Stroke

Fall 2000
Volume 11, Number 2

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Advances in treatment of stroke survivors with residual neurological deficits has lagged behind therapy for acute stroke and stroke prevention. Neural transplantation is an exciting new field of research for stroke patients. At the University of Pittsburgh we treated 12 patients with basal ganglia stroke and fixed motor deficits with a neuronal cell that have been shown in vivo to integrate, form synapses and processes and improve neurological function in animal models of stroke. There were no complications related to the implanted cells either at the time of surgery or in 12 to 22 months of follow up. Eight of the 12 patients had subjective improvements and 7 improved in European Stroke Scale scores at 12 months. PET scans at 12 months showed at least a 15% increase in local metabolism compared to baseline scans in 6 of 11 patients. These results are promising and further studies in patients with stroke are planned.

Introduction. The past decade has witnessed impressive advances in prevention and treatment of cerebrovascular disease. Despite these advances, stroke remains the leading cause of serious adult disability in the United States. It is estimated that there are over 4 million stroke survivors in this country. Physical therapy, occupational therapy, and speech therapy are the mainstay of rehabilitative efforts, but in many cases significant disabilities remain following stroke.

Background. Neural transplantation offers a new and promising approach for treatment of stroke survivors with residual deficits. Several different avenues of research in this area are in progress. Transplantation of adrenal tissue or fetal cells has been attempted in patients with Parkinson’s disease with limited success. The recent discovery of neural stem cells has stimulated interest in the possibility of improving neurological function by transplanting progenitor cells. Xenografts using porcine fetal cells are also under investigation. A phase I study using porcine fetal cells in patients with stroke was recently halted due to complications in one treated patient.

At the University of Pittsburgh, a neuronal cell line developed by Layton Bioscience (LBS) has been used to treat patients with fixed neurological deficits due to stroke. LBS neurons are derived from a cell line originally isolated from a lung metastasis in a 22-year-old male with a testicular germ cell tumor. When exposed to retinoic acid, one derived clone (NT2D1) has been shown to consistently differentiate into neuronal cells.1 The differentiated cells express proteins typical of neurons and have been shown in vivo to extend processes, produce neurotransmitters, form synapses, and integrate with host tissue.2-4 Preclinical studies in rats, mice, and monkeys using LBS neurons demonstrate that these cells survive in vivo and demonstrate no toxicity or tumorgenicity.2 In a rat model of stroke, transplantation of neuronal cells improved learning of an avoidance task and enhanced motor functions.5, 6

The precise mechanism of neurological improvement induced by implanting neuronal cells is uncertain. The neuron cells may provide a trophic influence, enhancing local cell function. Segmental connections lost due to the stroke may be reestablished. Neural transplantation may improve local oxygen tension or act by limiting glial scarring. Further basic research is needed to clarify these issues.

Phase I study. Based on the promising preclinical data, we initiated a phase I safety study of LBS neurons for treatment of patients with established motor stroke. Patients between 40 and 75 years of age were included with major deficits due to stroke involving the basal ganglia defined on CT or MRI. The stroke must have occurred 6 months to 6 years previously and the neurological deficits must have been stable for the previous 2 months. Exclusion criteria included coagulopathy or uncontrolled hypertension, concomitant major illness, serum creatinine *2.0, active malignancy, alcohol or drug abuse or inability to understand or cooperate with the study. Safety was assessed during hospitalization for the stereotactic procedure and at follow up visits occurring 1, 2, 4, 8, 12, 16, 24, 36, and 52 weeks following surgery. Efficacy was also assessed at each follow-up visit by the European Stroke Scale (ESS) and the National Institutes of Health Stroke Scale (NIHSS). In addition, functional disability and quality of life were assessed using the Barthel Index and SF-36 at baseline and 24 weeks. Positron Emission Tomography (PET) scans and MRI scans were performed prior to surgery and repeated at 4 and 24 weeks for MRI; 24 and 52 weeks for PET.

All patients were treated with Clyclosporine-A for 1 week prior and 8 weeks following surgery. LBS neurons were implanted under local anesthesia using standard stereotactic techniques through either a twist drill or burr hole craniostomy. The initial 4 patients all received 2 million cells separated into 3 20-microliter injections along a single needle pass. The subsequent 8 patients were randomized to receive either 2 million cells along one needle pass or 6 million cells along 3 needle passes. Evaluations at follow-up visits were performed by examiners blinded to the number of cells implanted.

Twelve patients were treated with LBS neurons. The mean age was 61 (range 44 to 74); 9 were male and 3 female. The mean time from stroke onset to treatment was 27 months (range 7 to 55 months). The basal ganglia was involved in all cases and in 4 patients there was also infarction of the cortex.

Safety Results. The procedure was completed successfully in all patients and there were no in hospital complications.

All patients were discharged the following morning. No hemorrhages occurred related to the stereotactic procedure. During follow up of 12 to 22 months 4 patients suffered medical events. In one, a single seizure occurred 6 months after surgery. Dilantin therapy was instituted without recurrence in an additional 16 months of follow up. Another patient developed renal insufficiency presumably related to cyclosporine. This reversed with discontinuation of the medication. One patient developed a urinary tract infection. In one case a recurrent stroke occurred involving the brainstem and remote from the site of neuronal implants. Follow-up MRIs showed no evidence of inflammation, mass effect, or new signal abnormality at the site of the implants.

Efficacy Results. Eight of the 12 patients noted subjective improvements including improved strength, improved gait, clearer speech, and reduced stiffness of the extremities. In this uncontrolled, unblinded study the significance of such reports is uncertain. Seven patients had improvement in the ESS of 3 to 15 points at 12 months, one showed no change, and 4 worsened by 1 to 13 points. As a group there was a slight increase in the ESS from a mean of 60 + 2.3 at baseline to 63 + 3.2 at 6 months (p=.046). The mean ESS change was 2.9. There was a trend toward greater improvement in the patients receiving 6 million cells, but the difference was not statistically significant. Only a minor change was seen in the NIHSS (7.8 + 0.9 baseline, 7.2 + 0.9 six months; p=.422) and no significant difference was found between those with 2 million and 6 million cells. Barthel index declined slightly, 78 + 3.8 at baseline to 72 + 4.6 in the 2 million cell group, but increased from 74 + 2.4 at baseline to 80 + 7.6 at 6 months in the 6 million cell group. The mean change was -4.3 for those receiving 2 million cells and 5.0 for those receiving 6 million cells. Changes in the SF-36 scores showed the same trends with slight worsening in the 2 million cell group and improvement from baseline to 6 months in the 6 million cell group.

PET Results. Results of PET scans were of particular interest. Six of 11 patients scanned at baseline and at 12 months showed at least a 15% increase in uptake of flourodeoxyglucose (FDG) at the implant site or ipsilateral adjacent brain. Five of these patients had 2 million cells implanted and one received 6 million cells. There was no clear correlation between increases in FDG uptake and either improvements in stroke scales or subjective changes.

The results of this phase I study demonstrate that implantation of LBS neurons is feasible and raised no safety concerns in patients with basal ganglia stroke. No complications occurred that could be directly attributed to the implanted cells in up to 22 months of follow up. The efficacy results must be interpreted with caution since there were no controls and the only blinding was to whether the patients received 2 or 6 million cells. However, given these limitations, several patients showed improvement in ESS scores. There was a trend toward greater increases in the patients receiving 6 million cells, but with the small number of patients in each group the significance of this trend is uncertain. The PET results are intriguing, but similar to the stroke scales, the significance is unknown. Increased FDG uptake may reflect inflammation or the process of cell necrosis although no changes were seen on MRI.

Summary. Additional studies with LBS neurons in patients with stroke are planned. Future studies should explore a greater range of cell numbers, larger patient numbers, additional sites, and more detailed assessment of neurological function. These results represent a first step toward the goal of effective treatment and improvement of neurological function in stroke survivors months to years after the initial event.

Address comments and questions to:

Lawrence R. Wechsler, MD
University of Pittsburgh Medical Center

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