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/microl and transplanted into two 0.5 microl 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/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl 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.

Figure 1. Calculation of THir neuron survival rates Following Transplantation

Survival Rate was calculated utilizing the following equations. *The number of THir neurons grafted was based on previous findings (Fawcett, et al., 1995).

Figure 2. Effect of Cell Suspension Concentration on Amphetamine Induced Asymmetry

Five mg/kg of d-amphetamine was administered to rats unilaterally lesioned with 6-OHDA. Rats receiving grafts of 100,000 and 200,000 cells/μl displayed significant recovery from baseline rotational asymmetry (P=0.007 and P=0.002, respectively); * indicates significant recovery from baseline (200,000 cells/μl); †indicates significant recovery from baseline (100,000 cells/μl).

Figure 3. Effect of Cell Suspension Concentration on the Absolute Number of Surviving THir Neurons Following Transplantation

Primary DA neurons were concentrated to 25,000, 50,000, 100,000 or 200,000 cells/μl prior to transplantation into the denervated rat striatum. Increasing the concentration of cells per μl resulted in a dose dependent increase in the total number of THir neurons per primary mesencephalic graft. * indicates a significant difference between the 200,000 cells/μl group (p≤0.05); †indicates a significant difference between the 100,000 cells/μl group (p≤0.05).

Figure 4. Effect of Cell Suspension Concentration on the Survival Rate of THir Neurons Following Transplantation

Primary DA neurons were concentrated to 25,000, 50,000, 100,000 or 200,000 cells/μl prior to transplantation into the denervated rat striatum. Increasing the concentration of cells per μl augmented the survival rate of THir neurons in primary mesencephalic grafts; *indicates a significant difference between the 200,000 cells/μl group (p≤0.05).

Figure 5. Effect of Cell Suspension Concentration on the Soma Size and Immunoreactivity of THir Neurons Following Transplantation

Primary DA neurons were concentrated to 25,000, 50,000, 100,000 or 200,000 cells/μl prior to transplantation into the denervated rat striatum. Grafts of more highly concentrated cell suspensions (200,000 cells/μl group; High Conc) displayed THir neurons with significantly larger soma size and TH staining intensity compared to grafts of lower concentrations (25,000, 50,000, and 100, 000 cells/μl groups; Low Conc). * indicates a significant difference between the High Concentration and Low Concentration groups (p=0.01).

Figure 6. Effect of Cell Suspension Concentration on the Soma Size and Immunoreactivity of THir Neurons Following Transplantation

A. 25,000 cells/μl. B. 50,000 cells/μl. C. 100,000 cells/μl. D. 200,000 cells/μl. Scale bar in A. equals 10μm.