On transplantation of fetal ventral mesencephalon with focus on dopaminergic nerve fiber formation

Poor cell survival and striatal reinnervation are two of the postulated limiting factors of fetal ventral mesencephalic (VM) grafting in Parkinson's disease. It has, however, been reported that an increased survival of VM dopaminergic neurons does not correlate with an enhanced target innervation. Instead, the population that was increased in number terminated within the VM graft.

In this thesis, different striatal innervation patterns were morphologically and functionally characterized, and traced to VM subpopulations displaying disparate histochemical traits, in order to achieve enhanced striatal reinnervation in volume and density in the rat model for Parkinson's disease. Since immature striatal tissue receives a more pronounced VM-derived innervation, factors associated with striatal immaturity were applied concomitantly with VM grafting by usage of polymeric bioactive rods. Knockout of the orphan nuclear receptor Nurr1 in mice causes a complete lack of tyrosine hydroxylase (TH)-expression in the ventral midbrain and of dopamine (DA) in the striatum. Therefore, Nurr1 may be implicated in the development of the nigrostriatal pathway. In vitro cultures of VM tissue from Nurr1 deficient mice were performed to obliterate putative endogenous inhibitory agents, and hence investigate the possibility of TH-induction. Furthermore, the presence of extra-cellular matrix (ECM) molecules was mapped within intrastriatal VM grafts to study the putative role of the ECM in guidance of dopaminergic outgrowth. The results confirm that striatal tissue, may be targeted by two different innervation patterns with different functional properties.

Patchy and dense DAergic outgrowth induced in immature striatal tissue possessed high functional capacity in terms of DA-release, while diffuse growth induced both in immature and mature striatal tissue possessed low functional capacity. Furthermore, it was shown that basic fibroblast growth factor (bFGF), expressed in striatal proliferative zones of the adult brain, improves nerve fiber density both within the striatal target and within the VM graft. Moreover, it was concluded that not all of the DA neurons within a VM graft innervate the striatal target and that the non-projecting neurons might belong to the A10 subpopulation of VM, based on calbindinD28k-immunoreactivity. It was demonstrated that it is possible to induce TH-expressing neurons of Nurr1 knockout VM tissue in in vitro cultures. These neurons, however, did not possess the capacity to form TH-positive nerve fiber bundles. It was clarified that the ECM molecule keratan sulfate (KS) is differentially expressed in VM grafts depending on the expression of calbindinD28k. CalbindinD28k-expressing neuronal clusters within VM grafts did not co-express KS, while calbindinD28k-negative clusters did. Moreover, tenascin was expressed in calbindinD28k- positive cell clusters.

In conclusion, the results from this thesis demonstrate that it might be important to induce an immature environment in the host striatum, in order to enhance the VMderived target innervation. Not all TH-expressing neurons within a VM graft have the potency to project to the target, and it was shown that neurons projecting to the striatum were calbindinD2ak-negative, indicating A9- origin. Nurr1 does not seem to be absolutely required for induction of a TH-expressing neuronal phenotype. However, Nurr1 is a prerequisite for the formation of nerve fiber bundles, indicating a malfunction of the induction of a nigrostriatal connection in Nurr1 knockout animals. KS seems to act as an attractant on dopaminergic outgrowth from VM grafts, and might be implicated in the guidance of the nigrostriatal pathway during development. Tenascin, on the contrary may hinder the outgrowth and instead cause terminalization within the transplant.

List of scientific papers

I. Vidal N, Bjorklund L, Stromberg I (1998). Morphological and functional evidence for enhanced growth and potassium-evoked dopamine release in striatal grafts innervated with a patchy growth pattern. An in oculo nigrostriatal cograft study. Cell Transplant. 7(2): 97-108.
https://pubmed.ncbi.nlm.nih.gov/9588592

II. Tornqvist N, Bjorklund L, Almqvist P, Wahlberg L, Stromberg I (2000). Implantation of bioactive growth factor-secreting rods enhances fetal dopaminergic graft survival, outgrowth density, and functional recovery in a rat model of Parkinsons disease. Exp Neurol. 164(1): 130-8.
https://pubmed.ncbi.nlm.nih.gov/10877923

III. Tornqvist N, Bjorklund L, Stromberg I (2001). Evidence for target-specific nerve fiber outgrowth from subpopulations of grafted dopaminergic neurons: a retrograde tracing study using in oculo and intracranial grafting. Exp Neurol. 169(2): 329-39.
https://pubmed.ncbi.nlm.nih.gov/11358446

IV. Tornqvist N, Hermanson E, Perlmann T, Stromberg I (2002). Generation of tyrosine hydroxylase-immunoreactive neurons in ventral mesencephalic tissue of Nurr1 deficient mice. Brain Res Dev Brain Res. 133(1): 37-47.
https://pubmed.ncbi.nlm.nih.gov/11850062

V. Tornqvist N, Stromberg I (2002). The localization of extracellular matrix molecules in intrastriatal ventral mesencephalic grafts in the rat model of Parkinsons disease. [Manuscript]