Stroke

Transplantation of human neuronal cells is a new approach for ameliorating functional deficits caused by central nervous system (CNS) disease or injury. Several investigators have evaluated the effects of transplanted fetal tissue, rat striatum, or cellular implants into small animal stroke models. One of the best studied models of brain ischemia is the murine hippocampal stroke that results in well-defined lesions, especially in the CA1 region. The standardization of this model is invaluable to the reliable testing of various experimental protective and regenerative therapies. Among them, cell transplantation of fetal hippocampal neurons has shown that they can survive and integrate in the ischemic brain. Methodological issues are still to be resolved because subsequent studies questioned the capacity of rat fetal neocortical tissues, implanted in an infarcted area, to integrate in the surrounding host tissue. However, it has been shown that the chronic ischemic region can support graft tissue.

Because the widespread clinical use of primary human tissue is likely to be extremely limited due to the ethical and logistical difficulties inherent in obtaining large quantities of fetal neurons, much effort has been devoted to developing alternate sources of human neurons for use in transplantation. One alternate source is the Ntera 2/cl.D1 (NT2) human embryonic carcinoma-derived cell line. These cells proliferate in culture and differentiate into pure, postmitotic human neuronal cells (LBS-neurons) upon treatment with retinoic acid (RetA). Thus, NT2 precursor cells appear to function as CNS progenitor cells with the capacity to develop diverse mature neuronal phenotypes. When transplanted, these neuronal cells survive, extend processes, express neurotransmitters, form functional synapses, and integrate with the host. The final product is a >95% pure population of human neuronal cells that appear virtually indistinguishable from terminally differentiated postmitotic neurons. The cells are capable of differentiation to express different neuronal markers characteristic of mature neurons, including all three neurofilament proteins (NFL, NFM, and NFH), microtubule-associated protein 2 (MAP2), the somal-dendritic protein, and t, the axonal protein. Their neuronal phenotype makes them a promising candidate for replacement in CNS disorders as a virtually unlimited supply of pure, postmitotic, terminally differentiated, human neuronal cells.

In support of different mechanisms for efficacy, animal transplantation studies of LBS-neurons revealed graft survival, a mature neuronal phenotype, and integration into the host brain in vivo.

In patients disabled by stroke, the concept of restoring function by transplanting human neuronal cells into the brain is innovative. Research in a rat model of transient focal cerebral ischemia demonstrated that transplantation of fetal tissue restored both cognitive and motor functions. Sanberg, Borlongan, and colleagues were the first to show that transplants of LBS-neurons could also reverse the deficits caused by stroke. The preclinical studies of LBS-neurons were carried out in a model of transient focal, rather than global, ischemia in order to maximize the chances of functional recovery. Animals that received transplants of LBS-neurons (and cyclosporine treatment) showed amelioration of ischemia-induced behavioral deficits throughout the 6-month observation period. They demonstrated complete recovery in the passive avoidance test, as well as normalization of motor function in the elevated body swing test. In comparison, control groups receiving transplants of rat fetal cerebellar cells, medium alone, or cyclosporine failed to show significant behavioral improvement. A second study that evaluated response in comparison to the number of cells transplanted, confirmed the efficacy of transplanted LBS-neurons in reversing the behavioral deficits resulting from transient ischemia in an MCA occlusion rat model.

The initial objectives of the first clinical study performed at the University of Pittsburgh were to demonstrate the safety and feasibility of the neuronal-cell implantation procedure. These goals were met, in that no adverse events related to the implantation have occurred in at least 36 months of follow-up in 12 patients (Fig. 3). The adverse events that did occur in these patients were thought to be unrelated to the implantation of the neuronal cells and can be considered typical of a population with known cardiovascular disease and advanced age. This study was also intended to provide some information on the efficacy of neuronal-cell implantation in improving stroke-related neurologic deficits. In both treatment groups, mean NIHSS (National Institutes of Health Stroke Scale) total scores decreased and mean ESS (European Stroke Scale) total scores increased; both changes indicated improvement. For the ESS, the increases tended to be larger in the group of four patients receiving 6 million cells, both in the total scores and in the composite motor subscale scores. Both the Barthel Index and the SF-36 scores decreased in the group receiving 2 million cells and increased in the group receiving 6 million cells. All outcomes measurements were consistent in identifying a trend toward improved scores in the group of patients who received 6 million neuronal cells. The PET scan results also provide a suggestion of efficacy, in that increased activity at the area of the stroke was seen in 6 patients.

The neuronal cells could improve neurologic function through a number of different mechanisms. These include the provision of neurotrophic support (acting as local pumps to support cell function), provision of neurotransmitters, reestablishment of local interneuronal connections, cell differentiation and integration, and improvement of regional oxygen tension. Transplanted cells also may act to limit the reactive glial response and to limit retrograde degeneration, although this may be less feasible in a chronic injury. We believe that axonal reconnections through the grafted cells (serving as a "bridge") over large distances are less likely, although this phenomenon has been observed in spinal cord injury models. Phase 2 dose-response trials in patients are ongoing to evaluate further the role of neurotransplantation for patients with chronic motor deficits caused by basal ganglia region infarction.

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