Análisis de la marcha: evaluación de un exoesqueleto aplicado a la marcha asistida

La marcha humana es el mecanismo de locomoción por el cual el cuerpo humano se traslada en línea recta gracias a una serie de movimientos coordinados de la pelvis y de las articulaciones del miembro inferior. Frecuentemente se encuentra influenciada por factores biomecánicos, anatómicos o patologías del sistema neuromusculoesquelético que modifican la forma de caminar de cada individuo. La lesión de médula espinal es una de las patologías que afectan el desarrollo normal de los patrones de la marcha por alteración de la movilidad, de la sensibilidad o del sistema nervioso autónomo. Aunque la lesión medular afecta otras funciones, además de la pérdida de función motora y sensorial, la recuperación de la capacidad de caminar es la mayor prioridad identificada por los pacientes durante la rehabilitación. Por ello, el desarrollo de dispositivos que faciliten la rehabilitación o compensación de la marcha es uno de los principales objetivos de diferentes grupos de investigación y empresas. En el contexto del proyecto Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients se ha desarrollado un dispositivo que combina una órtesis activa (exoesqueleto) y un andador motorizado. Este sistema, como otros dispositivos, tiene el movimiento humano como estándar de referencia, no obstante no se evalúa de manera habitual, cómo es el patrón de la marcha reproducido y su similitud o diferencias con la marcha humana, o las modificaciones o adaptaciones en la interacción con el cuerpo del paciente. El presente estudio trata de examinar las características de la marcha normal en diversos grupos de población, y las diferencias con el patrón de marcha lenta. Finalmente, se pretende evaluar qué modificaciones y adaptaciones sufre el patrón de marcha lenta teórico al ser reproducido por el exoesqueleto. La presente investigación consiste en un estudio cuantitativo transversal desarrollado en dos etapas: estudio 1 y estudio 2. En el estudio 1 se analizó el patrón de la marcha a velocidad libremente seleccionada (normal) y el patrón de la marcha a velocidad lenta (0.25m/s) en 62 sujetos distribuidos en grupos considerando el sexo y los percentiles 25, 50 y 75 de estatura de la población española. Durante el estudio 2 se analizó el patrón de la marcha lenta reproducido por el dispositivo Hybrid a diferentes porcentajes de peso corporal (30%, 50% y 70%) en diez sujetos seleccionados aleatoriamente de la muestra del estudio 1. En ambos estudios se obtuvieron variables espacio-temporales y cinemáticas mediante un sistema de captura de movimiento con 6 cámaras distribuidas a lo largo de un pasillo de marcha. Se calcularon las medias, las desviaciones estándar y el 95% de intervalo de confianza, y el nivel alfa de significación se estableció en α=0.05 para todas las pruebas estadísticas. Las principales diferencias en el patrón normal de la marcha se encontraron en los parámetros cinemáticos de hombres y mujeres, aunque también se presentaron diferencias entre los grupos en función de la estatura. Las mujeres mostraron mayor flexión de cadera y rodilla, y mayor extensión de tobillo que los hombres durante el ciclo normal, aunque la basculación lateral de la pelvis, mayor en las mujeres, y el desplazamiento lateral del centro de gravedad, mayor en los hombres, fueron los parámetros identificados como principales discriminantes entre sexos. La disminución de la velocidad de la marcha mostró similares adaptaciones y modificaciones en hombres y en mujeres, presentándose un aumento de la fase de apoyo y una disminución de la fase de oscilación, un retraso de los máximos y mínimos de flexoextensión de cadera, rodilla y tobillo, y una disminución del rango articular en las tres articulaciones. Asimismo, la basculación lateral de la pelvis y el movimiento vertical del centro de gravedad disminuyeron, mientras que el movimiento lateral del centro de gravedad y el ancho de paso aumentaron. Durante la evaluación del patrón de la marcha reproducido por el exoesqueleto se observó que las tres articulaciones del miembro inferior disminuían el rango de movimiento por la falta de fuerza de los motores para contrarrestar el peso corporal, incluso con un 70% de descarga de peso. Además, la transferencia de peso se encontró limitada por la falta de movimiento de la pelvis en el plano frontal y se sustituyó por un aumento de la inclinación del tronco y, por tanto, del movimiento lateral del centro de gravedad. Este hecho, junto al aumento del desplazamiento vertical del centro de gravedad, hizo del patrón de la marcha reproducido por el exoesqueleto un movimiento poco eficiente. En conclusión, se establecen patrones de marcha normal diferenciados por sexos, siendo la basculación lateral de la pelvis y el movimiento lateral del centro de gravedad los parámetros discriminantes más característicos entre sexos. Comparando la marcha a velocidad libremente seleccionada y la velocidad lenta, se concluye que ambos sexos utilizan estrategias similares para adaptar el patrón de la marcha a una velocidad lenta y se mantienen las características diferenciadoras entre hombres y mujeres. En relación a la evaluación del dispositivo Hybrid, se deduce que la falta de movimiento lateral de la pelvis condiciona la transferencia de peso y el aumento del rango de movimiento del centro de gravedad y, en consecuencia, tiene como resultado un patrón de la marcha poco eficiente. Este patrón no resultaría indicado para los procesos de rehabilitación o recuperación de la marcha, aunque podría considerarse adecuado para la compensación funcional de la bipedestación y la locomoción. ABSTRACT The human walking is a means of moving body forward using a repetitious and coordinated sequence of pelvis and lower limb motions. It is frequently influenced by biomechanical and anatomical factors or by musculoskeletal pathologies which modify the way of walking. The spinal injury is one of those pathologies which affect the normal pattern of walking, due to the alteration of the mobility, the sensory or the autonomic nervous system. Although the spinal injury affects many other body functions, apart from the motor and sensory ones, the main priority for patients is to recover the ability of walking. Consequently, the main objective of many research groups and private companies is the development of rehabilitation and compensation devices for walking. In this context, the Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients project has developed a device which integrates an exoskeleton and a motorized smart walker. This system, as other similar devices, has the human movement as standard reference. Nevertheless, these devices are not usually evaluated on the way they reproduce the normal human pattern or on the modifications and in the interactions with the patient’s body. The aim of the present study is to examine the normal walking characteristics, to analyze the differences between self-selected and low speed walking patterns, and to evaluate the modifications and adaptations of walking pattern when it is reproduced by the exoskeleton. The present research is a quantitative cross-sectional study carried out in two phases: study 1 and study 2. During the study 1, the self-selected and the low speed (0.25m/s) walking patterns were analyzed in sixty-two people distributed in groups, according to sex and 25th, 50th and 75th percentiles of height for Spanish population. The study 2 analyzed the low speed walking pattern reproduced by the Hybrid system in three conditions: 30%, 50% and 70% of body weight support. To do this, ten subjects were randomly selected and analyzed from the people of study 1. An optoelectronic system with six cameras was used to obtain spatial, temporal and kinematic parameters in both studies. Means, standard deviations and 95% confidence intervals of the study were calculated. The alpha level of significance was set at α=0.05 for all statistical tests. The main differences in normal gait pattern were found in kinematic parameters between men and women. The hip and the knee were more flexed and the ankle plantar flexion was higher in women than in men during normal gait cycle. Although the greater pelvic obliquity of women and the higher lateral movement of center of gravity of men were the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, both sexes showed similar adaptations and modifications. At low speed walking, men and women increased the stance phase ratio and decreased the swing phase ratio. The maximum and minimum peak flexion of hip, knee and ankle appeared after and the range of motion of them decreased during low speed walking. Furthermore, the pelvic obliquity and the vertical movement of the center of gravity decreased, whereas the lateral movement of center of gravity and step width increased. Evaluating the gait pattern reproduced by the exoskeleton, a decrease of lower limb range of motion was observed. This was probably due to the lack of strength of the engines, which were not able to control the body weight, even with the 70% supported. Moreover, the weight transfer from one limb to the contralateral side was restricted due to the lack of pelvis obliquity. This movement deficiency was replaced by the lateral torso sway and, consequently, the increase of lateral movement of the center of gravity. This fact, as well as the increase of the vertical displacement of the center of gravity, made inefficient the gait pattern reproduced by the exoskeleton. In conclusion, different gait patterns of both sexes have been determined, being pelvis obliquity and lateral movement of center of gravity the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, it was concluded that both sexes use similar strategies for adapting the gait pattern to a low speed, and therefore, the differentiating characteristics of normal gait are maintained. Regarding the Hybrid system evaluation, it was determined that the gait pattern reproduced by the exoskeleton is inefficient. This was due to the lack of pelvis obliquity and the increase of the center of gravity displacement. Consequently, whereas the walking pattern reproduced by the exoskeleton would not be appropriated for the rehabilitation process, it could be considered suitable for functional compensation of walking and standing.

Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents

Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5′-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/⋅OH formation from the SOD and SOD mutants. The relative ratios of DMPO/⋅17OH and DMPO/⋅16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/⋅17OH and 37% DMPO/⋅16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/⋅17OH (64%); DMPO/⋅16OH (36%) from A4V and DMPO/⋅17OH (62%); and DMPO/⋅16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/⋅OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/⋅OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/⋅OH or DEPMPO/⋅OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Low dimensionality of supraspinally induced force fields

Recent experiments using electrical and N-methyl-d-aspartate microstimulation of the spinal cord gray matter and cutaneous stimulation of the hindlimb of spinalized frogs have provided evidence for a modular organization of the frog’s spinal cord circuitry. A “module” is a functional unit in the spinal cord circuitry that generates a specific motor output by imposing a specific pattern of muscle activation. The output of a module can be characterized as a force field: the collection of the isometric forces generated at the ankle over different locations in the leg’s workspace. Different modules can be combined independently so that their force fields linearly sum. The goal of this study was to ascertain whether the force fields generated by the activation of supraspinal structures could result from combinations of a small number of modules. We recorded a set of force fields generated by the electrical stimulation of the vestibular nerve in seven frogs, and we performed a principal component analysis to study the dimensionality of this set. We found that 94% of the total variation of the data is explained by the first five principal components, a result that indicates that the dimensionality of the set of fields evoked by vestibular stimulation is low. This result is compatible with the hypothesis that vestibular fields are generated by combinations of a small number of spinal modules.

Primary structure and tissue distribution of two novel proline-rich γ-carboxyglutamic acid proteins

Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal γ-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational γ-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called proline-rich Gla proteins (PRGP1 and PRGP2). Unlike the γ-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 ± 2.0 nM; n1 = 170 ± 32 sites per cell vs. Kd1 = 7.2 ± 1.1 nM; n1 = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 ± 0.1 μM; n2 = 1,832 ± 232 sites per cell vs. Kd2 = 1.6 ± 0.1 μM; n2 = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.

The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis

The synthetic amino acid copolymer copolymer 1 (Cop 1) suppresses experimental autoimmune encephalomyelitis (EAE) and is beneficial in multiple sclerosis. To further understand Cop 1 suppressive activity, we studied the cytokine secretion profile of various Cop 1-induced T cell lines and clones. Unlike T cell lines induced by myelin basic protein (MBP), which secreted either T cell helper type 1 (Th1) or both Th1 and Th2 cytokines, the T cell lines/clones induced by Cop 1 showed a progressively polarized development toward the Th2 pathway, until they completely lost the ability to secrete Th1 cytokines. Our findings indicate that the polarization of the Cop 1-induced lines did not result from the immunization vehicle or the in vitro growing conditions, but rather from the tendency of Cop 1 to preferentially induce a Th2 response. The response of all of the Cop 1 specific lines/clones, which were originated in the (SJL/J×BALB/c)F1 hybrids, was restricted to the BALB/c parental haplotype. Even though the Cop 1-induced T cells had not been exposed to the autoantigen MBP, they crossreacted with MBP by secretion of interleukin (IL)-4, IL-6, and IL-10. Administration of these T cells in vivo resulted in suppression of EAE induced by whole mouse spinal cord homogenate, in which several autoantigens may be involved. Secretion of anti-inflammatory cytokines by Cop 1-induced suppressor cells, in response to either Cop 1 or MBP, may explain the therapeutic effect of Cop 1 in EAE and in multiple sclerosis.

EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract

Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.

The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury

The purine nucleoside inosine has been shown to induce axon outgrowth from primary neurons in culture through a direct intracellular mechanism. For this study, we investigated the effects of inosine in vivo by examining whether it would stimulate axon growth after a unilateral transection of the corticospinal tract. Inosine applied with a minipump to the rat sensorimotor cortex stimulated intact pyramidal cells to undergo extensive sprouting of their axons into the denervated spinal cord white matter and adjacent neuropil. Axon growth was visualized by anterograde tracing with biotinylated dextran amine and by immunohistochemistry with antibodies to GAP-43. Thus, inosine, a naturally occurring metabolite without known side effects, might help to restore essential circuitry after injury to the central nervous system.

Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/synaptobrevin and block of exocytosis.

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination

Demyelination is a common pathological finding in human neurological diseases and frequently persists as a result of failure of endogenous repair. Transplanted oligodendrocytes and their precursor cells can (re)myelinate axons, raising the possibility of therapeutic intervention. The migratory capacity of transplanted cells is of key importance in determining the extent of (re)myelination and can, at present, be evaluated only by using invasive and irreversible procedures. We have exploited the transferrin receptor as an efficient intracellular delivery device for magnetic nanoparticles, and transplanted tagged oligodendrocyte progenitor cells into the spinal cord of myelin-deficient rats. Cell migration could be easily detected by using three-dimensional magnetic resonance microscopy, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. The present results demonstrate that magnetic resonance tracking of transplanted oligodendrocyte progenitors is feasible; this technique has the potential to be easily extended to other neurotransplantation studies involving different precursor cell types.