Olfactory ensheathing cells (OECs) are non-myelinating glial cells that provide ensheathment for axons of the olfactory nerve in vivo. OECs have been shown to facilitate the regeneration of CNS axons, to assemble a myelin sheath around demyelinated axons, and it has been suggested OECs migrate very well within the microenvironment of the injured CNS. However, there has been no direct test of their migratory ability in vivo. The aims of this study were to determine whether: 1) OECs can be induced to migrate towards an ethidium bromide (EtBr)-induced focal (~1 mm long) demyelination of the spinal cord white matter; 2) OECs migrate away from a focal demyelination either into normal CNS tissue or towards a second demyelinated lesion; 3) microglial reactivity is required for the generation of the migratory signal(s) inducing OECs to migrate towards a focal demyelination; 4) OECs grafted into the subarachnoid space surrounding the spinal cord will migrate into the neuropil in the absence of demyelination. To achieve these aims, we developed an in vivo model for studying the migratory ability of OECs within the adult rat spinal cord. A small focal EtBr-induced demyelination of the dorsal funiculus (unilaterally) of the spinal cord was made at variable distances from the site of a DiI-labelled OEC graft. The major findings were: i) the strength of the migratory signal(s) inducing OECs to migrate increased as the demyelinated lesion was located closer to the grafting site; ii) the OEC migration towards a distal demyelinated lesion was greatly enhanced when the cells were grafted directly into a second demyelinated lesion; iii) the cell migration occurred along a migratory path containing many reactive astrocytes and microglia; iv) the migration of OECs was significantly reduced when the microglial reactivity was dampened using minocycline; and v) OECs survived grafting into cerebrospinal fluid (i.e. subarachnoid space) and migrated into the neuropil of the brain and spinal cord. The major conclusions are that OECs can respond to migratory signal(s) arising as a result of a focal EtBr-induced demyelination and that microglia are one potential source of these migratory signal(s).

Olfactory ensheathing cells (OECs) are non-myelinating glial cells that provide ensheathment for axons of the olfactory nerve in vivo. OECs have been shown to facilitate the regeneration of CNS axons, to assemble a myelin sheath around demyelinated axons, and it has been suggested OECs migrate very well within the microenvironment of the injured CNS. However, there has been no direct test of their migratory ability in vivo. The aims of this study were to determine whether: 1) OECs can be induced to migrate towards an ethidium bromide (EtBr)-induced focal (~1 mm long) demyelination of the spinal cord white matter; 2) OECs migrate away from a focal demyelination either into normal CNS tissue or towards a second demyelinated lesion; 3) microglial reactivity is required for the generation of the migratory signal(s) inducing OECs to migrate towards a focal demyelination; 4) OECs grafted into the subarachnoid space surrounding the spinal cord will migrate into the neuropil in the absence of demyelination. To achieve these aims, we developed an in vivo model for studying the migratory ability of OECs within the adult rat spinal cord. A small focal EtBr-induced demyelination of the dorsal funiculus (unilaterally) of the spinal cord was made at variable distances from the site of a DiI-labelled OEC graft. The major findings were: i) the strength of the migratory signal(s) inducing OECs to migrate increased as the demyelinated lesion was located closer to the grafting site; ii) the OEC migration towards a distal demyelinated lesion was greatly enhanced when the cells were grafted directly into a second demyelinated lesion; iii) the cell migration occurred along a migratory path containing many reactive astrocytes and microglia; iv) the migration of OECs was significantly reduced when the microglial reactivity was dampened using minocycline; and v) OECs survived grafting into cerebrospinal fluid (i.e. subarachnoid space) and migrated into the neuropil of the brain and spinal cord. The major conclusions are that OECs can respond to migratory signal(s) arising as a result of a focal EtBr-induced demyelination and that microglia are one potential source of these migratory signal(s).

Over the recent years, olfactory ensheathing cells (OECs) have gained world-wide attention due to their reputed potential in promoting spinal cord regeneration and repair. In order to isolate, identify, and characterize OECs in vitro and following implantation, researchers have used three OEC markers: p75NTR, GFAP, and S100. The downfall with using these specific proteins is that Schwann cells, which are located within the olfactory system, as well as migrate into the damaged spinal cord, also express these proteins. It is therefore impossible to distinguish OECs from phenotypically similar Schwann cells using these molecular markers. Recently proteomic analyses have revealed that OECs (derived from embryonic rat olfactory bulbs), but not Schwann cells (derived from adult rat sciatic nerves) express a variety of proteins. The main aim of this project is to determine if heat shock protein-27 (Hsp27), carbonic anhydrase-III (CA-III), and annexin-A3 (Anx3) markers label OECs but not Schwann cells, both in vivo and in vitro. Additional analyses were also done to determine if smooth muscle α-actin (SMA) and calponin (two smooth muscle-related markers previously shown to label mucosal OECs of adult rats) label bulbar OECs of adult rats and OECs of adult cats. Using immunohistochemistry we found that SMA labeled olfactory mucosal and bulbar OECs of adult rats and adult cats, Hsp27 labeled olfactory mucosal and bulbar OECs of adult rats and olfactory mucosal OECs of adult cats, while calponin labeled only olfactory mucosal OECs of adult rats. In addition, calponin and SMA did not label Schwann cells (in vivo and in vitro), while Hsp27 labeled this peripheral glial cell. Finally, CA-III did not label OECs of adult rats or adult cats, in vivo or in vitro, and Anx3 did not label OECs in vivo, but showed immunopositive labeling of OECs and Schwann cells in vitro. In conclusion, Hsp27, CA-III, and Anx3 cannot be used as OECs markers either because of their expression in both OECs and Schwann cells or their lack of expression in OECs. Discovering new molecular markers expressed only by OECs is essential in order to determine the properties, fate, and overall potential of OECs in promoting spinal cord regeneration. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-29 09:50:09.869

Olfactory ensheathing cells (OECs), the glia of the olfactory nerve, are promising candidates for patient-specific cell-mediated repair of both peripheral nerves and the spinal cord. The recent discovery that OECs originate from the neural crest, rather than the olfactory epithelium as previously thought, potentially means that homogeneous populations of OECs for repair could be expanded in culture from neural crest stem cells persisting in the patient's own skin and hair follicles. The first step towards this long-term goal is to understand the molecular mechanisms underlying neural crest differentiation into OECs, as opposed to Schwann cells (the glia of all other peripheral nerves), which are less effective in spinal cord repair. To identify transcription factors and signalling pathways that might be involved in OEC versus Schwann cell differentiation, I took an unbiased transcriptome profiling approach. Taking advantage of Sox10 expression throughout both OEC and Schwann cell development, I used laser-capture microdissection on cryosections of mouse embryos carrying a Sox10:H2BVenus transgene, to isolate OEC subpopulations (olfactory mucosal OECs, from the olfactory nerve, and olfactory nerve layer OECs, from the olfactory nerve layer surrounding the olfactory bulb) at different stages of development, and Schwann cells from trigeminal nerve branches on the same sections, for RNA-seq and cross-wise comparison of transcriptomes. Validation of candidate genes by in situ hybridisation revealed some contamination with adjacent cells from mesenchyme, olfactory epithelium or olfactory bulb, but also identified the expression in developing OECs of various genes previously reported to be expressed in adult OECs, and of over 20 genes previously unknown in OECs. Some of these genes are expressed by OECs but not Schwann cells; some are expressed by olfactory nerve layer OECs but not olfactory mucosal OECs, while some are expressed by olfactory mucosal OECs and Schwann cells but not olfactory nerve layer OECs. For a subset of the genes, I was also able to analyse OEC differentiation in mouse mutants. I also collected transcriptome data from neural crest-derived cells that persist on the olfactory nerve in Sox10-null embryos (in which neural crest-derived cells colonise the olfactory nerve, but normal OEC differentiation is disrupted). Comparison with wild-type OEC transcriptome data from the same embryonic stage identified genes whose expression is likely either downregulated or up-regulated in the absence of Sox10, supporting a role in normal OEC differentiation. Overall, these various transcriptomic comparisons (between OECs at different developmental stages, different OEC subpopulations, OECs versus Schwann cells, and OECs versus Sox10-null neural crest-derived cells on the olfactory nerve) have identified multiple transcription factor and signalling pathway genes, amongst others, that are expressed during OEC development in vivo (including some specific to different OEC subpopulations) and that may be important for OEC differentiation. Furthermore, some of these genes are not expressed by embryonic Schwann cells. This work provides a foundation for understanding how to promote OEC rather than Schwann cell differentiation from neural crest stem cells in culture, with the potential for clinical application in the future.

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. Olfactory ensheathing cells (OECs), specialized glial cells within the olfactory nerve, are thought to play an important role in the ongoing growth and replenishment of sensory connections in this system. To gain insight into novel molecules that could mediate OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments revealed very high expression of the fibulin-3 glycoprotein in OECs. To date, research on fibulin-3 has been limited and mainly focused on its involvement in Doyne honeycomb retinal dystrophy, vasculogenesis and tumor formation. As the extracellular matrix associated with OECs is thought to be an important contributor to a growth-permissive environment, the main aim of this thesis was to define a putative role for fibulin-3 during olfactory receptor neuron replacement and regeneration. This hypothesis was investigated in a series of in vitro and in vivo experiments that involved lentiviral vectors to manipulate fibulin-3 gene expression in OECs as well as the use of knock-out mice. Using genetically-modified OECs, experimental data showed that increased levels of fibulin-3 induced morphological changes in OECs and also impeded their migration. Lentiviral vector-mediated expression of fibulin-3 in OECs also had an inhibitory effect on neurite outgrowth from dorsal root ganglion explants. On the other hand, knock-down of fibulin-3 levels via siRNA technology resulted in reduced proliferation. Comparative lesioning experiments in fibulin-3 knock-out and wild-type mice allowed for further assessment of a role for fibulin-3 in olfactory nerve repair in vivo. Two experimental injury models, i.e. epithelial (Triton-X) lesioning and olfactory bulbectomy, were employed. The results obtained were in line with in vitro observations. A lack of fibulin-3 in knock-out mice resulted in a seemingly augmented regeneration of the olfactory epithelium at 10 days post-injury. However, at the latest recovery time point of 42 days post-injury, an impaired recovery of the olfactory epithelium from the experimental insults was observed. Although the precise mechanism for the latter phenomenon is not yet fully understood, our data point towards several factors which include vascular abnormalities and altered cell proliferation within the olfactory epithelium. Additionally, the precise protein distribution of another wide-spread family of extracellular matrix molecules, the laminins, was investigated in this thesis. It was of interest to investigate the spatiotemporal expression of laminin isoforms during iii olfactory nerve development and regeneration as these molecules may have distinct roles in promoting olfactory sensory neuron growth and patterning. In situ hybridization and immunohistochemical studies concluded that laminin-211 and laminin-411 were the most likely candidates to play such a role. In summary, this thesis provides new insights into the role of the extracellular matrix, fibulin-3 in particular, in regulating cell migration, division and axonal growth in the primary olfactory pathway. Such knowledge also gives a greater understanding of the molecular mechanisms by which OEC transplants may enhance axonal regeneration elsewhere in the CNS.

Disorders of the autonomic nervous system constitute serious complications of spinal cord injury (SCI) and their treatment is usually highly prioritised by spinal patients. Among these, autonomic dysreflexia and impaired thermoregulation are potentially life threatening conditions and require effective management. Olfactory ensheathing cells (OECs), progenitor cells and polymeric scaffolds have been tested in animal models of SCI and some of them have been considered for clinical trials. However, evaluation of the effect of such interventions on autonomic functions has received only rudimentary attention and would require a more thorough experimental assessment before the methods are utilised in human patients. This thesis tested two potential therapeutic strategies for autonomic dysreflexia and examined disorders of thermoregulatory functions in a rat model of spinal cord transection. Magnitude and duration of autonomic dysreflexia were evaluated with radio telemetry in spinalised animals treated with (i) implants of OECs and olfactory neurosphere-derived cells seeded in poly(lactic co glycolic) porous scaffolds or with (ii) transplants of OECs alone. (iii) Effects of SCI and of OECs on the morphology of sympathetic preganglionic neurons (SPNs; which are involved in pathogenesis of autonomic dysreflexia) stained for NADPH diaphorase were examined. (iv) Doppler ultrasonography and infrared thermography were used to assess responses of tail blood flow and surface temperature to cold. Transplants of OECs alone, but not in combination with olfactory neurosphere-derived cells and polymeric scaffolds, resulted in significantly shortened episodes of autonomic dysreflexia. This may be attributed to the alterations to the morphology of SPNs adjacent to the lesion: a transient increase in the morphometric features of the SPNs was evoked by spinal cord transection and this was further altered by transplantation of OECs. The thesis also showed that local responses of tail blood flow and temperature to cold were not abolished by complete SCI suggesting that temperature homeostasis could still be maintained in response to cold. It is hypothesised that OECs facilitate improved recovery from autonomic dysreflexia through alteration of the morphology of SPNs. Furthermore, it is suggested that the role of the tail in heat conservation can be regulated by mechanisms that are independent of the descendent neural control from supraspinal centres.

Disorders of the autonomic nervous system constitute serious complications of spinal cord injury (SCI) and their treatment is usually highly prioritised by spinal patients. Among these, autonomic dysreflexia and impaired thermoregulation are potentially life threatening conditions and require effective management. Olfactory ensheathing cells (OECs), progenitor cells and polymeric scaffolds have been tested in animal models of SCI and some of them have been considered for clinical trials. However, evaluation of the effect of such interventions on autonomic functions has received only rudimentary attention and would require a more thorough experimental assessment before the methods are utilised in human patients. This thesis tested two potential therapeutic strategies for autonomic dysreflexia and examined disorders of thermoregulatory functions in a rat model of spinal cord transection. Magnitude and duration of autonomic dysreflexia were evaluated with radio telemetry in spinalised animals treated with (i) implants of OECs and olfactory neurosphere-derived cells seeded in poly(lactic co glycolic) porous scaffolds or with (ii) transplants of OECs alone. (iii) Effects of SCI and of OECs on the morphology of sympathetic preganglionic neurons (SPNs; which are involved in pathogenesis of autonomic dysreflexia) stained for NADPH diaphorase were examined. (iv) Doppler ultrasonography and infrared thermography were used to assess responses of tail blood flow and surface temperature to cold. Transplants of OECs alone, but not in combination with olfactory neurosphere-derived cells and polymeric scaffolds, resulted in significantly shortened episodes of autonomic dysreflexia. This may be attributed to the alterations to the morphology of SPNs adjacent to the lesion: a transient increase in the morphometric features of the SPNs was evoked by spinal cord transection and this was further altered by transplantation of OECs. The thesis also showed that local responses of tail blood flow and temperature to cold were not abolished by complete SCI suggesting that temperature homeostasis could still be maintained in response to cold. It is hypothesised that OECs facilitate improved recovery from autonomic dysreflexia through alteration of the morphology of SPNs. Furthermore, it is suggested that the role of the tail in heat conservation can be regulated by mechanisms that are independent of the descendent neural control from supraspinal centres.

Chez le mammifère la fonction de reproduction est sous le contrôle des neurones hypothalamiques à GnRH-I. Au cours du développement embryonnaire ces neurones migrent de la fosse nasale vers le cerveau. De nombreuses études s’intéressent aux facteurs impliqués dans leur migration, mais l’influence de leur environnement cellulaire est très peu étudiée. Nous avons émis l’hypothèse que les neurones à GnRH-I d’origine extra-cérébrale possèdent un environnement gliale nécessaire à leur migration, connaissant le rôle de ces cellules dans l’ontogenèse neuronale du cerveau. Nos résultats montrent que 1) les neurones à GnRH-I sont associés à des cellules gliales au cours de leurs migrations nasale et télencéphalique 2) ces cellules gliales sont des progéniteurs des cellules gliales olfactives engainantes qui se différencient dans les régions rostrales au cours de la migration neuronale. 3) ces cellules expriment des gènes codant pour des facteurs impliqués dans la migration de ces neurones. 4) le transcriptome de ces cellules gliales est perturbé en présence d’un perturbateur endocrinien œstrogèno-mimétique, et touche des familles de gènes impliquées dans les molécules d’adhésions cellulaires nécessaire à la migration et à la régulation de l’activité des neurones à GnRH-I. / GnRH-I cells control reproduction functions in mammals. These cells are extra cerebral since they come from the nasal pit and migrate to the forebrain during embryonic development. Numerous studies have described the influence of different molecules on the migration of GnRH-1 neurons, however, the role of microenvironment cells remains poorly understood. Considering the role of glial cells in the forebrain’s neuronal migration, we had hypothesized that extra-cerebral GnRH-I neurons possess a glial environment necessary for their migration from the nose to the brain. Our results demonstrated that 1) GnRH-I neurons are associated with glial cells during their migration in the nasal septum and forebrain 2) These glial cells are progenitors of olfactory ensheathing cells, and differentiated within the rostral regions during neuronal migration. 3) These cells express genes encoding factors involved in GnRH-I neurons migration 4) Glial cells transcriptome are disrupted with estrogen-mimicking endocrine disruptor, and affects gene families involved in cell adhesion molecules necessary for migration and activity regulation of GnRH-I neurons

Lesões medulares resultam em uma perda irreversível da função abaixo do sítio da lesão. Esses comprometimentos são permanentes e ocorrem devido à perda de neurônios localmente e também dos tratos axonais ascendentes e descendentes da medula espinal. Na tentativa de criar um ambiente favorável à regeneração dos axônios lesionados, células da glia embainhante olfatória (GEO) vêm sendo transplantadas como estratégia de tratamento em animais submetidos a diferentes modelos experimentais de lesões medulares. Entretanto, um consenso sobre o potencial terapêutico desse tipo de transplante celular ainda precisa ser estabelecido. O objetivo do presente trabalho foi verificar a eficácia do transplante de lâmina própria (LP) olfatória (que possui células da GEO) e de LP respiratória (desprovido de células da GEO), quando implantadas imediatamente, 2 ou 4 semanas após a realização da transecção da medula espinal. Doze semanas após a realização dos implantes, os animais que receberam LP olfatória e respiratória apresentaram uma melhora sutil na função motora dos membros posteriores. Além disso, o transplante de LP olfatória quando realizado imediatamente após a lesão reduziu a hiperatividade do reflexo de retirada, enquanto o implante desse tipo de tecido 4 semanas pós-lesão produziu uma discreta depressão dependente de frequência do reflexo de Hoffman (um análogo elétrico do reflexo monossináptico de estiramento). Nas diferentes janelas terapêuticas utilizadas, o transplante de ambos os tipos de LP produziu resultados comparáveis em relação à preservação do tecido medular, brotamento de neuritos e regeneração de fibras mielínicas no local da lesão, indicando que o tempo decorrido antes da realização dos transplantes não parece limitar os efeitos regenerativos. Todavia, as fibras mielínicas observadas no sítio da transecção nos animais que receberam LP olfatória 2 e 4 semanas pós-lesão possuíam menor área, diâmetro e espessura da bainha de mielina quando comparados aos animais que receberam LP respiratória nesses mesmos períodos. O transplante imediato de LP olfatória e respiratória também favoreceu o restabelecimento das conexões entre as fibras axonais lesionadas com núcleos do tronco encefálico e até mesmo com a região do córtex somatossensorial, como indicado pela presença de neurônios nessas regiões marcados positivamente com um marcador axonal retrógrado. Um número maior de fibras positivas para 5-HT foi observado no coto proximal dos grupos transplantados com ambos os tipos de LP em comparação às regiões da lesão e do coto caudal. Fibras positivas para CGRP estavam presentes em número considerável no local da lesão. A recuperação locomotora e a regeneração axonal no local da lesão foram limitadas e comparáveis entre os grupos transplantados nos diferentes tempos com LP olfatória e respiratória, sugerindo que esses resultados não estão exclusivamente relacionados à presença de células da GEO nos enxertos utilizados. Um melhor entendimento sobre o potencial restaurativo desse tipo de transplante é necessário a fim de justificar a aplicação dessa terapia em humanos. / Spinal cord injury (SCI) results in an irreversible loss of function below the injury site. These permanent disabilities occur due to local neuronal death and loss of ascending and descending axons in the spinal cord. In attempt to create a favorable environment for the re-growth of injured axons, olfactory ensheathing cells (OECs) have been transplanted as a treatment strategy in animals submitted to different experimental models of SCI. However, a consensus on the efficacy of this cellular transplantation has yet to be reached. The main focus of the present study was explore the efficacy of olfactory lamina propria (OLP, graft containing OECs) or respiratory lamina propria (RLP, graft without OECs) when transplanted immediately, 2-week or 4-week after spinal cord transection. After 12 weeks of transplantation, animals with OLP and RLP grafts showed a subtle hindlimb motor improvement. Furthermore, the transplantation of OLP when performed immediately after injury reduced the withdrawal reflex over-responsiveness, while the implantation of this tissue 4 weeks post-injury produced a discrete frequency-dependent habituation of the Hoffman reflex (the electrical analogue of the classic tendon jerk reflex). In all therapeutic windows used, both lamina propria grafts produced comparable results for tissue sparing, fibers sprouting and re-growth of myelinated fibers at the lesion site, indicating that delayed transplantation approach does not seem to limit the regenerative effects. However, the myelinated fibers observed at the transection site of animals that received OLP 2 or 4 weeks after injury had a smaller myelinated fiber area, diameter and myelin sheath thickness when compared to those animals transplanted with RLP grafts in the same periods. The immediate transplantation of OLP and RLP also foster limited supraspinal axonal re-connection as shown by the presence of neurons stained by retrograde tracing in brainstem nuclei and in the somatosensory cortex. A larger number of 5-HT positive axons were found in the cranial stump of both lamina propria groups compared to the lesion and caudal regions. CGRP positive axons were present in considerable numbers at the SCI site. The locomotor recovery and axon reparative effects were limited and similar between groups transplanted at different times with OLP and RLP, suggesting that these results could not be exclusively related to OECs. In conclusion, a greater understanding of the restorative potential of these tissue grafts is necessary to strengthen the rationale for application of this treatment in humans.

Lesões medulares resultam em uma perda irreversível da função abaixo do sítio da lesão. Esses comprometimentos são permanentes e ocorrem devido à perda de neurônios localmente e também dos tratos axonais ascendentes e descendentes da medula espinal. Na tentativa de criar um ambiente favorável à regeneração dos axônios lesionados, células da glia embainhante olfatória (GEO) vêm sendo transplantadas como estratégia de tratamento em animais submetidos a diferentes modelos experimentais de lesões medulares. Entretanto, um consenso sobre o potencial terapêutico desse tipo de transplante celular ainda precisa ser estabelecido. O objetivo do presente trabalho foi verificar a eficácia do transplante de lâmina própria (LP) olfatória (que possui células da GEO) e de LP respiratória (desprovido de células da GEO), quando implantadas imediatamente, 2 ou 4 semanas após a realização da transecção da medula espinal. Doze semanas após a realização dos implantes, os animais que receberam LP olfatória e respiratória apresentaram uma melhora sutil na função motora dos membros posteriores. Além disso, o transplante de LP olfatória quando realizado imediatamente após a lesão reduziu a hiperatividade do reflexo de retirada, enquanto o implante desse tipo de tecido 4 semanas pós-lesão produziu uma discreta depressão dependente de frequência do reflexo de Hoffman (um análogo elétrico do reflexo monossináptico de estiramento). Nas diferentes janelas terapêuticas utilizadas, o transplante de ambos os tipos de LP produziu resultados comparáveis em relação à preservação do tecido medular, brotamento de neuritos e regeneração de fibras mielínicas no local da lesão, indicando que o tempo decorrido antes da realização dos transplantes não parece limitar os efeitos regenerativos. Todavia, as fibras mielínicas observadas no sítio da transecção nos animais que receberam LP olfatória 2 e 4 semanas pós-lesão possuíam menor área, diâmetro e espessura da bainha de mielina quando comparados aos animais que receberam LP respiratória nesses mesmos períodos. O transplante imediato de LP olfatória e respiratória também favoreceu o restabelecimento das conexões entre as fibras axonais lesionadas com núcleos do tronco encefálico e até mesmo com a região do córtex somatossensorial, como indicado pela presença de neurônios nessas regiões marcados positivamente com um marcador axonal retrógrado. Um número maior de fibras positivas para 5-HT foi observado no coto proximal dos grupos transplantados com ambos os tipos de LP em comparação às regiões da lesão e do coto caudal. Fibras positivas para CGRP estavam presentes em número considerável no local da lesão. A recuperação locomotora e a regeneração axonal no local da lesão foram limitadas e comparáveis entre os grupos transplantados nos diferentes tempos com LP olfatória e respiratória, sugerindo que esses resultados não estão exclusivamente relacionados à presença de células da GEO nos enxertos utilizados. Um melhor entendimento sobre o potencial restaurativo desse tipo de transplante é necessário a fim de justificar a aplicação dessa terapia em humanos. / Spinal cord injury (SCI) results in an irreversible loss of function below the injury site. These permanent disabilities occur due to local neuronal death and loss of ascending and descending axons in the spinal cord. In attempt to create a favorable environment for the re-growth of injured axons, olfactory ensheathing cells (OECs) have been transplanted as a treatment strategy in animals submitted to different experimental models of SCI. However, a consensus on the efficacy of this cellular transplantation has yet to be reached. The main focus of the present study was explore the efficacy of olfactory lamina propria (OLP, graft containing OECs) or respiratory lamina propria (RLP, graft without OECs) when transplanted immediately, 2-week or 4-week after spinal cord transection. After 12 weeks of transplantation, animals with OLP and RLP grafts showed a subtle hindlimb motor improvement. Furthermore, the transplantation of OLP when performed immediately after injury reduced the withdrawal reflex over-responsiveness, while the implantation of this tissue 4 weeks post-injury produced a discrete frequency-dependent habituation of the Hoffman reflex (the electrical analogue of the classic tendon jerk reflex). In all therapeutic windows used, both lamina propria grafts produced comparable results for tissue sparing, fibers sprouting and re-growth of myelinated fibers at the lesion site, indicating that delayed transplantation approach does not seem to limit the regenerative effects. However, the myelinated fibers observed at the transection site of animals that received OLP 2 or 4 weeks after injury had a smaller myelinated fiber area, diameter and myelin sheath thickness when compared to those animals transplanted with RLP grafts in the same periods. The immediate transplantation of OLP and RLP also foster limited supraspinal axonal re-connection as shown by the presence of neurons stained by retrograde tracing in brainstem nuclei and in the somatosensory cortex. A larger number of 5-HT positive axons were found in the cranial stump of both lamina propria groups compared to the lesion and caudal regions. CGRP positive axons were present in considerable numbers at the SCI site. The locomotor recovery and axon reparative effects were limited and similar between groups transplanted at different times with OLP and RLP, suggesting that these results could not be exclusively related to OECs. In conclusion, a greater understanding of the restorative potential of these tissue grafts is necessary to strengthen the rationale for application of this treatment in humans.