Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat.

Great interest exists in using cell replacement strategies to repair the damaged central nervous system. Previous studies have shown that grafting rat fetal spinal cord into neonate or adult animals after spinal cord injury leads to improved anatomic growth/plasticity and functional recovery. It is clear that fetal tissue transplants serve as a scaffold for host axon growth. In addition, embryonic Day 14 (E14) spinal cord tissue transplants are also a rich source of neural-restricted and glial-restricted progenitors. To evaluate the potential of E14 spinal cord progenitor cells, we used in vitro-expanded neurospheres derived from embryonic rat spinal cord and showed that these cells grafted into lesioned neonatal rat spinal cord can survive, migrate, and differentiate into neurons and oligodendrocytes, but rarely into astrocytes. Synapses and partially myelinated axons were detected within the transplant lesion area. Transplanted progenitor cells resulted in increased plasticity or regeneration of corticospinal and brainstem-spinal fibers as determined by anterograde and retrograde labeling. Furthermore, transplantation of these cells promoted functional recovery of locomotion and reflex responses. These data demonstrate that progenitor cells when transplanted into neonates can function in a similar capacity as transplants of solid fetal spinal cord tissue.

Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins.

Little axonal regeneration occurs after spinal cord injury in adult mammals. Regrowth of mature CNS axons can be induced, however, by altering the intrinsic capacity of the neurons for growth or by providing a permissive environment at the injury site. Fetal spinal cord transplants and neurotrophins were used to influence axonal regeneration in the adult rat after complete spinal cord transection at a midthoracic level. Transplants were placed into the lesion cavity either immediately after transection (acute injury) or after a 2-4 week delay (delayed or chronic transplants), and either vehicle or neurotrophic factors were administered exogenously via an implanted minipump. Host axons grew into the transplant in all groups. Surprisingly, regeneration from supraspinal pathways and recovery of motor function were dramatically increased when transplants and neurotrophins were delayed until 2-4 weeks after transection rather than applied acutely. Axonal growth back into the spinal cord below the lesion and transplants was seen only in the presence of neurotrophic factors. Furthermore, the restoration of anatomical connections across the injury site was associated with recovery of function with animals exhibiting plantar foot placement and weight-supported stepping. These findings suggest that the opportunity for intervention after spinal cord injury may be greater than originally envisioned and that CNS neurons with long-standing injuries can reinitiate growth, leading to improvement in motor function.

Intervention strategies to enhance anatomical plasticity and recovery of function after spinal cord injury.

Taken together, our studies indicate that (a) transplants mediate recovery of skilled forelimb movement as well as locomotor activity, (b) combinations of interventions may be required to restore reflex, sensory, and locomotor function to more normal levels after SCI, and (c) that remodeling of particular pathways may contribute to recovery of rather specific aspects of motor function. In conclusion, we suggest that it seems unlikely that any single intervention strategy will be sufficient to ensure regeneration of damaged pathways and recovery of function after SCI. Clearly, work from a number of laboratories indicates that the dogma that mature CNS neurons are inherently incapable of regeneration of axons after injury is no longer tenable. The issue, rather, is to identify and reverse the conditions that limit regeneration after SCI. After SCI, a hierarchy of "intervention-strategies" may be required to restore suprasegmental control leading to recovery of function. The hierarchy may be both temporal and absolute. For example, early interventions (such as the administration of methylprednisolone within hours of the injury) may be required to interrupt the secondary injury cascade and restrict the extent of damage after SCI. At the injury site itself, interventions to minimize the secondary injury effects may be followed by interventions to alter the environment at the site of injury to provide a terrain conducive to axonal elongation. For example, one might envision strategies to downregulate the expression of molecules that limit growth and upregulate the expression of those that support growth. Early after the injury, axotomized neurons may require neurotrophic support either for their survival or to initiate and maintain a cell body response supporting axonal elongation. There may be an absolute hierarchy as well. Particular populations of neurons may have very specific requirements for regenerative growth. For example, the conditions that enhance the regenerative growth of descending motor pathways may differ from those required by ascending sensory systems. One may also want to design strategies to restrict the plasticity of some pathways (e.g., nociceptive) and enhance the growth in other pathways. The demands on the CNS for anatomic reorganization after SCI may be far less formidable than one might at first imagine. If one assumes that recovery of function will require regenerative growth of large numbers of axons over long distances in a point-to-point topographically specific fashion, the idea of recovery of function becomes daunting. On the other hand, it has been shown in many studies and in many areas of the CNS that as little as 10% of a particular pathway can often subserve substantial function. Furthermore, regrowth over relatively short distances can have major functional consequences. For example, relatively modest changes in the level of SCI can have relatively profound effects on the functional consequences of injury. This is particularly true in cervical SCI: an individual with a C5/6 SCI is dramatically more impaired than one with C7/8 injury. One might envision relatively short distance growth across the injury site to re-establish suprasegmental control. Coupled with strategies to enhance the anatomic and functional reorganization of spinal cord circuitry caudal to the level of the injury, even modest long distance growth may have sufficient functional impact. One might imagine the ability to learn to "use" even modest quantities of novel inputs in functionally useful, appropriate ways.

Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat.

The capacity of CNS neurons for axonal regrowth after injury decreases as the age of the animal at time of injury increases. After spinal cord lesions at birth, there is extensive regenerative growth into and beyond a transplant of fetal spinal cord tissue placed at the injury site. After injury in the adult, however, although host corticospinal and brainstem-spinal axons project into the transplant, their distribution is restricted to within 200 micron of the host/transplant border. The aim of this study was to determine if the administration of neurotrophic factors could increase the capacity of mature CNS neurons for regrowth after injury. Spinal cord hemisection lesions were made at cervical or thoracic levels in adult rats. Transplants of E14 fetal spinal cord tissue were placed into the lesion site. The following neurotrophic factors were administered at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary-derived neurotrophic factor (CNTF), or vehicle alone. After 1-2 months survival, neuroanatomical tracing and immunocytochemical methods were used to examine the growth of host axons within the transplants. The neurotrophin administration led to increases in the extent of serotonergic, noradrenergic, and corticospinal axonal ingrowth within the transplants. The influence of the administration of the neurotrophins on the growth of injured CNS axons was not a generalized effect of growth factors per se, since the administration of CNTF had no effect on the growth of any of the descending CNS axons tested. These results indicate that in addition to influencing the survival of developing CNS and PNS neurons, neurotrophic factors are able to exert a neurotropic influence on injured mature CNS neurons by increasing their axonal growth within a transplant.

Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors.

There is little axonal growth after central nervous system (CNS) injury in adult mammals. The administration of antibodies (IN-1) to neutralize the myelin-associated neurite growth inhibitory proteins leads to long-distance regrowth of a proportion of CNS axons after injury. Our aim was: to determine if spinal cord lesion in adult rats, followed by treatment with antibodies to neurite growth inhibitors, can lead to regeneration and anatomical plasticity of other spinally projecting pathways; to determine if the anatomical projections persist at long survival intervals; and to determine whether this fibre growth is associated with recovery of function. We report here that brain stem-spinal as well as corticospinal axons undergo regeneration and anatomical plasticity after application of IN-1 antibodies. There is a recovery of specific reflex and locomotor functions after spinal cord injury in these adult rats. Removal of the sensorimotor cortex in IN-1-treated rats 2-3 months later abolished the recovered contact-placing responses, suggesting that the recovery was dependent upon the regrowth of these pathways.

Recovery of function after spinal cord injury: mechanisms underlying transplant-mediated recovery of function differ after spinal cord injury in newborn and adult rats.

Fetal spinal cord transplants placed into the site of spinal cord injury support axonal growth of host systems in both newborn and adult animals. The amount of axonal growth, however, is much more robust in the newborn animals. The current studies were designed to determine if the differences in the magnitude of the anatomical plasticity of host pathways in the presence of transplants is reflected in differences in recovery of function between the neonatal and adult operates. Newborn and adult rats received a midthoracic "overhemisection." Immediately following the hemisection embryonic (E14) spinal cord transplants were placed into the lesion site. All animals were trained and tested as adults, on a battery of qualitative and quantitative tests of motor function. Immunocytochemical methods were used to compare the extent of growth of descending (serotonergic and noradrenergic) and segmental (calcitonin gene-related peptide containing dorsal root axons) pathways in both groups. The growth of descending pathways into the transplants was substantially greater in density and spatial extent after lesions at birth than at maturity. The distribution of segmental dorsal root axons, in contrast, was similar in both groups. Fetal spinal cord transplants promoted recovery of motor function in both newborn and adult operates. The particular aspects of locomotor function which recover differ between the neonatal and adult operates, suggesting that the mechanisms underlying recovery of function must differ between the two groups.

Methods to assess the development and recovery of locomotor function after spinal cord injury in rats.

The ability to assess recovery of function after spinal cord injury is a very important part of spinal cord injury research. Recent progress has been made in a number of avenues of treatment designed to ameliorate the consequences of spinal cord injury and enhance recovery of function. This potential for intervention to modify the sequellae of spinal cord injury requires stringent criteria for methods used to evaluate the effects of injury and subsequent recovery of function. Methods which rely on composite ratings of an animal's overall performance, while appropriate for screening groups of animals with spinal cord injury, are not sufficient to demonstrate whether a particular treatment has had a specific effect on motor function or the degree to which function is affected. We have designed a series of sensitive quantitative methods to assess the recovery of locomotor function in rats. The methods examine specific reflex responses and specific components of motor behavior and are sensitive to subtle differences in the pattern of locomotion and individual limb movements. Several of the tests can be used to assess the development of locomotor function as well as the mature response. Postural reflex testing and locomotor function under conditions of graded difficulty are examined and the motor capacity of individual limbs is assessed. Animals are trained to cross runways, to walk on a treadmill, and to climb onto a platform. The animals' performance is videotaped for subsequent quantitative analysis. The pattern of overground and treadmill locomotion is also examined by footprint analysis. Spinal cord injury alters an animal's reflex responses and deficits are evident in locomotor function. Examples are given of the quantitative measurements obtained from analysis of the animals' performance on each of the tests. No single test is sufficient to assess recovery of function after spinal cord injury. Rather, a combination of tests, each examining particular components of normal and recovered motor function, is required. The methods used to assess recovery of locomotor function are specific, are sensitive, and allow individual limb movements to be isolated. Such specific methods allow one to begin to address the mechanisms underlying recovery of function following spinal cord injury.

Recovery of function after spinal cord hemisection in newborn and adult rats: differential effects on reflex and locomotor function.

It is often assumed that the response of the immature nervous system to injury is more robust and exhibits greater anatomical reorganization and greater recovery of function than in the adult. In the present experiments the extent of recovery of function after spinal cord injury at birth or at maturity was assessed. We used a series of quantitative tests of motor behavior to measure reflex responses and triggered movements and to examine different components of locomotion. Rats received a midthoracic "over-hemisection" at birth or as adults. The neonatal operates were allowed to mature and the adult operates were allowed to recover. The animals were trained to walk on a treadmill and to cross runways of varying difficulty. The animals were tested for reflex responses and triggered movements, videotaped while crossing the runways, and footprinted while walking on the treadmill. The adult operates had greater deficits in the reflex responses than the neonatal operates. The adult operates lost the contact placing response and had a decreased hopping response in the ipsilateral limb, while these responses were not impaired in the neonatal operates. Although the contact placing response in the neonatal operates was spared, a greater stimulus was necessary to induce the response than in control animals. In contrast, the neonatal operates had greater deficits in locomotion. Footprint analysis revealed that the animals' base of support was significantly greater after the neonatal injury than after the adult injury, and deficits in limb rotation were larger in the neonatal operates than in the adult operates. Both groups crossed the grid with a similar number of steps but the adult operates made significantly more errors with the hindlimb ipsilateral to the lesion than the contralateral one, while the neonatal operates made an equivalent number of errors with both limbs. The neonatal operates took longer to execute the climb test and used a different movement pattern than the adult operates. The neonatal operates had a different locomotor pattern than the adult operates. Despite greater recovery of reflex responses after spinal cord injury at birth, the pattern of locomotion exhibits greater deficits when compared with the same lesion in the adult. Just as the anatomical consequences of injury to the developing nervous system are not uniform, similarly, the behavioral consequences are also not uniform. Spinal cord injury before the mature pattern of locomotion has developed results in a different motor strategy than after injury in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis.

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin.

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.