Parkinson's disease is a common neurological disorder characterized by a dopamin-ergic deficit in the striatum, principally caused by the degeneration of the substantia nigra. This relatively focal pathology offers the option to treat the disorder with cell replacement therapy. Since the 1980s preclinical and clinical experiments with transplantation of fetal mesencephalic progenitor cells gathered evidence for possible efficacy. However, the use of grafts derived from fetal sources remains problematic and there is a need to find alternative sources for grafts.
Depending on the questions to be answered by an experiment, either naïve animals or suitable models for Parkinson's disease may serve as graft recipients.
1. Anesthetize animal with 100mg/kg ketamine + 10mg/kg xylazine. Redosing with xylazine is not recommended; if necessary, redose with ketamine alone.
2. Mount animal in stereotaxic frame.
3. Make a medial skin incision of approx. 15 mm length (Figure 23.3).
4. Use the four bulldog-type clamps to retract the skin from the operation area.
5. Carefully remove periosteum with bone scraper, without applying strong pressure.
6. Clean with disinfectant (i.e. povidone-iodine).
7. Identify bregma (Figure 23.3), measure coordinates [anteroposterior (AP), mediolateral (ML) and ventrodorsal (VD)] with tip of the injection needle. Be careful to only slightly touch the skull with the needle, in order to avoid blunting and/or bending the tip.
8. Identify lambda (Figure 23.3), and adjust incisor bar so that lambda and bregma are at same height.
9. Mark the burr hole position with a surgical skin marker:
Note: These coordinates are an example for a single graft in the striatum. Depending on the purpose of a study one might perform multiple micro-grafts or graft in different areas of the CNS. Coordinates can be derived from published materials (e.g. Paxinos and Watson, 2004). When starting a new experiment, some pilot experiments with injection of dye or ink at the calculated coordinates should be performed in order to correct for differences in strain, age, and gender.
10. Drill hole. Remove bone fragments and clean skull with disinfectant.
11. Load 10 pL syringe with cell suspension.
12. Lower injection needle to 6.0 mm below the bregma at the burr hole position.
Note: The injected volume is an example. Depending on the cell concentration in the graft suspension, one might want to vary the injected volume. It is not recommended, however, to drastically increase the injected volume at one site. If higher volumes are necessary, we would recommend transplanting to more than one site. In order to minimize damage through transplantation, one can, for example, put several deposits at different heights on one needle tract.
16. Slowly retract needle.
17. Suture skin.
18. Remove animal from stereotactic frame and return it to its cage.
19. Keep animals under observation until they are fully awake.
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