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Adam O. Hebb, Kari Hebb, Arun C. Ramachandran and Ivar Mendez

Object. Transplantation of fetal dopaminergic tissue is being investigated in animal models and clinical trials for its potential as a treatment for advanced Parkinson disease. At the same time, the availability of fetal tissue is limited, making its storage time prior to transplantation a key practical issue. Although it results in a smaller percentage of surviving cells, a longer storage time enables fetal tissue obtained over several days to be pooled for transplantation in a recipient. Glial cell line—derived neurotrophic factor (GDNF) has been shown to improve survival of human dopaminergic tissue that has been stored prior to transplantation. The objective of this study was to evaluate the effects on fetal dopaminergic tissue of GDNF-supplemented hibernation for extended periods of 6 to 15 days.

Methods. The ventral mesencephalon (VM) was harvested in a total of 27 14-day-old rat fetuses, and three VMs were cultured immediately (fresh control group). The remaining 24 VMs were divided sagittally along the midline to yield 48 equal pieces of hemimesencephalon. Twenty-four pieces were stored with GDNF-supplemented hibernation medium for 6, 9, 12, or 15 days, and the 24 “partner” hemimesencephalon pieces were stored in control hibernation medium for the same periods of time. Tissue was cultured for 48 hours and processed for tyrosine hydroxylase (TH) immunoreactivity and double-stained with cresyl violet. Cell counts for all cultures and the percentage of TH-immunoreactive cells were obtained. The percentage of TH-immunoreactive cells for the fresh control group was 6.3 ± 0.5%. The percentage of TH-immunoreactive cells in cultures derived from tissue stored in GDNF-supplemented medium was significantly increased at 6 and 9 days posthibernation compared with the fresh control group and the “partner” groups stored in hibernation medium only. No significant increase in the percentage of TH-immunoreactive cells was observed in the 12- and 15-day groups.

Conclusions. In this study the authors have demonstrated that fetal dopaminergic tissue can be safely stored for up to 9 days in GDNF-supplemented hibernation medium. Furthermore, the percentage of TH-immunoreactive cells is significantly increased after 6 and 9 days of storage in this medium, improving the yield of TH-immunoreactive cells prior to transplantation. These observations have practical clinical implications for collecting fetal dopaminergic cells and improving their survival after transplantation.

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Adam O. Hebb, Kari Hebb, Arun C. Ramachandran and Ivar Mendez

Object

Investigation of fetal dopaminergic tissue transplantation is being conducted in animal models and clinical trials as a potential treatment for advanced Parkinson disease (PD). Because the availability of fetal tissue is limited, however, the duration of its storage prior to transplantation is a key practical issue. Longer storage times may enable fetal tissue obtained over several days to be pooled together for transplantation in a recipient. Glial cell line–derived neurotrophic factor (GDNF) has been shown to improve survival of stored human dopaminergic tissue prior to transplantation. The objective of this study was to evaluate GDNF-supplemented hibernation of fetal dopaminergic tissue for extended periods of 6 to 15 days.

Methods

A total of 27 rat ventral mesencephalons (VMs) were obtained in gestation Day 14 rat fetuses, and three were cultured immediately (fresh-culture control group). The remaining 24 VMs were divided sagittally along the mid-line to form 48 equal pieces of hemimesencephalons. Twenty-four pieces were stored with GDNF-supplemented hibernation medium for 6, 9, 12, or 15 days, and the 24 “partner” hemimesencephalons were stored in control hibernation medium for the same periods of time. Tissue was cultured for 48 hours and processed for tyrosine hydroxylase (TH) immunoreactivity and cresyl violet. Cell counts for all cultures and percentage of TH-immunoreactive cells were obtained. The percentage of TH-positive cells for the fresh control group was 6.3 ± 0.5%; that measured in cultures derived from tissue hibernated in GDNF-supplemented medium was significantly increased at 6 and 9 days posthiber-nation compared with the fresh-culture control group and the partner groups stored in hibernation medium only. No significant increase in percentage of TH-immunoreactive cells was observed in the 12- and 15-day hibernation groups.

Conclusions

In summary the authors found that fetal dopaminergic tissue can safely be stored up to 9 days in GDNF-supplemented hibernation medium. Furthermore the percentage of TH-immunoreactive cells is significantly increased after 6 and 9 days of storage in this medium, improving the yield of TH-positive cells prior to transplantation. These observations may have important clinical implications for collecting fetal dopaminergic cells and improving their survival after transplantation.

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Ivar Mendez, Murray Hong, Stephen Smith, Alain Dagher and Jacques Desrosiers

✓ The authors present a simple, reliable, and safe system for performing neural transplantation in the human brain. The device consists of a transplantation cannula and microinjector system that has been specifically designed to reduce implantation-related trauma and to maximize the number of graft deposits per injection. The system was evaluated first in an experimental rat model of Parkinson's disease (PD). Animals in which transplantation with this system had been performed showed excellent graft survival with minimal trauma to the brain. Following this experimental stage, the cannula and microinjector system were used in eight patients with PD enrolled in the Halifax Neural Transplantation Program who received bilateral putaminal transplants of fetal ventral mesencephalic tissue. A total of 16 transplantation operations and 64 trajectories were performed in the eight patients, and there were no intraoperative or perioperative complications. Magnetic resonance imaging studies obtained 24 hours after surgery revealed no evidence of tissue damage or hemorrhage. Transplant survival was confirmed by fluorodopa positron emission tomography scans obtained 6 and 12 months after surgery.

As neural transplantation procedures for the treatment of neurological conditions evolve, the ability to deliver viable grafts safely will become critically important. The device presented here has proved to be of value in maximizing the number of graft deposits while minimizing implantation-related trauma to the host brain.

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Ivar Mendez, Murray Hong, Stephen Smith, Alain Dagher and Jacques Desrosiers

The authors present a simple, reliable, and safe system for performing neural transplantation in the human brain. The device consists of a transplantation cannula and microinjector system that has been specifically designed to reduce implantation-related trauma and to maximize the number of graft deposits for each injection. The system was evaluated first in an experimental rat model of Parkinson's disease (PD). Animal transplantation with this system showed excellent graft survival with minimal trauma to the brain. Following this experimental stage, the cannula and microinjector system was used in eight patients with PD enrolled in the Halifax Neural Transplantation Program who received bilateral putaminal transplants of fetal ventral mesencephalic tissue. A total of 16 transplantation operations and 64 trajectories were performed in the eight patients, and there were no intra- or perioperative complications. Magnetic resonance imaging studies obtained 24 hours after surgery revealed no evidence of tissue damage or hemorrhage. Transplant survival was confirmed on fluorodopa positron emission tomography scans 6 and 12 months after surgery.

As neural transplantation procedures for the treatment of neurological conditions evolve, the ability to deliver viable grafts safely will become of critical importance. The device presented here has been proven to be of value in maximizing the number of graft deposits while minimizing implantation-related trauma to the host brain.

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Vivek Mehta, Murray Hong, Julian Spears and Ivar Mendez

The goal of this study was to investigate the ability of fetal dopaminergic neurons to improve complex sensorimotor behavior.

The authors obtained ventral mesencephalic tissue from 14-day-old rat fetuses. The cells were exposed to glial cell line-derived neurotrophic factor (GDNF) prior to transplantation into rats with unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal pathway. Animals that received 400,000 cells exposed to GDNF demonstrated significant improvement in contralateral forelimb function and showed improvement in rotational behavior faster than animals that received cells not exposed to GDNF. Increasing the number of implanted cells to 800,000 exposed to GDNF did not result in any additional improvement in functional recovery.

As neural grafting procedures in the nervous system evolve and genetically engineered cells or stem cells replace fetal tissue, crucial questions about cell number and trophic regulation will need to be addressed. This study demonstrates that grafting of 400,000 cells exposed to GDNF before transplantation has a beneficial effect in the restoration of complex sensorimotor behavior.

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Vivek Mehta, Murray Hong, Julian Spears and Ivar Mendez

Object. The goal of this study was to investigate the ability of fetal dopaminergic neurons to improve complex sensorimotor behavior.

Methods. The authors obtained ventral mesencephalic tissue from 14-day-old rat fetuses. The cells were exposed to glial cell line—derived neurotrophic factor (GDNF) prior to transplantation into rats with unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal pathway. Animals that received 400,000 cells exposed to GDNF demonstrated significant improvement in contralateral forelimb function and showed improvement in rotational behavior faster than animals that received cells not exposed to GDNF. Increasing the number of implanted cells to 800,000 exposed to GDNF did not result in any additional improvement in functional recovery.

Conclusions. As neural grafting procedures in the nervous system evolve and genetically engineered cells or stem cells replace fetal tissue, crucial questions about cell number and trophic regulation will need to be addressed. This study demonstrates that grafting of 400,000 cells exposed to GDNF before transplantation has a beneficial effect in the restoration of complex sensorimotor behavior.

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Sean D. Christie, Ben Comeau, Tanya Myers, Damaso Sadi, Mark Purdy and Ivar Mendez

Object

Oxidative stress leading to lipid peroxidation is a major cause of secondary injury following spinal cord injury (SCI). The objectives of this study were to determine the duration of lipid peroxidation following acute SCI and the efficacy of short-and long-term administration of methylprednisolone on decreasing lipid peroxidation.

Methods

A total of 226 female Wistar rats underwent clip-compression induced SCI. In the first part of the study, spinal cords of untreated rats were assayed colorimetrically for malondialdehyde (MDA) to determine lipid peroxidation levels at various time points between 0 and 10 days. In the second part of the study, animals were treated with methylprednisolone for either 24 hours or 7 days. Control animals received equal volumes of normal saline. Treated and control rats were killed at various time points between 0 and 7 days.

Results

The MDA levels initially peaked 4 hours postinjury. By 12 hours, the MDA levels returned to baseline. A second increase was observed from 24 hours to 5 days. Both peak values differed statistically from the trough values (p < 0.008). The methylprednisolone reduced MDA levels (p < 0.04) within 12 hours of injury. No effect was seen at 24 hours or later.

Conclusions

The results of this study indicate that oxidative stress persists for 5 days following SCI in rats, and although methylprednisolone reduces MDA levels within the first 12 hours, it has no effect on the second lipid peroxidation peak.

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