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.
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.
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.
Although there is no spontaneous regeneration of mammalian spinal axons after injury, they can be enticed to grow if cAMP is elevated in the neuronal cell bodies before the spinal axons are cut. Prophylactic injection of cAMP, however, is useless as therapy for spinal injuries. We now show that the phosphodiesterase 4 (PDE4) inhibitor rolipram (which readily crosses the blood–brain barrier) overcomes inhibitors of regeneration in myelin in culture and promotes regeneration in vivo . Two weeks after a hemisection lesion at C3/4, with embryonic spinal tissue implanted immediately at the lesion site, a 10-day delivery of rolipram results in considerable axon regrowth into the transplant and a significant improvement in motor function. Surprisingly, in rolipram-treated animals, there was also an attenuation of reactive gliosis. Hence, because rolipram promotes axon regeneration, attenuates the formation of the glial scar, and significantly enhances functional recovery, and because it is effective when delivered s.c., as well as post-injury, it is a strong candidate as a useful therapy subsequent to spinal cord injury.
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 μm 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 aneurotropicinfluence on injured mature CNS neurons by increasing their axonal growth within a transplant.
Abstract 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 proteins 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.
