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. Author manuscript; available in PMC: 2011 Feb 2.
Published in final edited form as: Neurochem Res. 2010 Apr;35(4):531–539. doi: 10.1007/s11064-009-0093-7

Strain Difference in the Up-Regulation of FGF-2 Protein Following a Neurotoxic Lesion of the Nigrostriatal Pathway

David M Yurek 1,✉, Anita M Fletcher 2, Laura E Peters 3, Wayne A Cass 4
PMCID: PMC3032212  NIHMSID: NIHMS265701  PMID: 19921430

Abstract

Lesions of the nigrostriatal pathway are known to induce a compensatory up-regulation of various neurotrophic factors. In this study we examined protein content of basic fibroblast growth factor (FGF-2) in tissue samples taken from the ventral midbrain and striatum at two different time points following a neurotoxic lesion of the nigrostriatal pathway in two different rat strains, the out-bred Sprague–Dawley (SD) and inbred F344 × Brown Norway F1 hybrid (F344BNF1). Despite both rat strains having comparable lesions of the nigrostriatal pathway, we observed a difference in the temporal up-regulation of FGF-2 in ventral midbrain samples taken from the side ipsilateral to the lesion. Basic FGF was significantly up-regulated in ventral midbrain in SD rats 1 week post-lesion while we did not observe an up-regulation of FGF-2 in the lesioned ventral midbrain of F344BNF1 at this same time point. However, both strains showed a significant up-regulation of FGF-2 in the lesioned ventral midbrain 3 weeks post-lesion. Sprague–Dawley rats also appeared to be more sensitive to the lesion in terms of up-regulating FGF-2 expression. The differences reported here suggest currently unknown genetic differences between these two strains may be important factors for regulating the compensatory release of neurotrophic factors, such as FGF-2, in response to a neurotoxic lesion of the nigrostriatal pathway.

Keywords: Neurotrophic factor, Basic FGF, Dopamine, Parkinson’s disease, Substantia nigra, Sprague–Dawley, Fisher 344 × Brown Norway F1 hybrid

Introduction

Basic FGF is one member of a large family of fibroblast growth factors [1, 2]. Basic FGF, along with several other members of the FGF family, has been shown to be important signaling protein during the development of the central nervous system as well as regulating the maintenance of neural structures, such as the nigrostriatal pathway, in adult and/or damaged brain tissue [3, 4]. Basic FGF is localized to the substantia nigra of rat, monkey, and human [5, 6], and has been shown to promote the survival and growth of cultured dopaminergic neurons [7–10]. Several studies have shown that the trophic effect of FGF-2 on dopaminergic neurons is mediated by glia [11, 12].

Damage to the nigrostriatal pathway can induce a compensatory up-regulation of several neurotrophins or neurotrophic factors that are known to provide neurotrophic support for dopaminergic neurons. In previous studies, we and others reported a differential up-regulation of the neurotrophin brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor in the striatum and ventral midbrain of adult rats [13–16] or monkeys [17]. There are conflicting reports whether a similar phenomenon occurs for FGF-2 following a neurotoxic lesion of the nigrostriatal pathway. While qualitative and semi-quantitative techniques such as immunohistochemical, in situ hybridization, Western blots, PCR, or cDNA array techniques have been used to characterize FGF-2 expression in the nigrostriatal pathway, very few studies have performed quantitative measures of FGF-2 protein expression. Claus et al. [18] measured FGF-2 protein expression in the lesioned nigrostriatal pathway using Western blot analysis and observed no change in FGF-2 expression during a 28 day post-lesion period. Nakajima et al. [19] observed a slight but non-significant increase of FGF-2 protein expression in the striatum ipsilateral to a 6-OHDA lesion of the nigrostriatal pathway 3 weeks post-lesion using an enzyme-linked immunosorbent assay (ELISA) analysis. In contrast, results from other studies that have examined FGF-2 immunohistochemistry or measured FGF-2 mRNA levels in the lesioned nigrostriatal pathway show a persistent up-regulation of FGF-2 expression in the ventral midbrain that begins immediately after the lesion and is sustained for at least 2 weeks [20, 21]. In a mouse model of Parkinson’s disease (PD), an up-regulation of FGF-2 mRNA can be observed in the striatum 7–18 days following MPTP treatment [22, 23]. Similarly, cDNA array analysis of tissue samples taken from the striatum of rats 1 week following a 6-OHDA lesion showed a significant up-regulation of FGF-2 [24]. The results of these studies were obtained primarily from tissue samples taken from young adult rats and indicate that damage to the nigrostriatal pathway may elicit an increased expression of FGF-2 in brain tissue as a compensatory neurotrophic mechanism. In human brain, postmortem analysis of FGF-2 immunoreactivity in the substantia nigra (SN) of normal aged brain and brain from Parkinson’s patients are significantly different. In the normal aging human brain, 82% of nigral dopaminergic neurons are FGF-2 immunoreactive while only 12% of the remaining nigral dopaminergic neurons are FGF-2 immunoreactive in the brains of patients diagnosed with Parkinson’s disease [25, 26]. While this reduction of FGF-2 immunoreactivity in dopaminergic neurons of PD patients may be relevant to the progression of the disease, it is important to remember that glia may be the primary source of FGF-2 and it remains to be determined whether there is a reduction of FGF-2 in mesencephalic glia in PD patients.

Factors such as species, age, gender, or strain of rat can contribute to disparate results in experimental data that are often reported in the literature. For example, age and gender differences in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) have been reported in several brain structures [13, 14, 27, 28]. Dhabhar et al. [29] reported large differences in diurnal and stress corticosterone profiles for three genetically similar strains of rats: Fisher 344, Sprague–Dawley, and Lewis. In an animal model of ischemia-induced retinal neovascularization, hyperoxia-treated SD rats showed changes in pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) levels that were less in magnitude and of shorter duration than in Brown Norway rats [30]. Thus, it is clear from these studies that age, gender, and genetic factors can influence protein expression within the central nervous system. This may also explain why there is a disparity in the literature about the expression of FGF-2 in the nigrostriatal pathway following a neurotoxic lesion.

In this study we examined whether a neurotoxic lesion of the nigrostriatal pathway altered FGF-2 expression in the nigrostriatal pathway of two different rat strains: the widely used Sprague–Dawley (SD) and the inbred Brown Fisher 344 × Brown Norway F1 hybrid (F344BNF1).

Experimental Procedure

Animals

A total of 10 (4–5 months old) male Sprague–Dawley (SD; Harlan Farms) and 13 male (4–5 month old) Fischer 344 × Brown Norway F1 (F344BNF1) hybrid rats (NIA Aging Colony) were used in this study. Animals were housed in environmentally regulated rooms and had free access to food and water for the duration of the study. All animal procedures were conducted in strict compliance with approved institutional protocols and in accordance with the provisions for animal care and use described in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86–23, 1985).

6-Hydroxydopamine Lesions

All rats were given unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway; 6-OHDA (Sigma) was dissolved in 0.9% saline (containing 0.2% ascorbic acid) at a concentration of 3.0 µg/µl and stereotaxically injected into the nigrostriatal pathway of anesthetized rats at a rate of 1.0 µl/min for 2 min. Each rat received two injections of 6-OHDA: one in the vicinity of the medial forebrain bundle (AP −4.4, ML 1.2, DV −7.5) and the other in the rostral pars compacta of the substantia nigra (AP −5.3, ML 2.0, DV −7.5); all coordinates reported in this study represent millimeter adjustments from bregma (AP, ML) and below the dural surface (DV) with the top of the skull in a flat position. This technique routinely produces complete lesions of dopamine neurons in the A9 and A10 midbrain regions, and near complete denervation of dopaminergic fibers innervating the ipsilateral striatum.

Tissue Dissections

For striatal dissections, brains were removed from euthanized animals and placed upside down (ventral surface facing upwards) into a stainless steel brain matrix (ASI Instruments, RMB-4000C). Approximate stereotaxic coordinates are listed in parentheses [31]. The initial coronal cut was targeted at the level of the optic chiasm (~ AP 0.0) and a second cut was made 2.0 mm anterior to the initial cut. The 2.0 mm thick coronal slab formed by these two cuts was removed from the brain matrix and then divided into two pieces along the midline. Both the left and right striatum were dissected from the two pieces as follows. A rectangular piece of tissue was dissected by making the following cuts: (1) a cut parallel to the midline was made just lateral to the lateral ventricle, (2) a cut parallel to the midline was made medial to the lateral aspect of the corpus callosum, (3) a cut perpendicular to the midline was made just ventral to the dorsal aspect of the corpus callosum, and (4) a cut perpendicular to the midline was made just dorsal to the anterior commissure. For the ventral midbrain dissections we used the remaining brain piece in the matrix and made another coronal cut 5.0 mm posterior to the initial cut made for the striatal dissection (~ AP −5.0); a second coronal cut was made 2.0 mm posterior to this cut and the 2.0 mm coronal tissue slab that was formed between these two cuts was removed from the brain matrix. A cut along the midline divided the tissue slab into left and right hemispheric pieces. The dark pigment tissue that comprises the substantia nigra/VTA was dissected from each hemispheric piece.

Enzyme-Linked Immunosorbent Assay (ELISA) for FGF-2

Animals were euthanized either 1 or 3 weeks after the 6-OHDA lesions. Brains were removed, the striatum and ventral midbrain (substantia nigra/ventral tegmental area) of each hemisphere were dissected on ice, and the samples were then stored at −80°C. Subsequently, each tissue sample was homogenized in 1.5 ml volumes of homogenate buffer [400 mM NaCl, 0.1% Triton-X, 2.0 mM EDTA, 0.1 mM benzethonium chloride, 2.0 mM benzamidine, 0.1 mM PMSF, Aprotinin (9.7 TIU/ml), 0.5% BSA, 0.1 M phosphate buffer, pH = 7.4]. The homogenate was centrifuged for 10 min at 10,000 × g at 4°C. The homogenate was divided into 100 µl duplicate samples and the remaining homogenate was used in the HPLC assay described below. Human FGF-2 is 97% amino acid identical to rat FGF-2 [32]. Basic FGF concentration was determined using a sandwich ELISA format: FGF-2 from each sample was captured with a monoclonal antibody against human FGF-2 (1:200, R&D Systems, Minneapolis, MN); the captured FGF-2 was then bound to a second, specific monoclonal antibody against FGF-2 (1:200, R&D Systems, Minneapolis, MN). After washing, the amount of specifically bound antibody was bound to SA-HRP (1:250, R&D Systems, Minneapolis, MN) as a tertiary reactant. Unbound conjugate was removed by washing, and following an incubation period in TMB One (Promega, Madison, WI), the color change was measured in a microplate reader at 450 nm. The amount of FGF-2 was proportional to the color change generated in an oxidation–reduction reaction. Based on the standard curves, the reliability of the assays is .980–.999.

HPLC Analysis for Dopamine Content

Tissue levels of DA were measured using HPLC with electrochemical detection. An aliquot of sample in ELISA homogenization buffer was transferred to 0.22 µm pore size Millipore Ultrafree centrifugal filters and spun at 12,000 × g for 1 min. Forty microlitre of filtrate was then injected onto the HPLC column for separation and analysis as previously described [33]. Results were expressed as percent change in DA on the lesioned side compared to the contralateral side.

Immunohistochemistry

All rats were anesthetized with Fatal-Plus™ (0.88 ml/kg, IP; Vortech Pharmaceuticals, Dearborn, MI) and transcardially perfused with cold 0.9% saline followed by 4% buffered paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer). Brains were post-fixed overnight in 4% paraformaldehyde and transferred to 30% sucrose. Sections (40 µm) were cut using a sliding microtome and placed into a cryoprotectant solution at −20°C (47). For immunohistochemical detection of markers, free-floating sections were first rinsed in 0.1 M phosphate buffer (22 mM NaH2PO4 and 80 mM K2HPO4, pH = 7.2) followed by 3% H2O2 treatment to inhibit endogenous peroxidase activity. Sections were then rinsed in 0.1 M PO4 and 0.1 M PO4-Triton followed by an overnight incubation in primary antisera containing a monoclonal antibody against TH (1:4000; Chemicon). The sections were then incubated in an affinity-purified biotinylated goat anti- mouse IgG secondary antibody (1:400, Chemicon) and then incubated in an avidin–biotin-peroxidase complex (Vector Laboratories). Staining was completed by placing sections in a 0.003% H2O2 solution containing diaminobenzidine chromagen to visualize the reaction.

Statistics

Data from our HPLC analysis and ELISA analysis were analyzed using ANOVA, t-test or linear regression analysis.

Results

In these experiments we utilized two strains of rats, Sprague–Dawley (SD) and Fisher 344 × Brown Norway F1 hybrid (F344BNF1), and administered the neurotoxin 6-hydroxydopamine (6-OHDA) into two sites in order to create a near-complete lesion of dopamine neurons in the left nigrostriatal pathway. Tissue from the striatum and ventral midbrain was dissected from each hemisphere of the brain at either 1 or 3 weeks post-lesion. Subsequently, tissue samples were analyzed for FGF-2 protein and dopamine content.

Baseline measures for dopamine analysis were obtained from the side of the brain contralateral to the lesion and were as follows: for ventral midbrain samples, 581.1 ± 61.3 ng/g tissue weight (SD) and 491.5 ± 71.8 ng/g tissue weight (F344BNF1); for striatal samples, 6344.7 ± 216.1 ng/g tissue weight (SD) and 6810.1 ± 312.3 ng/g tissue weight (F344BNF1).

In tissue samples taken 1 week post-lesion, we observed large reductions of dopamine in striatal and ventral midbrain samples taken from the side ipsilateral to the lesion for both strain of rats. For SD rats, dopamine reductions in ventral midbrain samples ranged from 46.3 to 94.2% of control (contralateral) values with a mean reduction of 70.6 ± 8.2% (Fig. 1). For F344BNF1 rats, dopamine reductions in ventral midbrain samples ranged from 63.2 to 97.9% of control (contralateral) values with a mean reduction of 79.5 ± 3.7% (Fig. 1). At this same time point, loss of striatal dopamine was even greater. For SD rats, dopamine reductions in striatal samples ranged from 79.2 to 98.0% of control (contralateral) values with a mean reduction of 94.1 ± 3.0% (Fig. 1). For F344BNF1 rats, dopamine reductions in striatal samples ranged from 93.8 to 99.2% of control (contralateral) values with a mean reduction of 97.5 ± 0.5% (Fig. 1).

Fig. 1.

Fig. 1

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Loss of dopamine in the ventral midbrain or striatum 1 week following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum), the % dopamine loss = [1.0 – (amount of ipsilateral dopamine/amount of contralateral dopamine)] × 100. Bars represent the mean % dopamine loss (±SEM) for SD or F344BNF1 rats. Mean comparisons between SD (n = 5) and F344BNF1 (n = 7) were not statistically significant for ventral midbrain [t(9) = 1.36, P = 0.192] or striatal samples [t(9) = 1.45, P = 0.17]

In tissue samples taken 3 weeks post-lesion, we observed even larger reductions of dopamine in striatal and ventral midbrain samples taken from the side ipsilateral to the lesion for both strains of rats. For SD rats, dopamine reductions in ventral midbrain samples ranged from 77.1 to 96.8% of control (contralateral) values with a mean reduction of 89.7 ± 3.9% (Fig. 2). For F344BNF1 rats, dopamine reductions in ventral midbrain samples ranged from 71.2 to 98.4% of control (contralateral) values with a mean reduction of 90.5 ± 4.9% (Fig. 2). For SD rats, dopamine reductions in striatal samples ranged from 99.5 to 99.8% of control (contralateral) values with a mean reduction of 99.7 ± 0.1% (Fig. 2). For F344BNF1 rats, dopamine reductions in striatal samples ranged from 98.9 to 99.5% of control (contralateral) values with a mean reduction of 99.2 ± 0.12% (Fig. 2).

Fig. 2.

Fig. 2

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Loss of dopamine in the ventral midbrain or striatum 3 weeks following a unilateral 6-OHDA lesion of the left nigrostriatal pathway. The amount of dopamine in each sample was determined by HPLC analysis. For each brain structure (ventral midbrain or striatum), the % dopamine loss = [1.0 – (amount of ipsilateral dopamine/amount of contralateral dopamine)] × 100. Bars represent the mean % dopamine loss (±SEM) for SD or F344BNF1 rats. Mean comparisons between SD (n = 5) and F344BNF1 (n = 6) were not statistically significant for ventral midbrain [t(9) = 0.60, P = 0.558] or striatal samples [t(9) = 1.18, P = 0.26]

We also measured the DOPAC:DA ratio to examine dopamine turnover rate in the SN and striatum at each experimental time point. Figure 3 shows that neither strain showed an significant difference in the DOPAC:DA ratio between the lesion and intact SN at week 1 nor was DOPAC:DA significantly different in the lesioned SN between the strains. However, both strains showed an elevation of DOPAC:DA in the lesioned SN when compared to the intact SN at week 3. Three-way ANOVA (Strain, Week, Side) detected a significant Week × Side interaction [F(1,32) = 11.35, P = 0.002] while all other interactions were non-significant. The difference between DOPAC:DA in the lesioned SN between the strains at week 3 was not significant (P = 0.21). Figure 4 shows DOPAC:DA was significantly elevated in the lesion striatum when compared to the intact striatum for both strains at both time points. Three-way ANOVA (Strain, Week, Side) detected a significant effect of Side [F(1,32) = 5.16, P = 0.03] while the effect of Week was not significant [F(1,32) = 3.00, P = 0.09] and all other interactions were not significant.

Fig. 3.

Fig. 3

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DOPAC:DA ratio in the substantia nigra at 1 and 3 weeks post-lesion. The amount of dopamine (DA) and DOPAC in each sample was determined by HPLC analysis. Statistical analysis revealed a significant Week × Side interaction [F(1,32) = 11.35, P = 0.002]. The Strain × Side interaction was not significant [F(1,32) = 0.65, P = 0.43] indicating there was no statistical difference in mean DOPAC:DA values for the lesioned striatum between the two strains at both time points

Fig. 4.

Fig. 4

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DOPAC:DA ratio in the striatum at 1 and 3 weeks post-lesion. The amount of dopamine (DA) and DOPAC in each sample was determined by HPLC analysis. The only statistically significant effect was side [F(1,32) = 5.16, P = 0.03]. The Strain × Side interaction was not significant [F(1,32) = 0.045, P = 0.83] indicating there was no statistical difference in mean DOPAC:DA values for the lesioned striatum between the two strains at both time points

We performed an ELISA assay to determine the amount of FGF-2 protein in each sample by using a fraction of the same ventral midbrain and striatal samples that were used in the above neurochemical analysis. Table 1 summarizes FGF-2 protein content in tissue samples taken from both rat strains at two post-lesion time points.

Table 1.

FGF-2 protein content (ng/g tissue)

Post-lesion SD F344BNF1
Striatum Ventral midbrain Striatum Ventral midbrain
Ipsi. Contra. Ipsi. Contra. Ipsi. Contra. Ipsi. Contra.
1 Week 181 ± 4.1 177 ± 3.4 146 ± 3.9* 103 ± 6.7 164 ± 6.5 147 ± 5.7 126 ± 8.3 116 ± 12.3
3 Weeks 185 ± 2.8 199 ± 10.8 177 ± 15.6* 109 ± 8.6 183 ± 7.2 176 ± 10.1 177 ± 9.6* 129 ± 9.4
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*

P < 0.05 vs. contralateral side

The results presented in Table 1 show values for FGF-2 protein content in striatal tissue samples were statistically similar on the sides ipsilateral to and contralateral to the lesion at both post-lesion time points for both rat strains. The data also revealed an up-regulation of FGF-2 within the ventral midbrain at both post-lesion time points for SD rats while F344BNF1 rats only showed a significant up-regulation of FGF-2 at the 3 week post-lesion time point.

We also observed another difference between the two rat strains in terms of the magnitude of FGF-2 protein up-regulation in the ventral midbrain as a function of the amount of dopamine loss in ventral midbrain samples. Figure 5 is a scattergram showing the percent increase of FGF-2 protein expression plotted as a function of the amount of dopamine loss in each sample taken from the lesioned ventral midbrain; data were collapsed across time points for this analysis. Superimposed on these data are linear regression lines for each rat strain. Using this regression analysis we see lesions that produced only a 50% reduction of ventral midbrain dopamine would be expected to produce an approximate 35–40% increase of FGF-2 protein in the lesioned ventral midbrain of SD rats while > 95% loss of ventral midbrain dopamine is required to induce the same amount of FGF-2 up-regulation in F344BNF1 rats. The data presented in Table 1 and Fig. 5 suggest that SD rats are more sensitive to the neurotoxic effect of 6-OHDA than F344BNF1 rats in terms of up-regulating FGF-2 protein in response to lesion-induced neurodegeneration.

Fig. 5.

Fig. 5

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Correlation between dopamine loss in the ventral midbrain and increase of FGF-2 protein in the ventral midbrain. Each point represents an individual animal from both the 1 and 3 week post-lesion groups. Solid black dots represent SD rats (n = 10) and white dots represent F344BNF1 rats (n = 13). The % increase of FGF-2 in the ventral midbrain was calculated for each animal as follows: % increase of FGF-2 = [(amount of ipsilateral FGF-2 – amount of contralateral FGF-2)/amount of contralateral FGF-2)] × 100. The % loss of dopamine in the ventral midbrain was calculated for each animal as follows: % loss of dopamine = [1.0 – (amount of ipsilateral dopamine/amount of contralateral dopamine)] × 100. Regression analysis was performed on these data: the solid black line is the regression line for SD (r2 = 0.54) and the dashed line is the regression line for F344BNF1 (r2 = 0.61)

Discussion

Here we report that two different rat strains given comparable lesions of the nigrostriatal pathway resulted in a differential expression of FGF-2 in the lesioned ventral midbrain. At 1 week post-lesion, SD rats showed a significant elevation of FGF-2 in the lesioned ventral midbrain when compared to FGF-2 levels in the intact contralateral ventral midbrain; on the other hand, F344BNF1 rats did not show a significant increase in FGF-2 expression in the lesioned ventral midbrain at this same time point. While the lesions at the 1 week time point were statistically similar for the two strains, the mean percentage of dopamine loss in the ventral midbrain was actually slightly less for SD rats than that observed for F344BNF1 rats; this suggests that, if anything, as a group SD rats had less severe lesions than F344BNF1 rats. This would imply that the up-regulation of FGF-2 in lesioned SD rats was not due to a more severe lesion. Furthermore, when data were collapsed across time points and individual changes in FGF-2 were plotted as a function of dopamine loss in the ventral midbrain, regression analysis revealed that SD rats were more sensitive to the effects of the lesion than F344BNF1 rats in terms of up-regulating FGF-2 in the ventral midbrain.

The lesions induced in both rat strains were comparable and fairly severe. Our statistical analysis revealed no significant difference between the two rat strains at both post-lesion time points. However, it is clear that the amount of dopamine loss in the ventral midbrain for both rat strains was higher at 3 weeks post-lesion than it was at 1 week post-lesion. These data are consistent with the reports from other laboratories that used a similar technique of injecting 6-OHDA directly into the SN. For instance, using a lesion technique that produces near complete nigrostriatal pathway lesions, Jeon et al. [34] reported evidence of cell death in the SN as early as 12 h after 6-OHDA treatment that continued until 31 days after the treatment; while a large percentage of dopamine cells died during the first post-lesion week, there were a considerable number of dopamine neurons that continued to die during the second post-lesion week. This suggests cell death in the SN is a progressive event following intranigral 6-OHDA administration. On the other hand, in this same study they observed fiber degeneration in the nigrostriatal pathway was nearly complete 7 days after 6-OHDA treatment. We observed a similar phenomenon in this study. Dopamine loss in our ventral midbrain samples was higher at 3 weeks post-lesion than it was at 1 week post-lesion. Also, we observed > 95% loss of striatal dopamine at both 1 and 3 weeks post-lesion, and this is consistent with the report by Jeon et al. [34] that near complete terminal and fiber degeneration occurs by the 7th post-lesion day.

The F344BNF1 rat strain is a longer-lived rat strain when compared to the outbred SD or other inbred rat strains, and this strain was bred primarily for aging studies by the National Institute on Aging [35]. There is common ancestry between these two strains: SD is the maternal parent for Fisher 334. While the F344BNF1 rats may be ideal for aging studies, SD rats are used more commonly in the neuroscience research community. This serendipitous finding of a differential sensitivity and expression of FGF-2 in the ventral midbrain following a 6-OHDA lesion between these two rat strains reminds us of the importance of strain variability that occurs in the experimental setting. Had we used only the F344BNF1 rat strain in our studies, we may have concluded that a significant up-regulation of FGF-2 in the lesioned ventral midbrain does not occur until after the 1 week post-lesion time point. Despite this strain difference at the 1 week post-lesion time point, we did not observe a strain difference in FGF-2 protein expression in the lesioned ventral midbrain at the 3 week post-lesion time point.

There were several similarities and differences between our results and those reported by other laboratories. Gomide and Chadi reported findings similar to ours in unilaterally lesioned Wistar rats [21]. Two weeks following a unilateral 6-OHDA lesion of the nigrostriatal pathway, they observed a significant up-regulation of FGF-2 immunoreactivity in the ipsilateral substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) but not in the ipsilateral striatum. As a note to this study, when examining rat lineages we see that SD rats originated from female Wistar rats that were crossed to a hybrid male of unknown origin. Chadi et al. [20] reported that a unilateral 6-OHDA lesion of the nigrostriatal pathway induced an up-regulation of FGF-2 mRNA in the ipsilateral SN and VTA beginning 2 h after the lesion that persisted 2 weeks after the lesion but only a transient increase in FGF-2 mRNA expression occurred in the ipsilateral striatum 48 h following the lesion; this study utilized SD rats. Our results extend this finding and show an up-regulation of FGF-2 protein in the lesioned ventral midbrain to 3 weeks post-lesion. It is interesting that Claus et al. [18] reported no change in the expression of various isoforms of FGF-2 up to 4 weeks following a complete 6-OHDA lesion of the nigrostriatal pathway; however, the strain of rat used in this study was not reported and a less sensitive Western blot technique was used to measure FGF-2 protein expression.

We observed slightly elevated levels of FGF-2 in the ipsilateral striatum that were not significantly different than FGF-2 levels in the contralateral striatum for F344BNF1 rats at both 1 and 3 weeks post-lesion and for SD rats at 1 week post-lesion. These data are consistent with the report of a slight but non-significant elevation of FGF-2 protein measured in the ipsilateral striatum at 3 weeks post-lesion in male Wistar rats [19]. On the other hand, Nakagawa and Schwartz reported significant elevations of FGF-2 in the ipsilateral striatum 1 week post-lesion using cDNA array analysis; however, it should be noted that in this study the levels of FGF-2 met only the minimal criteria for up-regulation of activity and, in addition, the rat strain used in the study was not reported [24]. Thus, it is difficult to compare our data with those reported in two of the last three cited studies without knowing what strain of rat was used in those experiments.

While we did not observe significant changes in FGF-2 protein levels in striatum from 1 to 3 weeks post-lesion in either strain, the presence of FGF-2 within the microenvironment of the striatum may have implications for studies utilizing embryonic stem cells (ESC) for replacement therapies, particularly for the treatment of Parkinson’s disease (PD). FGF-2 is one of the primary growth factors used to keep ESC in their proliferative, undifferentiated state and removal of FGF-2 along with the addition of other growth and inductive factors is the process that drives these cells to differentiate into to cells with a dopaminergic phenotype [36, 37]; we have used FGF-2 to propagate neural precursors cells in vitro prior to differentiation and transplantation into a rodent model of PD [38]. The presence of FGF-2 within the degenerating striatum could very well hold any undifferentiated grafted cells in this state and may even promote their continued proliferation, which is an often unwanted side effect of ESC transplanted into the brain. While this point is made in passing, it is important to identify factors in the brain, such as FGF-2, which may impact the success of cellular replacement therapies using ESC.

In conclusion, we report a variable expression of FGF-2 protein within the nigrostriatal pathway following a neurotoxic lesion of the pathway that may be due to genetic factors that are different between the two strains of rats used in this study. While we report both strains eventually showed an up-regulation of FGF-2 protein in the lesioned ventral midbrain, we did notice a distinct temporal difference in FGF-2 expression following the lesion as well as a difference in sensitivity to the lesion. We believe these differences are important because they highlight how underlying genetic factors may influence experimental results. Unlike previous studies that have focused on the expression of FGF-2 mRNA in the 6-OHDA lesion model, this is one of the first studies to report on changes in FGF-2 protein expression using this model and is also one of the first studies to report a strain difference in the temporal up-regulation of FGF-2. Moreover, these findings may shed some light about why some laboratories find no changes in FGF-2 expression following a neurotoxic lesion while other laboratories report distinct changes in FGF-2 expression.

Acknowledgments

This research was supported by DOD grant DAMD 17-01-1-0786 (DMY) and NIH grants NS50311 (DMY) and AG17963 (WAC).

Contributor Information

David M. Yurek, Email: David.Yurek@uky.edu, Department of Neurosurgery, University of Kentucky College of Medicine, Health Sciences Research Building, Lexington, KY 40536-0305, USA.

Anita M. Fletcher, Department of Neurosurgery, University of Kentucky College of Medicine, Health Sciences Research Building, Lexington, KY 40536-0305, USA

Laura E. Peters, Department of Anatomy & Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536, USA

Wayne A. Cass, Department of Anatomy & Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536, USA

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