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While the majority of gene therapy studies in neurological indications have focused on direct gene transfer to the central nervous system (CNS), there is growing interest in the delivery of therapeutics using the cerebrospinal fluid (CSF) as a conduit. Historically, direct CNS routes-of-administration (RoAs) have relied on tissue dynamics, displacement of interstitial fluid, and regional specificity to achieve focal delivery into regions of interest, such as the brain. While intraparenchymal delivery minimizes peripheral organ exposure, one perceived drawback is the relative invasiveness of this approach to drug delivery. In this mini review, we examine the CSF as an alternative RoA to target CNS tissue and discuss considerations associated with the safety of performing such procedures, biodistribution of therapeutics following single administration, and translation of findings given differences between small and large animals. These factors will help delineate key considerations for translating data obtained from animal studies into clinical settings that may be useful in the treatment of neurological conditions.
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The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.
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Encéfalo , Gadolínio , Animais , Suínos , Encéfalo/diagnóstico por imagem , Encéfalo/cirurgia , Encéfalo/patologia , Imageamento por Ressonância Magnética/métodos , Técnicas Estereotáxicas , Espectroscopia de Ressonância MagnéticaRESUMO
In this study, we built upon our previous work to demonstrate the distribution and transport of AAV5-green fluorescent protein (GFP) following a single convection-enhanced delivery infusion into the nonhuman primate cerebellum, with no untoward side effects noted. Dosing under magnetic resonance imaging guidance revealed a sixfold larger volume of distribution compared with the volume of infusion, with no evidence of reflux underscoring the convective properties of the cerebellum and step design of the cannula. Postmortem tissue analysis, 4 weeks post-adeno-associated viral (AAV) delivery, revealed the robust presence of the transgene in situ, with GFP detection in secondary regions not directly targeted by the infusion, denoting distal transport of the vector. Irrespective of tropism, a twofold larger area of transgene expression was found and was corroborated against the presence of contrast on T1-weighted images. Different levels of transduction were detected between animals, which were negatively correlated with the level of antibody titer against the GFP construct, whereby the higher the antibody titer, the lower the level of transgene expression. These findings support the use of the posterior fossa as a potential target site for direct delivery of gene-based therapeutics for cerebellar diseases.
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Convecção , Dependovirus , Animais , Cerebelo , Dependovirus/genética , Estudos de Viabilidade , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , PrimatasRESUMO
Non-human primate (NHP) models are the closest approximation of human spinal cord injury (SCI) available for pre-clinical trials. The NHP models, however, include broader morphological variability that can confound experimental outcomes. We developed subject-specific finite element (FE) models to quantify the relationship between impact mechanics and SCI, including the correlations between FE outcomes and tissue damage. Subject-specific models of cervical unilateral contusion SCI were generated from pre-injury MRIs of six NHPs. Stress and strain outcomes were compared with lesion histology using logit analysis. A parallel generic model was constructed to compare the outcomes of subject-specific and generic models. The FE outcomes were correlated more strongly with gray matter damage (0.29 < R2 < 0.76) than white matter (0.18 < R2 < 0.58). Maximum/minimum principal strain, Von-Mises and Tresca stresses showed the strongest correlations (0.31 < R2 < 0.76) with tissue damage in the gray matter while minimum principal strain, Von-Mises stress, and Tresca stress best predicted white matter damage (0.23 < R2 < 0.58). Tissue damage thresholds varied for each subject. The generic FE model captured the impact biomechanics in two of the four models; however, the correlations between FE outcomes and tissue damage were weaker than the subject-specific models (gray matter [0.25 < R2 < 0.69] and white matter [R2 < 0.06] except for one subject [0.26 < R2 < 0.48]). The FE mechanical outputs correlated with tissue damage in spinal cord white and gray matters, and the subject-specific models accurately mimicked the biomechanics of NHP cervical contusion impacts.
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Fenômenos Biomecânicos/fisiologia , Encéfalo/fisiopatologia , Vértebras Cervicais/lesões , Simulação por Computador , Análise de Elementos Finitos , Traumatismos da Medula Espinal/fisiopatologia , Animais , Encéfalo/diagnóstico por imagem , Vértebras Cervicais/diagnóstico por imagem , Simulação por Computador/tendências , Análise de Elementos Finitos/tendências , Humanos , Macaca mulatta , Masculino , Primatas , Traumatismos da Medula Espinal/diagnóstico por imagem , Estresse MecânicoRESUMO
Inhibitory extracellular matrices form around mature neurons as perineuronal nets containing chondroitin sulfate proteoglycans that limit axonal sprouting after CNS injury. The enzyme chondroitinase (Chase) degrades inhibitory chondroitin sulfate proteoglycans and improves axonal sprouting and functional recovery after spinal cord injury in rodents. We evaluated the effects of Chase in rhesus monkeys that had undergone C7 spinal cord hemisection. Four weeks after hemisection, we administered multiple intraparenchymal Chase injections below the lesion, targeting spinal cord circuits that control hand function. Hand function improved significantly in Chase-treated monkeys relative to vehicle-injected controls. Moreover, Chase significantly increased corticospinal axon growth and the number of synapses formed by corticospinal terminals in gray matter caudal to the lesion. No detrimental effects were detected. This approach appears to merit clinical translation in spinal cord injury.
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Condroitinases e Condroitina Liases/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Axônios/patologia , Condroitinases e Condroitina Liases/administração & dosagem , Condroitinases e Condroitina Liases/efeitos adversos , Substância Cinzenta/patologia , Mãos/inervação , Mãos/fisiopatologia , Injeções Intralesionais , Macaca mulatta , Masculino , Microglia/patologia , Neurônios Motores/patologia , Desempenho Psicomotor , Tratos Piramidais/patologia , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologia , Suínos , Sinapses/patologia , Resultado do TratamentoRESUMO
Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF. In particular, MRI revealed differential patterns of infusate distribution depending on the route of delivery. Gadoteridol coverage analysis showed that cerebellomedullary cistern delivery was a reliable and effective route of injection, achieving broad infusate distribution in the brain and spinal cord, and was even greater when combined with lumbar injection. In contrast, intracerebroventricular injection resulted in strong cortical coverage but little spinal distribution. Lumbar injection alone led to the distribution of MRI contrast agent mainly in the spinal cord with little cortical coverage, but this delivery route was unreliable. Similarly, vector clearance analysis showed differences between different routes of delivery. Overall, our data support the value of monitoring CSF injections to dissect different patterns of gadoteridol distribution based on the route of intrathecal administration.
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This study explored the feasibility of intraparenchymal delivery (gadoteridol and/or Serotype 5 Adeno-Associated Viral Vector-enhanced Green Fluorescent Protein [AAV5-eGFP]) into the cerebellum of nonhuman primates using real-time magnetic resonance imaging-guided convection enhanced delivery (MRI-CED) technology. All animals tolerated the neurosurgical procedure without any clinical sequela. Gene expression was detected within the cerebellar parenchyma at the site of infusion and resulted in transduction of neuronal cell bodies and fibers. Histopathology indicated localized damage along the stem of the cannula tract. These findings demonstrate the potential of real-time MRI-CED to deliver therapeutics into the cerebellum, which has extensive reciprocal connections and may be used as a target for the treatment of neurological disorders.
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Cerebelo/metabolismo , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/métodos , Animais , Convecção , Dependovirus/genética , Gadolínio/efeitos adversos , Terapia Genética/efeitos adversos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos/efeitos adversos , Infusões Intraventriculares , Macaca fascicularis , Imageamento por Ressonância Magnética , Masculino , Compostos Organometálicos/efeitos adversosRESUMO
The goal of developing computational models of spinal cord injury (SCI) is to better understand the human injury condition. However, finite element models of human SCI have used rodent spinal cord tissue properties due to a lack of experimental data. Central nervous system tissues in non human primates (NHP) closely resemble that of humans and therefore, it is expected that material constitutive models obtained from NHPs will increase the fidelity and the accuracy of human SCI models. Human SCI most often results from compressive loading and spinal cord white matter properties affect FE predicted patterns of injury; therefore, the objectives of this study were to characterize the unconfined compressive response of NHP spinal cord white matter and present an experimentally derived, finite element tractable constitutive model for the tissue. Cervical spinal cords were harvested from nine male adult NHPs (Macaca mulatta). White matter biopsy samples (3â¯mm in diameter) were taken from both lateral columns of the spinal cord and were divided into four strain rate groups for unconfined dynamic compression and stress relaxation (post-mortem <1-hour). The NHP spinal cord white matter compressive response was sensitive to strain rate and showed substantial stress relaxation confirming the viscoelastic behavior of the material. An Ogden 1st order model best captured the non-linear behavior of NHP white matter in a quasi-linear viscoelastic material model with 4-term Prony series. This study is the first to characterize NHP spinal cord white matter at high (>10/sec) strain rates typical of traumatic injury. The finite element derived material constitutive model of this study will increase the fidelity of SCI computational models and provide important insights for transferring pre-clinical findings to clinical treatments. STATEMENT OF SIGNIFICANCE: Spinal cord injury (SCI) finite element (FE) models provide an important tool to bridge the gap between animal studies and human injury, assess injury prevention technologies (e.g. helmets, seatbelts), and provide insight into the mechanisms of injury. Although, FE model outcomes depend on the assumed material constitutive model, there is limited experimental data for fresh spinal cords and all was obtained from rodent, porcine or bovine tissues. Central nervous system tissues in non human primates (NHP) more closely resemble humans. This study characterizes fresh NHP spinal cord material properties at high strains rates and large deformations typical of SCI for the first time. A constitutive model was defined that can be readily implemented in finite strain FE analysis of SCI.
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Força Compressiva , Elasticidade , Modelos Neurológicos , Estresse Mecânico , Substância Branca/química , Animais , Análise de Elementos Finitos , Humanos , Macaca mulattaRESUMO
PURPOSE: To evaluate the accuracy of percutaneous fluoroscopic injection into the spinal cord of a spine phantom utilizing integrated navigational guidance from fused flat panel detector CT (FDCT) and MR datasets. Conventional and convection-enhanced delivery (CED) techniques were evaluated. MATERIALS AND METHODS: FDCT and MR datasets of a swine thoracic spine phantom were co-registered using an integrated guidance system and surface to spinal cord target trajectory planning was performed on the fused images. Under real-time fluoroscopic guidance with pre-planned trajectory overlay, spinal cord targets were accessed via a coaxial technique. Final needle tip position was compared with a pre-determined target on 10 independent passes. In a subset of cases, contrast was injected into the central spinal cord with a 25G spinal needle or customized 200 µm inner diameter step design cannula for CED. RESULTS: Average needle tip deviation from target measured 0.92±0.5 mm in the transverse, 0.47±0.4 mm in the anterior-posterior, and 1.67±1.2 mm in the craniocaudal dimension for an absolute distance error of 2.12±1.12 mm. CED resulted in elliptical intramedullary diffusion of contrast compared with primary reflux observed with standard needle injection. CONCLUSIONS: These phantom feasibility data demonstrate a minimally invasive percutaneous approach for targeted injection into the spinal cord utilizing real-time fluoroscopy aided by overlay trajectories derived from fused MRI and FDCT data sets with a target error of 2.1 mm. Intramedullary diffusion of injectate in the spinal cord is facilitated with CED compared with standard injection technique. Pre-clinical studies in large animal models are warranted.
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Imageamento por Ressonância Magnética/normas , Agulhas/normas , Imagens de Fantasmas/normas , Software/normas , Medula Espinal/diagnóstico por imagem , Tomografia Computadorizada por Raios X/normas , Animais , Fluoroscopia/métodos , Fluoroscopia/normas , Humanos , Imageamento por Ressonância Magnética/métodos , Suínos , Tomografia Computadorizada por Raios X/métodosRESUMO
We grafted human spinal cord-derived neural progenitor cells (NPCs) into sites of cervical spinal cord injury in rhesus monkeys (Macaca mulatta). Under three-drug immunosuppression, grafts survived at least 9 months postinjury and expressed both neuronal and glial markers. Monkey axons regenerated into grafts and formed synapses. Hundreds of thousands of human axons extended out from grafts through monkey white matter and synapsed in distal gray matter. Grafts gradually matured over 9 months and improved forelimb function beginning several months after grafting. These findings in a 'preclinical trial' support translation of NPC graft therapy to humans with the objective of reconstituting both a neuronal and glial milieu in the site of spinal cord injury.
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Regeneração Nervosa , Células-Tronco Neurais/transplante , Medula Espinal/fisiopatologia , Animais , Axônios/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Humanos , Macaca mulatta , Imageamento por Ressonância Magnética , Masculino , Células-Tronco Neurais/citologia , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Resultado do TratamentoRESUMO
The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-ß. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected.
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Oligodendroglia/efeitos dos fármacos , Piperazinas/farmacologia , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doenças Desmielinizantes/patologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oligodendroglia/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Ensaio Radioligante , Ratos , Receptor de Fator de Crescimento Neural/biossíntese , Receptor trkA/metabolismo , Recuperação de Função FisiológicaRESUMO
The development of a non-human primate (NHP) model of spinal cord injury (SCI) based on mechanical and computational modeling is described. We scaled up from a rodent model to a larger primate model using a highly controllable, friction-free, electronically-driven actuator to generate unilateral C6-C7 spinal cord injuries. Graded contusion lesions with varying degrees of functional recovery, depending upon pre-set impact parameters, were produced in nine NHPs. Protocols and pre-operative magnetic resonance imaging (MRI) were used to optimize the predictability of outcomes by matching impact protocols to the size of each animal's spinal canal, cord, and cerebrospinal fluid space. Post-operative MRI confirmed lesion placement and provided information on lesion volume and spread for comparison with histological measures. We evaluated the relationships between impact parameters, lesion measures, and behavioral outcomes, and confirmed that these relationships were consistent with our previous studies in the rat. In addition to providing multiple univariate outcome measures, we also developed an integrated outcome metric describing the multivariate cervical SCI syndrome. Impacts at the higher ranges of peak force produced highly lateralized and enduring deficits in multiple measures of forelimb and hand function, while lower energy impacts produced early weakness followed by substantial recovery but enduring deficits in fine digital control (e.g., pincer grasp). This model provides a clinically relevant system in which to evaluate the safety and, potentially, the efficacy of candidate translational therapies.
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Contusões/patologia , Modelos Animais de Doenças , Traumatismos da Medula Espinal/patologia , Animais , Vértebras Cervicais , Contusões/cirurgia , Macaca mulatta , Masculino , Traumatismos da Medula Espinal/cirurgiaRESUMO
Non-human primate (NHP) models of spinal cord injury better reflect human injury and provide a better foundation to evaluate potential treatments and functional outcomes. We combined finite element (FE) and surrogate models with impact data derived from in vivo experiments to define the impact mechanics needed to generate a moderate severity unilateral cervical contusion injury in NHPs (Macaca mulatta). Three independent variables (impactor displacement, alignment, and pre-load) were examined to determine their effects on tissue level stresses and strains. Mechanical measures of peak force, peak displacement, peak energy, and tissue stiffness were analyzed as potential determinants of injury severity. Data generated from FE simulations predicted a lateral shift of the spinal cord at high levels of compression (>64%) during impact. Submillimeter changes in mediolateral impactor position over the midline increased peak impact forces (>50%). Surrogate cords established a 0.5 N pre-load protocol for positioning the impactor tip onto the dural surface to define a consistent dorsoventral baseline position before impact, which corresponded with cerebrospinal fluid displacement and entrapment of the spinal cord against the vertebral canal. Based on our simulations, impactor alignment and pre-load were strong contributors to the variable mechanical and functional outcomes observed in in vivo experiments. Peak displacement of 4 mm after a 0.5N pre-load aligned 0.5-1.0 mm over the midline should result in a moderate severity injury; however, the observed peak force and calculated peak energy and tissue stiffness are required to properly characterize the severity and variability of in vivo NHP contusion injuries.
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Simulação por Computador , Modelos Animais de Doenças , Modelos Teóricos , Traumatismos da Medula Espinal , Animais , Macaca mulattaRESUMO
Gene therapy has emerged as a potential avenue of treatment for many neurological disorders. Technological advances in imaging techniques allow for the monitoring of real-time infusions into the brain of rodents, nonhuman primates, and humans. Here, we discuss the use of magnetic resonance imaging (MRI) as a tool in the delivery of adeno-associated viral (AAV) particles into brain of nonhuman primates.
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Encéfalo/virologia , Vetores Genéticos/administração & dosagem , Imageamento por Ressonância Magnética/instrumentação , Animais , Dependovirus/genética , Terapia Genética/métodos , Macaca fascicularis , Macaca mulatta , Imageamento por Ressonância Magnética/métodosRESUMO
Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma.
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Axônios/fisiologia , Regeneração Nervosa/fisiologia , Splicing de RNA/fisiologia , RNA/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Ligases/fisiologia , Camundongos , Compressão Nervosa , Nervo Óptico/patologia , Células Ganglionares da Retina/fisiologia , Nervo Isquiático/patologiaRESUMO
Recent preclinical advances highlight the therapeutic potential of treatments aimed at boosting regeneration and plasticity of spinal circuitry damaged by spinal cord injury (SCI). With several promising candidates being considered for translation into clinical trials, the SCI community has called for a non-human primate model as a crucial validation step to test efficacy and validity of these therapies prior to human testing. The present paper reviews the previous and ongoing efforts of the California Spinal Cord Consortium (CSCC), a multidisciplinary team of experts from 5 University of California medical and research centers, to develop this crucial translational SCI model. We focus on the growing volumes of high resolution data collected by the CSCC, and our efforts to develop a biomedical informatics framework aimed at leveraging multidimensional data to monitor plasticity and repair targeting recovery of hand and arm function. Although the main focus of many researchers is the restoration of voluntary motor control, we also describe our ongoing efforts to add assessments of sensory function, including pain, vital signs during surgery, and recovery of bladder and bowel function. By pooling our multidimensional data resources and building a unified database infrastructure for this clinically relevant translational model of SCI, we are now in a unique position to test promising therapeutic strategies' efficacy on the entire syndrome of SCI. We review analyses highlighting the intersection between motor, sensory, autonomic and pathological contributions to the overall restoration of function. This article is part of a Special Issue entitled SI: Spinal cord injury.
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Modelos Animais de Doenças , Informática Médica , Plasticidade Neuronal , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal , Animais , Humanos , Macaca mulatta , Atividade Motora , Traumatismos da Medula Espinal/fisiopatologia , Pesquisa Translacional Biomédica , Resultado do TratamentoRESUMO
When nanoparticles/proteins are infused into the brain, they are often transported to distal sites in a manner that is dependent both on the characteristics of the infusate and the region targeted. We have previously shown that adeno-associated virus (AAV) is disseminated within the brain by perivascular flow and also by axonal transport. Perivascular distribution usually does not depend strongly on the nature of the infusate. Many proteins, neutral liposomes and AAV particles distribute equally well by this route when infused under pressure into various parenchymal locations. In contrast, axonal transport requires receptor-mediated uptake of AAV by neurons and engagement with specific transport mechanisms previously demonstrated for other neurotropic viruses. Cerebrospinal fluid (CSF) represents yet another way in which brain anatomy may be exploited to distribute nanoparticles broadly in the central nervous system. In this study, we assessed the distribution and perivascular transport of nanoparticles of different sizes delivered into the parenchyma of rodents and CSF in non-human primates.
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Many studies have demonstrated that adeno-associated virus serotype 9 (AAV9) transduces astrocytes and neurons when infused into rat or nonhuman primate (NHP) brain. We previously showed in rats that transduction of antigen-presenting cells (APC) by AAV9 encoding a foreign protein triggered a full neurotoxic immune response. Accordingly, we asked whether this phenomenon occurred in NHP. We performed parenchymal or intrathecal infusion of AAV9 encoding green fluorescent protein (GFP), a non-self protein derived from jellyfish, or human aromatic L-amino acid decarboxylase (hAADC), a self-protein, in separate NHP. Animals receiving AAV9-GFP into cisterna magna (CM) became ataxic, indicating cerebellar pathology, whereas AAV9-hAADC animals remained healthy. In transduced regions, AAV9-GFP elicited inflammation associated with early activation of astrocytic and microglial cells, along with upregulation of major histocompatibility complex class II (MHC-II) in glia. In addition, we found Purkinje neurons lacking calbindin after AAV9-GFP but not after AAV9-hAADC delivery. Our results demonstrate that AAV9-mediated expression of a foreign-protein, but not self-recognized protein, triggers complete immune responses in NHP regardless of the route of administration. Our results warrant caution when contemplating use of serotypes that can transduce APC if the transgene is not syngeneic with the host. This finding has the potential to complicate preclinical toxicology studies in which such vectors encoding human cDNA's are tested in animals.
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Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Dependovirus , Vetores Genéticos , Inflamação/genética , Inflamação/imunologia , Animais , Sistema Nervoso Central/patologia , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dependovirus/genética , Dependovirus/imunologia , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Proteínas de Fluorescência Verde/genética , Humanos , Inflamação/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Transdução Genética , TransgenesRESUMO
This study presents a computational tool for auto-segmenting the distribution of brain infusions observed by magnetic resonance imaging. Clinical usage of direct infusion is increasing as physicians recognize the need to attain high drug concentrations in the target structure with minimal off-target exposure. By co-infusing a Gadolinium-based contrast agent and visualizing the distribution using real-time using magnetic resonance imaging, physicians can make informed decisions about when to stop or adjust the infusion. However, manual segmentation of the images is tedious and affected by subjective preferences for window levels, image interpolation and personal biases about where to delineate the edge of the sloped shoulder of the infusion. This study presents a computational technique that uses a Gaussian Mixture Model to efficiently classify pixels as belonging to either the high-intensity infusate or low-intensity background. The algorithm was implemented as a distributable plug-in for the widely used imaging platform OsiriX®. Four independent operators segmented fourteen anonymized datasets to validate the tool's performance. The datasets were intra-operative magnetic resonance images of infusions into the thalamus or putamen of non-human primates. The tool effectively reproduced the manual segmentation volumes, while significantly reducing intra-operator variability by 67±18%. The tool will be used to increase efficiency and reduce variability in upcoming clinical trials in neuro-oncology and gene therapy.
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Meios de Contraste/farmacocinética , Compostos Heterocíclicos/farmacocinética , Interpretação de Imagem Assistida por Computador , Neuroimagem , Compostos Organometálicos/farmacocinética , Software , Algoritmos , Animais , Meios de Contraste/administração & dosagem , Dependovirus/genética , Gadolínio/administração & dosagem , Gadolínio/farmacocinética , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Compostos Heterocíclicos/administração & dosagem , Infusões Intraventriculares , Imageamento por Ressonância Magnética , Variações Dependentes do Observador , Compostos Organometálicos/administração & dosagem , Primatas , Putamen/metabolismo , Tálamo/metabolismoRESUMO
The present study builds on previous work showing that infusion of adeno-associated virus type 9 (AAV9) into the cisterna magna (CM) of nonhuman primates resulted in widespread transduction throughout cortex and spinal cord. Transduction efficiency was severely limited, however, by the presence of circulating anti-AAV antibodies. Accordingly, we compared AAV9 to a related serotype, AAV7, which has a high capsid homology. CM infusion of either AAV7 or AAV9 directed high level of cell transduction with similar patterns of distribution throughout brain cortex and along the spinal cord. Dorsal root ganglia and corticospinal tracts were also transduced. Both astrocytes and neurons were transduced. Interestingly, little transduction was observed in peripheral organs. Our results indicate that intrathecal delivery of either AAV7 or AAV9 directs a robust and widespread cellular transduction in the central nervous system and other peripheral neural structures.