[1]
124w
It is well known that Parkinson's disease (PD) is a chronic progressive degenerative disorder characterized by the selective degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta. The loss of dopaminergic neurons afferents to the striatum and puta-men results in extrapyramidal motor dysfunction, including tremor, bradykinesia, rigidity and postural instability (Olanov et al. 2003). Numerous investigations have been reported the mechanisms responsible for dopaminergic neuron degeneration in PD. Oxidative stress, excitotoxicity, depletion of endogenous antioxidants, decreased expression of trophic factors and dysfunction of protein degradation system are believed to participate in the cascade of events leading to dopaminergic neuronal loss (Jenner 2003;Olanov et al. 2003). In the brain, however, there are 10 times more glial cells than neurons (Kimelberg and Norenberg 1989).
[2]
35w
Mouse model of PD is generated by intoxication of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropridine) that damages selectively the nigrostriatal dopaminergic system. Neuronal degeneration is followed by an abundant oxidative stress and inflammatory responses involving astrocytes, microglia and lymphocytes.
[3]
156w
On the other hand, oligodendrocytes are the cells responsible for myelin synthesis and assembly around axon in the central nervous system (CNS). Oligodendrocytes loss and demyelination in the CNS have been reported in a variety of myelin disorders such as multiple sclerosis (Holgado et al. 2001;Hisahara et al. 2003). Furthermore, it is suggested that the oligodendrocytes are vulnerable to various factors that can easily cause cell death, including inflammatory cytokines, viruses and brain-blood barrier disruption. A previous study reported that the myelin-basic protein immunoreactivity in the sciatic nerve was markedly decreased in normal aging (Melcangi et al. 1998). A recent interesting study also suggests that age-related degeneration of oligodendrocytes had occurred in the hippocampus of senescence-accelerated mouse (Tanaka et al. 2005). Moreover, oligodendroglial injury has been shown to occur rapidly in response to ischemia (Irving et al. 1997;McCracken et al. 2002;Gresle et al. 2006). However, little is known about the damage of oligodendrocytes after MPTP neurotoxicity.
[4]
27w
We therefore conducted the present study to clarify the alteration of oligodendrocytes in comparison with that of astorocytes and microglia in the mouse striatum after MPTP treatment.
[1]
189w
Glial cells have many functions in the CNS that impact the fate of the local neuronal population. It is known that two classes of glial cells, astrocytes and microglia, have different roles in the pathogenesis after MPTP treatment. In general, astrocytes act as protective agents through several mechanisms including their ability to buffer the cellular milieu as well as through their production of neurotrophic factor. Furthermore, GFAP-null mice are known to be highly susceptible to brain damage, such as cerebral ischemia (Nawashiro et al. 2000). In contrast, astrocytes upregulate a number of molecules when activated, but it remains controversial whether the astocytic activation is beneficial or detrimental (Ridet et al. 1997). On the other hand, the activated microglia have been shown to secrete cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1b IL-1b), and IL-6, that participate in the cascade of oxidative stress and inflammation (Liu and Hong 2003;Ferger et al. 2004). These cytokines have been shown to play a role in the modulation of neuronal death, including that found in animal models of PD (Hirrlinger et al. 2002). However, little is known about the alteration of oligodendrocytes after MPTP toxicity.
[2]
81w
In the present study, four administrations of MPTP at 2-h intervals to mice produced the remarkable loss of TH protein levels in the striatum after 3 and 7 days. By contrast, our Western blot of GFAP and Iba-1 protein in the striatum showed a significant increase of GFAP and Iba-1 protein levels 3 and 7 days after MPTP treatment. These findings were consistent with our previous reports with an immunohistochemical study (Muramatsu et al. 2003;Kato et al. 2004;Kurosaki et al. 2004).
[3]
91w
It is known that damage to the mammalian brain induces migration of activated microglia to be damaged site, phagocytosis, astrocytic activation=proliferation, and astrocyte-mediated formation of a fibrous scar. Several previous studies suggest that microglial cells are a source of astrocytic growth factors, including IL-1b and mitogens that stimulate further increased number of microglia (Guillian et al. 1988;Laping et al. 1994). Microglial cells are also activated and proliferated, secreting various cytokines damaging to oligodendrocytes (Hartung et al. 1992;Felzien et al. 2001). However, the precise role for these findings is not yet understood.
[4]
134w
In cases of diseases such as Alzheimer's disease and multiple sclerosis, oligodendrocytes and white matter are affected significantly. Oligodendroglial injury has also been occurred in response to cerebral ischemia. Irving et al. (1997) observed structural alterations to the oligodendrocyte cytoskeleton within 20 and 40 min of middle cerebral artery occlusion (MCAO) in rats. These changes were assessed by increases in immunoreactivity to the microtubule associated protein, Tau-1, which has been found be a sensitive marker for oligodendroglial damage in sereveral studies involving focal cerebral ischemia in rats (Dewar and Dawson 1995;Imai et al. 2001;McCracken et al. 2002). The levels of CNPase were also decreased in the patient's brain in cases of Alzheimer's disease and Down's syndrome (Vlkolinsky et al. 2001). From these findings, it is conceivable that oligodendrocytes may be damaged after MPTP treatment.
[5]
82w
In the present study, a significant decrease in the area of CNPase-positive profiles as an oligodendrocyte marker was observed in the striatum 3 and 7 days after MPTP treatment. Furthermore, our Western blot of CNPase protein showed a significant decrease in the striatum 3 and 7 days after MPTP treatment. These findings demonstrate that oligodendrocytes may be damaged by inflammatory cytokines produced by glial cells after MPTP treatment. However, further studies are needed to investigate the precise mechanisms responsible for our findings.
[6]
29w
In conclusion, the present study demonstrates that oligodendrocytes in the striatum are damaged after MPTP treatment. Thus our present findings provide valuable information for the pathogenesis of Parkinson's disease.
[1]
113w
Male C57BL=6 mice (Nihon SLC Co., Shizuoka, Japan), 8 weeks of age, were used in this study. The animals were housed in a controlled environment (23 AE 1 C, 50 AE 5% humidity) and were allowed food and tap water ad libitum. The room lights were on between 8:00 and 20:00. The mice were injected intraperitoneally (i.p.) four times with MPTP (20 mg=kg) at 2 h intervals, the total dose per mouse being 80 mg=kg, as described previously (Muramatsu et al. 2003;Kurosaki et al. 2004). Control animals received i.p. four injections of physiological saline. All experiments were performed in accordance with the Guidelines for Animal Experiments of the Tokushima University School of Medicine.
[2]
317w
The mice were killed by cervical dislocation 3 and 7 days after MPTP treatment. The striatal tissues were homogenized in HEPES-buffered sucrose (0.32 M sucrose containing 4 mg=ml pepstatin, 5 mg=ml aprotinin, 20 mg=ml trypsin inhibitor, 4 mg=ml leupeptin, 0.2 mM phenylmethanesulfonyl fluroride, 2 mM EDTA, 2 mM EGTA, and 20 mM HEPES, pH 7.2) using a microtube homogenizer. Protein concentrations were determined using a BCA kit (PIERCE, IL, USA). The homogenates were solubilized in Laemmli's sample buffer. Ten micrograms of protein from each sample were separated on 5-20% SDS-PAGE gel using constant current. Separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (ATTO, Tokyo, Japan) for 1 h with semi-dry blotting system. The PVDF membranes were incubated for 1 h at room temperature with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 0.5% skim milk, followed by overnight incubation at 4 C with desired antibodies. The anti-tyrosine hydroxylase (TH) antibody (1:3000, Chemicon International, Inc., Temecula, CA, USA) as a marker of dopaminergic neurons, anti-glial fibrillary acidic protein (GFAP) antibody (1:2000, Sigma, Saint Louis, MO, USA) as a marker of reactive astrocytes, anti-Iba-1 antibody (1:2000, Wako Pure Chemicals, Osaka, Japan) as a marker of microglia and anti-2 0 , 3 0cyclic nucleotide 3 0 -phosphodiesterase (CNPase) antibody (1:2000, Sigma, St Louis, MO, USA) as a marker of oligodendrocyte were diluted in TBST containing 0.5% skim milk. Membranes were washed six times for 5 min at room temperature and incubated with horseradish peroxidase-conjugated secondary antibody in TBST containing 0.5% Skim milk for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescent autoradiography (ECL Kit, Amersham, IL, USA), according to manufacturer's instructions. Anti-actin antibody (Sigma, Saint Louis, MO, USA) was used as a house keeping protein to confirm that equal amounts of protein were loaded in each line. Optical densities were determined using a computerized image analysis system (Dolphin-DOC, Kurabo, Osaka, Japan). Each group consisted of 4-5 mice.
[3]
92w
The mice were anesthetized with sodium pentobarbital (50 mg=kg, i.p.) 3 and 7 days after MPTP treatment and the brains were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) following a heparinized saline flush. The brains were removed 1 h after perfusion fixation at 4 C and were immersed in the same fixative until they were embedded in paraffin. Paraffin sections (5 mm) of striatum were used for immunohistochemistry. Adjacent sections stained with Cresyl violet in all mice were examined with a light microscope at a magnification of Â400.
[4]
165w
For immunohistochemical studies, a Vectastain elite ABC kit (Vector Labs., Burlingame, CA, USA) and anti-CNPase antibody were used. Briefly, the paraffin sections were washed for 5 min in 0.01 M phosphate-buffered saline (PBS, pH 7.4) and treated with 0.3% hydrogen peroxide in 0.01 M PBS including 10% methanol. The paraffin sections were then washed three times for 5 min each time in 0.01 M PBS, followed by 30 min of preincubation with 10% normal horse serum. The brain sections were then incubated with anti-CNPase antibody (1:200) including 0.3% Triton X-100 overnight at 4 C. After a 5-min rinse in 0.01 M PBS, the sections were incubated with biotinylated secondary antibody for 60 min and then with avidin-biotin peroxidase complex for 30 min at room temperature. Immunoreactivity was visualized using enzyme substrate kits (Vector Labs., Burlingame, CA, USA), as described previously (Watanabe et al. 2004). Negative control sections were treated in the same way as described above except for the antibody against anti-CNPase antibody was omitted.
[5]
62w
For stainings for CNPase, area of expression of the CNPase-immunopositive profiles in three stained sections was evaluated under a light microscope at a magnification of Â400 without the examiner knowing the experimental protocols, using a computer-associated image analyzer software (WinRoof Version 5, Mitani Corporation, Fukui, Japan), as described previously (Hayakawa et al. 2007;Tanaka et al. 2007). Each group consisted of 5-6 mice.
[6]
35w
All values were expressed as the means AE S.D. and statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Fisher's PLSD multiple comparison test (Stat View version 5.0, SAS Institute Inc., USA).
[7]
139w
As shown in Fig. 1A, C, four administrations of MPTP at 2-h intervals to mice produced the remarkable loss of TH protein levels in the striatum after 3 and 7 days. In contrast, our Western blot of GFAP protein in the striatum showed a significant increase of GFAP protein levels 3 and 7 days after MPTP treatment, as shown in Fig. 1B, D. The levels of Iba-1 protein were also increased significantly in the striatum 3 and 7 days after MPTP treatment, as shown in Fig. 2A, C. On the other hand, the levels of CNPase protein were decreased significantly in the striatum 3 and 7 days after MPTP treatment, as shown in Fig. 2B, D. In addition, actin protein was detected as a house keeping protein to confirm that equal amounts of protein were loaded in each line.
[8]
215w
Representative photographs of CNPase immunostaining in the striatum are shown in Fig. 2A. The area of expression of CNPase immunopositive profiles was decreased significantly in the striatum 3 and 7 days after MPTP treatment, as shown in Fig. 2B. Actin protein was detected as a house keeping protein to confirm that equal amounts of protein were loaded in each line. (C) TH protein levels were expressed as % of vehicle (means AE S.D.) using ratios to actin protein levels. (D) GFAP protein levels were expressed as % of vehicle (means AE S.D.) using ratios to actin protein levels. Ã p < 0.01 compared with control group (Fisher's PSLD multiple comparison test). n ¼ 4-5 Fig. 2. Immunoblotting analysis of Iba-1 and CNPase protein levels in the mouse striatum 3 and 7 days after MPTP treatment. (A, B) Western blot analysis. Actin protein was detected as a house keeping protein to confirm that equal amounts of protein were loaded in each line. (C) Iba-1 protein levels were expressed as % of vehicle (means AE S.D.) using ratios to actin protein levels. (D) CNPase protein levels were expressed as % of vehicle (means AE S.D.) using ratios to actin protein levels. Ã p < 0.01 compared with control group (Fisher's PSLD multiple comparison test). n ¼ 4-5
[9]
4w
Oligodendrocytes and MPTP neurotoxicity