Experimental subjects consisted of seven adult male St. Kitts/African green monkeys (Chlorocebus sabaeus
) from the colony at the St. Kitts Biomedical Research Foundation, St. Kitts, West Indies. All studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. To produce a dopamine-depleted host environment, all of the monkeys were treated with five 0.4 mg/kg intramuscular doses of MPTP over a five day period, as described in detail (Redmond et al., 2007
). Tissue for grafting into the SN was obtained and dissected freshly from fetuses of embryonic age 44 days, and for striatal co-grafting from embryonic age 52–56 (Sladek, 1995
) based upon ultrasonography and direct measurements after hysterotomy vs prior morphometric tables. Four monkeys (Group1: X222, X200, X092, and X104) received unilateral small solid grafts of ventral mesencephalic tissue (VM) into the rostral mesencephalon immediately dorsal to or within the host substantia nigra. Kopf Instruments stereotaxic devices (Tujunga, CA, USA) were used to target coordinates AP 11.1, Lateral 3.5, and Vertical 12.1 mm from actual ear bar zero), and vector delivered GDNF was injected in the ipsilateral striatum via a 22 gauge needle attached to a Hamilton Syringe (Reno, NV, USA) driven by a microprofusion pump (Stoelting Instruments, Wood Dale IL, USA) at a rate of 1 µl/minute (). Two of these animals also received GDNF in the contralateral striatum (X222, X200). Prior to sacrifice FG was injected into the identical striatal targets on the ipsilateral and contralateral sides to the SN grafts in these two animals, but only into the contralateral side in the other two animals to serve as controls (X092, X104) for potential spread of the label. Three additional animals (Group2: W586, W587, W626) received striatal and nigral co-grafts at increasing distances apart. The nigral grafts were positioned similarly to those in the first group whereas the striatal grafts were located more rostral along the trajectory of the ascending pathway to the striatum from the SN (each 2.5 mm more rostral and 1 mm more dorsal). Two of these three animals received FG injections bilaterally into the striatum, the remaining animal received a single FG injection as a control for the contralateral nigral graft ().
Figure 1 The positions of graft, vector and retrograde tracer injection are illustrated schematically for Group 1 animals. The cerebrum is shown as an oval while the rostral brain stem is represented by a tube. In this and the following diagram the presence or (more ...)
Group 2 animals are shown schematically as in . The relative distances between the co-grafts and the FG injections are indicated. MGE medial ganglionic eminence, LGE lateral ganglionic eminence.
All of the monkeys were without any observable signs of parkinsonism at the time of the grafting experiments and remained asymptomatic throughout the experiment, but based upon prior data of a large number of monkeys treated with identical MPTP doses, had dopamine depletions in the striatum of at least 50% of normals. Group 1 was sacrificed at approximately 6 months after graft implantation while brains for Group 2 were removed after 22 months.
The GDNF gene was delivered using two different vectors – modified adeno-associated virus, serotype 2/, with mouse phosphoglycerate kinase 1 promoter/GDNF (rAAV2/PGK-GDNF) vector, constructed as previously described (Rabinowitz et al., 2002
), or the same PGK/GDNF cassette delivered by a modified lentiviral vector derived from equine infectious anemia virus (rEIAV/PGK/GDNF) (Deglon et al., 2000
). The AAV2 vector was confirmed to express GDNF in vivo in this species for at least two years, whereas the expression of GDNF from the EIAV vector was significantly attenuated after 3 months. Injections were made bilaterally in the mid to rostral portion of the caudate (stereotaxic coordinate, AP 23.1, Lateral 4.0 mm, vertical 19.0 from ear bar zero), at a rate of 1 µl/minute) at the same surgery when fetal VM tissue was implanted. X200 and X222 were injected bilaterally into the caudate with 5 µl rAAV2/GDNF, and X092 and X104 were injected with 10 µl EIAV/GDNF into the right caudate (ipsilateral to the VM graft in the SN) and near the SN on the left side (see –). The titers of the AAV2 batch (10e11 vg/ml) and EIAV batch (3x10e8 transducing units/ml) were roughly comparable assuming one transducing unit per 300 physical particles for AAV2.
For Group1, FG was injected 7 to 20 days before sacrifice, 10 µl of a 2% solution, infused at a rate of 0.5 µl/minute standard targets for the caudate and putamen.. The striatal/VM co-graft monkeys in Group2 were injected with 5 µl of 2% FG into anterior and middle caudate, and 10 µl into posterior caudate and posterior putamen. Stereotaxic target coordinates for FG injections were for caudate, AP 23.0, 21.0, and 19.0, Lateral 4.0, Vertical 19.0, and for putamen, AP 21.1, Lateral 10.0, Vertical, 19.0.
Animals were killed by pentobarbital overdose and perfused with ice-cold physiological saline, followed by buffered 4% paraformaldehyde solution before brain removal, 12 hours post fixation in paraformaldehyde solution, and storage in 30% sucrose., Brains were then frozen and sectioned into 50 µm thick parasagittal sections using a sliding blade microtome and stored in a cryoprotectant solution. Sections were processed immunohistochemically using double fluorescence labeling for tyrosine hydroxylase (TH) and fluorogold (FG). Sections were incubated with TH antibody (1:1000, Millipore Cat.#MAB318, mouse monoclonal against TH purified from PC12 cells) and FG antibody (1:5000, Millipore Cat.#AB153, rabbit polyclonal against fluorogold). Visualization for FG-labeled cells was with Cy2-conjugated Goat Anti-Rabbit IgG (Jackson Immunoresearch Cat.#111-225-144) and for TH-labelled cells was with Texas Red Anti-Mouse IgG (Vector Laboratories Cat.#TI2000). TH staining was consistent with the classic morphology and distribution of dopamine cells in primate tissue and Cy2 fluorescence was absent in tissue that had not been exposed to FG labeling.
Cell counts were performed using an unbiased optical fractionator sterological method whereby digital images were obtained in selected areas using an Olympus AX-70 microscope equipped with an automated z-stage and MicroSuiteBiological software. Visualization of TH- or FG-labeled cells was done immunohistochemically with diaminobenzidine-nickel sulfate as the chromagen.