Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

Purpose: The purpose of this study was to investigate the role of the vasculature and immune system in models of glaucoma. Vascular changes have been implicated in glaucoma. As well there is mounting evidence that the immune system plays a role in the disease. It is my hypothesis that the vasculature and immune system play a role in the retinal response to injury in models of glaucoma. Methods: Immunohistochemistry, in vivo retinal imaging (Bright field, fluorescent, optical coherence tomography), Slit2 injections and Evan’s blue labeling were used to investigate vascular and immune changes associated with retinal ganglion cell death after optic nerve crush up to 28 days after injury. Histology, immunohistochemistry, and intravascular labeling were utilized to investigate the role of the vascular degeneration and the systemic immune response to elevated intraocular pressure in 8-16 week old AP-2β Neural Crest Cell Knockout (AP-2β NCC KO) mice. Results: The vascular and immune responses to optic nerve crush were not found to play a significant role in the response to retinal ganglion cell death. Conversely the role of vascular degeneration and immune cell recruitment to the retinas of AP-2β NCC KO mice demonstrated that these factors played a significant role in the retinal response to injury. Conclusion: The vasculature and immune system play a varied role in the response to retinal injury and neurodegeneration depending on the model being studied. The vascular and immune changes were of minimal significance in acute optic nerve crush injury. On the other hand, the chronic injury associated with elevated intraocular pressure in AP-2β NCC KO mice was associated with significant vascular degeneration and systemic immune cell infiltration. / Thesis / Doctor of Philosophy (PhD)

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

Made available in DSpace on 2016-01-26T12:51:11Z (GMT). No. of bitstreams: 1 joaoariskouyoumdjian_dissert.pdf: 613490 bytes, checksum: d3e0f432c81a706e636cdcbf67d2676d (MD5) Previous issue date: 1998-07-31 / Carpal tunnel syndrome: clinical, epidemiological and nerve conduction studies in 668 cases Between January/89 and June/96, 1,059 carpal tunnel syndrome hands (CTS) from 668 patients were studied. None had been previously operated and all had bilateral conduction studies; peripheral neuropathy was excluded. The patients were selected with sensory median/radial difference (MRD) ³ 1.0 ms that strongly supports electrodiagnosis of CTS (standard deviation > 6) after simultaneous stimulation on wrist and recording on thumb. The age ranged from 17 to 83 years (mean 47.5) and 91.3% were female; the complaints were bilateral in 72% and nocturnal/awakening in 85.3%; pain, numbness and paraesthesia occurred in 64.4%; pain as the only symptom was rare but proximal extension was frequent (39.4%); all fingers were symptomatic in 42.5%, followed by middle, middle-ring, thumbindex- middle and then index-middle-ring ones; there was no correlation with traumatic past history on wrist. The duration CTS symptoms ranged from 1 to > 120 months without precise correlation with the severity of conduction abnormalities on median nerve. MRD ³ 1.0 ms correlates in 95% with median distal motor latency > 4.25 ms (80 mm distance) and with median distal sensory latency to index finger ³ 3.01 ms, middle finger ³ 3.14 ms and ring finger ³ 3.26 ms, all of them 140 mm distance, antidromic and onset-measured. The results have brought new values for the limit of normality in our EMG laboratory since MRD ³ 1.0 ms is very sensitive for CTS diagnosis. / Foram estudados 668 pacientes (1,059 mãos) com síndrome do túnel do carpo (stc) entre janeiro de 1989 e junho de 1996, o critério de seleção e inclusão dos pacientes baseou-se na diferença de latência sensitiva 1,0 ms entre os potenciais de ação sensitivos (pas) dos nervos mediano e radial após estimulação simultânea no punho e resgistro no i dedo (dmr), representando diferença maior que 6 desvios-padrão. Todos os casos tiveram estudo eletrofisiológico bilateral, sendo excluídos casos com cirurgia prévia ou evidência de neuropatia periférica. Nota de Resumo A idade variou de 17 a 83 anos com a média de 47,5 anos; 91,3% eram do sexo feminino; 72% referiam sintomatologia bilateral e 85,3% referiam sintomatologia bilateral e 85,3% no período noturno/matinal; dor, dormência e formigamento foram conjuntamente referidos por 64,4%, sendo que dor como sintoma isolado foi raro; além da mão, houve extensão do quadro álgico para outros territórios em 39,4%; 0s sintomas ocorreram em todos dos dedos em 42,5% seguindo do III, III-IV, I-II-III e II-III-IV dedos; não houve correlação precisa com antecedentes treumáticas no punho. A duração da sintomalogia foi ampla, variando de 1 a 120 meses; não houve correlação entre tempo de sintomatologia e gravidade da compressão do nervo mediano no campo. Valor de dmr 1,0 ms correlacionou-se em 95% dos casos com latência distal motora do nervo mediano 4,25 ms (80mm) e com latência distal sensitiva punho - ii dedo 3,14 ms (vs 44,6 m/s) e punho - iv dedo 3,26 ms (vc 42,9 m/s), todos com 140 mm de distância e latância medida no início do pas. Nota de Resumo Os resultados estabelecem novos critérios anormais para o diagnóstico eletrofisiológico de stc em uma população brasileira para a condução motora (latência distal motora do nervo mediano) e sensitiva (latência distal sensitiva do nervo mediano após dmr 1,0 ms (diagnóstico seguro de stc em praticamente 100% dos casos suspeitos).

Les nerfs périphériques sont sujets à de nombreuses pathologies et l’étiologie des neuropathies périphériques (NP) est vaste (troubles métaboliques, infections, toxines, blessures physiques et mutations génétiques, ect.). Par exemple, les NP d’origine traumatique sont courantes et sont caractérisées par une dégénérescence dite Wallérienne des fibres nerveuses. Autre exemple, la maladie de Charcot-Marie-Tooth 1A (CMT1A) qui est la NP génétique héréditaire la plus fréquente. Elle est caractérisée par une surexpression de la protéine PMP22 impliquée dans le maintien de la gaine de myéline. Actuellement, il n’existe pas de traitement pharmacologique de ces deux affections des nerfs. Récemment, l’intérêt pour le rôle des antioxydants alimentaires, tels que la curcumine, a suscité de nombreuses recherches. Cette molécule est depuis longtemps utilisée en médecine asiatique pour ces propriétés thérapeutiques. Cependant, elle possède une très faible biodisponibilité et nécessite donc l’emploi de doses très élevées pour obtenir des effets bénéfiques. Dans une première étude, nos résultats ont montré que des faibles doses de curcumine administrées localement et en continu, améliorent la récupération fonctionnelle, les paramètres électrophysiologiques et histologiques, et l’expression des principales protéines de la myéline dans un modèle d’écrasement du nerf sciatique chez le rat. Ces effets bénéfiques ont été attribués aux propriétés antioxydantes de la curcumine. Dans une seconde étude, nos résultats ont montré qu’une faible dose de nanocristaux de curcumine (Nano-Cur), injectée en IP, améliorent le phénotype, les paramètres électrophysiologiques et histologiques dans un modèle transgénique de rat CMT1A. Dans cette étude, les effets positifs ont été attribués aux propriétés antioxydantes des Nano-Cur, couplés à l’activation de la voie de dégradation associée au réticulum endoplasmique, permettant la réduction de la surexpression nocive de PMP22 chez les rats CMT1A. L'ensemble de ces résultats démontrent que, l’administration de faibles doses de curcumine constitue un traitement prometteur dans la réparation des nerfs périphériques. / Peripheral nerves are subject to many pathologies and the etiology of peripheral neuropathies (PN) is vast (metabolic disorders, infections, toxins, physical injuries and genetic mutations, etc.). For example, PN of traumatic origin are common and are characterized by a called Wallerian degeneration of nerve fibres. Another example is Charcot-Marie-Tooth disease 1A (CMT1A), which is the most common hereditary genetic PN. It is characterized by an overexpression of the PMP22 protein involved in maintaining the myelin sheath. Currently, there is no pharmacological treatment for these two nerve disorders. Recently, interest in the role of dietary antioxidants, such as curcumin, has led to much research. This molecule has long been used in Asian medicine for its therapeutic properties. However, it has a very low bioavailability and therefore requires the use of very high doses to obtain beneficial effects. In a first study, our results showed that low doses of curcumin administered locally and continuously improve functional recovery, electrophysiological and histological parameters, and expression of major myelin proteins in a rat sciatic nerve crush model. These beneficial effects have been attributed to the antioxidant properties of curcumin. In a second study, our results showed that a low dose of curcumin nanocrystals (Nano-Cur), injected in IP, improves phenotype, electrophysiological and histological parameters in a transgenic model of CMT1A rats. In this study, the positive effects were attributed to the antioxidant properties of the Nano-cur, coupled with the activation of the endoplasmic reticulum associated degradation pathway, allowing the reduction of harmful overexpression of PMP22 in CMT1A rats. All these results show that the administration of low doses of curcumin is a promising treatment for peripheral nerve repair

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Introduction: Les implants dans le système nerveux périphérique (SNP) peuvent potentiellement restaurer les capacités sensorielles et motrices chez les patients avec des amputations des membres supérieures. Cependant, la réaction à un corps étrangers affecte significativement la fonction à long-terme et la biocompatibilité de ces systèmes avec le temps. Le dendrimère (DND) et la Poly-D-Lysine (PDL) sont deux polymères synthétiques qui peuvent potentiellement améliorer la performance de ces implants. Pour cette étude, notre objectif est de déterminer si ces polymères peuvent promouvoir la formation d’éléments présynaptiques sur des surfaces synthétiques in vivo dans un modèle animal. Méthodes: Pour l’étude in vivo, nous avons utilisé un modèle d’écrasement du nerf sciatique chez le rat. Des billes enduites de DND et PDL et contrôle ont été injectées dans le nerf sciatique aux sites d’écrasement et 5 mm distaux au site d’écrasement. Après 4, 6 et 8 semaines, les nerfs ont été retirés et marqués avec des anticorps spécifiques au neurofilament et à la synaptophysine. Nous avons ensuite compté le nombre d’éléments présynaptiques retrouvant sur la surface de chaque bille pour toutes les conditions. Pour l’étude de l’électrode, deux électrodes ont été implantées dans le nerf sciatique du rat. Nous avons ensuite effectué des enregistrements nerveux à chaque semaine, et le potentiel d’action dans le nerf a été mesuré en variant uniquement la largeur de l’impulsion. Résultats: L’étude in vivo a démontré que les billes enduites de DND pouvaient promouvoir une accumulation significative de synaptophysine sur leurs surfaces comparé aux billes contrôles de 4 à 8 semaines. À 4 semaines, les billes dans la condition DND avaient également une accumulation de synaptophysine significativement supérieure à celles dans la condition PDL pour le site distal à l’écrasement. L’étude de l’électrode a démontré que les deux électrodes pouvaient stimuler et acquérir des signaux nerveux du nerf sciatique jusqu’à 1 et 2 semaines respectivement avant de ne plus fonctionner. Conclusion: Les résultats de notre étude suggèrent que DND possède une propriété à promouvoir la synaptogenèse qui est supérieure à PDL in vivo et que notre modèle d’électrode peut être utilisé pour évaluer la stabilité du signal des implants SNP. / Background: Implants in the peripheral nervous system (PNS) can potentially restore sensory feedback, improve motor control and alleviate phantom-limb pain in upper-limb amputees. However, nervous system implants have poor long-term function and biocompatibility when implanted into the body due to foreign body reaction. Dendrimer (DND) and Poly-D-Lysine (PDL) are two synthetic polymers with properties that could improve the performance of these interfaces. In my masters’ research, my objective is to determine whether these synthetic polymers could promote the formation of presynaptic elements on artificial surfaces in vivo making intraneural implants more biocompatible and long-lasting. Methods: In the coated microsphere in vivo experiment, a nerve crush injury model in the rat was used for the study. PDL-coated, DND-coated and uncoated beads were injected into the rat sciatic nerve at the crush site and 5 mm distal to the crush site. The nerves were then harvested after 4, 6 and 8 weeks and stained for neurofilament and synaptophysin. Synaptophysin puncta were then counted on the bead surface for each group. Additionally, in a proof-of-concept experiment, two uncoated electrodes were implanted into the rat sciatic nerve. Nerve recordings were then performed every week, and the threshold nerve potential in the sciatic nerve was measured by only varying the pulse duration of the stimulation. Results: The coated microsphere in vivo experiment demonstrated that DND-coated microspheres had a significantly higher number of synaptophysin puncta around their surface from 4 to 8 weeks compared to uncoated beads. At 4 weeks, the DND condition also showed a significantly higher number of synaptophysin puncta around its microbeads vs. the PDL condition for the distal site. In the uncoated electrode in vivo experiment, the results showed that the two implants could stimulate and record threshold nerve potentials in the rat sciatic nerve for one week and two weeks respectively before being non-functional. Conclusion: Our study showed for the first time that DND has a stable synapse-promoting property that is superior to PDL in vivo and that our electrode design can be used to assess the long-term signal stability of peripheral nerve implants.