Grafting of nigral tissue hibernated with tirilazad mesylate and glial cell line-derived neurotrophic factor

Transplantation of embryonic ventral mesencephalon is a potential therapy for patients with Parkinson's disease. As only around 5-10% of embryonic dopaminergic neurons survive grafting into the adult striatum, it is considered necessary to use multiple donor embryos. To increase the survival of the grafted dopaminergic neurons, the clinical transplantation program in Lund currently employs the lipid peroxidation inhibitor, tirilazad mesylate, in all solutions used during tissue storage, preparation, and transplantation. However, the difficulty in obtaining a sufficient number of donor embryos still remains an important limiting factor for the clinical application of neural transplantation. In many clinical transplantation programs, it would be a great advantage if human nigral donor tissue could be stored for at least 1 week. This study was performed in order to investigate whether storage of embryonic tissue at 4 degrees C for 8 days can be applied clinically without creating a need to increase the number of donors. We compared the survival of freshly grafted rat nigral tissue, prepared according to the clinical protocol, with tissue transplanted after hibernation. Thus, in all groups tirilazad mesylate was omnipresent. One group of rats was implanted with fresh tissue and three groups with hibernated tissue with or without addition of glial cell line-derived neurotrophic factor (GDNF) in the hibernation medium and/or the final cell suspension. Earlier studies have suggested that GDNF improves the survival of hibernated nigral transplants. We found no statistically significant difference between the groups regarding graft survival after 3 weeks. However, there was a nonsignificant trend for fewer surviving dopaminergic neurons in grafts from hibernated tissue compared to fresh controls. Furthermore, we show that the addition of GDNF to the hibernation medium and/or to the final cell suspension does not significantly increase the survival of the dopaminergic neurons.     

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.     

Promotion of survival and regeneration of nigral dopamine neurons in a rat model of Parkinson's disease after implantation of embryonal carcinoma-derived neurons genetically engineered to produce glial cell line-derived neurotrophic factor

OBJECT: The P19 embryonal carcinoma-derived cell line consists of undifferentiated multipotential cells, which irreversibly differentiate into mature neurons after exposure to retinoic acid (RA). In the present study, the authors genetically engineered P19 cells to produce glial cell line-derived neurotrophic factor (GDNF), and grafted the cells in a rat model that had been rendered parkinsonian. METHODS: Undifferentiated P19 cells were grown in vitro and transduced with GDNF complementary DNA. The level of GDNF released from the transduced cells was measured using an enzyme-linked immunosorbent assay, and its neurotrophic activities were assessed by testing the effects on rat embryonic dopamine (DA) neurons in culture. After having been exposed to RA for 48 hours and allowed to differentiate into postmitotic neurons, the GDNF gene-transduced cells were implanted into the midbrain of immunosuppressed rats. A unilateral nigrostriatal lesion was then induced by intrastriatal infusions of 6-hydroxydopamine. Immunohistochemical analyses performed 4 weeks postgrafting revealed that the GDNF-producing cells expressed several neuronal markers without evidence of overgrowth. The grafts expressed GDNF protein and prevented the death of nigral DA neurons. Furthermore, the GDNF-producing cells implanted 4 weeks after nigrostriatal lesions restored the expression of tyrosine hydroxylase in injured DA neurons and induced their dendritic sprouting. CONCLUSIONS: The results indicate that the P19 cell line transduced with the GDNF gene can stably secrete functional levels of GDNF, even after being converted to postmitotic neurons. Because it is has been established that GDNF exerts trophic effects on DA neurons, the means currently used to deliver GDNF into the brain could be a viable strategy to prevent the death of nigral DA neurons in cases of Parkinson's disease.     

人GDNF基因的腺病毒载体构建及其在人神经干细胞中的表达

目的：观察构建的含人胶质细胞源性生长因子（GDNF）基因腺病毒载体对人神经干细胞的感染及其基因表达情况,为脊髓损伤的基因及神经干细胞治疗提供前期实验依据。方法：从12周龄流产人新鲜胚胎中提取人神经干细胞并进行培养,行Nestin免疫荧光染色进行检验。将全长558bp编码人GDNF的cDNA克隆到重组腺病毒载体质粒,并在人胚肾细胞（HEK293细胞）中包装出含有目的基因hGDNF的腺病毒,然后用该腺病毒感染人神经干细胞,应用荧光显微镜和Western-blot检测病毒感染及外源基因的表达情况,并进行GFAP和Tubulin免疫荧光染色检测神经干细胞向神经细胞分化情况。结果：Nestin免疫荧光染色显示所培养细胞为阳性染色红色,表明其具有神经干细胞性状;感染48h后观察到神经干细胞中有大量的增强绿色荧光蛋白（EGFP）表达,以及hGDNF蛋白高表达;感染后的神经干细胞有长的伪足伸出,呈GFAP和Tubulin染色阳性,表明促进了神经干细胞向神经元的分化。结论：腺病毒对神经干细胞具有较高感染效率,可作为一种良好的基因导入载体,实现外源基因hGDNF在神经干细胞内的有效表达,并可为神经干细胞分化为神经元提供更有利条件。     

Fetal grafting for Parkinson's disease: Expression of immune markers in two patients with functional fetal nigral implants

In a number of centers throughout the world, fetal nigral transplantation is being performed for the treatment of Parkinson's disease (PD). Clinical results have been inconsistent. One parameter that differs among transplant studies is the degree and manner by which patients are immunosuppressed following transplantation. Indeed, the role of the immune system following fetal grafting in humans is not well understood. Recently, two patients from our open label trial that received fetal nigral implants have come to autopsy. These patients were immunosuppressed with cyclosporin for 6 mo posttransplantation and survived for a total of 18 mo postgrafting. Robust survival of grafted dopamine-containing cells was observed in both cases. Immunostaining for HLA-DR revealed a dense collection of cells within grafts from both cases. HLA-DR staining was rarely observed within the host including nongrafted regions of the striatum. A more detailed analysis of immune markers was performed in Case 2. Numerous pan macrophages, T-cells, and B-cells were observed within graft sites located in the postcommissural putamen. In contrast, staining for these immune cells was not observed within the ungrafted anterior putamen. These findings suggest that even in healthy appearing functional nigral implants, grafts are invaded by host immune cells that could compromise their long-term viability and function. Alternatively, immune cells are known to secrete trophic factors, which may ultimately favor graft survival and function. Further work is needed to understand the role of the immune system in fetal grafting.     

Unilateral intraputamenal glial cell line-derived neurotrophic factor in patients with Parkinson disease: response to 1 year of treatment and 1 year of withdrawal

OBJECT: Glial cell line-derived neurotrophic factor (GDNF) infused unilaterally into the putamen for 6 months has been previously shown to improve significantly motor functions and quality of life measures in 10 patients with Parkinson disease (PD) in a Phase I trial. In the present study the authors report the safety and efficacy of continuous treatment for a minimum of 1 year. After the trial was halted by the drug sponsor, the patients were monitored for an additional 1 year during which the effects of drug withdrawal were evaluated. METHODS: During the extended study period, patients received a 30-microg/day unilateral intraputamenal infusion of GDNF at a basal infusion rate supplemented with pulsed boluses every 6 hours at a convection-enhanced delivery rate to increase tissue penetration of the protein. When the study was stopped, the delivery system was reprogrammed to deliver sterile saline at the basal infusion rate of 2 microl/hour. The Unified Parkinson's Disease Rating Scale (UPDRS) total scores after 1 year of therapy were improved by 42 and 38% in the off- and on-medication states; the motor UPDRS scores were also improved 45 and 39%, respectively. Benefits from treatment were lost by 9 to 12 months after the cessation of GDNF infusion. The UPDRS scores returned to their baseline and the patients required higher levels of conventional antiparkinsonian drugs to treat symptoms. After 11 months of treatment, the delivery system had to be removed in one patient because of risk of infection. Seven patients developed antibodies to GDNF but without evident clinical sequelae. There was no evidence for GDNF-induced cerebellar toxicity, as evaluated by magnetic resonance imaging and clinical testing. CONCLUSIONS: The unilateral administration of GDNF results in significant, sustained bilateral benefits in patients with PD. These improvements are lost within 9 months of drug withdrawal. Safety concerns with GDNF therapy can be closely monitored and managed.     

Long-term hibernation of human fetal striatal tissue does not adversely affect its differentiation in vitro or graft survival: implications for clinical trials in Huntington's disease

Transplantation of human fetal CNS tissue is a promising therapy for neurodegenerative conditions such as Huntington's disease (HD), but its widespread adoption is limited by restricted tissue availability. One method of overcoming this problem would be to store the tissue in hibernation medium, an approach that we reported previously for human fetal striatal tissue stored for up to 24 h. We now demonstrate the feasibility of storing such tissue for up to 8 days in hibernation medium. When either fresh or 8-day hibernated striatal cells were cultured under standard conditions for 4 days, the proportion of DARPP-32-positive neurons did not differ significantly, although the total number of cells was significantly less from tissue that had been hibernated. Six weeks after transplantation into cyclosporin A-immunosuppressed unilateral quinolinic acid-lesioned rats, there was no significant difference between fresh and hibernated grafts, both in terms of graft volume and extent of striatal phenotypic markers. This study therefore clearly demonstrates that hibernation of human fetal striatal tissue for up to 8 days is not deleterious to its differentiation in culture or survival following transplantation, and is therefore an appropriate method of storage for this tissue.