C .W. Shults et al. Brain Research 883 2000 192 –204
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behavioral effects and the loss of striatal dopaminergic for 5 min. The cells were centrifuged and resuspended at a
fibers and nigral dopaminergic neurons in MPTP-treated final concentration of 100,000 cells ml in PBS with
D
- mice [7]. A trophic effect of FGF-2 on the nigrostriatal
glucose 1.0 g l and 2 normal rat serum. dopaminergic system is not surprising in light of the
Female Sprague–Dawley rats, which weighed approxi- presence of both FGF-2 and the FGF receptor-1 in the
mately 225 g, were housed under a 12 h light dark cycle neurons of the substantia nigra pars compacta SNpc
with free access to food and water and were cared for [4,6,20].
according to NIH guidelines. Prior to surgical procedures, A focus of our research has been to develop methods by
the animals were anesthetized with a mixture of ketamine, which trophic factors, such as FGF-2, can be used effec-
xylazine and acepromazine. Twenty-four animals were tively in the treatment of Parkinson’s disease. The cardinal
used in the study. The animals received one of three pathological feature of Parkinson’s disease is loss of
treatments: burr hole only two of these animals died prior dopaminergic neurons in the SNpc and their axons to the
to lesioning with 6-OHDA n56, implantation of b-gal- striatum [17]. Pertinent to Parkinson’s disease are the
fibroblasts n58, or implantation of FGF-2-fibroblasts observations that most of the neurons in the human SNpc
n58. The burr hole was made at: anterior 20.5 mm and are immunoreactive for FGF-2 and in Parkinson’s disease
lateral right 22.4 mm from bregma. The cells were there is disproportionate loss of FGF-2-immunoreactive
implanted using a 10 ml Hamilton syringe with a 28-gauge neurons in the SNpc [47].
needle. The cells were implanted at two depths: 6.4 mm Although certain trophic factors, such as FGF-2, hold
and 4.8 mm from the top of the skull. At each site 1.5 ml promise as treatments in Parkinson’s disease, the optimal
of cells 150,000 were implanted. The cells were injected method of using trophic factors as treatments for Parkin-
at a rate of 0.5 ml min, and the needle was left in place for son’s disease remains uncertain. Some of the issues that
5 min after each injection. must be addressed in development of trophic factors as
Approximately 2 weeks later, the animals received a treatments for neurological disorders include: delivery to
single, intrastriatal injection of 6-OHDA. Twenty-five the central nervous system, limitation of delivery to
micrograms of 6-OHDA hydrobromide RBI-Sigma, St. specific regions of the central nervous system, and delivery
Louis, MO, which was dissolved in 1.5 ml of normal of the optimal dose of trophic factor. A promising tech-
saline with 0.2 ascorbic acid, was injected at a depth of nique for sustained delivery of a trophic factor to a specific
5.6 mm ventral to the skull at the same anterior posterior region of the central nervous system is to genetically
and medial lateral coordinates at which the cells had been engineer cells, such as fibroblasts, to produce trophic
injected. The injection site was chosen to be midway factors and implant the cells into specific brain region, e.g.,
between the two implant sites. 6-OHDA was injected over the striatum [13]. We have found that fibroblasts ge-
5 min, and the needle was left in place for an additional 5 netically engineered to produce FGF-2 and implanted into
min before withdrawal of the needle. the striatum can protect the nigrostriatal system from
Beginning the following week, the animals were tested intrastriatal injections of 6-OHDA.
weekly for apomorphine-induced rotation 0.1 mg kg for 30 min and amphetamine-induced rotation 1.3 mg kg of
amphetamine sulfate for the 20–60 min epoch following
2. Materials and methods injection.
After 3 weeks of behavioral testing, the animals were 2.1. Effects in 6-OHDA lesion model
anesthetized and perfused with 4 paraformaldehyde in phosphate buffer PB. The brains were sectioned at 25
Fibroblasts from Fischer 344 rats were genetically mm intervals. Sections from the forebrains and midbrains
engineered to express either FGF-2 or b-galactosidase were stained for Nissl substance at 100 mm and 200 mm
b-gal, as previously described [37]. The b-gal fibroblasts intervals, respectively. At 200 mm intervals sections from
were used after passage 10, and the FGF-2 fibroblasts were the forebrains and midbrains were immunolabeled for TH.
used after passage 11. The cells were grown to confluence The sections were rinsed three times in PBS, quenched in
2
in 75 cm flasks in medium consisting of Dulbecco’s
0.9 H O 1 NaOH PBS for 20 min, and rinsed five
2 2
modified Eagle’s medium with
D
-glucose 1.0 g l, 10 times in PBS. After a 1 h incubation in 20 normal horse
fetal bovine serum, fungizone 2 mg ml, gentamicin sulfate serum 0.25 Triton X-100 PBS, sections were incubated
50 mg ml, G418 400 mg ml, glutamine 1 mM. After the in a 1:5000 dilution of mouse anti-TH monoclonal anti-
fibroblasts had reached confluence, they were dissociated body Chemicon, Temecula, CA overnight at 48C. After
by first removing the medium, rinsing with Dulbecco’s three rinses in Triton X-100 PBS, sections were incubated
phosphate-buffered saline PBS with 1.0 g l
D
-glucose in a biotinylated anti-mouse IgG at a 1:250 dilution
and 2 normal rat serum, and then treatment with trypsin Vector, Burlingame, CA for 1 h, followed by three rinses
EDTA solution 226 mg l EDTA tetrasodium, 1.0 g l in PBS. Finally, the sections were incubated in the avidin–
D
-glucose, 400 mg l potassium chloride, 8 g l sodium biotin complex substrate ABC Elite, Vector, Burlingame,
chloride, 500 mg l trypsin, 580 mg l sodium bicarbonate CA and developed with a nickel ammonium-intensified
194 C
DAB reaction. After five rinses in PBS, sections were performed using the 34 objective. The evaluator outlined
mounted on gelatin-coated slides, air-dried, dehydrated and the region with GFAP-IR fibers, measured that region in
coverslipped. the three sections and summed the areas for the three
Nissl-stained sections of the striatum from each animal sections.
were evaluated by an observer, who was blinded to the Estimation of number of TH-IR neurons in the SNpc
treatment, to determine whether the injection track was in was achieved by using the optical fractionator procedure
the proper location and whether the brain was free of [49] with the assistance of a semiautomatic stereology
changes suggesting infection or other abnormality. One of system StereoInvestigator version 3.0, MicroBrightField,
the animals that received a burr hole only had a congenital Inc, Brattleboro, VT. The sections from the midbrains
abnormality of the cortex, and this animal was not included were 25 mm in thickness, and every eighth section was
in the analyses. The final number of animals in each group immunolabeled and evaluated. Analysis began at the
was: burr hole — 5, b-gal-fibroblasts — 8, and FGF-2- rostral border of the SNpc approximately 24.8 mm caudal
fibroblasts — 8. to bregma [35] and continued to the caudal extent of the
In the five consecutive, TH-immunolabeled striatal SNpc. Seven or eight sections were analyzed in each brain.
sections, we used an image analysis system to determine Images were acquired on an Olympus BH2 microscope
the area in the injected striatum that lacked TH-IR fibers Tokyo, Japan equipped with appropriate filter sets using
and the total area of the injected striatum. The system has a single-chip CCD camera and displayed by using the
been previously described [43]. An observer outlined the StereoInvestigator software driving a Ludl X–Y–Z motor-
regions of the striatum that were devoid of TH-IR axons ized stage Ludl Electronic Products, Ltd., Hawthorn, NY.
and the total area of the striatum. The percentage of the Rough boundaries to delimit the optical fractionator area
striatum that was devoid of TH-IR fibers was calculated sampling fraction were drawn by using a 310 objective
for each animal. In addition, in three sections, which were and subsequently were sampled by using a 360 objective
centered on the implant, we measured the width of the oil; SPlanApo, 1.4 N.A.. The area evaluated included
region that lacked TH-IR fibers at its greatest length and both the SNpc and the substantia nigra pars lateralis. A
averaged this length for each animal. All quantitative guard focus height of 2 mm was set in the software, which
studies of anatomical changes, such as this one, were would then focus through each sample region for sections
carried out by an observer, who was unaware of the determined to be on average 9 mm in thickness with
treatment group assignment. markings made on cells meeting the sampling criteria
In each of the Nissl-stained sections in which a graft interactively throughout the focus session. Two dimension-
was present, we used the image analysis system to measure al counting rules state that cells that lie entirely within the
the cross-sectional area of the graft. The cross-sectional sampling frame are counted, whereas those that lie outside
areas were summed for each animal. Injection of 6-OHDA are not. For cells that intersect the sampling frame, those
and destruction of the dopaminergic axons typically caused that intersect the green lines are counted, whereas those
inflammation, which could obscure the grafted cells. The that intersect the red lines are excluded. By using these
graft size was only measured in areas in which there was counting rules, one counts real cells in a volumetric sub-
no inflammation. In two animals, which had received sample of the entire tissue without making assumptions
grafts of b-gal-fibroblasts, in some of the sections the graft regarding the size, shape, or orientation of cells. This raw
had detached from the section during the staining process. count of cells is used to estimate total neuronal number
Although the cavity left outlined the graft site, we ex- using mathematical calculation from tissue volume.
cluded these animals in the analysis. In two animals, which received grafts of FGF-2 fibroblasts, the implanted cells
2.2. FGF-2 were obscured by inflammatory cells or the needle track,
and these two animals were not included in this analysis. 2.2.1. Expression in intact rats
Although, the grafts were obscured in the sections avail- To determine the time-course of expression of FGF-2
able and could not be adequately evaluated, the number of and reaction to the implants, in 18 intact female Sprague–
dopaminergic neurons in the SN and preservation of Dawley rats approximately 225 g we implanted FGF-2-
dopaminergic axons in the striatum was similar to that of fibroblasts n59 and b-gal-fibroblasts n59 by the
the other animals that received FGF-2-fibroblasts. technique described above. At 1, 2 and 5 weeks, three
In three sequential striatal sections, which were 200 mm animals from each group were sacrificed. Two of the
apart and in which the implant was clearly visible, we animals in each group were fixed with paraformaldehyde
labeled the tissue for glial fibrillary acidic protein GFAP as described above, and the other one was fixed with
using a monoclonal antibody against GFAP Amersham, periodate–lysine–paraformaldehyde
fixative [29].
The Newark, NJ at a dilution of 1:1000 by the technique
brains were immunolabeled for FGF-2-IR material using a described above. We used the image analysis system to
monoclonal antibody Upstate Biotechnology, Lake Placid, measure the area of increased GFAP-IR material along the
NY at a concentration of 12.5 mg ml. injection track in these three sections. Measurements were
We noted the presence of FGF-2-IR material not only in
C .W. Shults et al. Brain Research 883 2000 192 –204
195
the implanted cells but also in cells adjacent to the implant, was taken for cell counting. The cell suspension was
so we sought to further define the cell type containing the centrifuged at 6003g for 5 min at room temperature. The
FGF-2-IR material by performing labeling of the tissue for dissociation solution was aspirated and the cells were
both FGF-2 and GFAP using fluorescent immunohisto- resuspended in 1 ml of PBS and transferred to two 1.5 ml
chemistry. Striatal sections were immunolabeled with a Eppendorf tubes. The cells were then centrifuged at 6003g
mouse monoclonal raised against FGF-2 Upstate Bio- for 5 min. In one of the tubes the PBS was aspirated and
technology, Lake Placid, NY at a concentration of 12.5 the cells were resuspended in 100 ml of lysis buffer. The
mg ml and a rabbit polyclonal antiserum raised against lysis buffer was composed of: 137 mM NaCl, 20 mM Tris
GFAP Sigma, St. Louis, MO, 1:80 dilution. The sections pH 8.0, 1 NP-40, 10 glycerol, 1 mM phenylmethyl-
were subsequently incubated with fluorescein-labeled an- sulfonyl fluoride, 10 mg ml aprotinin, 1 mg ml leupeptin
tiserum raised in goat against rabbit IgG Jackson Im- and 0.5 mM sodium vanadate. The tubes were kept on ice
munoresearch, West Grove, PA, 1:100 dilution and for 30 min and were vortexed for 10 s at 5 min intervals.
Rhodamine Red-X-labeled antiserum raised in goat against The contents were sonicated on ice three times for 5 s. The
mouse IgG Jackson Immunoresearch, 1:100 dilution. We tubes were then centrifuged at 12,0003g for 15 min at
evaluated seven animals FGF-2-fibroblasts sacrificed at 48C. The supernatant was transfered to an Eppendorf tube
weeks 1 1 and 5 2 and b-gal-fibroblasts sacrificed at and was assayed immediately for FGF-2-immunoreactive
weeks 1 2, 2 1 and 5 1. The double-labeled sections material using the FGF-2 ELISA system RD, Min-
were examined using a Zeiss Laser Scanning Microscope neapolis, MN and assayed for protein content by the BCA
LSM510 with Argon Krypton laser. technique Sigma Chemicals, St. Louis, MO.
In these sections we also immunolabeled the tissue for Statistical analyses were performed with GB-STAT,
OX-42, which recognizes microglia and macrophage as version 5.0.8 Silver Spring, MD. Rotational data was
well as monocytes and neutrophils [39]. The immuno- analyzed by application of two factor treatment and time
labeling was described above for TH. The monoclonal repeated measures analysis of variance ANOVA and post
antibody was obtained from Serotec Raleigh, NC, and hoc Newman–Keuls tests. Other analyses were performed
sections were incubated overnight with the antibody at a by application of one factor treatment randomized
1:250 dilution. ANOVA with post hoc Newman–Keuls tests.
2.2.2. Measurement of FGF-2 produced by the FGF-2 fibroblasts
3. Results
