancy index R (h) . The most significant changes in the brain functional network of SCI patients occurred mainly in the lower spectral contents. These changes were related to an improved propagation of communication between the closest cortical areas rather than to a different level of redundancy. This evidence strengthens the hypothesis of the need for a higher functional interaction among the closest ROIs as a mechanism to compensate the lack of feedback from the peripheral nerves to the sensomotor areas.

Analgesia em pós-operatório de toratomias : estudo prospectivo e randomizado

Introdução: A dor é um importante fator de incremento da morbidade e mortalidade em pacientes submetidos a procedimentos cirúrgicos que incluem toracotomias. Diversos fatores contribuem para que esses pacientes apresentem um alto grau de dor no pós-operatório, entre os quais a secção da pele, músculos e pleura, retração dos músculos e ligamentos pelo afastador de Finochietto, irritação da pleura e nervos intercostais pelos drenos tubulares torácicos e fraturas ocasionais dos arcos costais. O aumento das taxas de morbidade e mortalidade é dado principalmente à respiração superficial decorrente da pouca mobilidade da parede torácica e conseqüente à dor e pela perda da efetividade do principal mecanismo de eliminação de secreções da árvore traqueobrônquica (tosse), resultando em atelectasias, inadequado gradiente ventilação / perfusão, hipoxemia e pneumonia. Uma vez caracterizada a necessidade de atenuação da dor como fator primordial na melhora dos índices de morbidade e mortalidade no período pós-operatório de cirurgia torácica, torna-se imperiosa uma análise das terapêuticas disponíveis na atualidade para tanto. Objetivos: Avaliar a utilização de três diferentes métodos de analgesia: 1. bloqueio peridural com morfina (BPM); 2. morfina parenteral (MP); e 3. bloqueio intercostal extrapleural contínuo com lidocaína” (BIC), em pacientes submetidos a procedimentos que incluíram toracotomias em sua execução, além de analisar o custo financeiro desses métodos. Materiais e métodos: Trata-se de um estudo prospectivo, randomizado, no qual foram analisados 79 pacientes, submetidos a toracotomias, subdivididos de forma aleatória em três grupos, de acordo com a modalidade terapêutica instituída: 25 pacientes no grupo BIC, 29 pacientes no grupo BPM e 25 pacientes no grupo MP. Cada paciente foi observado e analisado por profissionais de enfermagem previamente treinados. As variáveis analisadas foram a dor e a sedação. (quantificadas através de escores e analisadas através do método de Kruskal-Wallis com correção pelo teste de Dunn), além do custo financeiro de cada método e da necessidade de administração de opióides adicionais. Resultados: As variáveis dor e sedação foram obtidas através das seguintes medianas, respectivamente: grupos BIC (2,5 e 0); BPM (4 e 0) e MP (3,5 e 0). O custo financeiro foi de US$ 78,69 para o grupo BIC; US$ 28,61 para o grupo BPM e US$ 11,98 para o grupo MP. A necessidade adicional de opióide foi de 4,2 mg/dia para o grupo BIC; 5,7 mg/dia para o grupo BPM e 10,7 mg/dia para o grupo MP. Conclusões: A intensidade da dor foi significativamente menor no grupo BIC, quando comparado ao grupo MP. Não foram identificadas diferenças significativas de intensidade da dor quando comparados os grupos BIC versus BPM e BPM versus MP. A intensidade de sedação foi significativamente maior no grupo MP quando comparado aos grupos BIC e BPM. Não foram evidenciadas diferenças significativas quanto à sedação entre os grupos BIC e BPM. O custo financeiro do grupo MP foi sensivelmente menor quando comparado aos grupos BIC e BPM. A necessidade adicional de morfina foi significativamente maior no grupo MP, quando comparados aos grupos BIC e BPM.

Avaliação das capacitações de Hanseníase: enfermeiros opinião de médicos e enfermeiros das equipes de saúde da família

MORENO,Cléa Maria da Costa,ENDERS,Bertha Cruz, SIMPSON, Clélia Albino. Avaliação das capacitações de Hanseníase: enfermeiros opinião de médicos e enfermeiros das equipes de saúde da família. Revista Brasileira de Enfermagem, Brasília, v.61,n.esp.p. 671-5.2008.