Intracranial and intraocular grafting of fetal ventral mesencephalic and striatal tissues : neuronal survival and nerve growth characteristics

Abstract

Parkinson's disease is caused by degeneration of dopaminergic neurons in the ventral mesencephalon. Various animal models and clinical trials has shown that grafts of fetal ventral mesencephalic tissue can survive and functionally reinnervate the dopamine-depleted striatum, leading to long-term improvements of therapeutic value, but still symptomatic relief is far from complete. The aim of this thesis was to enhance survival of grafted fetal dopaminergic neurons and to characterise nerve growth formed by grafted and adult dopaminergic neurons regarding density and extent of nerve fiber outgrowth.

One way to increase dopaminergic neuronal survival is to increase trophic support to the grafted tissue. Glial cell line-derived neurotrophic factor (GDNF) mRNA was shown to be expressed in the developing striatum, thus making GDNF an interesting factor to study regarding trophic support for grafted dopaminergic neurons. The results showed that GDNF increased survival of intraocularly grafted dopaminergic neurons. Another approach to promote survival of grafted neurons is to reduce oxidative stress during dissection and grafting by the use of a free radical scavenger or a dissection medium with high buffering capacity. Dulbecco's modified Eagle medium (DMEM), used at dissection, increased the survival of intraocularly grafted fetal dopamine neurons three-fold compared to Hank's balanced salt solution (HBSS), a commonly used dissection medium.

The addition of the antioxidant U 74006F (tirilazad mesylate, Freedox) to DMEM resulted in an additional increase in survival of grafted dopamine neurons. During development of the striatal dopaminergic innervation, the striatum first becomes innervated by dense patches of dopaminergic fibers, and later, a more widespread pattern fills up the areas between the patches. Using fetal striatal and cortical tissue as targets for host derived dopaminergic growth, it was possible study these growth patterns.

The formation of different growth patterns was dependent on the implantation site. Striatal grafts placed in dorsal striatum received dense patches of dopaminergic nerve fibers, but large portions of the grafts remained uninnervated. Striatal grafts to ventral striatum or globus pallidus received the same type of dense patches of dopaminergic nerve fibers but in addition, the large uninnervated portions seen in grafts placed in dorsal striatum became partially filled with a less dense, widespread nerve fiber network. These two growth patterns resembled those seen during normal development of the striatal dopaminergic innervation, indicating that these different types of growth still could be induced in the adult animal.

Fetal cortical grafts placed into dorsal striatum received a widespread dopaminergic innervation similar to that found in striatal grafts placed in globus pallidus and ventral striatum, but with the exception that no dense patches occurred. It was also shown that increased striatal dopaminotrophic activity, created by chronic haloperidol treatment, was selective for the widespread growth type while the patchy growth was unaffected. When a fetal ventral mesencephalic graft, placed in the lateral ventricle, reinnervated a dopamine deafferented adult striatum, solely the widespread growth pattern occurred in the striatum. However, using intraocular double-grafts of ventral mesencephalic and striatal tissues, the age of the striatal target was shown to determine the type of growth pattern formed by the dopamine neurons.

Patchy dopaminergic growth was induced in immature striatal tissue while diffuse growth dominated when the striatal tissue had been left to mature prior to implantation of the ventral mesencepha1ic tissue. Furthermore, patchy growth innervated the total volume of a striatal target, while widespread growth terminated prior to complete reinnervation of the striatal target. The density of the patchy growth was also much higher than the widespread. Enhanced survival of grafted dopamine neurons after treatment with U-74006F did not enhance nerve fiber formation or change the growth pattern in the striatal target, but a hyperinnervation of the ventral mesencephalic graft was found. Using a retrograde tracer to mark neurons innervating adult dopamine-depleted striatum, it was shown that a subpopulation of the dopaminergic neurons dit not send their axons into the striatum, which indicates that an increase in survival of grafted neurons does not necessarily lead to an enhanced innervation of the striatal target.

This thesis demonstrates that it is possible to enhance survival of grafted dopaminergic neurons by using the trophic factor GDNF or the lazaroid U-74006F and that the extent and density of growth into the striatal target seem to be dependent on the type of growth pattern formed by the dopaminergic graft.