<p>Schwann cells (SCs) are PNS glia with numerous neuron-supporting functions, including myelination of axons. Although lesser discussed, SCs also fulfill many important roles after peripheral nerve injury (PNI) contributing significantly to the PNS regeneration process. Clusters of congregated SCs (Bands of Bungner) precede axon regeneration and facilitate the growth of extending axons to their distal targets which is particularly important in the lesion area of severed nerves. While this phenomenon occurs naturally, recovery from PNI can still be inadequate, especially in nerve transection or large gap injuries. Current treatments for nerve transection injuries are limited to coaptation of the nerve via sutures or nerve grafts. However, poor functional outcomes or donor site morbidity remain unaddressed problems. At the cellular level, axon pathfinding and extension relies heavily on the interaction between SCs and axonal growth cones. Depletion or removal of SCs at the lesion has been implicated to poor functional outcomes. With their pivotal role throughout nerve regeneration, we theorize axon regeneration can be improved by augmenting the SC population at the site of injury by encouraging migration to the lesion and via expression of morphological phenotypes that imitate the Bands of Bungner. </p> <p>DC electric fields (EFs) have been well studied in the past as a method to modulate cell orientation and migration and within the context of the nervous system, have been used to promote regeneration in lesioned spinal cords. However, very little work has investigated the effects of electrical stimulation on glia, such as SCs. Existing literature is lacking with regards to various aspects of SC responses, including direction of alignment. We hypothesize electrical stimulation can modulate SC behavior to reinforce/replicate behaviors observed within Bands of Bungner, which may be developed into a treatment for victims suffering peripheral nerve injury. </p> <p>We begin the current study with a thorough investigation into electric field modulated SC behavior. Using conventional 2D cell culture we demonstrate SC sensitivity to EFs by analyzing alignment, morphology and migration data. We employed EFs within the physiologic range. Waveforms used were constant DC as well as a 50% duty cycle DC and an oscillating DC. The latter two may prove more appropriate <i>in vivo</i> due to reduced accumulation of cytotoxic byproducts generated at the electrode interfaces. </p> <p>Our results highlight the sensitivity of SCs to DC electric fields of varying waveforms. SCs showed a strong propensity to align perpendicular to the field and display some cathodal migration in 2D cultures. Additional studies with variable cell density revealed cell-cell interaction further enhanced the alignment response. To more closely replicate the nerve microenvironment, a 3D cell culture model of PNI was created. Embedded in matrices, we found SCs displayed weaker migratory and alignment responses compared to 2D results. The direction of galvanotaxis was reversed, with SCs migrating toward the anode. Both alignment and migratory responses have potential applications for PNI. The galvanotactic behavior of SCs could be used to boost the SC population, increasing the number of Bands of Bungner. Cell alignment would be particularly advantageous at the lesion where axon regeneration is most difficult without the physical guidance of endoneurial tubes.</p> <p>This study characterizes SC behavior in applied EFs using conventional 2D and 3D cell culture techniques. We found SCs are sensitive to electric stimulation, supporting the idea that applied EFs could be used to indirectly promote regeneration in damaged peripheral nerve by modulating SC response after injury. Potential applications include generating an EF across damaged nerves to align SCs, especially in the lesioned area, using EFs to induce SC migration to the lesion to increase the number of cells guiding severed axons, and pre-aligning SCs in synthetic nerve grafts.</p>

Noch immer ist die Behandlung von Neuropathien mit den gängigen therapeutischen Mitteln für viele Patienten sehr unbefriedigend. Als erfolgsversprechender therapeutischer Ansatz werden zur Zeit Wege erforscht, welche direkt in die molekularen Entstehungsmechanismen pathologischer Veränderungen und regenerationsfördernder Mechanismen eingreifen, um dadurch eine Heilung von Nervenschäden zu ermöglichen. Bisher sind die Erkenntnisse über diese Mechanismen nicht vollständig genug, um daraus eine sichere Behandlungsmöglichkeit abzuleiten. Wegweisende Erkenntnisse deuten sich allerdings durch Studien von unterschiedlichen Vertretern des Zytokinnetzwerkes an - darunter auch TNF-alpha - welche als molekulare Ursache neuropathischer Veränderungen diskutiert werden. In dieser Studie wurde an Knockoutmäusen der Einfluss des jeweiligen TNF-alpha-Rezeptors auf morphologische Veränderungen nach CCI (Chronic constriction injury) und Crush-Verletzung des N. ischiadicus untersucht. Nach 3,7,15 und 36 Tagen (CCI) bzw. 3,7 und 28 Tagen (Crush) wurden in Methylenblau gefärbten Semidünnschnitten intakte und degenerierte Nervenfasern, Makrophagen, Angioproliferation, Ödembildung udn Veränderung des Anteils nicht neuronaler Zellen lichtmikroskopisch beurteilt. Zusätzlich wurden Mac-1+ Makrophagen immunzytochemisch erfasst. Die Ergebnisse zeigten in beiden Modellen und bei beiden Knockouttypen eine starke axonale Schädigung, die von einer großen endoneuroalen Makrophagenansammlung begleitet war. Bei TNF-R1-/- Mäusen war eine stärkere und verlängerte Degeneration mit entsprechend höheren Makrophagenzahlen sichtbar. In den Immunzytochemischen Färbungen wiesen die TNF-R1-/- Mäuse hingegen den geringsten Makropahgenanteil auf.Trotz der starken Schädigung war die anschließende Regeneration im Gegensatz zu WT und TNF-R2-/- Mäusen besser. Die Ödembildung war bei den TNF-R2-/- nach CCI besonders stark ausgeprägt und von einer schlechten Regeneration gefolgt. Während die gefundenen Daten auf eine Beteiligung beider Rezeptoren während degenerativer Prozesse hindeuten, scheint insbesondere TNF-R2 regenerationsfördernde Effekte zu vermitteln. / Current Treatment of neuropathic disorders is still dissatisfactory for many patients. A promising approach is the investigation of agents that directly interfere with molecular development of pathologic changes and regeneration. Up to now, consolidated findings of the underlying mechanisms are not yet sufficent to allow therapeutic intervention. Pathbreaking findings come from studies investigating different agents of the cytokine network - as e.g. TNF-alpha - that are discussed as molecular cause of neuropathic changes. This study investigated the influence of both TNF-alpha-receptors on morphologic changes after CCI (chronic constriction injury) and crush-injury of the sciatic nerve of TNF-R-knockoutmice. After 3,7,15 and 36 days (CCI), and 3,7 and 28 respectively (Crush),intact and degenerating nerve fibers, macrophages, angioproliferation, development of edema and changes in the amount of non-neuronal cells were acquired by light microscopy of toluidin-stained semithin sections. Additionally Mac-1+ macrophages were acquired via immuncytochemically stained sections. The results showed strong axonal damage in both knockout-types accompanied by large amounts of endoneurial macrophages. TNF-R1-/-mice showed a longer degeneration phase including respectively higher amounts of macrophages. In contrast the TNF-R1-/-mice revealed the fewest amount of macrophages in immunocytochemical sections. Despite the strong damage better nerve regeneration was observed compared to WT and TNF-R2-/-mice. Formation of edema was pronounced in TNF-R2-/- after CCI and followed by poorly regeneration. Whereas these findings point to a participation of both receptors in degeneration, TNF-R2 seems to support regeneration.

Made available in DSpace on 2016-01-26T12:51:39Z (GMT). No. of bitstreams: 1 nagibpezatiboer_tese.pdf: 2425172 bytes, checksum: 4c714f51e7b7b8d75dfe136b995e6b72 (MD5) Previous issue date: 2012-10-17 / There are many varieties of lesions of the peripheral nerves derived from acquired or congenital degenerate diseases which can affect the central nervous system and can cause loss of sensory and motor functions. The aim of this work was to make an experimental stereological study of the axons after end-to-side neurorrhaphies and after side-to-side neurorrhaphies to compare the axon, endoneural and myelin sheath volume densities (as well as the muscle mass) in the fibular and tibial nerves with contact anastomosis between 0.25cm and 0.50 cm. After approval by the Ethics Committee, twenty male Wistar rats were divided into four distinct groups of five rats each for the anastomosis between the tibial and fibular nerves (G1= end-to-side neurorrhaphy; G2= 0.25cm side-to-side neurorrhaphy; G3= 0.50cm side-to-side neurorrhaphy and G4= control group of normality and denervation). After 180 days, nervous fragments were fixed in 10% formaldehyde included in paraffin and stained with Masson's trichrome tecnique for the stereological study by the software Image Pro Plus ® version 6.0 with the adoption of Weibel stereological grid to set the axon, endoneural and myelin sheath volume densities. The Mann-Whitney (U Test) was applied for the statistical analysis. Also tibial cranial muscles were removed and then weighed in a precision scale. There was no statistically significant difference between the groups, but there were significant differences comparing the 3 groups (G1, G2 e G3) to the control group (G4). The axon, enduneral and myelin sheath volume densities of tibial and fibular nerves as well as the muscle mass during postoperative period keep egalitarian for both end-to-side and side-to-side neurorrhaphies of anastomosis area of contact in experimental stereological study comparing neurorrhaphies. Therefore, both end-to-side neurorrhaphies (which recover and prevent the motor atrophy) and side-to-side l neurorrhaphies which is independent on the gap of the nerve stumps are recommended as surgical treatment options. / Introdução: Há grande variedade de lesões que acometem nervos periféricos, derivadas de doenças adquiridas ou degenerativas congênitas que afetam a parte central do sistema nervoso e que ocasionam perda de funções sensoriomotoras. Objetivo: O objetivo deste trabalho foi perfazer estudo experimental estereológico axonal pós-neurorrafias término-lateral ou látero-lateral para comparar densidade de volume de axônios, de endouneuro e de bainha de mielina (bem como massa muscular) em nervos fibular e tibial, com anastomose de contato entre 0,25cm e 0,50 cm. Material e Método: Após aprovação do comitê de ética, foram utilizados 20 ratos machos da raça Wistar divididos em 04 grupos de 05 ratos cada, para realização de anastomose entre nervos fibular e tibial (G1=Neurorrafia término-lateral; G2=Neurorrafia látero-lateral de 0,25cm; G3= Neurorrafia látero-lateral de 0,50cm e G4= Controle de normalidade). Após 180 dias, fragmentos de nervos foram fixados em formaldeído a 10%, incluídos em parafina e corados com Tricômico de Masson para estudo estereológico pelo programa Image Pro Plus versão 6.0, com grade de Weibel para determinação dos números absolutos e da densidade de volume de axônios, endoneuro e bainha de mielina e aplicação do teste não paramétrico de Mann-Whitney. Também o músculo tibial cranial foi removido e pesado em balança de precisão. Resultados: Não houve diferença estatisticamente significante entre os grupos, mas sim de todos os 03 grupos de estudo (G1, G2 e G3) em relação ao grupo controle (G4). Conclusões: Em estudo experimental estereológico comparativo entre neurorrafias, a densidade de volume de axônios, endoneuro e bainha de mielina de nervos tibial e fibular do mesmo modo que a massa muscular pós-cirúrgica se mantém equitativa em neurorrafias término-lateral e látero-lateral, independentemente da área de anastomose de contato. Pode-se inferir, como opções de reparo cirúrgico, tanto a neurorrafia término-lateral que recupera e previne a atrofia de placa motora quanto a neurorrafia látero-lateral que independe da distância entre os cotos nervosos.

Este estudo analisou experimentalmente as propriedades de tração no local da neurorrafia em nervos ciáticos seccionados dos porcos. Trata-se de um estudo comparativo entre quatro técnicas de suturas. Foram utilizados 25 nervos ciáticos distribuídos em cinco grupos com cinco nervos cada, de acordo com o tipo de técnica de sutura. Grupo 1: grupo controle composto por nervos, sem lesão; Grupo 2: com nervos lesionados submetidos à sutura epineural; Grupo 3: com nervos lesionados submetidos à sutura epineural e reforço com duas suturas extra lesionais; Grupo 4: composto por nervos lesionados submetidos à sutura epineural e reforço com Ultrapro® (Tela Cirúrgica Sintética não Absorvível) e Grupo 5: com nervos lesionados submetidos à sutura epineural reforçada com duas veias com suturas extra lesionais. A força máxima (N), deformação de ruptura (mm) e energia na força máxima (N.mm) foram medidos em ensaios mecânicos de tração em máquina universal de teste. Os resultados mostraram que não houve diferença estatística entre os Grupos 3 e 4 e Grupos 2 e 5. A sutura do Grupo 3 apresentou maior resistência a tração, maior capacidade de absorver energia. Conclui-se que a técnica de sutura utilizada no Grupo 3 alcançou melhor desempenho. / This study aimed to analyze experimentally, in the laboratory, the tensile properties at the neurorrhaphy site on sectioned sciatic nerves of pigs. A comparative study of four suture techniques. Twenty-five sciatic nerves were used. They were randomly distributed into 5 groups with 5 nerves each, according to the type of suturing technique. Group 1, Control group, consisting of nerves, without injury. Group 2, with injured nerves submitted to epineural suture. Group 3, with injured nerves submitted to epineural suture and reinforcement using 2 extralesional sutures. Group 4, composed of injured nerves submitted to epineural suture and reinforcement with Ultrapro® (Synthetic Non-Absorbable Surgical Patch). And Group 5, with injured nerves submitted to reinforced epineural suture with two veins using extralesional sutures. Maximum force (N), rupture deformation (mm), and energy at maximum force (N.mm) were measured in mechanical trials using a universal test machine. The results showed that there was no statistical difference between Groups 3 and 4 and between Groups 2 and 5. The Group 3 suture presented greater tensile strength, greater capacity to absorb energy. It was concluded that the suture technique used in group 3 achieved better performance.

Made available in DSpace on 2017-07-10T14:17:16Z (GMT). No. of bitstreams: 1 Dissertacao_ Jhenifer Karvat.pdf: 2486983 bytes, checksum: 33eaca14028c159e971fc3ae3f9fc94f (MD5) Previous issue date: 2016-02-26 / This thesis consists of an introduction and general literature review, as well as two scientific articles. Injury to the peripheral nerves can occur by crushing, compression and transection, resulting in pain, reduction or loss of sensory and motor in the innervated area. Among the most common disorders is compression the sciatic nerve, responsible for the innervation of the back leg thigh and foot. Various forms of treatment can be used for this dysfunction, and within the physical therapy there is cryotherapy, however there is a lack in the literature regarding cryotherapy as a treatment for peripheral nerve injury. Thus, this study has the aim to evaluate treatment with cryotherapy in an experimental model of sciatic nerve compression in Wistar rats. 42 rats were randomly separated in groups: G1 - control; G2, G3 and G4 - groups that were submitted to sciatic nerve compression without receiving any treatment, being euthanized on the 3rd, 8th and 15th postoperative (PO), respectively; G5, G6 and G7 - groups that were submitted to sciatic nerve compression and treated with cryotherapy, euthanized at 3rd, 8th and 15th PO, respectively. The sciatic nerve compression was performed using a hemostat for 30 seconds. For treatment with cryotherapy in G5, G6 and G7, the animals were placed on the injured hind limb (right) immersed in a container with water and ice at 5 °C for 20 minutes, the first intervention immediately after the injury. G5 was only submitted to a treatment session, while G6 and G7 returned to the treatment of the 3rd to the 7th PO. Functional assessments were performed using the Sciatic Functional Index (SFI), functional disability test, nociception and edema, which occurred in stages: pre-injury, 2nd PO and the corresponding day euthanasia of each injured group, and G1 rated at the same time that the groups submitted to injury, however euthanized in 15th PO. After the intervention period, the animals were under anesthesia and the sciatic nerve collected for morphological analysis. It was used ANOVA mixed model for statistical analysis with 5% significance level. According to the results, there was a decrease of SFI after injury, independent of cryotherapy. The functional disability test showed an increase in paw rise time after injury. Nociception held at the injury site showed decreased the withdrawal threshold of the injured groups, however there was increase in the threshold in groups related to the assessments of the 8th and 15th PO. Nociception held in the plantar region was similar to the previous one, but the withdrawal threshold did not increase. In the evaluation of edema animals showed edema after the injury in the groups evaluated on 2nd PO corresponding the G1, G4 and G7, and on 3nd PO, which is not reduced. Regarding morphological analysis, G1 had nerve fibers with standard morphology, while the injured groups have nerve degeneration, and G6 showed a slight recovery of the nerve fibers, and present regeneration in the G4 and G7. We conclude that the cryotherapy protocol used was not effective to recover all the functional parameters analyzed, however, there was a slight improvement in the morphology of the group euthanized on the 8th PO. / Esta dissertação é composta por uma introdução geral, pela revisão de literatura geral e por dois artigos científicos. Lesões dos nervos periféricos podem ocorrer por esmagamento, compressão e transecção, o que resulta em dor e em redução, ou perda, da sensibilidade e da motricidade no território inervado. Entre as afecções mais comuns está a compressão do nervo isquiático, responsável pela inervação da parte posterior da coxa, da perna e do pé. Diversas formas de tratamento podem ser utilizadas para essa disfunção, sendo que, dentro da fisioterapia existe a crioterapia, embora haja uma carência na literatura a respeito dessa modalidade no tratamento da lesão nervosa periférica. Dessa maneira, o objetivo do estudo foi avaliar o tratamento com crioterapia em modelo experimental de compressão do nervo isquiático de ratos Wistar. Foram utilizados 42 ratos distribuídos aleatoriamente nos grupos: G1 controle; G2, G3 e G4 grupos que foram submetidos à compressão do nervo isquiático, mas não receberam tratamento, eutanasiados no 3º, 8º e 15º pós-operatório (PO), respectivamente; G5, G6 e G7 grupos que foram submetidos à compressão do nervo isquiático e tratados com crioterapia, eutanasiados no 3º, 8º e 15º PO, respectivamente. A compressão do nervo isquiático foi realizada com o auxílio de pinça hemostática, durante 30 segundos. Para o tratamento com crioterapia em G5, G6 e G7, os animais foram posicionados com o membro pélvico lesionado (direito) imerso em recipiente com água e gelo a 5ºC por 20 minutos, sendo a primeira intervenção imediatamente após a lesão. G5 foi submetido apenas a uma sessão de tratamento, enquanto G6 e G7 retornaram ao tratamento do 3º ao 7º PO. As avaliações funcionais foram realizadas por meio do índice funcional do isquiático (IFC), teste de incapacidade funcional, nocicepção e edema, que aconteceram nos seguintes momentos: pré-lesão, 2º PO e no dia correspondente a eutanásia de cada grupo lesionado, sendo G1 avaliado nos mesmos momentos que os grupos submetidos à lesão, porém, eutanasiado no 15º PO. Após o período de intervenção, o nervo isquiático foi coletado e processado para a análise morfológica. Os dados obtidos foram analisados por meio de ANOVA mista, com nível de significância de 5%. De acordo com os resultados, houve diminuição do IFC após a lesão, independente da crioterapia. O teste de incapacidade funcional mostrou aumento do tempo de elevação da pata após a lesão. A nocicepção realizada no local da lesão mostrou diminuição do limiar de retirada dos grupos lesionados, entretanto, houve aumento do limiar nos grupos relacionados às avaliações do 8º e 15º PO. Já a nocicepção realizada na região plantar foi semelhante a anterior, porém, o limiar não aumentou. Além disso, os animais apresentaram edema no 2º PO correspondente a G1, G4 e G7 e no 3º PO, o qual não reduziu. Em relação à análise morfológica, G1 apresentou fibras nervosas com aspecto morfológico normal, enquanto que nos grupos lesionados houve degeneração nervosa. Houve uma discreta recuperação das fibras nervosas em G6, bem como regeneração em G4 e em G7. Conclui-se que o protocolo de crioterapia utilizado não foi eficaz na recuperação de todos os parâmetros funcionais analisados, entretanto, houv

A lesão nervosa periférica acarreta atrofia do músculo esquelético por ele inervado, levando a perda de função. E estimulação elétrica tem sido usada na prática clínica como recurso terapêutico complementar, embora existam controvérsias na literatura a respeito da sua eficácia na manutenção do trofismo muscular e dos seus efeitos no tecido conjuntivo associado ao tecido muscular. Assim sendo, este trabalho avaliou os efeitos da estimulação elétrica do músculo tibial anterior em um modelo experimental de axonotmese leve ou severa do nervo ciático de ratos. Após a lesão, um grupo de animais foi tratado com estimulação elétrica (3 vezes por semana) com parâmetros obtidos por eletrodiagnóstico, enquanto que outro grupo de animais lesados constituíram o grupo controle. Foram analisados parâmetros biomecânicos (tensão, rigidez e deformação relativa), parâmetros do eletrodiagnóstico e parâmetros histológicos (volume absoluto do músculo, volume absoluto de tecido muscular propriamente dito, volume absoluto do colágeno fibrilar e volume médio do miócito). Os resultados mostram que a estimulação elétrica promove um aumento da tensão e da rigidez nos dois tempos experimentais; em relação ao eletrodiagnóstico, os animais que receberam estimulação elétrica apresentaram valores de reobase, cronaxia, acomodação e coeficiente de acomodação significativamente diferentes dos animais não tratados, com valores que tendem à normalidade. Os parâmetros histológicos mostram que: 1) há uma diminuição do volume absoluto muscular e do tecido muscular nos animais lesados tanto nos animais controles como nos tratados, em relação aos não-lesados; 2) há um aumento progressivo do colágeno fibrilar ao longo do tempo tanto nos tratados como nos controles; nos animais tratados a quantidade de fibras de colágeno aos 8 dias é maior quando comparada aos controles, porém aos 30 dias a quantidade de colágeno tecidual é menor nos animais tratados se aproximando mais aos valores encontrados no grupo não-lesado; 3) para lesões severas a estimulação elétrica é capaz de prevenir parcialmente a atrofia da célula, uma vez nestes animais os valores de diâmetro do miócito foi maior nos animais tratados que nos controles. Assim sendo, os dados obtidos sugerem que a estimulação elétrica tem um efeito modulador na deposição do colágeno fibrilar e no trofismo muscular, principalmente nas lesões severas. Os resultados dos testes biomecânicos refletem os achados estruturais. Além disso, os resultados obtidos sugerem que a estimulação elétrica pode atuar positivamente na regeneração neural, pois os parâmetros do eletrodiagnóstico são próximos aqueles de músculos com inervação íntegra. / Peripheral nerve injury results in atrophy of skeletal muscles innervated by it, leading to loss of function. Electrical stimulation has been used in clinical practice as a therapeutic supplement, although there is controversy in the literature regarding its effectiveness in maintaining muscle mass and its effects on the connective tissue associated with the muscle. Therefore, this study evaluated the effects of electrical stimulation of the tibialis anterior muscle in an experimental model of mild or severe axonotmesis of the sciatic nerve of rats for 8 or 30 days. After injury, a group of animals was treated with electrical stimulation (3 times per week) and parameters being obtained by electrodiagnostic, while another group of injured animals was used as controls. We analyzed the biomechanical parameters (tension, stiffness and relative deformation), electrodiagnostic parameters and histological parameters (absolute volume of muscle, the absolute volume of muscle tissue proper, the absolute volume of the fibrillar collagen and average volume of myocytes). The results show that electrical stimulation promotes an increase in tension and stiffness in both experimental times. In relation to electrodiagnostic, animals receiving electrical stimulation showed values of rheobase, chronaxie, accommodation and accommodation coefficient significantly different from untreated animals, with values that tend to normality. The histological parameters show that: 1) there is a decrease in the absolute volume of muscle tissue and muscle proper in the injured animals both in controls and in treated animals, compared to non-injured, 2) there is a progressive increase in fibrillar collagen over time in both treated and controls, in treated animals collagen deposition at 8 days is higher compared to controls, but at 30 days the amount of collagen tissue is lower and more similar to the values found in the non-injured; 3) for severe injuries electrical stimulation is able to partially prevent the atrophy of cells, since higher values of diameter of myocytes was found in treated animals in comparison to controls. Therefore, the data suggest that electrical stimulation has a modulating effect on deposition of fibrillar collagen which can regulate the muscle mass, especially in severe injuries, as evidenced by the results of the biomechanical tests. Moreover, the results suggest that electrical stimulation can play a positive role in neural regeneration, because the electrodiagnostic parameters are similar to those of muscles with intact innervation

Este estudo analisou experimentalmente as propriedades de tração no local da neurorrafia em nervos ciáticos seccionados dos porcos. Trata-se de um estudo comparativo entre quatro técnicas de suturas. Foram utilizados 25 nervos ciáticos distribuídos em cinco grupos com cinco nervos cada, de acordo com o tipo de técnica de sutura. Grupo 1: grupo controle composto por nervos, sem lesão; Grupo 2: com nervos lesionados submetidos à sutura epineural; Grupo 3: com nervos lesionados submetidos à sutura epineural e reforço com duas suturas extra lesionais; Grupo 4: composto por nervos lesionados submetidos à sutura epineural e reforço com Ultrapro® (Tela Cirúrgica Sintética não Absorvível) e Grupo 5: com nervos lesionados submetidos à sutura epineural reforçada com duas veias com suturas extra lesionais. A força máxima (N), deformação de ruptura (mm) e energia na força máxima (N.mm) foram medidos em ensaios mecânicos de tração em máquina universal de teste. Os resultados mostraram que não houve diferença estatística entre os Grupos 3 e 4 e Grupos 2 e 5. A sutura do Grupo 3 apresentou maior resistência a tração, maior capacidade de absorver energia. Conclui-se que a técnica de sutura utilizada no Grupo 3 alcançou melhor desempenho. / This study aimed to analyze experimentally, in the laboratory, the tensile properties at the neurorrhaphy site on sectioned sciatic nerves of pigs. A comparative study of four suture techniques. Twenty-five sciatic nerves were used. They were randomly distributed into 5 groups with 5 nerves each, according to the type of suturing technique. Group 1, Control group, consisting of nerves, without injury. Group 2, with injured nerves submitted to epineural suture. Group 3, with injured nerves submitted to epineural suture and reinforcement using 2 extralesional sutures. Group 4, composed of injured nerves submitted to epineural suture and reinforcement with Ultrapro® (Synthetic Non-Absorbable Surgical Patch). And Group 5, with injured nerves submitted to reinforced epineural suture with two veins using extralesional sutures. Maximum force (N), rupture deformation (mm), and energy at maximum force (N.mm) were measured in mechanical trials using a universal test machine. The results showed that there was no statistical difference between Groups 3 and 4 and between Groups 2 and 5. The Group 3 suture presented greater tensile strength, greater capacity to absorb energy. It was concluded that the suture technique used in group 3 achieved better performance.

Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.