Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats

Abstract

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors.

Introduction

In Parkinson's disease, progressive deterioration of motor function results from the loss of nigrostriatal dopamine neurons and consequent dopamine depletion in the caudate and putamen [12]. Because treatment with the dopamine precursor l-dopa loses its effectiveness after several years, alternative therapeutic strategies such as neurotransplantation are being pursued 14, 15, 16, 17, 22, 28, 35, 37. A major problem with this approach is that up to 95% of the transplanted dopamine neurons die after transplantation 3, 23.

Previous work with embryonic mesencephalon has shown that dopamine neurons have a better rate of survival when kept intact as a tissue block rather than dissociated as single cells 13, 19. In vitro studies have shown that monolayers of mesencephalic cells do not support dopamine neuron survival as well as do multi-layered mesencephalon cultures [13]. Similarly, densely plated cells survive better than cells plated sparsely [30]. In vivo studies of transplants in the rat model of Parkinson's disease have shown that tissue blocks of mesencephalon lead to better graft survival and better behavioral improvement when compared to dispersed mesencephalic tissue [19].

The growth factor GDNF has been shown to improve dopamine neuron survival and neurite outgrowth in vitro [26]and to protect against 6-OHDA toxicity in vivo [20]. Other growth factors such as IGF-I and bFGF also improve dopamine neuron survival in vitro [21]and in vivo [36]. With in vitro experiments using embryonic mesencephalon, our lab has shown that GDNF reduces the rate of apoptosis in rat [6]and human dopamine neurons [7]and that a combination of bFGF and IGF-I is additive in reducing apoptosis in rat dopamine neurons [39].

Transplants of embryonic mesencephalon undergo apoptotic cell death in vivo 29, 40. We have recently found that dopamine cell survival can be improved by simple preincubation of transplanted tissue with the growth factor combination of GDNF, IGF-I and bFGF [40]. Others have noted that cografts of fetal striatum augment the effects of ventral mesencephalic transplants in rats 4, 10, 38and non-human primates [34].

In the present experiments, we have compared survival of grafts of dissociated mesencephalic tissue with transplants of tissue kept intact by extrusion of mesencephalic fragments into tissue strands. We have also sought to improve survival of transplanted embryonic dopamine cells through a combination of growth factors and striatal cografts that might reduce the rate of apoptosis in the transplants.