1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GAGA(A))-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were sensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 ± 4.0 μM to 18.0 ± 5.0 μM). The maximum response to GABA was depressed by 34.0 ± 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABA(A)-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.

No description available.

A la suite d'un traumatisme nerveux les neurones périphériques ont la capacité de régénérer. La repousse est possible grâce à l'environnement permissif et les aptitudes intrinsèques des neurones périphériques à entamer un processus régénératif. Cette capacité intrinsèque se traduit par des remaniements cellulaires et moléculaires induits notamment par la modification de l'expression de nombreux gènes. Ma thèse a porté sur l'étude de l'un d'entre eux : CaMKIα (Calcium-Calmodulin-dependent kinase Iα), dont nous avons montré l'induction de l'expression dans les neurones de ganglions rachidiens dorsaux par une lésion du nerf sciatique. Cette kinase, jamais encore décrite dans le système nerveux périphérique adulte, est impliquée dans le développement neuronal au niveau central. Nous avons établi que l'expression de CaMKIα est spécifiquement induite à la suite de différents types de traumatismes mécaniques du nerf sciatique (sections, compressions chroniques ou aiguës) dans une population restreinte de neurones lésés, majoritairement myélinisés. La localisation subcellulaire de CaMKIα, à la fois dans le corps cellulaire des neurones et dans les fibres du nerf sciatique, évoque un transport axonal de la kinase vers le site de lésion. L'inhibition de la voie de signalisation de CaMKIα par traitement pharmacologique ou l'utilisation de siRNA dirigés contre CaMKIα induit in vitro une chute significative de la vitesse de pousse des neurites des neurones lésés. L'ensemble de ces résultats suggère que l'induction de CaMKIα contribue à la régénération axonale post-lésionnelle des neurones périphériques. / Peripheral neurons have the capacity to regenerate after injury. This regeneration is allowed by thefavorable environment generated by the cellular components of the system and intrinsic aptitudes ofthe peripheral neurons to enter this process. These intrinsic abilities are manifested as cellular changes and molecular alterations including transcriptional and post-transcriptional modifications. Prior to my work, our laboratory carried out transcriptomic analysis on dorsal root ganglia after nerve injury. This allowed us to highlight a set of genes induced in response to peripheral nerve lesion. My thesis focused on one of them: CaMKIα (Calcium-Calmodulin-dependent kinase Iα). This kinase, not previously described in the adult peripheral nervous system, has been shown to be involved in central nervous system neuronal development. We have shown that CaMKIα is specifically induced following different kinds of mechanical lesions of the sciatic nerve (sections and acute or chronic crush) in a restricted, predominantly myelinated, population of injured neurons. The subcellular location of CaMKIα, both in the soma and nerve fibers suggest an axonal transit of the kinase to the injury site. The inhibition of the CaMKIα signaling pathway by a pharmacological compound or RNA silencing in vitro induced a significantly decreased velocity of neurite growth in injured neurons. Taken together, these results suggest that the induction of CaMKIα contributes to the post injury axonal regeneration of peripheral neurons.

The effect of interleukin-1 beta (IL-1β) on the electrical properties of sensory neurons was assessed at comparable levels and exposure times to those found in animal models of neuropathic pain. Experiments involved whole cell current- or voltage-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined medium, neuron enriched cultures. 5-6 days exposure to 100 pM IL-1β produced neuron specific effects. These included an increase in the excitability of medium diameter and small diameter isolectin B4 (IB4)-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large diameter neurons, while no effect was found in small diameter IB4-negative neurons. Further characterization of changes in medium and small IB4-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Using appropriate voltage protocols and/or ion substitutions, it was found that neuron specific changes in several ionic currents, including alterations in hyperpolarization activated inward current (IH) and decreases in various K+ currents contribute to the increased excitability produced by IL-1β. Overall, these studies revealed that: 1. The effects of long-term exposure of DRG neurons to IL-1β are reflective of the enduring increase in primary afferent excitability reported after peripheral nerve injury. This expands the recognized role of IL-1β in acute inflammatory pain to neuropathic pain. 2. Hyperexcitability in medium neurons exposed to IL-1β likely includes mixed populations of neurons corresponding to nociceptive and non-nociceptive primary afferent fibres and, therefore, has relevance to hyperalgesia and allodynia, respectively. 3. The responsiveness of small IB4-positive neurons, but not IB4-negative, to prolonged IL-1β exposure is consistent with the suggestion that small IB4-negative afferents are involved in inflammatory pain, while small IB4-positive afferents are involved neuropathic pain. 4. The identification of receptor mediated effects and several contributing ionic mechanisms, may have relevance to the development of new therapeutic approaches to neuropathic pain. 5. IL-1β can contribute to increased neuronal excitability by mechanisms that are independent of IL-1RI signalling. This should be taken into account when targeting IL-1β, or more specifically IL-1RI, in the management of neuropathic pain.

No description available.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nos gânglios sensitivos, os corpos celulares dos neurônios encontram-se circundados por células gliais denominadas células satélites. Estudos recentes apontam para uma possível comunicação entre neurônios e células satélites através da liberação de ATP e ativação de receptores P2X7 presentes nas células gliais. Além disto, células satélites adjacentes podem estar conectadas através de junções comunicantes (“gap junctions”). Até o presente, a comunicação entre células satélites e neurônios tem sido implicada na cronificação da dor e em processos inflamatórios. Nesse estudo buscamos avaliar o papel da comunicação entre neurônios e células satélites através da ativação dos receptores P2X7 assim como das junções comunicantes em modelos de dor aguda. Em culturas primárias de gânglios da raiz dorsal, verificamos que a administração de capsaicina leva a um aumento de cálcio em neurônios e em seguida em células satélites sendo que a resposta das células satélites foi bloqueada por A740003, um antagonista seletivo para receptores P2X7, indicando que os nociceptores quando ativados liberam ATP que, por sua vez, ativa receptores P2X7 nas células gliais. Para avaliar o papel desta comunicação celular in vivo, o antagonista P2X7 ou o bloqueador de junções comunicantes, carbenoxolona, foram administrados por via intraganglionar (L5) e foram avaliados os efeitos das injeções intraplantares de capsaicina, mentol e formalina em ratos. Tanto o A740003 quanto a carbenoxolona reduziram a nocicepção induzida por capsaicina e mentol. No teste da formalina, ambas as substâncias afetaram apenas a segunda fase do teste, considerada a fase inflamatória. Capsaicina ativa seletivamente receptores TRPV1 e mentol ativa receptores TRPM8, e possivelmente receptores TRPA1, que são expressos majoritariamente em neurônios nociceptivos associados a fibras C. Além disto, estudos de outros autores indicam a primeira fase do teste da formalina envolve principalmente a ativação de fibras do tipo Aδ enquanto que a segunda fase envolve a ativação de fibras Aδ e C. Considerando estes dados juntamente como os presentes resultados, podemos sugerir que a comunicação entre células satélites e neurônios ocorre também na dor aguda, mas apenas quando esta depende da ativação de fibras C. Deste modo, a comunicação entre neurônios e células satélites, via liberação de ATP e ativação de receptores P2X7, assim como uma comunicação entre células satélites adjacentes através de junções comunicantes parecem estar envolvidos em um processamento rápido do sinal doloroso no gânglio da raiz dorsal. / In sensory ganglia, the cellular bodies of neurons are surrounded by glial cells called satellite cells. Recent studies point to a possible communication between neurons and satellite cells through the release of ATP and activation of P2X7 receptors present in glial cells. In addition, adjacent satellite cells may be connected through gap junctions. Still today, the communication between satellite cells and neurons has been implicated in chronic pain and in inflammatory processes. In this study we sought to evaluate the role of communication between neurons and satellite cells through the activation of the P2X7 receptors as well as of the communicating junctions in acute pain models. In primary cultures of dorsal root ganglia, we found that the administration of capsaicin leads to an increase of calcium in neurons and then in satellite cells. The response of satellite cells was blocked by A740003, a selective antagonist for P2X7 receptors, indicating that nociceptors when activated release ATP, which in turn activates P2X7 receptors in the glial cells. To assess the role of this in vivo cellular communication, the P2X7 antagonist or the gap junction blocker, carbenoxolone, were administered by intraganglionar injection (L5) and the effects of intraplantar injections of capsaicin, menthol or formalin in rats were evaluated. Both A740003 and carbenoxolone reduced nociception induced by capsaicin and menthol. In the formalin test, both substances affected only the second phase of the test, considered the inflammatory phase. Capsaicin selectively activates TRPV1 receptors while menthol activates TRPM8 receptors, and possibly TRPA1 receptors, which are expressed mainly in nociceptive neurons associated with C fibers. In addition, studies by other authors indicate that the first phase of the formalin test involves primarily the activation of Aδ fibers whereas the second phase involves the activation of Aδ and C fibers. Considering these data together with the present results, we can suggest that the communication between satellite cells and neurons also occurs in acute pain, but only, when it depends on the activation of C fibers. Thus, communication between neurons and satellite cells, via release of ATP and activation of P2X7 receptors, as well as communication between adjacent satellite cells through gap junctions seems to be involved in a rapid processing of the pain signal in the dorsal root ganglion. / Dissertação (Mestrado)

Orientador: Carlos Amilcar Parada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T20:52:22Z (GMT). No. of bitstreams: 1 Araldi_Dioneia_D.pdf: 2812057 bytes, checksum: ba0136e00ebc1ab271d4e1ed908bcd8c (MD5) Previous issue date: 2012 / Resumo: A liberação de Interleucina-1? (IL-1?) no tecido periférico estimula a síntese de Prostaglandinas (PGs), especialmente, da Prostaglandina-E2 (PGE2), que leva a sensibilização dos nociceptores aferentes primários induzindo a hiperalgesia inflamatória. Recentemente demonstramos que a IL-1? pode ativar diretamente o receptor de Interleucina-1 (IL-1R) do nociceptor aferente periférico e levar a liberação de PGE2 associada ao desenvolvimento da hiperalgesia. A IL-1? também é liberada no Gânglio da Raiz Dorsal (GRD), entretanto a função que a IL-1? desempenha no GRD para o desenvolvimento da hiperalgesia inflamatória ainda não está clara. Portanto, o objetivo deste estudo foi investigar se a liberação de IL-1? e a ativação do Receptor de Interleucina-1 Tipo I (IL-1RI) no GRD estão envolvidos no desenvolvimento da hiperalgesia inflamatória. A administração de IL-1Ra (antagonista natural de receptor IL- 1, 6 ?g) no GRD de ratos preveniu a hiperalgesia mecânica (avaliada por meio do von Frey Eletrônico) induzida pela administração intraplantar (i.pl) de Adjuvante Completo de Freund (CFA, 100 ?L), Carragenina (Cg, 100 ?g) ou IL-1? (0,5 pg), mas não pela administração i.pl de PGE2 (100 ng), avaliadas 3 horas após suas administrações. Além disso, a administração i.pl periférica de CFA ou Cg aumentaram as concentrações de IL-1? (avaliadas por ELISA) no GRD. O tratamento ganglionar (GRD-L5) com oligonucleotídeo (ODN) antisense contra IL-1RI (30 ?g/dia durante 4 dias) reduziu de maneira significativa a expressão de IL-1RI no GRD-L5 e a hiperalgesia mecânica induzida por CFA, Cg e IL-1?, mas não pela PGE2, administradas no tecido periférico da pata. Também verificamos a hipótese de que a prévia ativação do receptor neuronal, IL-1RI, no tecido periférico é importante para a liberação de IL-1? no GRD e para a subsequente hiperalgesia induzida por PGE2. A IL-1? (0,5 pg/pata) co-administrada com a dose sub-limiar de PGE2 (10 ng/pata) em patas traseiras tratadas com indometacina induziu uma proeminente hiperalgesia, que foi prevenida pelo prétratamento com ODN antisense contra IL-1RI ou IL-1Ra (6 ?g) administrados no GRD. Além disso, o IL-1Ra reduziu a expressão de COX-2 em células do GRD. Para confirmar a ativação do IL-1RI em células do GRD, administramos Cg ou CFA no tecido periférico o que levou ao aumentou da expressão de IRAK-1 e IRAK-4 em células do GRD. Os resultados deste estudo sugerem que o desenvolvimento da hiperalgesia inflamatória depende da ativação do receptor IL-1RI neuronal no tecido periférico que, em partes, induz a liberação de IL-1? no GRD e subsequente ativação da COX-2. Os dados aqui apresentados oferecem novas perpectivas sobre a participação das células do GRD nos mecanismos envolvidos na hiperalgesia inflamatória e revelam novos e interessantes alvos para o controle das hiperalgesias inflamatórias / Abstract: The release of Interleukin-1? (IL-1?) in the peripheral tissue stimulates the synthesis of Prostaglandins, specially, Prostaglandin-E2 (PGE2) that ultimately sensitize the peripheral afferent nociceptor inducing inflammatory hyperalgesia. We have recently demonstrated that IL-1? can directly activate IL-1R receptor of peripheral afferent nociceptor to induce release of PGE2 associated to development of hyperalgesia. IL-1? is also released in Dorsal Root Ganglion (DRG), however the role that IL-1? in DRG plays to development of inflammatory hyperalgesia is not yet elucidated. Therefore, the aim of this study was to investigate whether IL-1? released in the DRG and the activation of Interleukin-1 Receptor Type I (IL-1RI) is involved in the development of the inflammatory hyperalgesia. Administration of IL-1Ra (IL-1 receptor antagonist, 6 ?g) in the DRG of rats prevented the mechanical hyperalgesia (measured with Electronic von Frey) induced by intraplantar (i.pl) administration of Complete Freund's Adjuvant (CFA, 100 ?L), Carrageenan (Cg, 100 ?g) or IL- 1? (0.5 pg), but not by PGE2 (100 ng), measured 3 hours after their administrations. Also, peripheral i.pl administration of CFA or Cg induced an increase in IL-1? concentrations (measured by ELISA) in the DRG. Ganglionar (DRG-L5) treatment with oligonucleotides (ODN) antisense against IL-1RI (30 ?g/day for four days) reduced the expression of IL-1RI in the DRG-L5 and the mechanical hyperalgesia induced by CFA, Cg, and IL-1?, but not by PGE2 administered in peripheral tissue. We also verified the hypothesis that previous activation of neuronal IL-1RI in the peripheral tissue is important to the release of IL-1? in the DRG and to the subsequent PGE2-induced hyperalgesia. IL-1? (0.5 pg/paw) co-administrated with a sub-threshold dose of PGE2 (10 ng/paw), in hind paws treated with indomethacin, greatly induces hyperalgesia, which was prevented by pre-treatment with ODN antisense against IL-1RI or IL-1Ra (6 ?g) administrated in DRG. Also, IL-1Ra administrations reduced the COX-2 expression in DRG cells. To confirm IL-1RI activation in DRG cells, it was observed that IRAK-1 and IRAK-4 expression was increased in DRG neurons after administration of Cg or CFA in the peripheral tissue. These findings suggest that the development of inflammatory hyperalgesia depends on neuronal IL-RI activation in the peripheral tissue that, in turn, induces the release of IL- 1? in the DRG and subsequent COX-2 activation. These data provide new insights about the participation of DRG cells in the mechanisms underlying inflammatory hyperalgesia and reveal new interesting targets to control inflammatory hyperalgesia / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Indiana University-Purdue University Indianapolis (IUPUI) / Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.

In der retrospektiven, monozentrischen Studie soll die Sicherheit von magnet-resonanztomographischen (MRT) Untersuchungen bei Patienten mit implantiertem Neurostimulator untersucht werden. Von November 2011 bis einschließlich März 2019 wurden am SRH Wald-Klinikum Gera insgesamt 171 MRT-Untersuchungen an Patienten mit implantiertem rücken-marknahem Neurostimulator (Spinal Cord Stimulation, SCS; Dorsal Root Ganglion Stimulation, DRG-S) durchgeführt. Zudem konnten 22 MRT-Anforderungen ohne Durchführung einer MRT-Untersuchung identifiziert und die Gründe für die nicht erfolgte MRT-Untersuchung ausgewertet werden. Aufgrund initial geringer Fallzahl wurde in Absprache mit der zuständigen Ethikkommission der Auswertungszeitraum für MRT-Untersuchungen an Patienten mit DRG-S bis einschließlich Oktober 2020 erweitert, hierdurch konnten elf weitere Untersuchungen ausgewertet werden. Von den im Beobachtungszeitraum in unserem Klinikum implantierten Patienten haben 28% in der eigenen Einrichtung mindestens eine MRT-Untersuchung erhalten. Retrospektiv konnte eine Vorhersagbarkeit des MRT-Bedarfs nur in 22% der Anforde-rungen zum Zeitpunkt der Implantation unterstellt werden. Aber selbst in Kenntnis anatomischer Besonderheiten bleibt eine prospektive Vorhersagbarkeit des künftigen MRT-Bedarfs sehr vage. MRT-Untersuchungen für Patienten mit rückenmarknahem Neurostimulator sind nur selten innerhalb der Zulassungskriterien möglich. Hauptgründe sind die fehlende Zulassung von einzelnen Komponenten, die Elektrodenlage und Einschränkungen bezüglich der untersuchbaren Körperregionen bzw. der technischen Voraus-setzungen für eine MRT-Bildgebung. Im Ergebnis unserer Studie sind bei adäquater 4 Implantatvorbereitung, Patientenaufklärung und Beachtung von definierten Abbruch-kriterien (deutliche Erwärmung der Implantate, Auftreten von sensomotorischen Stimulationssensationen, neue Schmerzen im Bereich der Implantate) MRT-Unter-suchungen auch für diese Patientengruppe relativ sicher. In unserem Patientengut, welches sowohl die epidurale Rückenmarkstimulation (156 Fälle) als auch die Dorsal Root Ganglion Stimulation (26 Fälle) umfasst, traten ein schwerwiegendes uner-wünschtes Ereignis (0,6%), zwölf möglicherweise implantatbezogene unerwünschte Ereignisse (7,0%) und drei unerwünschte Ereignisse ohne Zusammenhang mit dem Implantat (1,8%) auf. Nach unserer Kenntnis ist dies die erste wissenschaftliche Arbeit, die MRT-Unter-suchungen bei Patienten mit Systemen zur Dorsal Root Ganglion Stimulation erfasst. Auch handelt es sich um die erste Publikation hinsichtlich von Systemen zur Spinal Cord Stimulation des Herstellers Abbott Laboratories und übernommener Gesell-schaften (St. Jude Medical, Inc., Advanced Neuromodulation Systems, Inc.). Zum MRT-Bedarf von Patienten mit implantiertem Neurostimulator gibt es bisher nur zwei US-amerikanische Arbeiten; auch die Vorhersagbarkeit des künftigen MRT-Bedarfs wurde von uns erstmals untersucht.:1. Bibliographische Beschreibung 2. Abkürzungsverzeichnis 3. Einführung 3.1 Begriffsbestimmung: Neuromodulation, Neurostimulation 3.2 Spinal Cord Stimulation (SCS) 3.3 Dorsal Root Ganglion Stimulation (DRG-S) 3.4 Magnetresonanztomographie (MRT) 3.5 Problematik von MRT-Untersuchungen bei Patienten mit Neurostimulator und im bzw. nahe am zentralen Nervensystem platzierten Elektroden 3.6 Ziel der Arbeit 4. Publikationen 4.1 Publikation 1: MRT-Bedarf und Vorhersagbarkeit 4.2 Publikation 2: MRT-Sicherheit von SCS-Systemen 4.3. Ergänzende Daten zu MRT-Untersuchungen mit SCS-Systemen 4.3.1 Materialien und Methoden 4.3.2 Analyse der MRT-Anmeldungen ohne folgende MRT-Untersuchung 4.3.3 Ergänzende demographische Daten 4.3.4 MRT-Diagnostik mit unvollständigen oder defekten SCS-Systemen 4.3.5 Analyse der technischen Parameter 4.3.6 MRT-Diagnostik mit SCS-Systemen anderer Hersteller 4.4 Publikation 3: MRT-Sicherheit von DRG-S-Systemen 5. Zusammenfassung der Arbeit 6. Literaturverzeichnis 7. Anlagen 7.1 Erklärung über die eigenständige Abfassung der Arbeit 7.2 Darstellung des eigenen Beitrags 7.3 Lebenslauf 7.4 Publikationsverzeichnis 7.4.1 Beiträge in Fachzeitschriften 7.4.2 Posterbeiträge 7.4.3 Wissenschaftliche Vorträge 7.5 Danksagung

Peripheral nerve trauma is a common cause of considerable functional morbidity, and healthcare expenditure. Particularly in the ~15% of injuries unsuitable for primary repair, standard clinical management results in inadequate sensory restitution in the majority of cases, despite the rigorous application of complex microsurgical techniques. This can largely be explained by the failure of surgical management to adequately address the neurobiological hurdles to optimal regeneration. Most significant of these is the extensive sensory neuronal death that follows injury, and which is accompanied by a reduction in the regenerative potential of axotomised neurons, and in the supportive capacity of the Schwann cell population if nerve repair is delayed. The present study aimed to accurately delineate the timecourse of neuronal death, in order to identify a therapeutic window during which clinically applicable neuroprotective strategies might be adopted. It then proceeded to investigate means to increase the regenerative capacity of chronically axotomised neurons, and to augment the Schwann cells’ ability to promote that regenerative effort. Unilateral sciatic nerve transection in the rat was the model used, initially assessing neuronal death within the L4&5 dorsal root ganglia by a combination of morphology, TdT uptake nick-end labelling (TUNEL), and statistically unbiased estimation of neuronal loss using the stereological optical disector technique. Having identified 2 weeks, and 2 months post-axotomy as the most biologically relevant timepoints to study, the effect upon neuronal death of systemic treatment with acetyl-L-carnitine (ALCAR 10, or 50mg/kg/day) or N-acetyl-cysteine (NAC 30, or 150mg/kg/day) was determined. A model of secondary nerve repair was then adopted; either 2 or 4 months after unilateral sciatic nerve division, 1cm gap repairs were performed using either reversed isografts, or poly-3-hydroxybutyrate (PHB) conduits containing an alginate-fibronectin hydrogel. Six weeks later nerve regeneration and the Schwann cell population were quantified by digital image analysis of frozen section immunohistochemistry. Sensory neuronal death begins within 24 hours of injury, but takes 1 week to translate into significant neuronal loss. The rate of neuronal death peaks 2 weeks after injury, and neuronal loss is essentially complete by 2 months post-axotomy. Nerve repair is incompletely neuroprotective, but the earlier it is performed the greater the benefit. Two clinically safe pharmaceutical agents, ALCAR & NAC, were found to virtually eliminate sensory neuronal death after peripheral nerve transection. ALCAR also enhanced nerve regeneration independently of its neuroprotective role. Plain PHB conduits were found to be technically simple to use, and supported some regeneration, but were not adequate in themselves. Leukaemia inhibitory factor enhanced nerve regeneration, though cultured autologous Schwann cells (SC’s) were somewhat more effective. Both were relatively more efficacious after a 4 month delay in nerve repair. The most profuse regeneration was found with recombinant glial growth factor (rhGGF-2) in repairs performed 2 months after axotomy, with results that were arguably better than were obtained with nerve grafts. A similar conclusion can be drawn from the result found using both rhGGF-2 and SC’s in PHB conduits 4 months after axotomy. In summary, these findings reinforce the significance of sensory neuronal death in peripheral nerve trauma, and the possibility of its` limitation by early nerve repair. Two agents for the adjuvant therapy of such injuries were identified, that can virtually eliminate neuronal death, and enhance regeneration. Elements in the creation of a bioartificial nerve conduit to replace, or surpass autologous nerve graft for secondary nerve repair are presented.