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1.
Int J Mol Sci ; 24(12)2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37373396

RESUMO

The spinal cord has a poor ability to regenerate after an injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treating a spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface on the other side. When the cells are cultured on OPF via chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to that of the multichannel scaffold control, which is likely due to the greater number of axons growing across it. The immune cell number (microglia or hemopoietic cells: 50-120 cells/mm2 in all conditions), scarring (5-10% in all conditions), and ECM deposits (Laminin or Fibronectin: approximately 10-20% in all conditions) were equal in all conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery. This study provides a hydrogel scaffold construct that can be used in vitro for cell characterization or in vivo for future neuroprosthetics, devices, or cell and ECM delivery.


Assuntos
Organofosfonatos , Traumatismos da Medula Espinal , Ratos , Animais , Hidrogéis/química , Organofosfonatos/metabolismo , Cicatriz/patologia , Ratos Sprague-Dawley , Regeneração Nervosa , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Axônios/patologia , Alicerces Teciduais/química
2.
Neuroscientist ; 28(2): 163-179, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-33089762

RESUMO

Evidence from preclinical and clinical research suggest that neuromodulation technologies can facilitate the sublesional spinal networks, isolated from supraspinal commands after spinal cord injury (SCI), by reestablishing the levels of excitability and enabling descending motor signals via residual connections. Herein, we evaluate available evidence that sublesional and supralesional spinal circuits could form a translesional spinal network after SCI. We further discuss evidence of translesional network reorganization after SCI in the presence of sensory inputs during motor training. In this review, we evaluate potential mechanisms that underlie translesional circuitry reorganization during neuromodulation and rehabilitation in order to enable motor functions after SCI. We discuss the potential of neuromodulation technologies to engage various components that comprise the translesional network, their functional recovery after SCI, and the implications of the concept of translesional network in development of future neuromodulation, rehabilitation, and neuroprosthetics technologies.


Assuntos
Traumatismos da Medula Espinal , Medula Espinal , Humanos , Recuperação de Função Fisiológica
3.
NPJ Regen Med ; 6(1): 66, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34671050

RESUMO

Here, we report the effect of newly regenerated axons via scaffolds on reorganization of spinal circuitry and restoration of motor functions with epidural electrical stimulation (EES). Motor recovery was evaluated for 7 weeks after spinal transection and following implantation with scaffolds seeded with neurotrophin producing Schwann cell and with rapamycin microspheres. Combined treatment with scaffolds and EES-enabled stepping led to functional improvement compared to groups with scaffold or EES, although, the number of axons across scaffolds was not different between groups. Re-transection through the scaffold at week 6 reduced EES-enabled stepping, still demonstrating better performance compared to the other groups. Greater synaptic reorganization in the presence of regenerated axons was found in group with combined therapy. These findings suggest that newly regenerated axons through cell-containing scaffolds with EES-enabled motor training reorganize the sub-lesional circuitry improving motor recovery, demonstrating that neuroregenerative and neuromodulatory therapies cumulatively enhancing motor function after complete SCI.

4.
Spinal Cord ; 59(3): 319-327, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33139846

RESUMO

STUDY DESIGN: Animal study. OBJECTIVES: Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have recently been shown to hold great therapeutic potential for spinal cord injury (SCI). However, majority of the studies have been done using human cells transplanted into the rat with immunosuppression; this may not represent the outcomes that occur in humans. Herein, we present the therapeutic effect of using rat UC-MSCs (rUC-MSC) without immunosuppression in a rat model of SCI. SETTING: Mayo Clinic, Rochester, MN, USA. METHODS: Twelve female rats were randomly divided into two groups, control, and rUC-MSC group, and then subjected to a T9 moderate contusion SCI. Next, 2 × 106 rUC-MSCs or ringer-lactate solution were injected through the tail vein at 7 days post injury. Rats were assessed for 14 weeks by an open-field Basso, Beattie, and Bresnahan (BBB) motor score as well as postmortem quantification of axonal sparing/regeneration, cavity volume, and glial scar. RESULTS: Animals treated with rUC-MSCs were found to have early and sustained motor improvement (BBB score of 14.6 ± 1.9 compared to 10.1 ± 1.7 in the control group) at 14 weeks post injury (mean difference: 4.55, 95% CI: 2.04 to 7.06; p value < 0.001). Total cavity volume in the injury epicenter was significantly reduced in the rUC-MSC group; control: 33.0% ± 2.1, rUC-MSC: 25.3% ± 3.8 (mean difference: -7.7% (95% CI: -12.3 to -2.98); p value < 0.05). In addition, spinal cords from rats treated with rUC-MSCs were found to have a significantly greater number of myelinated axons, decreased astrogliosis, and reduced glial scar formation compared to control rats. CONCLUSIONS: Our study indicates that intravenous injection of allogenic UC-MSCs without immunosuppression exert beneficial effects in subacute SCI and thus could be a useful therapy to improve the functional capacity among patients with SCI.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Traumatismos da Medula Espinal , Animais , Feminino , Humanos , Ratos , Recuperação de Função Fisiológica , Medula Espinal , Traumatismos da Medula Espinal/terapia , Cordão Umbilical
5.
J Tissue Eng Regen Med ; 13(5): 857-873, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30808065

RESUMO

Hydrogel scaffolds provide a beneficial microenvironment in transected rat spinal cord. A combinatorial biomaterials-based strategy provided a microenvironment that facilitated regeneration while reducing foreign body reaction to the three-dimensional spinal cord construct. We used poly lactic-co-glycolic acid microspheres to provide sustained release of rapamycin from Schwann cell (SC)-loaded, positively charged oligo-polyethylene glycol fumarate scaffolds. The biological activity and dose-release characteristics of rapamycin from microspheres alone and from microspheres embedded in the scaffold were determined in vitro. Three dose formulations of rapamycin were compared with controls in 53 rats. We observed a dose-dependent reduction in the fibrotic reaction to the scaffold and improved functional recovery over 6 weeks. Recovery was replicated in a second cohort of 28 animals that included retransection injury. Immunohistochemical and stereological analysis demonstrated that blood vessel number, surface area, vessel diameter, basement membrane collagen, and microvessel phenotype within the regenerated tissue was dependent on the presence of SCs and rapamycin. TRITC-dextran injection demonstrated enhanced perfusion into scaffold channels. Rapamycin also increased the number of descending regenerated axons, as assessed by Fast Blue retrograde axonal tracing. These results demonstrate that normalization of the neovasculature was associated with enhanced axonal regeneration and improved function after spinal cord transection.


Assuntos
Células Imobilizadas , Microesferas , Células de Schwann , Sirolimo , Regeneração da Medula Espinal , Alicerces Teciduais/química , Animais , Linhagem Celular , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Feminino , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Ratos , Ratos Endogâmicos F344 , Células de Schwann/metabolismo , Células de Schwann/patologia , Células de Schwann/transplante , Sirolimo/química , Sirolimo/farmacocinética , Sirolimo/farmacologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual
6.
J Tissue Eng Regen Med ; 12(1): e398-e407, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28296347

RESUMO

Positively-charged oligo[poly(ethylene glycol)fumarate] (OPF+ ) is a biodegradable hydrogel used for spinal cord injury repair. We compared scaffolds containing primary Schwann cells (SCs) to scaffolds delivering SCs genetically modified to secrete high concentrations of glial cell-derived neurotrophic factor (GDNF). Multichannel OPF+ scaffolds loaded with SCs or GDNF-SCs were implanted into transected rat spinal cords for 4 weeks. GDNF-SCs promoted regeneration of more axons into OPF+ scaffolds (2773.0 ± 396.0) than primary SC OPF+ scaffolds (1666.0 ± 352.2) (p = 0.0491). This increase was most significant in central and ventral-midline channels of the scaffold. Axonal remyelination was quantitated by stereologic analysis. Increased myelination of regenerating axons was observed in the GDNF-SC group. Myelinating cell and axon complexes were formed by host SCs and not by implanted cells or host oligodendrocytes. Fast Blue retrograde tracing studies determined the rostral-caudal directionality of axonal growth. The number of neurons that projected axons rostrally through the GDNF-SC scaffolds was higher (7929 ± 1670) than in animals with SC OPF+ scaffolds (1069 ± 241.5) (p < 0.0001). The majority of ascending axons were derived from neurons located more than 15 mm from the scaffold-cord interface, and were identified to be lumbosacral intraspinal motor neurons. Transected animals with GDNF-SC OPF+ scaffolds partially recovered locomotor function at weeks 3 and 4 following surgery. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Axônios/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hidrogéis/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Células de Schwann/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Alicerces Teciduais/química , Animais , Axônios/efeitos dos fármacos , Fumaratos/química , Humanos , Polietilenoglicóis/química , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos
7.
Tissue Eng Part A ; 21(13-14): 2099-114, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25891264

RESUMO

Positively charged oligo[poly(ethylene glycol) fumarate] (OPF+) scaffolds loaded with Schwann cells bridge spinal cord injury (SCI) lesions and support axonal regeneration in rat. The regeneration achieved is not sufficient for inducing functional recovery. Attempts to increase regeneration would benefit from understanding the effects of the scaffold and transplanted cells on lesion environment. We conducted morphometric and stereological analysis of lesions in rats implanted with OPF+ scaffolds with or without loaded Schwann cells 1, 2, 3, 4, and 8 weeks after thoracic spinal cord transection. No differences were found in collagen scarring, cyst formation, astrocyte reactivity, myelin debris, or chondroitin sulfate proteoglycan (CSPG) accumulation. However, when scaffold-implanted animals were compared with animals with transection injuries only, these barriers to regeneration were significantly reduced, accompanied by increased activated macrophages/microglia. This distinctive and regeneration permissive tissue reaction to scaffold implantation was independent of Schwann cell transplantation. Although the tissue reaction was beneficial in the short term, we observed a chronic fibrotic host response, resulting in scaffolds surrounded by collagen at 8 weeks. This study demonstrates that an appropriate biomaterial scaffold improves the environment for regeneration. Future targeting of the host fibrotic response may allow increased axonal regeneration and functional recovery.


Assuntos
Fumaratos/farmacologia , Polietilenoglicóis/farmacologia , Implantação de Prótese , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Alicerces Teciduais/química , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteína Básica da Mielina/metabolismo , Fenótipo , Proteoglicanas/metabolismo , Ratos Sprague-Dawley , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/transplante , Fatores de Tempo
8.
Transfusion ; 55(5): 1013-20, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25413276

RESUMO

BACKGROUND: There are no effective treatments that slow the progression of neurodegenerative diseases. A major challenge of treatment in neurodegenerative diseases is appropriate delivery of pharmaceuticals into the cerebrospinal fluid (CSF) of affected individuals. Mesenchymal stromal cells (MSCs-either naïve or modified) are a promising therapy in neurodegenerative diseases and may be delivered directly into the CSF where they can reside for months. In this preclinical study, we evaluated the safety of intrathecal autologous MSCs in a rabbit model. STUDY DESIGN AND METHODS: Autologous adipose-derived MSCs (or artificial CSF) were delivered intrathecally, either with single or with repeated injections into the foramen magnum of healthy rabbits and monitored for 4 and 12 weeks, respectively. RESULTS: Rabbits tolerated injections well and no definitive MSC-related side effects were observed apart from three rabbits that had delayed death secondary to traumatic foramen magnum puncture. Functional assessments and body weights were equivalent between groups. Gross pathology and histology did not reveal any abnormalities or tumor growth. Complete blood count data were normal and there were no differences in CSF interleukin-6 levels in all groups tested. CONCLUSION: Our data suggest that intrathecal delivery of autologous MSCs is safe in a rabbit model. Data from this study have supported two successful investigational new drug applications to the Food and Drug Administration, resulting in the initiation of two clinical trials using autologous MSCs in amyotrophic lateral sclerosis and multiple system atrophy.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/terapia , Animais , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Modelos Animais de Doenças , Feminino , Humanos , Injeções Espinhais , Interleucina-6/sangue , Interleucina-6/metabolismo , Masculino , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/terapia , Tamanho do Órgão , Coelhos
9.
Tissue Eng Part A ; 20(21-22): 2985-97, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24854680

RESUMO

The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.


Assuntos
Axônios/patologia , Transplante de Células-Tronco Mesenquimais/instrumentação , Neovascularização Fisiológica/fisiologia , Poliésteres/química , Polietilenoglicóis/química , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Alicerces Teciduais , Animais , Células Cultivadas , Análise de Falha de Equipamento , Feminino , Regeneração Tecidual Guiada/instrumentação , Regeneração Tecidual Guiada/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Regeneração Nervosa/fisiologia , Desenho de Prótese , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/fisiopatologia , Resultado do Tratamento
10.
Spine J ; 14(9): 2172-7, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24509005

RESUMO

BACKGROUND CONTEXT: Traumatic injuries occurring at the conus medullaris of the spinal cord cause permanent damage both to the central nervous system and to the cauda equina nerve roots. PURPOSE: This proof-of-concept study was to determine whether implanting the nerve roots into a biodegradable scaffold would improve regeneration after injury. METHODS: All experimental works involving rats were performed according to the approved guidelines by the Mayo Clinic Institutional Animal Care and Use Committee. Surgical procedures were performed on 32 Sprague-Dawley rats. Four ventral cauda equina nerve roots were reimplanted either directly into the ventral cord stump or through a poly(lactic-co-glycolic acid) (PLGA) scaffold. These experimental groups were compared with a control group in which the nerves were inserted into a muscle fascia barrier that was placed between the spinal cord and the nerve roots. Animals were sacrificed at 4 weeks. RESULTS: There was no difference in motor neuron counts in the spinal cord rostral to the injury in all treatment groups, implying equal potential for the regeneration into implanted nerve roots. One-way analysis of variance testing, with Tukey post hoc test, showed a statistically significant improvement in axon regeneration through the injury in the PLGA scaffold treatment group compared with the control (p<.05, scaffold n=11, control n=11). CONCLUSIONS: This pilot study demonstrated that a PLGA scaffold improved regeneration of axons into peripheral nerve roots. However, the number of regenerating axons observed was limited and did not lead to functional recovery. Future experiments will employ a different scaffold material and possible growth factors or enzymes to increase axon populations.


Assuntos
Materiais Biocompatíveis , Cauda Equina/cirurgia , Regeneração Nervosa , Reimplante/métodos , Traumatismos da Medula Espinal/cirurgia , Alicerces Teciduais , Animais , Ácido Láctico , Projetos Piloto , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica
11.
J Neurosci Methods ; 211(2): 179-84, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22960163

RESUMO

Techniques used to produce partial spinal cord injuries in animal models have the potential for creating variability in lesions. The amount of tissue affected may influence the functional outcomes assessed in the animals. The recording of somatosensory evoked potentials (SSEPs) may be a valuable tool for assessing the extent of lesion applied in animal models of traumatic spinal cord injury (SCI). Intraoperative tibial SSEP recordings were assessed during surgically induced lateral thoracic hemisection SCI in Sprague-Dawley rats. The transmission of SSEPs, or lack thereof, was determined and compared against the integrity of the dorsal funiculi on each side of the spinal cord upon histological sectioning. An association was found between the presence of an SSEP signal and presence of intact dorsal funiculus tissue. The relative risk is 4.50 (95% confidence interval: 1.83-11.08) for having an intact dorsal funiculus when the ipsilateral SSEP was present compared to when it was absent. Additionally, the amount of spared spinal cord tissue correlates with final functional assessments at nine weeks post injury: BBB (linear regression, R²=0.618, p<0.001) and treadmill test (linear regression, R²=0.369, p=0.016). Therefore, we propose intraoperative SSEP monitoring as a valuable tool to assess extent of lesion and reduce variability between animals in experimental studies of SCI.


Assuntos
Modelos Animais de Doenças , Potenciais Somatossensoriais Evocados/fisiologia , Monitorização Intraoperatória/métodos , Traumatismos da Medula Espinal/fisiopatologia , Animais , Feminino , Ratos , Ratos Sprague-Dawley
12.
Biomaterials ; 32(32): 8077-86, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21803415

RESUMO

The transected rat thoracic (T(9/10)) spinal cord model is a platform for quantitatively comparing biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ɛ-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF + hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF + polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.


Assuntos
Teste de Materiais/métodos , Polímeros/química , Regeneração da Medula Espinal , Medula Espinal/patologia , Alicerces Teciduais/química , Animais , Axônios/patologia , Comportamento Animal , Contagem de Células , Cistos/patologia , Feminino , Neuroglia/patologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/cirurgia
13.
J Neurosci Methods ; 154(1-2): 256-63, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16466800

RESUMO

We have characterized a method of labeling of axons in the post-mortem spinal cord using a silastic disc holding pins coated with DiI and DiO at the rostral and caudal ends of the cord. We optimized the DiI and DiO tracing techniques under different conditions of fixative concentration (1% versus 4% paraformaldehyde, PF), at room temperature (RT) versus 37 degrees C for up to 24 weeks. Crystal coated pins embedded in a silastic disc provided a novel method of dye application. Confocal microscopy of longitudinal sections showed DiI and DiO labeled both the axonal membrane and myelin sheath. DiI diffused significantly longer distances than DiO. Both dyes migrated greater distances at 37 degrees C compared with RT. No significant difference of dye labeling was found between 1% and 4% PF fixation. After prolonged incubation there was evidence that dye diffused through the aqueous medium and produced circumferential labeling of the cord. Placing a wax seal around the labeling site prevented this non-contiguous labeling. Labeling of myelin sheaths at extended distances into the cord suggested that dye could migrate between cells with prolonged incubation periods. Our data suggested that higher temperature facilitated dye diffusion along the axons, and demonstrated that with caution DiI and DiO could be used as specific tracers in the same spinal cords.


Assuntos
Axônios/fisiologia , Carbocianinas , Corantes , Medula Espinal/anatomia & histologia , Fixação de Tecidos/métodos , Animais , Difusão , Feminino , Fixadores , Formaldeído , Microscopia Confocal , Regeneração Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Temperatura
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