Role of the ASPP Family in the Regulation of p53-Mediated Apoptotic Death of Retinal Ganglion Cells after Optic Nerve Injury

Le glaucome est la première cause de cécité irréversible à travers le monde. À présent il n’existe aucun remède au glaucome, et les thérapies adoptées sont souvent inadéquates. La perte de vision causée par le glaucome est due à la mort sélective des cellules rétiniennes ganglionnaires, les neurones qui envoient de l’information visuelle de la rétine au cerveau. Le mécanisme principal menant au dommage des cellules rétiniennes ganglionnaires lors du glaucome n’est pas bien compris, mais quelques responsables putatifs ont été proposés tels que l’excitotoxicité, le manque de neurotrophines, la compression mécanique, l’ischémie, les astrocytes réactifs et le stress oxidatif, parmis d’autres. Indépendamment de la cause, il est bien établi que la perte des cellules rétiniennes ganglionnaires lors du glaucome est causée par la mort cellulaire programmée apoptotique. Cependant, les mécanismes moléculaires précis qui déclenchent l’apoptose dans les cellules rétiniennes ganglionnaires adultes sont mal définis. Pour aborder ce point, j’ai avancé l’hypothèse centrale que l’identification de voies de signalisations moléculaires impliquées dans la mort apoptotique des cellules rétiniennes ganglionnaires offrirait des avenues thérapeutiques pour ralentir ou même prévenir la mort de celles-ci lors de neuropathies oculaires telles que le glaucome. Dans la première partie de ma thèse, j’ai caractérisé le rôle de la famille de protéines stimulatrices d’apoptose de p53 (ASPP), protéines régulatrices de la famille p53, dans la mort apoptotique des cellules rétiniennes ganglionnaires. p53 est un facteur de transcription nucléaire impliqué dans des fonctions cellulaires variant de la transcription à l’apoptose. Les membres de la famille ASPP, soit ASPP1, ASPP2 et iASPP, sont des protéines de liaison de p53 qui régulent l’apoptose. Pourtant, le rôle de la famille des ASPP dans la mort des cellules rétiniennes ganglionnaires est inconnu. ASPP1 et ASPP2 étant pro-apoptotiques, l’hypothèse de cette première étude est que la baisse ciblée de ASPP1 et ASPP2 promouvrait la survie des cellules rétiniennes ganglionnaires après une blessure du nerf optique. Nous avons utilisé un modèle expérimental bien caractérisé de mort apoptotique neuronale induite par axotomie du nerf optique chez le rat de type Sprague Dawley. Les résultats de cette étude (Wilson et al. Journal of Neuroscience, 2013) ont démontré que p53 est impliqué dans la mort apoptotique des cellules rétiniennes ganglionnaires, et qu’une baisse ciblée de ASPP1 et ASPP2 par acide ribonucléique d’interference promeut la survie des cellules rétiniennes ganglionnaires. Dans la deuxième partie de ma thèse, j’ai caractérisé le rôle d’iASPP, le membre anti-apoptotique de la famille des ASPP, dans la mort apoptotique des cellules rétiniennes ganglionnaires. L’hypothèse de cette seconde étude est que la surexpression d’iASPP promouvrait la survie des cellules rétiniennes ganglionnaires après axotomie. Mes résultats (Wilson et al. PLoS ONE, 2014) démontrent que le knockdown ciblé de iASPP exacerbe la mort apoptotique des cellules rétiniennes ganglionnaires, et que la surexpression de iASPP par virus adéno-associé promeut la survie des cellules rétiniennes ganglionnaires. En conclusion, les résultats présentés dans cette thèse contribuent à une meilleure compréhension des mécanismes régulateurs sous-jacents la perte de cellules rétiniennes ganglionnaires par apoptose et pourraient fournir des pistes pour la conception de nouvelles stratégies neuroprotectrices pour le traitement de maladies neurodégénératives telles que le glaucome.

Fibular Nerve Injury After Small Saphenous Vein Surgery

Superficial nerve injuries are very common during varicose vein surgery. In contrast, deep nerve injuries are rare and reported especially when surgery involves the small saphenous vein (SSV). The deep motor nerves most commonly injured are the tibial nerve and the peroneal nerve, which are directly or indirectly affected by extrinsic compression, stretching, or healing process involvement. In this report, two cases of common fibular nerve injury after SSV stripping are described, including treatment used and patient outcomes. Nerve damage mechanisms, anatomy, and prevention strategies are also discussed. In conclusion, fibular nerve damage may occur during SSV stripping. Preventive measures include careful preoperative ultrasonographic investigation of the anatomy of the vein, determining location of the saphenopopliteal joint, and careful dissection far from fibular nerve and restricted to the popliteal fossa.

Increased susceptibility to development of anhedonia in rats with chronic peripheral nerve injury: Involvement of sleep deprivation?

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

Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: modulation in the spared nerve injury model of neuropathic pain

Neuronal hyperexcitability following peripheral nerve lesions may stem from altered activity of voltage-gated sodium channels (VGSCs), which gives rise to allodynia or hyperalgesia. In vitro, the ubiquitin ligase Nedd4-2 is a negative regulator of VGSC α-subunits (Na(v)), in particular Na(v)1.7, a key actor in nociceptor excitability. We therefore studied Nedd4-2 in rat nociceptors, its co-expression with Na(v)1.7 and Na(v)1.8, and its regulation in pathology. Adult rats were submitted to the spared nerve injury (SNI) model of neuropathic pain or injected with complete Freund's adjuvant (CFA), a model of inflammatory pain. L4 dorsal root ganglia (DRG) were analyzed in sham-operated animals, seven days after SNI and 48 h after CFA with immunofluorescence and Western blot. We observed Nedd4-2 expression in almost 50% of DRG neurons, mostly small and medium-sized. A preponderant localization is found in the non-peptidergic sub-population. Additionally, 55.7 ± 2.7% and 55.0 ± 3.6% of Nedd4-2-positive cells are co-labeled with Na(v)1.7 and Na(v)1.8 respectively. SNI significantly decreases the proportion of Nedd4-2-positive neurons from 45.9 ± 1.9% to 33.5 ± 0.7% (p<0.01) and the total Nedd4-2 protein to 44% ± 0.13% of its basal level (p<0.01, n=4 animals in each group, mean ± SEM). In contrast, no change in Nedd4-2 was found after peripheral inflammation induced by CFA. These results indicate that Nedd4-2 is present in nociceptive neurons, is downregulated after peripheral nerve injury, and might therefore contribute to the dysregulation of Na(v)s involved in the hyperexcitability associated with peripheral nerve injuries.

Iatrogenic sciatic nerve injury in eighteen dogs and nine cats (1997-2006)

OBJECTIVE: To report clinical features associated with iatrogenic peripheral nerve injury in dogs and cats admitted (1997-2006) to a referral teaching hospital. STUDY DESIGN: Retrospective study. ANIMALS: Dogs (n=18), 9 cats. METHODS: Patients had acute signs of monoparesis attributable to sciatic nerve dysfunction that developed after treatment. Neurologic examination and electrodiagnostic testing were performed. Surgical therapy was used for nerve entrapment and delayed reconstructive surgery used in other cases. RESULTS: Of 27 nerve injuries, 25 resulted from surgery (18 with treatment of pelvic injuries). Iliosacral luxation repair resulted in tibial (4 cats) and peroneal (3 dogs) nerve dysfunction. Other causes were intramedullary pinning of femoral fractures (3), other orthopedic surgery (cemented hip prosthesis [2] and tibial plateau-leveling osteotomy [1]), and perineal herniorrhaphy [1]. Nerve injury occurred after intramuscular injection (1 cat, 1 dog). Immediate surgical treatment was removal of intramedullary nails, extruded cement, or entrapping suture. Delayed nerve transplantation was performed in 2 dogs. Within 1 year, 13 patients recovered completely, clinical improvement occurred in 7, and there was no improvement in 7. Five of the 7 dogs that did not recover had acetabular or ilium fracture. CONCLUSION: Iatrogenic sciatic nerve injury occurred most commonly during treatment of pelvic orthopedic diseases and had a poor prognosis. Clinical variation in sciatic nerve dysfunction in dogs and cats can be explained by species anatomic differences. CLINICAL RELEVANCE: Iatrogenic sciatic nerve injury leads to severely debilitating locomotor dysfunction with an uncertain prognosis for full-functional recovery.

Nerve Injury-Induced Neuropathic Pain Causes Disinhibition of the Anterior Cingulate Cortex

Neuropathic pain caused by peripheral nerve injury is a debilitating neurological condition of high clinical relevance. On the cellular level, the elevated pain sensitivity is induced by plasticity of neuronal function along the pain pathway. Changes in cortical areas involved in pain processing contribute to the development of neuropathic pain. Yet, it remains elusive which plasticity mechanisms occur in cortical circuits. We investigated the properties of neural networks in the anterior cingulate cortex (ACC), a brain region mediating affective responses to noxious stimuli. We performed multiple whole-cell recordings from neurons in layer 5 (L5) of the ACC of adult mice after chronic constriction injury of the sciatic nerve of the left hindpaw and observed a striking loss of connections between excitatory and inhibitory neurons in both directions. In contrast, no significant changes in synaptic efficacy in the remaining connected pairs were found. These changes were reflected on the network level by a decrease in the mEPSC and mIPSC frequency. Additionally, nerve injury resulted in a potentiation of the intrinsic excitability of pyramidal neurons, whereas the cellular properties of interneurons were unchanged. Our set of experimental parameters allowed constructing a neuronal network model of L5 in the ACC, revealing that the modification of inhibitory connectivity had the most profound effect on increased network activity. Thus, our combined experimental and modeling approach suggests that cortical disinhibition is a fundamental pathological modification associated with peripheral nerve damage. These changes at the cortical network level might therefore contribute to the neuropathic pain condition.

Laser therapy and pain-related behavior after injury of the inferior alveolar nerve: possible involvement of neurotrophins

Nerve-related complications have been frequently reported in dental procedures, and a very frequent type of occurrence involves the inferior alveolar nerve (IAN). The nerve injury in humans often results in persistent pain accompanied by allodynia and hyperalgesia. In this investigation, we used an experimental IAN injury in rats, which was induced by a Crile hemostatic clamp, to evaluate the effects of laser therapy on nerve repair. We also studied the nociceptive behavior (von Frey hair test) before and after the injury and the behavioral effects of treatment with laser therapy (emitting a wavelength of 904 nm, output power of 70 Wpk, a spot area of *0.1 cm2, frequency of 9500 Hz, pulse time 60 ns and an energy density of 6 J/cm2). As neurotrophins are essential for the process of nerve regeneration, we used immunoblotting techniques to preliminarily examine the effects of laser therapy on the expression of nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF). The injured animals treated with laser exhibited an improved nociceptive behavior. In irradiated animals, there was an enhanced expression of NGF (53%) and a decreased BDNF expression (40%) after laser therapy. These results indicate that BDNF plays a locally crucial role in pain-related behavior development after IAN injury, increasing after lesions (in parallel to the installation of pain behavior) and decreasing with laser therapy (in parallel to the improvement of pain behavior). On the other hand, NGF probably contributes to the repair of nerve tissue, in addition to improving the pain-related behavior.

Long term peripheral nerve regeneration using a novel PCL nerve conduit.

The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.

Extracellular matrix components in peripheral nerve repair: how to affect neural cellular response and nerve regeneration?

Peripheral nerve injury is a serious problem affecting significantly patients' life. Autografts are the "gold standard" used to repair the injury gap, however, only 50% of patients fully recover from the trauma. Artificial conduits are a valid alternative to repairing peripheral nerve. They aim at confining the nerve environment throughout the regeneration process, and providing guidance to axon outgrowth. Biocompatible materials have been carefully designed to reduce inflammation and scar tissue formation, but modifications of the inner lumen are still required in order to optimise the scaffolds. Biomicking the native neural tissue with extracellular matrix fillers or coatings showed great promises in repairing longer gaps and extending cell survival. In addition, extracellular matrix molecules provide a platform to further bind growth factors that can be released in the system over time. Alternatively, conduit fillers can be used for cell transplantation at the injury site, reducing the lag time required for endogenous Schwann cells to proliferate and take part in the regeneration process. This review provides an overview on the importance of extracellular matrix molecules in peripheral nerve repair.

Nerve guidance conduits from aligned nanofibers: improvement of nerve regeneration through longitudinal nanogrooves on a fiber surface

A novel fibrous conduit consisting of well-aligned nanofibers with longitudinal nanogrooves on the fiber surface was prepared by electrospinning and was subjected to an in vivo nerve regeneration study on rats using a sciatic nerve injury model. For comparison, a fibrous conduit having a similar fiber alignment structure without surface groove and an autograft were also conducted in the same test. The electrophysiological, walking track, gastrocnemius muscle, triple-immunofluorescence, and immunohistological analyses indicated that grooved fibers effectively improved sciatic nerve regeneration. This is mainly attributed to the highly ordered secondary structure formed by surface grooves and an increase in the specific surface area. Fibrous conduits made of longitudinally aligned nanofibers with longitudinal nanogrooves on the fiber surface may offer a new nerve guidance conduit for peripheral nerve repair and regeneration.

Muscle reinnervation in one or two stages?: experimental study in rats with end-to-side nerve graft

OBJETIVO: Comparar a reinervação muscular com enxerto de nervo em um e dois tempos operatórios, utilizando a neurorrafia término-lateral (NTL) sem lesão do nervo doador. MÉTODOS: Vinte ratos foram distribuídos em quatro grupos. O grupo 1 (G1), um estágio, recebeu o enxerto que foi suturado ao nervo tibial (NT), por meio de NTL, e seu coto livre foi suturado por NTL ao coto distal do nervo peroneal (NP), seccionado a um centímetro do NT, na mesma cirurgia. O grupo 2 (G2), dois estágios, recebeu o enxerto de nervo na primeira cirurgia, como já descrito. Dois meses depois, na segunda cirurgia, o NP foi seccionado e seu coto distal ligado ao coto distal do enxerto como em G1. O grupo controle de normalidade (Gn) recebeu o enxerto da mesma forma, apenas. E o grupo controle de denervação (Gd), além de receber o enxerto, teve o NP seccionado e seus cotos sepultados na musculatura adjacente, com a finalidade de denervar o músculo tibial cranial (MTC), alvo deste estudo. Os parâmetros utilizados para avaliar a reinervação do MTC foram massa muscular, diâmetro mínimo da fibra muscular e área. RESULTADOS: O grupo G2 apresentou superioridade (p<0,0001) em relação ao G1 na massa do MTC, no diâmetro mínimo e na área das fibras musculares. Na comparação entre os quatro grupos, estes mesmos parâmetros tiveram sua expressão máxima em Gn e mínima em Gd, como era esperado. CONCLUSÃO: A reinervação muscular em dois estágios apresenta melhor resultado quando comparada à técnica em um tempo.

Muscle graft as a substitute for peripheral nerve graft in rats

OBJETIVO: Avaliar a aplicabilidade do uso de músculo autógeno, tratado de diversas maneiras, em substituição aos enxertos de nervo. MÉTODOS: Os ratos foram separados em sete grupos que receberam, como tratamento a uma lesão nervosa padronizada, os seguintes tipos de enxertos: músculo fresco, músculo fixado com formol 10%, músculo congelado em freezer, músculo congelado em refrigerador, músculo denervado, nervo periférico e um grupo ficou sem qualquer tratamento. Foi avaliado o aspecto histológico das fibras nervosas no segmento reparado. RESULTADOS: A avaliação do segmento nervoso reparado mostrou que existiam axônios em quase todos os grupos, mas a metodologia empregada não possibilitou caracterizar adequadamente as diferenças entre os grupos. CONCLUSÃO: Este estudo mostrou a migração de axônios por meio de todos os enxertos utilizados.