Induction of microglial toll-like receptor 4 by prothrombin kringle-2: a potential pathogenic mechanism in Parkinson’s disease

Microglia-mediated neuroinflammation may play an important role in the initiation and progression of dopaminergic (DA) neurodegeneration in Parkinson’s disease (PD), and toll-like receptor 4 (TLR4) is essential for the activation of microglia in the adult brain. However, it is still unclear whether patients with PD exhibit an increase in TLR4 expression in the brain, and whether there is a correlation between the levels of prothrombin kringle-2 (pKr-2) and microglial TLR4. In the present study, we first observed that the levels of pKr-2 and microglial TLR4 were increased in the substantia nigra (SN) of patients with PD. In rat and mouse brains, intranigral injection of pKr-2, which is not directly toxic to neurons, led to the disruption of nigrostriatal DA projections. Moreover, microglial TLR4 was upregulated in the rat SN and in cultures of the BV-2 microglial cell line after pKr-2 treatment. In TLR4-deficient mice, pKr-2-induced microglial activation was suppressed compared with wild-type mice, resulting in attenuated neurotoxicity. Therefore, our results suggest that pKr-2 may be a pathogenic factor in PD, and that the inhibition of pKr-2-induced microglial TLR4 may be protective against degeneration of the nigrostriatal DA system in vivo.


Results
Activation of microglia and an increase in microglial TLR4 in the SN of patients with PD. Microglial activation in the brains of patients with PD has been well described in previous reports 3,4 . To assess microglial activation in the SN of the patients with PD used in the current study (Table 1), we examined the morphological changes in microglia by immunostaining with anti-ionized calcium-binding adapter molecule 1 (Iba1). Our observations showed that decreases in neuromelanin-positive neurons (DA neurons) were confirmed in SN sections from patients with PD compared with sections from age-matched controls (Fig. 1a), and many microglia that were stained with anti-Iba1 had an activated morphology characterized by enlarged cell bodies with short processes (Fig. 1b). Consistent with decreases in neuromelanin-positive neurons, western blot analysis showed that tyrosine hydroxylase (TH) expression, a marker for DA neurons, was significantly decreased in the SN of patients with PD compared with age-matched controls (p = 0.001, Fig. 1c). However, the total level of Iba1 and OX-42, which also serves as a marker of microglial levels, was not significantly different between patients with PD and age-matched controls (Fig. 1c), even though there was a significant increase in tumor necrosis factor-alpha (TNF-α ) as a neurotoxic inflammatory cytokine, which could be produced by activated microglia, in the SN of patients with PD compared with age-matched controls (Extended data Fig. 1). These observations suggest that microglia-mediated neuroinflammation, which is capable of inducing neurodegeneration, may be caused by the activation state of the microglia, but not by the total number of microglia in the SN of human brains.
TLR4 is a crucial receptor for microglia-mediated neuroinflammation [13][14][15] , and its activation contributes to the production of neurotoxic inflammatory cytokines, which results in neurodegeneration in the adult brain [16][17][18][19] . However, it is still unclear whether the levels of microglial TLR4 are increased in the brains of patients with PD. To assess the levels of TLR4 expression in PD brains and controls, we measured its expression by western blotting and performed immunofluorescence double staining for Iba1 and TLR4. Western blotting indicated that there was a significant increase in TLR4 expression in SN samples from individuals with PD compared with age-matched controls (p = 0.005, Fig. 1c), even though there was no significant increase in the overall levels of Iba1 and OX-42 in the SN of patients with PD (Fig. 1c). Moreover, immunofluorescence staining confirmed the increase in TLR4 expression observed by western blotting, and further demonstrated that TLR4 was mainly localized within Iba1-positive cells (marked In SN sections from patients with PD, many microglia stained with anti-Iba1 showed an activated morphology, characterized by enlarged cell bodies with short processes. Insets show representative resting and activated microglia in the SN of an age-matched control and a patient with PD, respectively. Scale bar, 20 μ m. (c) Western blot analysis showed that TH expression was significantly decreased in the SN samples from patients with PD compared with age-matched controls (CON), but there was not a significant difference in the levels of either Iba1 or OX-42. *p = 0.001 vs. CON (n = 4, each group). In addition, western blot analysis of TLR4 showed that patients with PD exhibited a significant increase in its expression in the SN compared with CON. & p = 0.005 vs. CON (n = 4, each group). (d) Immunofluorescence double staining for Iba1 (microglia, red) and TLR4 (green) in the SN of patients with PD and age-matched controls. Brown arrowheads and yellow arrows indicate the location of DA neurons and TLR4-positive cells merged with microglia, respectively, in SN sections of human brain. Increases in TLR4 expression and activated microglia were observed in the SN of patients with PD compared with age-matched controls, and increased TLR4 expression was mainly localized within microglia. Scale bar, 30 μ m. with yellow arrows, Fig. 1d), suggesting that there was a significant increase in microglial TLR4 in the SN of patients with PD compared with age-matched controls.
Increase in pKr-2 expression in the SN of patients with PD. It is largely unknown whether there are any endogenous molecules capable of inducing neurodegeneration through microglial activation without a direct neurotoxic effect. We recently reported that pKr-2 was able to induce the death of DA neurons in the rat SN through microglial activation, even though pKr-2 itself was not directly toxic to neurons 7 . However, it is still unclear whether pKr-2 levels are upregulated in the brains of patients with PD, and whether an increase in pKr-2 expression induces degeneration of the nigrostriatal DA system in the adult brain. To first evaluate whether there is an increase in pKr-2 expression in the SN of patients with PD, we compared immunohistochemical staining of pKr-2 in human postmortem SN tissues from control brains and PD brains. Specifically, the sections were immunostained with either an antibody raised against prothrombin fragment 2, which is a synonym for pKr-2 (70R-10587), or an antibody raised against prothrombin, which recognizes an epitope in the kringle-2 domain of prothrombin (AHP-5013). The results for both antibodies demonstrated increased immunoreactivity in SN sections from patients with PD compared with age-matched control sections (Red arrows, Fig. 2a,b). In addition to immunohistochemical staining, western blotting using the AHP-5013 antibody indicated that pKr-1-2 levels were significantly increased in the SN of PD brains compared with those of age matched controls (p = 0.018, Extended data Fig. 2), suggesting that pKr-2 levels might be increased in the SN of patients with PD. Moreover, immunofluorescence staining confirmed the increase in pKr-2 expression in the SN of patients with PD (marked with arrows, Fig. 2c), and further demonstrated that some of the pKr-2 expression was localized within Iba1-positive cells (marked with yellow arrows, Fig. 2c). This suggests that pKr-2 might be translocated within microglia, even though further research is needed to elucidate the mechanisms involved in the intracellular translocation of pKr-2 in the adult brain.
Upregulation of pKr-2 disrupts nigrostriatal DA projections in the adult brain. To investigate whether upregulation of pKr-2 contributes to the degeneration of nigrostriatal DA projections, we unilaterally injected pKr-2 (48 μ g in 4 μ L) or vehicle (phosphate-buffered saline [PBS], 4 μ L) into the SN of Brown arrowheads and red arrows in the higher magnification images within each dotted rectangle indicate neuromelaninpositive neurons (DA neurons) and pKr-2 immunoreactivity, respectively, in the SN of human brains. Note that sections from patients with PD showed an increase in pKr-2 expression in the SN compared with agematched controls. Scale bars, 40 μ m and 20 μ m, respectively. (c) Immunofluorescence staining showed that some of increased pKr-2 (green), which is marked with yellow arrows in the SN from PD-affected brains, was localized within Iba1-positive cells (red). All arrows indicate pKr-2 immunoreactivity. Scale bar, 10 μ m. rats, as previously described 7 . Similar to our previous study 7 , a significant loss of TH-immunopositive (ip) neurons and fibers was observed in the SN and striatum (STR), respectively, a week after injection of pKr-2 (Fig. 3a). In addition to the observed neurotoxicity, there were more non-neuronal cells stained with cresyl violet in the pKr-2-treated SN than in the intact control SN (blue arrowheads in the high-magnification image of the SN, Fig. 3a), suggesting microglial activation by pKr-2 treatment 7 . Quantitatively, vehicle (PBS)-treated controls showed no signs of neurotoxicity (Fig. 3b,c). However, the number of TH-ip neurons and the optical density (OD) of striatal TH were significantly reduced by 47% The amount of dopamine in rat striatal tissues was measured by ELISA, and the levels were quantitatively expressed as a percentage of the value for the contralateral control for each sample. # p < 0.001 vs. contralateral/PBS controls (n = 4, each group). (e) HPLC analysis of dopamine and its metabolites, DOPAC and HVA in the STR of rat brains shows that pKr-2 treatment significantly reduced striatal dopamine levels compared with PBS controls (p = 0.018; n = 4, each group). By contrast, the levels of striatal HVA were significantly increased by pKr-2 treatment compared with PBS controls (p = 0.029), and the levels of striatal DOPAC also showed a similar pattern in pKr-2-treated rats, even though this was not significantly different compared with PBS controls (p = 0.087).
Scientific RepoRts | 5:14764 | DOi: 10.1038/srep14764 (p = 0.002, Fig. 3b) and 32% (p = 0.026, Fig. 3c), respectively, in pKr-2-treated brain tissue compared with contralateral controls. Similar to the immunostaining data for TH-ip fibers in the STR, the levels of striatal dopamine measured using an enzyme-linked immunosorbent assay (ELISA) seven days after injection of pKr-2 were significantly decreased compared with controls (p < 0.001, Fig. 3d). To clarify the changes in striatal dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), that were induced by pKr-2 treatment, we further examined the levels in the STR seven days after injection of pKr-2 using reversed-phase high performance liquid chromatography (HPLC) with an electrochemical detector 20 . HPLC analysis revealed that pKr-2 treatment reduced striatal dopamine levels to 47% of those in PBS-treated controls (p = 0.018, Fig. 3e). By contrast, the levels of striatal HVA were significantly increased by pKr-2 treatment compared with PBS controls (p = 0.029, Fig. 3e), and a similar pattern of increase in DOPAC was observed in pKr-2-treated rats compared with PBS-treated controls, even though this was not significantly different (p = 0.087, Fig. 3e). This suggested that there might be a compensatory increase in the turnover of dopamine following a partial nigrostriatal lesion 21,22 . Moreover, similar to the levels in PBS-treated controls, the increased levels of DA metabolites were reduced at 2 weeks after pKr-2 treatment (Extended data Fig. 3), indicating that there was functional loss in the nigrostriatal DA system. Taken together, these results suggest that an increase in pKr-2 expression may contribute to disruption of the nigrostriatal DA projections and act as a PD-inducing stimulus in the adult brain.

pKr-2 treatment induces an increase in microglial TLR4 in vitro and in vivo.
Our results showed that the levels of pKr-2 ( Fig. 2) and microglial TLR4 (Fig. 1c,d) were increased in the SN of patients with PD. However, it is still unclear whether there is a correlation between microglial TLR4 and pKr-2-induced neurotoxicity. To investigate this question, the levels of TLR4 were measured in cultures of the BV-2 microglial cell line by western blotting and in the SN of rat brains by western blotting and immunofluorescence double staining after pKr-2 exposure. In BV-2 microglial cell line cultures, treatment with 50 μ g/mL pKr-2 7 significantly upregulated the levels of TLR4 compared with untreated and PBS-treated controls (p = 0.002 and 0.006 vs. untreated and PBS-treated controls, respectively, Fig. 4a), suggesting that pKr-2 treatment directly increased microglial TLR4 expression. Consistent with the increase in TLR4 in vitro, an intranigral injection of pKr-2 (48 μ g in 4 μ L) significantly upregulated its expression in the rat SN 3 h and 24 h post-injection, compared with PBS-treated controls, as demonstrated by western blotting (p < 0.001, Fig. 4b) and immunofluorescence staining for TLR4 and OX-42 ( Fig. 4c; 24 h post-injection). These results suggest that an increase in pKr-2 expression may lead to the induction of TLR4 in microglia in the SN of patients with PD.
pKr-2-induced TLR4 contributes to degeneration of the nigrostriatal DA system in the adult brain. To further ascertain whether pKr-2-induced microglial activation and neurotoxicity is correlated with the induction of TLR4, pKr-2 (24 μ g in 2 μ L) was injected into the SN of C57BL/6 wild-type (WT) control mice or TLR4 knockout (KO) mice, confirmed by genotyping and western blotting for TLR4 (Fig. 5a). PBS-treated WT mice, which showed no evidence of neurotoxicity and neuroinflammation, were used as controls for the effects of surgical damage to DA neurons and fibers in the mouse brain (Extended data Fig. 4a-c). To evaluate a possible link between pKr-2-induced TLR4 and microglial activation, SN sections from WT control and TLR4 KO mice were immunostained with Iba1 antibodies three days after an intranigral injection of PBS or pKr-2 ( Fig. 5b) because pKr-2-induced microglial activation occurred prior to the apparent lesion of DA neurons in the SN in vivo 7 . In the PBS-treated WT SN, most Iba1-ip microglia displayed a resting morphology characterized by small cell bodies with long, thin and many ramified processes. However, pKr-2 treatment in WT mice caused microglial activation, characterized by enlarged cell bodies with short processes. By contrast, the morphological transformation of microglia resulting from pKr-2 treatment was attenuated in TLR4-deficient mice. Consistent with the morphological activation of microglia, western blot analysis showed significant increases in neurotoxic inflammatory cytokines mediated by microglia, such as interleukin-1 beta (IL-1β ) and TNF-α 7 , in the SN of WT mice three days after injection of pKr-2 compared with PBS-treated controls (p < 0.001 and p = 0.031, respectively, Fig. 5c); these effects were significantly diminished in TLR4-deficient mice. In addition, western blot analysis of Iba1 expression in the SN revealed a significant increase in its levels in the SN of WT mice at three days after injection of pKr-2 (p < 0.001 vs. PBS-treated controls, Extended data Fig. 5), and the increase was significantly diminished in TLR4-deficient mice compared with WT mice (p = 0.009 vs. pKr-2-treated WT mice, Extended data Fig. 5). PBS-treated WT control mice showed no increase in the levels of IL-1β and TNF-α (Extended data Fig. 4d).
Consistent with inhibition of pKr-2-induced microglial activation in TLR4-deficient mice (Fig. 5b,c), we observed that the subsequent loss of TH-ip neurons and fibers in the SN and STR, respectively, was significantly attenuated in TLR4 KO mice compared with WT mice (Fig. 6a). When quantified and expressed as a percentage of DA neurons in the counting area of the ipsilateral SN relative to the contralateral control SN one week after an intranigral injection of pKr-2, only 59% of DA neurons were preserved in the SN of the pKr-2-treated WT mice (p < 0.001 vs. intact controls, Fig. 6b). In contrast, 79% of DA neurons were preserved in TLR4 KO mice (p = 0.037 vs. intact controls, Fig. 6b) with a significant difference compared with the pKr-2-treated WT mice (p = 0.042, Fig. 6b). Moreover, there was no significant difference in the OD of TH-ip fibers between the contralateral control STR and pKr-2-treated ipsilateral STR of TLR4 KO mice (p = 0.249 vs. intact controls, Fig. 6c), even though this treatment induced a decrease in the OD by 40% in WT mice (p < 0.001 vs. intact controls, Fig. 6c). Consistent with the results for immunostaining with TH antibodies, the levels of striatal dopamine assessed by ELISA were significantly decreased in the pKr-2-treated WT mice (p < 0.001 vs. intact controls, Fig. 6d), but not in the TLR4 KO mice (p = 0.402 vs. intact controls, Fig. 6d). Taken together, our results suggest that pKr-2-induced microglial TLR4 may be a major mediator of pKr-2-induced neuroinflammation and neurotoxicity in the adult brain, even though TLR4 deficiency did not completely prevent the neurotoxicity induced by pKr-2 treatment.

Discussion
Although the etiology of PD is not yet understood, accumulating evidence implicates microglia, which are the resident immune cells in the brain. They are crucial mediators of the brain inflammatory processes that lead to neurotoxicity, and microglial activation contributes to the initiation and progression of PD 4,7,23 . In the healthy brain, microglia are in a resting state that is characterized morphologically by small cell bodies with thin, ramified processes. In addition, resting microglia phenotypically show low expression of inflammatory molecules associated with immune functions 7,23,24 . However, under neuropathological conditions, activated microglia produce a spectrum of potentially neurotoxic molecules that contribute to the death of DA neurons 7,23-25 . In addition to neurotoxic cytokines, activated microglia generate reactive oxygen species, leading to oxidative stress in DA neurons 25,26 . Similar to these results, our observations showed that many microglia in the SN of patients with PD exhibited an increase in TLR4 expression with activated morphology (Fig. 1b-d). However, there was no significant difference in the total levels of Iba1 and OX-42, suggesting that the number of microglia might be similar between patients with PD and age-matched controls, as demonstrated by western blot analysis (Fig. 1c). These results suggest that the activation state of microglia rather than the number of microglia in the SN may mediate microglia-induced neurotoxicity towards DA neurons, and that control of microglial activation may be useful for preventing the degeneration of nigrostriatal DA projections in the adult brain.
Among the prothrombin domains, pKr-2 is produced by the cleavage of pKr-1-2 by active thrombin, which is generated following the activation of factor Xa in the prothrombinase complex 7 . Prothrombin, an endogenous source of thrombin, is expressed in the human brain 27 and accumulates in the SN of human PD brains 28 . Additionally, increased thrombin levels have also been found in the brain in other neurodegenerative diseases such as AD 29,30 . Moreover, disruption of the blood-brain barrier (BBB), resulting in cerebrovascular disturbances, may be one of features shown in patients with PD and AD 31 ; therefore, prothrombin and thrombin upregualtion may be mainly mediated by BBB breakdown, due to disruption of the tight junctions, in the brain of patients with PD. In a previous study, we showed that pKr-2 caused no direct neurotoxicity, but could activate microglia, and the resulting production of inflammatory cytokines from pKr-2-activated microglia contributed to the death of DA neurons in vivo and in vitro 7 . However, it was not determined whether pKr-2 expression is upregulated in the brains of patients with PD, and we did not assess the potential disruption of the nigrostriatal DA pathway, which is a representative phenotype of PD. Here, we present the first report showing that patients with PD have increases in pKr-2 and pKr-1-2 expression in the SN (Fig. 2a-c and Extended data Fig. 2, respectively), and the increase in pKr-2 expression disrupts the nigrostriatal DA system in the adult murine brain (Fig. 3,6 and Extended data Fig. 3). Moreover, some of pKr-2 expression were co-localized within activated microglia as shown in Fig. 2c, even though its expression was not localized within DA neurons (data not shown), suggesting that pKr-2 might be upregulated in neurodegenerative diseases due to an increase in active thrombin originated from blood, even though there have been no studies so far that have reported the upregulation of pKr-2 and cell types of pKr-2 production or secretion in human brains.
The pattern of TLR4 expression in the brain is controversial. Lehnardt et al. reported that TLR4 is expressed only in microglia 13 . By contrast, TLR4 expression in other brain cells such as astrocytes, oligodendrocytes, and neurons has been reported by many research groups 32,33 , and TLR4 may have different roles in glia and neurons 34 . However, there are many reports suggesting that microglia are important for TLR4-mediated immune responses, which may be involved in brain diseases such as AD 18 and PD 19 . Moreover, an increase in TLR4 expression has been found in α -synuclein-overexpressing transgenic mice and patients with human multiple system atrophy 35 , suggesting the possibility that TLR4 is upregulated in the brains of patients with PD. This is consistent with results showing that upregulation of TLR4 contributes to neurotoxicity in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated animal models of PD [36][37][38] . However, alterations in TLR4 expression in patients with PD had not yet been demonstrated, and it was unclear whether there are any endogenous molecules capable of inducing DA neurodegeneration through microglial activation via TLR4 induction, but in the absence of any direct neurotoxic effect.
To clarify whether TLR4 expression is increased in the brains of patients with PD, we measured its expression in the postmortem human SN from control and PD-affected brains by western blotting and immunofluorescence double staining. We found that that TLR4 levels were greater in the SN of patients with PD compared with age-matched controls (Fig. 1c,d), indicating that there is indeed an increase in TLR4 expression in PD brains, even though the total levels of microglia were not significantly different between patients with PD and age-matched controls (Fig. 1c). Moreover, the increased TLR4 expression was mainly localized within Iba1-positive microglia (Fig. 1d), suggesting that an increase in microglial TLR4 might be crucial for the pathogenesis of PD, and the discovery of endogenous molecules involved in the induction of microglial TLR4 might be useful for guiding the development of knowledge-based targeted therapeutics for PD. In the present study, we also found that pKr-2-induced neurotoxicity and microglial activation was correlated with the induction of TLR4 in microglia. Consistent with increases in both pKr-2 (Fig. 2a-c) and microglial TLR4 (Fig. 1c,d) in the SN of patients with PD, pKr-2 treatment increased microglial TLR4 in cultures of the BV-2 microglial cell line (Fig. 4a) and the SN of rat brains (Fig. 4b,c). Moreover, pKr-2-induced microglial activation and neurotoxicity were significantly diminished in TLR4-deficient mice compared with WT mice (Fig. 5b,c and 6).
We cannot exclude the possibility that another mechanism may be involved in pKr-2-induced neurotoxicity, because TLR4 deficiency did not completely reverse the neurodegeneration induced by pKr-2 treatment (Fig. 6b). In addition, the difference on the microglial activation following to pKr-2 upregulation in human (Fig. 1c) and murine brains (Extended data Fig. 5) is still unclear. However, our observations in the present study show that pKr-2-induced TLR4 overexpression may be an important mechanism for microglial activation, which could contribute to the degeneration of the nigrostriatal DA system in the adult brain. Therefore, our findings strongly suggest that pKr-2 may have a role in the pathogenesis of PD, and the control of pKr-2 expression and pKr-2-induced TLR4 overexpression in microglia may be crucial for protecting the nigrostriatal DA system against PD.
pKr-2 treatment of BV-2 microglial cell line cultures. BV-2 microglial cells were plated in 6-well plates and maintained in DMEM supplemented with 5% heat-inactivated FBS and 1% penicillin-streptomycin in 5% CO 2 at 37 °C. The cells were treated with pKr-2 (50 μ g/mL) 7 or vehicle (4 μ l) for 6 h, and then harvested for western blot analysis. This is because the upregulation of inflammatory biomolecules derived from activated microglia was apparent between 3 h and 24 h after pKr-2 treatment 7 .
Immunohistochemical staining procedures. The postmortem brain sections obtained from the VBBN were deparaffinized and subjected to citrate antigen retrieval prior to immunohistochemistry, and then washed in cold PBS for 15 min, and incubated in blocking solution [0.5% Triton X-100, 1% bovine serum albumin (BSA), 0.05% Tween 20, 0.1% cold fish gelatin, and 0.05% sodium azide in PBS] for 1 h at room temperature. For immunohistochemical staining, the sections were incubated with primary antibodies against pKr-2 (1:200) diluted in 0.1 M PBS containing 0.5% BSA (0.5% BSA blocking buffer) at 4 °C overnight, and then incubated with biotinylated secondary antibodies for 1 h at room temperature, followed by avidin-biotin reagents (Vector Laboratories) for 1 h. The signal was detected by incubating sections in 0. Mouse and rat brains were prepared as described previously [39][40][41] with some modifications. Animals were transcardially perfused and fixed, and brain sections (30-μ m thick) were rinsed in PBS, and then Scientific RepoRts | 5:14764 | DOi: 10.1038/srep14764 incubated for 48 h with the following primary antibodies: rabbit anti-TH (1:1000), rabbit anti-Iba1 (1:1000), mouse anti-OX-42 (1:500), and rabbit anti-TLR4 (1:200). After incubation, the sections were processed as described above. For Nissl staining, SN tissue samples were mounted on gelatin-coated slides and stained with 0.5% cresyl violet (Sigma).

Western blot analysis.
Animal tissues for western blotting were prepared as previously described 21,39 .
Briefly, animal SN tissues were removed and sliced using a brain matrix (Roboz Surgical Instrument Co., Gaithersburg, MD). Animal or human tissue samples were homogenized and centrifuged at 4 °C for 20 min at 14,000 × g. The supernatant was transferred to a fresh tube and the concentration was determined using a BCA kit. Proteins separated by gel electrophoresis were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) using an electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA), and the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-TH (1:1000), rabbit anti-Iba1 (1:1000), mouse anti-OX-42 (1:400), rabbit anti-TLR4 (1:200), goat anti-TLR4 (1:1000), mouse anti-prothrombin (1:100), goat anti-IL-1β (1:200), and goat anti-TNF-α (1:100). After washing, the membranes were incubated with secondary antibodies (1:5000) for 1 h, and then the blots were finally developed with ECL western-blotting detection reagents (Amersham Biosciences, Piscataway, NJ). For quantitative analyses, the density of the bands was measured using a Computer Imaging Device and accompanying software (Fuji Film, Tokyo, Japan), and the levels were quantitatively expressed as the density normalized to the housekeeping protein band for each sample.
Measurement of dopamine and its metabolites in the STR. Brain tissues for the determination of striatal levels of dopamine and its metabolites were prepared as previously described 20,21 with some modifications. Each brain was placed in a rat/mouse brain matrix, 2.0 mm thick coronal sections were cut through the forebrain, and then the sections were placed flat on a chilled glass plate. The STR sections were dissected using a 2.0 mm tissue punch, and the tissues were frozen immediately on dry ice. Dopamine levels were quantitatively measured using a commercial ELISA kit according to the manufacturer's instructions (CUSABIO; Wuhan, Hubei, China). Briefly, animal STR issues were homogenized with PBS and stored at − 20 °C overnight. After two freeze-thaw cycles, the homogenates were centrifuged at 5,000 × g and 4 °C for 5 min. The supernatants were then analyzed using the ELISA kit. The OD of the standards and samples were measured at 450 nm using Softmax software (Molecular Devices, Sunnyvale, CA).
To clarify whether pKr-2 treatment disrupts the nigrostriatal DA system in vivo, HPLC was used to measure the levels of dopamine and its metabolites such as DOPAC and HVA in additional rat STR samples, as previously described 20 . Briefly, the isolated tissues were homogenized and centrifuged at 9000 rpm for 20 min in 400 μ L of 0.1 M perchloric acid and 0.1 mM EDTA. Then 10 μ L samples of supernatant were injected into an autosampler at 4 °C (Waters 717 plus autosampler) and eluted through a μ Bondapak C18 column (3.9 × 300 mm × 10 μ m; ESA Biosciences, Chelmsford, USA) with a mobile phase for catecholamine analysis (Chromosystems, Munich, Germany). The peaks of dopamine and its metabolites were analyzed using an ESA CoulochemII electrochemical detector and integrated using a commercially available software program (Breeze, Waters Corp., Milford, MA). All samples were normalized for protein content as spectrophotometrically determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories).
Stereological estimation. As previously described 7,39 , the total number of TH-ip neurons were counted for the various animal groups using the optical fractionator method performed on an bright field microscope (Olympus Optical, BX51, Tokyo, Japan) using Stereo Investigator software (MBF Bioscience, Williston, VT).
Quantitative determination of striatal TH immunoperoxidase staining density. Densitometric analysis of the mouse or rat STR was carried out as previously described 21,39 with some modifications. Briefly, an average of 6 coronal sections of the STR were captured at a 1.25× magnification, and the OD was measured using the Science Lab 2001 Image Gauge (Fujifilm, Tokyo, Japan). To control for variations in background illumination, the density of the corpus callosum was subtracted from the density of the STR for each section. TH-ip fiber innervation in the STR was quantitatively expressed as a percentage by comparing the OD on the ipsilateral side with the contralateral control side.