京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21623号 / 医博第4429号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Despite recent advances in surgical procedures and immunosuppressive regimes, early pancreatic graft dysfunction, mainly specified as ischemia–reperfusion injury (IRI)— Remains a common cause of pancreas graft failure with potentially worse outcomes in simultaneous pancreas-kidney transplantation (SPKT). Anesthetic conditioning is a widely described strategy to attenuate IRI and facilitate graft protection. Here, we investigate the effects of different volatile anesthetics (VAs) on early IRI-associated posttransplant clinical outcomes as well as graft function and outcome in SPKT recipients. Methods: Medical data of 105 patients undergoing SPKT between 1998–2018 were retrospectively analyzed and stratified according to the used VAs. The primary study endpoint was the association and effect of VAs on pancreas allograft failure following SPKT; secondary endpoint analyses included “IRI- associated posttransplant clinical outcome” as well as long-term graft function and outcome. Additionally, peak serum levels of C-reactive protein (CRP) and lipase during the first 72 h after SPKT were determined and used as further markers for “pancreatic IRI” and graft injury. Typical clinicopathological characteristics and postoperative outcomes such as early graft outcome and long-term function were analyzed. Results: Of the 105 included patients in this study three VAs were used: isoflurane (n = 58 patients; 55%), sevoflurane (n = 22 patients; 21%), and desflurane (n = 25 patients, 24%). Donor and recipient characteristics were comparable between both groups. Early graft loss within 3 months (24% versus 5% versus 8%, p = 0.04) as well as IRI-associated postoperative clinical complications (pancreatitis: 21% versus 5% versus 5%, p = 0.04; vascular thrombosis: 13% versus 0% versus 5%; p = 0.09) occurred more frequently in the Isoflurane group compared with the sevoflurane and desflurane groups. Anesthesia with sevoflurane resulted in the lowest serum peak levels of lipase and CRP during the first 3 days after transplantation, followed by desflurane and isoflurane (p = 0.039 and p = 0.001, respectively). There was no difference with regard to 10-year pancreas graft survival as well as endocrine/metabolic function among all three VA groups. Multivariate analysis revealed the choice of VAs as an independent prognostic factor for graft failure three months after SPKT (HR 0.38, 95%CI: 0.17–0.84; p = 0.029). Conclusions: In our study, sevoflurane and desflurane were associated with significantly increased early graft survival as well as decreased IRI-associated post-transplant clinical outcomes when compared with the isoflurane group and should be the focus of future clinical studies evaluating the positive effects of different VA agents in patients receiving SPKT.

Abdominal aortic aneurysm (AAA) is a prevalent disease in developed countries. While endovascular aneurysm repair is fairly successful, it has shortcomings. Polymeric endoluminal paving and sealing is a method that has previously been developed to treat a range of diseases. Our goal is to further develop this technique to treat AAA, a process we have named polymeric endo-aortic paving (PEAP). We hypothesize that PEAP will overcome many of the limitations associated with EVAR by providing a minimally invasive treatment which can be used on patients with complicated AAA geometries and reducing incidence of migration and endoleak. Additionally, we plan to incorporate drug delivery into PEAP to improve efficacy. The purpose of this work was to evaluate a potential graft material for PEAP and to develop a thrombus mimic which will aid in further PEAP development. Blends of polycaprolactone/polyurethane (PCL/PU) were assessed by characterizing their mechanical, thermoforming, and degradation properties. PCL/PU grafts have a similar stiffness to aortic tissue and can be thermoformed at temperatures approaching 37 degrees C. Blending PCL with PU significantly reduces PCL's degradation. An anisotropic hyperelastic strain energy function was developed for the PCL/PU blends and finite element modeling (FEM) was used to show that stress reduction on the AAA wall that can be achieved by PEAP is similar to current EVAR. Stiffness varies throughout the AAA thrombus, and thrombus mimics were developed that have similar stiffness, components, and structure to native AAA thrombus.

Varje år genomförs omkring 700 organtransplantationer i Sverige. Patienter som genomgått en organtransplantation har en ständig risk för avstötning. Avstötning innebär att det transplanterade organet stöts bort och slutar fungera på grund av kroppens immunförsvar. Oavsett var transplantationen utförs så följs patienterna upp på sin hemort. Därför är det av stor vikt att som sjuksköterska inneha kunskaper om patienters upplevelser om avstötning. Syftet med litteraturstudien var därför att beskriva patienters upplevelser om avstötning efter en organtransplantation. För att uppnå syftet med studien utfördes en litteraturstudie, där fyra kategorier framstod i resultatet: Det ständiga hotet, En oönskad livssituation, Fångad i behandlingsregimen och Att finna styrka i vardagen. Avstötning är en upplevelse av ett ständigt hot som även kan medföra att patienter upplever ett emotionellt trauma. Patienterna känner sig tvungna att följa den utvalda behandlingsregimen för att undvika en avstötning. Efter en upplevd avstötning behöver patienterna använda sig av olika strategier för att kunna hantera vardagen. Ytterligare forskning om ämnet behövs för att kunna möta denna patientgrupps vårdbehov. / Each year about 700 organ transplants are performed in Sweden. Patients who have had an organ transplant are at constant risk of graft rejection. Graft rejection means that the transplanted organ is rejected and stops functioning because of the body’s immune system. Regardless of where the transplant is performed the patients are followed up at their domicile. Therefore it is of great importance that the nurse has knowledge about patients’ experiences in conjunction with graft rejection. The purpose of this study was to describe patients' experiences of graft rejection after organ transplantation. To achieve the purpose of this study a literature review was conducted, in which four categories emerged in the results: The constant threat, An undesirable situation in life, Caught in the treatment regimen, and To find strength in everyday life. Rejection is an experience of a constant threat that may cause patients to experience an emotional trauma. Patients feel obliged to follow the chosen treatment regimen to prevent rejection. After an experienced rejection, patients need to use different strategies to cope with everyday life. Further research on the topic is needed to meet the care needs of this patient group.

Electrospinning is a polymer processing technique which allows for the production of nano to micro size fibers and scaffolds which can be composed of numerous synthetic biodegradable materials and natural biopolymers. Natively, elastin and collagen are the main components of vascular tissue. Arranged in a tri-layered structure, they create a specific mechanical environment that can withstand the rigors of circulation. The goal of this study was to develop a mechanically ‘biomimicking’ vascular graft composed of three distinct layers through the process of electrospinning. We hypothesize that the use of bioactive agents such as elastin, collagen, and silk to supplement poly(caprolactone) at specified ratios for each layer would provide a finely tuned vascular replacement. This was accomplished by establishing cross-linking parameters for the biopolymer materials and then assessing the mechanical properties of individual materials and eventually a whole tri-layered graft. Additionally, while mechanical testing can lead to a good graft, a replacement graft requires excellent cellular properties as well to promote cell infiltration, proliferation, and migration. Therefore, the conclusion of this study examines the integrin binding characteristics of the electrospun biopolymers. First, the results from the preliminary cross-linking study examined the dissipation of soluble elastin when uncross-linked v. cross-linked. It was determined through this initial study that synthetic scaffolds blended with soluble proteins such as elastin require a fixation in order to retain their protein mass within the scaffold. Retaining this mass, incrementally changed the material properties of the blended scaffolds. This initial study was then carried further to establish optimal cross-linking parameters using two different types of reagents: carbodiimide and genipin. It was found that lower cross-linking molarities produced excellent results based on assays performed to assess cross-linking percentages and rate of reaction. Some differences in mechanical properties were seen, but they did not constitute a choice of one cross-linker over the other. The next portion of this study aimed to design a tri-layered graft. This was performed with the aid of mathematical analysis to observe circumferential wall stresses based on simple tensile properties. A series of tri-layered grafts were electrospun using poly(caprolactone), elastin, and collagen. The medial layers of these grafts were changed while the intima and adventitia remained constant. Differences were demonstrated as the elastin content of the medial layer decreased, proving that each layer had an affect on the overall graft properties and that it was possible to tune graft mechanics. A larger tri-layered study looked to evaluate changes in the adventitial and medial layers while keeping the intimal layer constant using poly(caprolactone), elastin, collagen, and silk fibroin. In this study, differences were exhibited under compliance and burst strength testing, narrowing the scope of material choices. Results from a 4 week degradation study with the best tri-layered grafts revealed no evidence of degradation, but did generate some positive compliance results for two of the grafts. Finally, integrin binding and protein analysis portrayed results that were indicative of the existence of ligand binding sites for collagen scaffolds and the possibility of a small amount of ligand sites on silk. Elastin, however, displayed low to non-existent adhesion. These studies produced results that allowed us to continuously narrow the scope of materials as the experiment progressed towards an optimized tri-layered vascular graft.

A técnica de levantamento de seio maxilar apresenta como uma possível complicação a perfuração da membrana sinusal. A utilização de dispositivos ultrassônicos apresenta-se como uma interessante ferramenta, particularmente segura e eficiente para realização das ostectomias de parede sinusal e divulsão de membrana sinusal. O presente trabalho teve por objetivo comparar duas técnicas que utilizam a cirurgia ultrassônica para realização de enxerto autógeno para levantamento de seio maxilar. Dez coelhos foram utilizados no estudo, sendo que um deles não foi submetido a procedimento cirúrgico. Os nove coelhos operados tiveram os seios maxilares preenchidos com enxertos autógenos coletados de díploe externa de calota craniana, nas formas particulado do lado direito e raspado do lado esquerdo, ambos com aparelho ultrassônico. Os dados de densidade óssea nos seios maxilares esquerdo e direito, obtidos por meio de tomografia computadorizada nos sentidos transversal e longitudinal, registrados 90 dias após a realização dos enxertos foram comparados estatisticamente. Cortes histológicos das áreas enxertadas foram realizados com finalidade descritiva. Não houve diferenças estatisticamente significantes entre as técnicas de enxerto que utilizaram osso raspado e particulado coletado por meio de dispositivo ultrassônico da calota craniana de coelhos. O aspecto histológico observado após 90 dias foi semelhante nos dois grupos, mostrando tecido ósseo maduro em meio a tecido conjuntivo frouxo. Os procedimentos transoperatórios nos levaram a concluir que o ultrassom piezoelétrico mostrou-se um instrumento seguro na abordagem cirúrgica do seio maxilar de coelhos, permitindo a manutenção da integridade da membrana sinusal durante as manobras cirúrgicas. / Maxillary sinus lifting is a technique which presents as a possible complication the sinus membrane perforation. The use of ultrasonic devices is presented as an interesting tool, particularly safe and efficient to perform ostectomies of sinus wall and dissection of sinus membrane. This study aimed to compare two techniques that used ultrasound surgery to perform autogenous graft for maxillary sinus lifting. Ten rabbits were used in the study, one of which did not undergo surgery. The other nine rabbits had their maxillary sinuses filled with autogenous bone grafts collected from the external skull diploe in particulate form on the right side and shaved on the left side, both with ultrasonic device. Data on bone density in left and right maxillary sinus, obtained by computed tomography in transverse and longitudinal sections, recorded 90 days after the grafts, were compared statistically. Histological sections of the grafted areas were performed with descriptive purposes. There were no statistically significant differences between the two techniques that used shaved and particulate bone collected by means of ultrasonic device of the skull of rabbits. Histological aspects after 90 days were similar in both groups, showing mature bone through a fibrous connective tissue. Operatory assessment procedures leads us to conclude that the piezoelectric ultrasound showed as a safe tool in the surgical approach of the maxillary sinus of rabbits, allowing the maintenance of sinus membrane integrity during surgical procedures.

O objetivo desta pesquisa em humanos é avaliar clínica e histologicamente os resultados obtidos de implantes instalados imediatamente à exodontia (Grupo 1) e de implantes tardios instalados em alvéolos regenerados (Grupo 2). Nos dois grupos, matriz óssea liofilizada xenógena foi utilizada associada à membrana de colágeno bovino reabsorvível e/ou membrana não reabsorvível de politetrafluoretileno expandido (PTFEe), seguindo-se os princípios da Regeneração Tecidual Guiada. Vinte pacientes, dez de cada grupo, foram acompanhados clinicamente durante 10 anos, submetidos a análises clínicas em intervalo de 1, 3, 5 e 10 anos e à análise histológica do osso alveolar neoformado após 6 meses da enxertia. Os pacientes foram avaliados clinicamente quanto à presença de sinais ou sintomas de doença periodontal através de sondagem. A avaliação histológica das lâminas previamente confeccionadas foi realizada através de classificação qualitativa de formação óssea e de possíveis remanescentes de biomaterial. Os aspectos clínicos (profundidade de sondagem e índice de sangramento marginal) nos Grupos 1 e 2 foram semelhantes e, através de uma análise subjetiva, pôde-se verificar que os pacientes dos Grupos 1e 2 realizaram uma boa manutenção da higiene bucal promovendo, dessa maneira, um índice insignificante de presença de sinais e sintomas de doença periodontal. Quanto às análises histológicas, os Grupos 1 e 2 se equiparam. Por essa razão, a apresentação da análise histológica da região com neoformação óssea, depois do período determinado, revelou o grau de maturação do osso remodelado na superfície perimplantar onde foi colocado o enxerto xenógeno, condizente com o quadro esperado pela osseointegração. Os enxertos xenogênicos associados à membrana possibilitam a manutenção do rebordo para a colocação de implantes e reabilitação protética, sejam no mesmo momento da extração e colocação imediata dos implantes ou em casos de preenchimento de alvéolos após exodontia e instalação tardia de implantes. / The aim of this research is to evaluate the clinical and histological findings of installed implants immediately after exodontics (Group 1) and later after the alveolus regeneration (Group 2). In both groups, xenogeneous lyophilized bone matrix was used in association with resorbable bovine collagen membrane and/or nonresorbable expanded politetrafluoretane membrane (PTFE - e) following the guided tissue regeneration principles. 20 patients, 10 in each group, were clinically followed during 10 years, submitted to clinical analysis during intervals of 1, 3, 5 and 10 years, and to histological analysis of the neoformed alveolar bone after 6 months of grafting. The patients were clinically evaluated as for signs and symptoms of periodontal disease by means of probing depth. The histological evaluation of the previously prepared laminae were performed after qualitative classification of bone formation and of possible biomaterial remnants. The clinical aspects (probing of depth and marginal bleeding index) in groups 1 and 2 were similar. After a subjective analysis, it could be verified that the patients in both groups performed an acceptable maintenance of oral hygiene, promoting, in such way, an insignificant index of periodontal disease signs and symptoms. The histological analysis of both groups were equivalent. For this reason, the histological analysis of the neoformed bone region after the determined period revealed the degree of maturation of the remodelated bone at the periimplant surface where the xenogeneous graft was placed, consistent with the expected osseointegration scenario. The xenogenic grafts associated with membranes perform the maintenance of the alveolar ridge for implant installation and prosthetic rehabilitation, either immediately after extraction or later after extraction and alveolus filling.

Com o avanço da tecnologia e a conseqüente produção de equipamentos mais sofisticados, a microcirurgia vem ganhando cada vez mais espaço no campo da investigação experimental referente a recuperação de nervos periféricos. É sabido que no caso de uma simples secção do nervo, sem perda tecidual, a neurorrafia término-terminal é a técnica mais aconselhável. Porém, em uma situação onde ocorre perda de tecido nervoso ou, quando não se têm mais o coto distal do nervo, outras técnicas devem ser empregadas, mesmo porque, não se pode de forma alguma tracionar o nervo numa tentativa de coaptá-lo novamente. Assim várias técnicas de tubulização utilizando-se de materiais biológicos (vasos, nervos, músculos, pericárdio, veia e músculo combinado, moléculas de adesão, etc) ou não biológicos (tubos de polietileno, silicone, etc) estão sendo testados com resultados ainda insatisfatórios, principalmente sob o ponto de vista funcional. É sabido que em um trauma sem perda tecidual, numa neuropraxia, por exemplo, o nervo recupera espontaneamente de forma satisfatória. É sabido também que em um feixe vásculo-nervoso, o nervo periférico encontra-se em íntimo contato com a adventícia de artérias e veias. A adventícia dos vasos é constituída por tecido conjuntivo frouxo, rico em adipócitos. Assim, em um trauma, os neuritos oriundos do coto proximal do nervo lesado, ficam diretamente em contato com esses adipócitos. Seguindo este raciocínio, e com base em trabalhos anteriores onde foi usada veia preenchida com músculo esquelético a fresco como enxerto, decidimos testar a possibilidade de crescimento axonal por meio de enxerto venoso preenchido por tecido adiposo autólogo. Para tanto foi utilizada a veia jugular externa do rato preenchido com tecido adiposo in natura retirado das adjacências da referida veia, na tentativa de se recuperar o nervo ciático e um de seus ramos, o nervo fibular comum. A certificação do sucesso da recuperação do nervo foi feita por meio da análise dos nervos (Ciático e Fibular Comum), e dos músculos Extensor Longo dos Dedos (EDL) e Tibial Cranial (TC), sempre comparando com os respectivos grupos controles. Técnicas de microcirurgia, microscopia, morfometria, e análise fisiológica (foot print), além da utilização do Laser de Baixa Potência (LBP), foram empregadas nesta investigação. / With technology advance and the resultant production of more sophisticated equipment, microsurgery has been obtaining more attention in the experimental investigation field referred to peripheral nerves repair. It is know that in case of simple nerve section, without tissue loss, the end-to-side-neurorraphy is the most recommended technique. However, in each situation where nerve tissue loss occurs or, when there is no nerve distal stump anymore, other techniques should be used, precisely because the nerve cannot be put on traction to try nerve coaptation. Thus, many tubulization techniques which use biological materials (veins, nerves, muscles, pericardium, veins and muscles combined, adherence molecule) or non-biological materials (polyethylene tubes, silicon, among others) are being tested and the results has been unsatisfactory yet, especially concerning functional point of view. It is known that in trauma without tissue loss, like neuropraxy, for example, the nerve recovers spontaneously in a satisfactory way. It is also known that in a vascular-nervous bundle, the peripheral nerve is very closed to arteries and veins adventitious layer. The vein adventitious is built by weak conjunctive tissue, which is rich in fat cells. Thus, in a trauma, the neurites, derived from proximal stump of injured nerve, have direct contact with the fat cells. Following this argumentation and based on previous works that used vein fulfilled with fresh skeleton muscle as graft, we have decided to test the possibility of axon growth through vein graft fulfilled with autologous fat tissue. To achieve this, it was used rats external jugular vein fulfilled with in natura fat tissue removed from the adjacencies of the referred vein, in a trial to recover the sciatic nerve and one of its branches, the common peroneal nerve. The certification of successful nerve recovering was done through the analysis of nerves (Sciatic and Common Peroneal Nerve) and muscles (Extensor Digitorium Longus - EDL and Tibial Cranial Muscle TC), always comparing them with the respective control groups. Techniques of microsurgery, microscopy, morphometry and physiological analysis (foot print) were employed, besides the use of Low-Power Laser (LPL).

O transplante de órgãos é hoje uma opção de tratamento de várias doenças terminais. Apesar de todos os progressos no campo do transplante, o principal problema enfrentado ainda é a rejeição. As principais moléculas responsáveis pela resposta alogeneica e subsequente rejeição ao enxerto, são os antígenos leucocitários humanos (HLA, do inglês Human Leucocyte Antigens). Porém, existem evidências que anticorpos dirigidos a antígenos não-HLA estão associados com rejeição de transplantes. Neste estudo, foi investigada a presença de anticorpos anti-célula endotelial (AACE) em 11 pacientes que perderam seus rins transplantados devido à rejeição humoral irreversível e em 2 com perda por trombose de veia renal. A ausência de anticorpos anti-HLA contra o doador foi verificada antes do transplante, da rejeição e antes e depois da transplantectomia, através da realização de provas cruzadas usando as técnicas mais sensíveis. Anticorpos não-HLA presentes em nove eluatos reagiram com EAHy.926. Eluatos positivos e negativos contra linhagem EAHy.926 foram testados contra cortes histológicos de 6 rins sadios para detecção de quais estruturas renais são reconhecidas por esses anticorpos. A reação foi avaliada pelo método de imunofluorescência indireta. Dos 13 eluatos testados, 4 (isotipo IgG) e 5 (isotipo IgM) reagiram com forte fluorescência nos glomérulos e endotélio arterial, mas não foi verificada reação na cápsula de Bowman e no epitélio tubular. Não foi observado polimorfismo na reatividade dos eluatos. Em onclusão, verificamos que os anticorpos não-HLA têm um importante papel na rejeição humoral. Estes estão reconhecendo antígenos de um sistema provavelmente não-polimórfico nas células de endoteliais presentes, principalmente, nos capilares glomerulares. / The transplant of organs is today an option of treatment to several terminal diseases. In spite of all the progress in the field of the transplants, the rejection remains a problem to be solved. The main target molecules for the allogenic response and subsequent allograft rejection are the human leukocyte antigens (HLA). However, there are growing evidences that non-HLA antibodies are associated with transplant rejection. In this study it was investigated the presence of anti-endothelial cell antibodies (AECA) in 11 patients who had early lost their transplanted kidney by irreversible humoral rejection and in 2 ones from renal venal thrombosis. The absence of anti-HLA antibodies against the donor was verified by the negativity of crossmatches performed using the most sensitive assays, at the transplant, at the rejection, and before and after the transplantectomy Antibodies from 9 eluates bound to EAHy.926. Positive and negatives eluates were tested against frozen sections from 6 normal kidneys in order to define the structures to which they were reactive. The reactivity was identified by indirect immunofluorescence method. From 13 eluates evaluated, 4 (isotipe IgG) and 5 (isotipe IgM) reacted to the glomerulus and renal arterial endothelium with intense fluorescence but they did not react to the Bowmans capsule and tubular epithelium. No polymorphism was observed in eluates reactivity. In conclusion, we have shown that non-HLA antibodies may represent a cause of the humoral rejection. These antibodies are probably recognizing antigens of a nonpolymorphic system in endothelial cells present, mainly, in the glomerular capillaries.

Análise da expressão de neurotrofinas durante a regeneração de nervo periférico de rato por enxerto venoso Enxertos de veias têm sido empregados para preencher lacunas em nervos periféricos transeccionados para melhor recuperação funcional. No entanto, vários inconvenientes, como a constrição do enxerto secundário foram observados. Uma nova alternativa para esta técnica foi desenvolvida. Simplesmente invertendo a veia de dentro para fora, chamado do Inside- out vein graft. As neurotrofinas são uma família de fatores neurotróficos conhecidos por desempenhar um papel significativo na regeneração de nervos periféricos. A família da neurotrofina é constituído por fator de crescimento nervoso (NGF), fator neurotrófico derivado do cérebro (BDNF), Neurotrofina-3 (NT-3) e Neurotrofina-4 (NT-4). No campo da neurobiologia, vários autores têm utilizado a técnica de PCR a fim de obter mais informações sobre os nervos regenerados. Neste estudo, foi utilizada a técnica de biologia molecular para explorar o papel e o nível das neurotrofinas durante a regeneração de nervos periféricos com enxerto de veia. O nervo isquiático de ratos foi seccionado e reparado com enxerto de veia invertida (IOVG) e técnicas de enxerto de veia padrão (SVG). No grupo controle, os ratos foram operados e o nervo isquiático foi mantido intacto. Os animais foram sacrificados após 6 e 12 semanas e os enxertos foram colhidos para observar o nível das neurotrofinas. Músculos EDL e Sóleo foram excisados e pesados para determinar a diferença de peso entre os grupos. Um pequeno segmento dos cotos distais de ambos os grupos SVG e IOVG também foram excisados e foram processados histologicamente para examinar a quantidade de axónios regenerados. Além disso, um outro pequeno segmento do coto distal foi processado para RT-PCR para analisar o nível das neurotrofinas nesta área.A tecnica do walk track analysis foi realizada para determinar o índice funcional do nervo isquiático nos grupos. Em 6 semanas, não ocorreu crescimento neuronal significativo no coto distal dos dois tipos de enxertos, porém um crescimento foi observado em 12 semanas. Não houve diferença significativa na massa muscular entre IOVG e SVG em ambos os períodos de tempo. No entanto, um aumento significativo na massa muscular foi observado a partir de 6 a 12 semanas nos grupos IOVG e SVG. Um aumento significativo na produção de NT-3 foi observado no grupo de SVG em ambos, enxerto e o coto distal quando comparados a partir de 6 a 12 semanas, no entanto, não houve aumento observado no nível de neurotrofinas dos outros tipos (NGF e NT-4) . Surpreendentemente, não houve aumento significativo da NT-3 no grupo IOVG. Conclui-se que, entre as neurotrofinas avaliadas neste estudo, não há nenhuma diferença significativa no seu nível de RNAm entre os dois grupos, exceto NT-3. Finalmente, uma vez que o nível de RNAm de NT-3 aumenta significativamente entre 6 e 12 semanas no grupo SVG e não no IOVG, observado por estas duas técnicas de nível molecular, estudos adicionais necessitam serem feitos para decifrar o mecanismo exato. / Vein grafts have been employed to bridge the gap in transected peripheral nerves to produce better functional recovery. However several disadvantages such as secondary graft constriction were observed and a new alternative to this technique was developed by simply reversing the vein inside out. Both inside out and standard vein grafts were successfully used in recovering the sensory segmental defect in humans. Neurotrophins are a family of eurotrophic factors known to play an important role in the regeneration of peripheral nerves. The neurotrophin family consists of Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and Neurotropinh-4 (NT-4). In the neurobiology field, several authors have been using PCR technique in order to gain more information regarding regenerated nerves. In this study, we employed this molecular biology technique to explore the role and level of the neurotrophins during the peripheral nerve regeneration with vein graft. The sciatic nerve of rats were sectioned and repaired with Inside out vein graft (IOVG) and standard vein graft techniques (SVG). In the control group the rats were sham operated wherein the sciatic nerve was kept intact. The animals were euthanized at 6 and 12 weeks and the grafts were harvested to observe the level the neurotrophins. EDL and Sol muscles were excised and measured to determine any weight difference between the groups. A small segment of the distal stumps from both the SVG and IOVG groups were also excised and were subjected to histological process to examine the amount of regenerated axon. In addition, another small segment of the distal stump was processed for RT-PCR to further examine the level of the neurotrophins in this area. At 6 weeks, no significant neuronal growth was observed in the distal stump of both graft types but a distinct growth was seen at 12 weeks. Walk track analysis showed poor motor function recovery in the experimental groups during both time intervals. Morphometric analysis demonstrated no significant differences in the amount of myelination between both the groups. There was no significant difference in the muscle mass between IOVG and SVG in both time periods. However, a significant increase in both the muscle mass was observed from 6 to 12 weeks in the IOVG and SVG groups. A significant increase in the production of NT-3 was observed in SVG group in both the distal stump and graft segment when compared from 6 to 12 weeks; however there was no observed increase in the level of other neurotrophins (NGF and NT-4). Surprisingly, no significant increase of NT-3 was noticed in the IOVG group. We conclude that amongst the neurotrophins evaluated in this study, there is no significant difference in their mRNA level between both groups except NT-3. Also, since mRNA level of NT-3 increases significantly between 6 and 12 weeks in SVG and not in IOVG, it suggests that the mechanism by which these two techniques operate at a molecular level may differ and further studies need to be done to decipher the exact mechanism.