3.4.1 Obtaining material for intrastriatal engraftment
i) Time-mating of SPRD rats and preparation of the embryonic tissue (performed by A. Nobre, PhD, Dr. M. Hohmann, M. Wesemann, Dipl.-Biol., Dr. O.
Baron, I. Kalve)
SPRD rat females purchased from Charles River were paired with breeder males just prior to the end of the light cycle. The following morning, each female was examined for the presence of vaginal plug composing of coagulated secretions from male accessory sex glands and indicating that coitus and ejaculation had occurred. The day the plug was observed was considered day 0 of gestation. These time-mated SPRD rats were exposed to CO2 for 3 minutes on the calculated embryonic day 12 (E12) and the isolated fetuses collected in cold phosphate-buffered saline (PBS).
Subsequently, the intact brain was removed. The E12 VM tissue was then dissected as described by Nikkhah et al. (Nikkhah et al., 1994a, Nikkhah et al., 1994b) based on the cell suspension technique according to Björklund et al. (Bjorklund et al., 1983a). The protocol previously used in our lab described by Timmer et al. (Timmer et al., 2006) was modified in abandoning of trypsin.
There were about 100,000 (depending on the age: 70,000–150,000) cells/VM obtained from the 12-day-old embryos having a crown-rump length of about 6 mm (Nobre, 2009, Cesnulevicius, 2006).
ii) Cell culture – the old (mono-layer) and the novel (co-layer) protocols (performed by A. Nobre, PhD, Dr. M. Hohmann, M. Wesemann, Dipl.-Biol.)
For in vitro differentiation of DA neurons three cell-culture media were used (see in Appendix 7.1) – adhesion, proliferation, and differentiation mediums respectively.
The proliferation medium resembled the composition of the attachment medium omitting fetal calf serum and B27 supplement. The differentiation medium contained no mitogen FGF-2. Cultures were maintained at 37oC in humidified 5% CO2–95% air incubator under normal oxygen conditions (20%). In order to detach the cells, the surface was rinsed with phosphate-buffered saline (PBS) once, and the cells were removed with trypsin / EDTA (PAA) incubation for 3–4 minutes. Trypsinization was stopped by adding attachment medium, and the cells were mechanically removed with a cell scraper. The cell aggregates were transferred into falcon tubes and triturated using a shaped glass pipette (three to five times). The latter three steps were very critical, especially for transplantation purpose. The cells were centrifuged for 5 min with 1000 rpm, and the pellet was resuspended in 1 ml of attachment medium. During these steps (particularly the cell scraping), about 10% of the cells got lost. The cells were counted, and the viability was controlled using trypan blue exclusion (viability: more than 98%).
The primary VM progenitor cells were cultured as already described in our lab (Timmer et al., 2006). The cells were counted using a cell-counting chamber (hemocytometer), adjusted to defined densities and plated on polyornithine (0.1mg/ml) / laminin (6 µg/ml) coated multi-well culture plates. So the freshly prepared cell suspensions were cultured for 1 day in attachment medium, followed by 3 days in proliferation medium.
For grafting experiments, the cells were initially seeded 400,000 cells per 6-well. On the 4th day in vitro (DIV4) sister-cultures were detached and total cell number estimated, followed by nucleofection (see 3.5.1. iii)). Afterwards the medium was switched to differentiation medium for 2 – 6 days (Ratzka et al., 2012). As for reseeding of DIV4 sister-cultures post-nucleofection, cells were either seeded on polyornithine / laminin coated plates for the mono-layer method as previously described by a former doctoral student in our lab (Cesnulevicius et al., 2006) or on top of the bottom-layer cultures in a certain ratio 1:3, 1:6 or 1:8 for the co-layer protocol respectively.
iii) Nucleofection (performed by A. Nobre, PhD, Dr. M. Hohmann, M. Wesemann, Dipl.-Biol.)
For nucleofection, primary mesencephalic cells were adjusted to a volume of 2,000,000. After centrifugation (1000 rpm for 5 minutes) the medium was removed and the cells were re-suspended in 100 µl of nucleofection solution and 5 µg of plasmid DNA. The Basic Nucleofector Kit for primary mammalian neurons (Amaxa) with the program A-033 of the Nucleofector device (Amaxa) was used as described by Cesnulevicius et al. (Cesnulevicius et al., 2006). After the pulse, 900 µl of RPMI 1640 medium (Biochrom AG, Berlin)containing 10% FCS was immediately added to neutralize the nucleofectionsolution.
iv) Expression vector used (performed by Dr. A. Ratzka)
The following DNA plasmid constructs were used for transfection: pCAGGS-EGFP, pCAGGS-EGFP-FLAG, and pCAGGS-BDNF-FLAG. pCAGGS-EGFP plasmid was a gift from Dr. Whiteford Alexopoulou (Alexopoulou et al., 2008). The pCAGGS-EGFP-FLAG and pCAGGS-BDNF-pCAGGS-EGFP-FLAG plasmids were constructed by Dr. A. Ratzka (Institute of Neuroanatomy, Hanover Medical School). The CAG-promoter is composed of a chicken β-actin promoter + cytomegalovirus (CMV) enhancer. In case of two latter plasmids it is possible to detect EGFP protein and BDNF via c-terminal 3xFLAG tag by immunocytochemistry, immunohistochemistry, and western blotting.
The decision to use the respective plasmid backbone was made based on preliminary in vitro experiments regarding the time and expression level following transfection of primary E12 VM neuronal progenitor cultures (Nobre, 2009).
In preliminary in vivo studies in 6-OHDA neurotoxin lesion rats other DNA plasmid constructs have been used, e.g. CDNF-CMV-14 (R403) and p3xFLAG-RGFP-CMV-14 (R405).
3.4.2 Microtransplantation
Not earlier than 8 weeks after 6-OHDA lesioning surgery and after having allocated animals in equal experimental groups according to the drug-induced rotational asymmetry, microtransplantation surgery was performed using a pulsed air pressure system connected to a glass micropipette (see Introduction 1.5.3.).
On the transplantation day, the cells were detached, resuspended with 0.05% DNase / DMEM and adjusted to a final density of cells – 100,000 cells/µl for Exp I – III and 130,000 cells/µl for Exp IV – VI.
After i.p. injection of Chloral hydrate 3.6% (3.6 dissolved in 100 ml dH2O), the animals were fixed in the stereotactic apparatus (Fig. 9A) and the tooth-bar was adjusted to 0,0 mm. The stereotactic transplantation into the denervated striatum was performed using a glass capillary attached to a 2µl Hamilton syringe as described by Nikkhah et al. (Nikkhah et al., 1994b). After rinsing the capillary with cell-free medium, single cell suspension was placed into the right striatum. The grafts consisted of two deposits (each 2 x 1µl) over two implantation tracks, one medial and one lateral, and the injection rate was 1μl/minute. After implantation of each deposit (the lower graft implanted first along each of the tracks), the capillary was kept in place for 3 minutes prior to slow retraction and rinsed with medium after each track respectively. This was followed by suturing the wound.
The slightly modified stereotactic coordinates (+0,5 mm in case of the craniocaudal coordinate) have been previously described by Timmer et al. (Timmer et al., 2004).
For implantation parameters see Table 5.
Table 5. Transplantation parameters for intrastriatal grafting given in mm with reference to the Bregma and dura (Paxinos and Watson, 1998)
Grafts on the right side
Before the in vivo experiments involving stereological quantification of the surviving DA neurons in the intrastriatal grafts (Exp IV to VI) were started, preliminary tests simulating the transplantation situation in a toxin-lesioned rat brain were applied to the pulsed air pressure system connected to a glass micropipette used for the microtransplantations. The cells were detached, resuspended with 0.05% DNase / DMEM and adjusted to a final density of 100,000 cells/µl. One μl deposits were then injected in separate eppendorf tubes and the number of cells counted afterwards. As a result, the final suspension used for intrastritatal engrafting was adjusted to
130,000 cells/μl. Such adjustment was supported by data published by Nikkhah et al.
(2009) depicting that graft size can be tailored effectively by varying the density of the final cell suspension at least between 11,000 and 320,000 cells/μl, resulting in comparable survival of the DA neurons in the range of 2-4%, when using the microtransplantation approach.
The animals were not immunosuppressed as it is not necessary for intraparenchymal allografts of fetal mesencephalic cell suspensions in this Parkinson model (Brandis et al., 1998).
3.5 Transcardial perfusion of the experimental animals, postfixation of the