A Bayesian model predicts the response of axons to molecular gradients

Axon guidance by molecular gradients plays a crucial role in wiring up the nervous system. However, the mechanisms axons use to detect gradients are largely unknown. We first develop a Bayesian “ideal observer” analysis of gradient detection by axons, based on the hypothesis that a principal constraint on gradient detection is intrinsic receptor binding noise. Second, from this model, we derive an equation predicting how the degree of response of an axon to a gradient should vary with gradient steepness and absolute concentration. Third, we confirm this prediction quantitatively by performing the first systematic experimental analysis of how axonal response varies with both these quantities. These experiments demonstrate a degree of sensitivity much higher than previously reported for any chemotacting system. Together, these results reveal both the quantitative constraints that must be satisfied for effective axonal guidance and the computational principles that may be used by the underlying signal transduction pathways, and allow predictions for the degree of response of axons to gradients in a wide variety of in vivo and in vitro settings.

Co-Signaling by Neurotrophic Factors and the Extracellular Matrix for Axonal Growth and Neuronal Survival

Neurotrophic factors (NTFs) and the extracellular matrix (ECM) are important regulators of axonal growth and neuronal survival in mammalian nervous system. Understanding of the mechanisms of this regulation is crucial for the development of posttraumatic therapies and drug intervention in the injured nervous system. NTFs act as soluble, target-derived extracellular regulatory molecules for a wide range of physiological functions including axonal guidance and the regulation of programmed cell death in the nervous system. The ECM determines cell adhesion and regulates multiple physiological functions via short range cell-matrix interactions. The present work focuses on the mechanisms of the action of NTFs and the ECM on axonal growth and survival of cultured sensory neurons from dorsal root ganglia (DRG). We first examined signaling mechanisms of the action of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) on axonal growth. GDNF, neurturin (NRTN) and artemin (ART) but not persephin (PSPN) promoted axonal initiation in cultured DRG neurons from young adult mice. This effect required Src family kinase (SFK) activity. In neurons from GFRalpha2-deficient mice, NRTN did not significantly promote axonal initiation. GDNF and NRTN induced extensive lamellipodia formation on neuronal somata and growth cones. This study suggested that GDNF, NRTN and ARTN may serve as stimulators of nerve regeneration under posttraumatic conditions. Consequently we studied the convergence of signaling pathways induced by NTFs and the ECM molecule laminin in the intracellular signaling network that regulates axonal growth. We demonstrated that co-stimulation of DRG neurons with NTFs (GDNF, NRTN or nerve growth factor (NGF)) and laminin leads to axonal growth that requires activation of SFKs. A different, SFK-independent signaling pathway evoked axonal growth on laminin in the absence of the NTFs. In contrast, axonal branching was regulated by SFKs both in the presence and in the absence of NGF. We proposed and experimentally verified a Boolean model of the signaling network triggered by NTFs and laminin. Our results put forward an approach for predictable, Boolean logics-driven pharmacological manipulation of a complex signaling network. Finally we found that N-syndecan, the receptor for the ECM component HB-GAM was required for the survival of neonatal sensory neurons in vitro. We demonstrated massive cell death of cultured DRG neurons from mice deficient in the N-syndecan gene as compared to wild type controls. Importantly, this cell death could not be prevented by NGF the neurotrophin which activates multiple anti-apoptotic cascades in DRG neurons. The survival deficit was observed during first postnatal week. By contrast, DRG neurons from young adult N-syndecan knock-out mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Corneal nerves in refractive surgery and dry eye

This study aimed to investigate the morphology and function of corneal sensory nerves in 1) patients after corneal refractive surgery and 2) patients with dry eye due to Sjögren's syndrome. A third aim was to explore the possible correlation between cytokines detected in tears and development of post-PRK subepithelial haze. The main methods used were tear fluid ELISA analysis, corneal in vivo confocal microscopy, and noncontact esthesiometry. The results revealed that after PRK a positive correlation exists between the regeneration of subbasal nerves and the thickness of regenerated epithelium. Pre- or postoperative levels of the tear fluid cytokines TGF-β1, TNF-α, or PDGF-BB did not correlate with the development of corneal haze objectively estimated by in vivo confocal microscopy 3 months after PRK. After high myopic LASIK, a discrepancy between subjective dry eye symptoms and objective signs of dry eye was observed. The majority of patients reported ongoing dry eye symptoms even 5 years after LASIK, although no objective clinical signs of dry eye were apparent. In addition, no difference in corneal sensitivity was observed between these patients and controls. Primary Sjögren's syndrome patients presented with corneal hypersensitivity, although their corneal subbasal nerve density was normal. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased number of antigen-presenting cells among subbasal nerves were observed, implicating the presence of an ongoing inflammation. Based on these results, the relationship between nerve regeneration and epithelial thickness 3 months after PRK appears to reflect the trophic effect of corneal nerves on epithelium. In addition, measurement of tear fluid cytokines may not be suitable for screening patients for risk of scar (haze) formation after PRK. Presumably, at least part of the symptoms of "LASIK-associated dry eye" are derived from aberrantly regenerated and abnormally functioning corneal nerves. Thus, they may represent a form of corneal neuropathy or "phantom pain" rather than conventional dry eye. Corneal nerve alterations and inflammatory findings in Sjögren's syndrome offer an explanation for the corneal hypersensitivity or even chronic pain or hyperalgesia often observed in these patients. In severe cases of disabling chronic pain in patients with dry eye or after LASIK, when conventional therapeutic possibilities fail to offer relief, consultation of a physician specialized in pain treatment is recommended.

Estudo da vascularização dos nervos periféricos e da sua influência na regeneração nervosa : estudo experimental e no cadáver humano

RESUMO: Na clínica, a recuperação funcional que se segue a uma lesão nervosa raramente é atingida na sua totalidade. A reinervação, quer motora, quer sensitiva, ocorre geralmente com maior ou menor deficit. Interessa, então, identificar os factores que podem interferir na regeneração nervosa. O neurónio é a unidade anatómica fundamental do sistema nervoso periférico e é muito vulnerável à isquemia pela grande distância que existe entre o corpo neuronal e a extensão do axónio, que pode ser de apenas alguns milímetros ou até atingir um metro. É, por isso, fundamental o estudo da vascularização do nervo periférico e da sua influência na regeneração nervosa. O resultado deste estudo pode levar ao desenvolvimento de técnicas cirúrgicas que criem as condições que garantam, por sua vez, a revascularização precoce do nervo periférico em caso de lesão, ou mesmo em caso de doenças, nas quais a vascularização do nervo está alterada como, por exemplo, na neuropatia diabética. O estudo da vascularização do nervo periférico realizou-se através da investigação da vascularização do nervo mediano do cadáver humano, pela investigação da vascularização do nervo isquiático do rato Wistar e do Plexo Braquial (PB) do mesmo. A vascularização do PB do rato não é muito diferente daquela que é reportada na espécie humana, existindo uma homologia entre o rato e o Homem no que diz respeito à morfologia e à vascularização do PB. Através da comparação angiomorfológica entre o nervo isquiático do rato e o nervo mediano humano, concluíu-se que a microvascularização do nervo isquiático do rato e do mediano humano são muito semelhantes, o que suporta a utilização do rato como modelo experimental de lesões do nervo mediano humano. Para a avaliação da influência da vascularização na regeneração nervosa foi feita a análise da eficácia de enxerto de tubo de membrana amniótica humana imunologicamente inerte, de enxerto de veia jugular externa autóloga e de auto-enxerto de nervo, na reparação de um defeito de 10 milímetros no nervo isquiático do rato, na presença de um fornecimento vascular axial, comparando-se com os mesmos procedimentos em estudos realizados anteriormente, sem suprimento vascular. Os ratos foram avaliados funcionalmente através do estudo das pegadas, da electroneurografia e da força de flexão ao nível do tornozelo, e estruturalmente, através das avaliações histológicas e morfométricas, da taxa de recuperação do peso dos músculos gastrocnémio e solhear e da marcação axonal retrógrada com True Blue às 4, 8 e 12 semanas. Os nervos reconstruídos apresentaram uma arquitectura normal, incluindo a arquitectura vascular. A membrana amniótica foi bem tolerada, persistindo imunologicamente em torno do nervo até à 12.ª semana. Concluiu-se também que, na presença de um suprimento vascular axial local, a membrana amniótica humana e as veias autólogas são, pelo menos, tão eficazes como os auto-enxertos nervosos na reconstrução de defeitos nervosos de 10 milímetros.--------------------------------------ABSTRACT: At the clinic, the functional recovery that follows a nerve lesion is rarely achieved in full. The neuron is very vulnerable to ischemia that’s why it is essential to study the vascularization of the peripheral nerve and its influence on the nerve’s regeneration. The outcome of this study may lead to the development of surgical techniques that create the conditions which are necessary to ensure an early revascularization in case of a peripheral nerve injury. This study investigated the vascularization of the median nerve of the human cadaver and the vascularization of the sciatic nerve of the Wistar rat and his Brachial Plexus (BP) through animal experimentation. The mouse's BP vascularization is not so different from the one that is reported in the human species. An angiomorphological comparison between the mouse sciatic nerve and the human median nerve concluded that the microvascularizations are very similar, which supports the use of the mouse as an experimental model for the study of median nerve’s lesions. To evaluate the influence of vascularization in the nerve’s regeneration, it was made an assessment of the effectiveness of the human amniotic immuno-inert membrane grafts, of the autologous external jugular vein grafts and of the nerve auto-graft in the repair of a defect of 10 mm on the sciatic nerve of the rat, in the presence of an axial vascular supply, comparing these with the same procedures that were adopted in the previous studies, without vascular supply. The rats were functionally assessed and structurally evaluated (through histological and morphometric evaluations) at the 4.th, 8.th and 12.th weeks. The nerves reconstructed presented a normal architecture, including vascular architecture. The amniotic membrane was well-tolerated immunologically, persisting around the nerve until the 12.th week. As a result, it was also concluded that in the presence of a local axial vascular supply, the human amniotic membrane and the autologous veins are, at least, as effective as the nerve auto-grafts in the reconstruction of the nerve’s defects of 10 mm.

La régénération axonale suivant l'axotomie du nerf sciatique et stimulation électrique directe et transcutanée chez la souris

La stimulation électrique directe (SED), pour une heure, améliore la régénération de nerfs périphériques chez le rat après la réparation. Cliniquement, ceci augmenterait le temps opératoire, rehaussant les risques de complications périopératoires. Objectif: Cette étude examine si la stimulation électrique transcutanée (SETC) est aussi efficace à améliorer la régénération de nerfs périphériques que la stimulation électrique directe. Méthode: Le nerf sciatique droit de 28 souris a été axotomisé. Une réparation par microsuture est effectuée. Quatre groupes sont étudiés : (1) sham; (2) suture seulement; (3) suture et SED; (4) suture et SETC. La stimulation est appliquée pour 1 heure à 20 Hz. Les souris sont étudiées pour un total de 12 semaines. La récupération sciatique est évaluée aux semaines 0, 1, 2 et aux 2 semaines par la suite par analyse de démarche sur la poutre. Résultats: La cinématique post-récupération démontre un index fonctionnel sciatique et angle de décollement significativement améliorés pour les groupes SED et SETC aux semaines 8, 10 et 12. Conclusions: 12 semaines après l’axotomie du nerf sciatique, la récupération fonctionnelle est significativement améliorée avec la SED et la SETC. Donc, la SETC est aussi bénéfique pour la promotion de la régénération nerveuse et réinnervation musculaire fonctionnelle que la SED.

HISTOLOGIC EVALUATION OF END-TO-SIDE NEURORRHAPHY. EXPERIMENTAL STUDY IN RATS

Objective: The aim of this study was to histologically compare the axonal sprouting after end-to-side neurorrhaphy with or without epineurotomy. Methods: twenty male Wistar rats were used, divided into two groups of 10 rats each. A 1.0cm segment of the tibial nerve E was dried and sutured on the opposite side, where it was sutured into the sciatic nerve D. In Group I, the suture was made directly in the epineurium and in Group II, epineurotomy was performed. After 4 weeks, histological evaluation was carried out of the transposed segment and the sciatic nerve distal to the suture. Results: the results showed a small number of remyelinated fibers, varying from 7 to 51 fibers in Group I and from 10 to 91 fibers in Group II. The Mann-Whitney U test was used, with p=0.311, showing there is no statistically significant difference between the two groups. There was no positive relation between the number of remyelinated fibers in the graft and in the suture site distal to the sciatic lesion. Conclusion: lateral-ending neurorrhaphy, with or without epineural window, does not promote efficient remyelinization. Level of Evidence: Level II, prospective comparative study.

Implante de tubo de silicone com e sem colágeno na regeneração de nervos em eqüinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Plasticidade de células tronco mesenquimais de medula óssea de ratos na presença de meio condicionado do nervo facial e do fator de crescimento fibroblástico 2

A number of evidences show the influence of the growth of injured nerve fibers in Peripheral Nervous System (PNS) as well as potential implant stem cells (SCs) to make it more suitable for nerve regeneration medium. In this perspective, this study aimed to evaluate the plasticity of mesenchymal stem cells from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants (D-10) and fibroblast growth factor-2 (FGF-2). In this perspective, the cells were cultivated only with DMEM (group 1), only with D-10(group 2), only with FGF-2(group 3) or with D-10 and FGF-2(group 4). The growth and morphology were assessed over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200 on the fourth day of cultivation. Cells cultured with conditioned medium alone or combined with FGF-2 showed distinct morphological features similar apparent at certain times with neurons and glial cells and a significant proliferative activity in groups 2 and 4 throughout the days. Cells cultived only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200. On average, area and perimeter fo the group of cells positive for GFAP and the área of the cells immunostained for OX-42 were higher than those of the group 4. This study enabled the plasticity of mesenchymal cells (MCs) in neuronal and glial nineage and opened prospects for the search with cell therapy and transdifferentiation

ESTUDO DO EFEITO DE GANGLIOSIDEOS EXOGENOS NA REGENERACAO NERVOSA APOS COMPRESSAO E NEURORRAFIA NO NERVO CIATICO DO RATO

The authors studied the possible inductive effect of exogenous gangliosides administrated intra-peritoneal and intra-muscular, in 33 rats, after compression and sutures of sciatic nerve. In the animals suffering compression of the sciatic nerve only, the myelin sheath was preserved with good regeneration of the nerve. There was no statistics difference between the two groups with or without the administration of gangliosides. In the group of rats where section of the sciatic nerve and suture were performed, neuroma of amputation as well as dense cicatricial tissue with inflammatory reaction of foreign body type was noted around the sutures. In conclusion, the formation of neuroma of amputation associated with severe inflammatory reaction had an important role in determining the regeneration of the nerve.

Avaliação experimental dos efeitos do anestésico local ropivacaína em nervos ciáticos

This study was undertaken to investigate the effects of ropivacaine after intrafascicular injection into the sciatic nerves of albino rabbits. Twenty adult albine rabbits were used, following sedation with intramuscular ketamine (50 mg/kg) for nerve exposure by lateral incision. We considered three experimental groups: Group I:sciatic nerve control; Group II: intrafascicular injection with 0.2 mL of physiologic saline solution in the left nerves and intrafascicular injection with 0.2 mL of local anesthetic ropivacaine into the rigth nerves. The specimens were colected at 48 h after drugs administration; Group III. intrafascicular injection with 0.2 mL of physiologic saline solution in the left nerves and intrafascicular injection with 0.2 mL of local anesthetic ropivacaine in the rigth nerves. The specimens were colected at 7 days after drugs administration. The sciatic nerves were removed from these animals and fixed in Karnowisky solution for 24 hours. After partial dehydration up to 95% ethanol, they were embedded in historesin (Leica). The tissue was then sectioned at 1-2μm. Sections were stained with haematoxylin-eosin (HE); toluidine blue (TB) or picrosirius-haematoxylin (PSH). Comparing with control group the histological evidence of inflammatory reaction (migration of macrophagic cells and eosinophils-appeared soon after injection, with intense proliferation of perineurial cells. The results show that after 7 days of intrafascicular injection there was a severe fibrosis and an increase on perineurial vascularization. In group 2 the inflammatory reaction was noted near the local of the injection. Furthermore in this experiment we observed an increase on the number of epineurial lipoblasts and adipocytes. This study demonstrated that the toxic effects of ropivacaine are transient. In many cases there was an initial fascicular recover and axonal regeneration after 7 days of the injection.

Estudo comparativo experimental da injeção perineural do álcool benzílico 0,75% e do álcool etílico absoluto nos nervos palmares de eqüinos

The clinical and histopathological effects of two alcoholic neurolytics were studied in horses. Normal horses were shod with a designed shoe adaptted with 5 screws to produce solar pain. After gait and lameness score analysis, the palmar nerve of 5 horses was injected with 5 ml of 0,75% benzyl alcohol (Group A) and 5 horses were injected with 5 ml of absolute ethyl alcohol (Group B). The animals were submitted to regular lameness evaluation and solar sensibility tests during next six months. The solar sensitivity returned 5 months latter in the group injected with benzyl alcohol 0,75%, while in the group injected with absolute ethyl alcohol, the sole was still desensitized 6 months latter. The histopathological findings showed that the nerve injected with benzyl alcohol 0,75%, resulted in axonotmesis, characterized by axonal nerve degeneration, with possibilities for the nerve conduction recovery. The perineural injection of ethyl alcohol absolute, resulted in neurotmesis with difficult nerve regeneration. It was concluded that chemical neurolysis with alcohol is an option for temporary or permanent nerve blocks in horses.

Neurorrafia do ramo bucal dorsal do nervo facial em coelhos com proteção de segmento intestinal alógeno

18 rabbits, New Zealand, males, adults were used for clinical and histological evaluation of repair dorsal buccal branch of facial nerve after 15, 30 and 60 days postoperatively (PO). The animals were divided into two groups for transection and 10-0 nylon monofilament epineural suture of buccal branch. In animals in Group I, the nerve was coated with protection of jejunum allograft preserved in glycerin 98% and in group II was applied epineural suture. Both groups occurred the return of movement of the upper lip from the eighth week. There was infiltrated cellular and giant cells with fibrosis unsystematic and collagen fibers of the allograft jejunum joing to the connective tissue. At 15 and 30 days of PO, the distal nerve stumps of both groups were found with degeneration wallerian and in 60 days, regenerated fibers. The repair of the dorsal buccal branch of facial nerve with the allograft wasn't significantly different between the control rabbits as to the assessment of histological and functional recovery.

Fator de crescimento nervoso em cola de fibrina no reparo término-lateral de nervos em ratos

Purpose: To determine the effects of end-to-side nerve repair performed only with fibrin glue containing nerve growth in rats. Methods: Seventy two Wistar rats were divided into six equal groups: group A was not submitted to nerve section; group B was submitted to nerve fibular section only. The others groups had the nerve fibular sectioned and then repaired in the lateral surface of an intact tibial nerve, with different procedures: group C: ETS with sutures; group D: ETS with sutures and NGF; group E: ETS with FG only; group F: ETS with FG containing NGF. The motor function was accompanied and the tibial muscle mass, the number and diameter of muscular fibers and regenerated axons were measured. Results: All the analyzed variables did not show any differences among the four operated groups (p>0.05), which were statistically superior to group B (p<0.05), but inferior to group A (p>0.05). Conclusion: The end-to-side nerve repair presented the same recovery pattern, independent from the repair used, showing that the addition of nerve growth factor in fibrin glue was not enough for the results potentiating.