Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons

Abstract

The poor survival rate (5–20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/μl and transplanted into two 0.5 μl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/μl group exhibited significantly higher survival rates (5.48 ± 0.83%) compared to the 25,000 cells/μl (2.81 ± 0.39%) and 50,000 cells/μl (3.36 ± 0.51%) groups (p = 0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/μl group was significantly larger than that of the 25,000 cells/μl (p < 0.0001) and 50,000 cells/μl groups (p = 0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.

Introduction

Patients with Parkinson's disease (PD) present clinically with bradykinesia, rigidity, and resting tremor (Olanow and Tatton, 1999). While pathology occurs throughout the brain in sporadic PD, these symptoms can mainly be attributed to the degeneration of dopaminergic neurons in the substantia nigra resulting in a loss of striatal dopamine (DA) (Bergman and Deuschl, 2002). Therefore, current therapies center around restoring DA levels in the striatum of affected individuals. Pharmaceutical replacement of DA with levodopa is highly effective as a symptomatic treatment in the early stages of the disease; however, due to the waning efficacy and side effects associated with this therapy, it is not a viable long-term treatment option (Jenner, 2004). While animal studies point to the transplantation of embryonic ventral mesencephalic cells as a potential alternative means of restoring DA levels in Parkinson's patients, human trials have yielded mixed results (Brundin et al., 1987, Freed et al., 2001, Olanow et al., 2003, Yurek and Sladek, 1990). Freed et al., 2001, observed a decrease in the total Unified Parkinson's Disease Rating Scale (UPDRS) scores in a subset of younger patients (≤60 years old); however, the transplantation of primary DA neurons in older individuals (>60 years old) did not yield an improvement in the total UPDRS scores (Freed et al., 2001). A more recent study has shown similar results when comparing preoperative motor scores indicating that it may be the level of disability, rather than age, that influences the level of symptomatic relief derived from the transplantation of primary DA cells (Olanow et al., 2003). Additionally, the long-term side effects of this therapy include debilitating “off period” graft-induced dyskinesias, which are more prevalent in the subset of patients who displayed improvements in Parkinsonian symptoms (Freed et al., 2001, Olanow et al., 2003). The limited ability of transplanted DA neurons to survive and properly integrate with the host striatum are likely contributing factors to both the lack of efficacy and side effects associated with the transplantation of embryonic ventral mesencephalic cells.

Numerous factors contribute to the compromised survival rate (5–10%) of DA neurons during the post-transplantation interval including trophic factor withdrawal, hypoxia, and oxidative stress (Sortwell, 2003). An additional factor implicated in the low survival rate of transplanted cells is anoikis, i.e. loss of cell–cell and extracellular matrix (EMC) contact (Meredith et al., 1993, Raff, 1992). Anoikis may play a role in the initiation of apoptosis during the dissociation of the ventral mesencephalon into cell suspension and following transplantation of embryonic tissue into the striatum. These and other insults may also decrease the functional capacity of the surviving tyrosine hydroxylase immunorecative (THir) neurons. Treatment of transplanted DA neurons with neurotrophic factors such as BDNF, NT-3, GDNF, aFGF, and bFGF has been shown to increase the number and functional capacity of grafted THir neurons (Giacobini et al., 1993, Granholm et al., 1997, Rosenblad et al., 1996, Timmer et al., 2004, Yurek et al., 1996). However, this approach is only able to increase survival rates to approximately 30–35%, still leaving poor survival as a major obstacle preventing transplantation of primary mesencephalic tissue from becoming a viable therapy for PD (Sortwell, 2003).

While neurotrophins and growth factors have been widely investigated for their ability to improve the efficacy of embryonic ventral mesencephalic grafts, parameters of the transplant procedure itself can also impact survival. Nikkhah and colleagues demonstrated that decreasing the volume of cell suspension implanted, while holding the concentration of the cell suspension constant, led to an increase in the survival rate of grafted DA neurons (Nikkhah et al., 1994). Given that optimal volume parameters were defined in the Nikkhah study, we hypothesized that an optimal concentration may also exist. The current investigation examines the ability of cell suspension concentration to affect the survival rate and functionality of grafted THir neurons.