**4. Nuroprotective role of microgliae**

Neurotrophic effects of microglial activation were reported in cell-culture studies (Elkabes et al., 1996, 1998; Miwa et al., 1997; Nagata et al., 1993a; Nakajima et al., 2001) and in studies using animal models of neurodegeneration (Hashimoto et al., 2005; Imai et al., 1999, 2007; Rabchevsky and Streit, 1997; Suzuki et al., 2001).

Fig. 1. Morphological changes due to MPTP administration in the SN. Immunostaining for tyrosine hydroxylase (TH)-positive dopaminergic (A9) neurons (green) and CD 11b-positive activated microglia (red) in the SN from mice treated with saline, MPTP, and LPS-MPTP are shown. In neonatal (P8) mice (**A**-**C**), dopaminergic (A9) neurons in the SN were decreased in MPTP-treated mice, whereas these neurons in the LPS-MPTP-treated mice were recovered, compared from MPTP-treated mice. The activated microglia had increased in number in the entire SN in mice treated with LPS-MPTP, as compared with saline- or MPTP-treated mice. In aged (60w) mice (**D**-**F**), numbers of the dopaminergic (A9) neurons were decreased in the order of saline, MPTP, and LPS-MPTP treatments. In the MPTP- and LPS-MPTP-treated mice, numbers of the activated microglia were increased with their accumulation in the SNc. P8 refers to postnatal day 8; and 60w, to 60-day-old mice (Sawada H. et al., 2007, J Neurosci Res, Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and Sons).

Neonatal microglia are activated macrophage colony-stimulating factor (M-CSF) dependently from late gestation up to two weeks after birth, and are very proliferative and easily activated under normal circumstances (Sawada M. et al., 1990; Thery et al., 1990). Sawada H. et al. (2007) showed that in MPTP-administered neonatal mice their microglia activated by treatment with systemic LPS showed neurotrophic potential toward dopaminergic neurons. Neonatal (postnatal day 7) mice treated with MPTP showed decreases in the number of dopaminergic (A9) neurons in the SN (Fig. 1 and 2), TH activity, and the levels of DA and the metabolite 3,4-dihydroxyphenylacetic acid in the midbrain. However, cell viability of dopaminergic (A9) neurons and these markers increased in mice treated with MPTP and LPS, along with marked LPS-induced activation of microglia (Fig. 1 and 2). A modest activation of microglia and a significant decrease in the number of

Neurotrophic effects of microglial activation were reported in cell-culture studies (Elkabes et al., 1996, 1998; Miwa et al., 1997; Nagata et al., 1993a; Nakajima et al., 2001) and in studies using animal models of neurodegeneration (Hashimoto et al., 2005; Imai et al., 1999, 2007;

Fig. 1. Morphological changes due to MPTP administration in the SN. Immunostaining for tyrosine hydroxylase (TH)-positive dopaminergic (A9) neurons (green) and CD 11b-positive activated microglia (red) in the SN from mice treated with saline, MPTP, and LPS-MPTP are shown. In neonatal (P8) mice (**A**-**C**), dopaminergic (A9) neurons in the SN were decreased in MPTP-treated mice, whereas these neurons in the LPS-MPTP-treated mice were recovered, compared from MPTP-treated mice. The activated microglia had increased in number in the entire SN in mice treated with LPS-MPTP, as compared with saline- or MPTP-treated mice. In aged (60w) mice (**D**-**F**), numbers of the dopaminergic (A9) neurons were decreased in the order of saline, MPTP, and LPS-MPTP treatments. In the MPTP- and LPS-MPTP-treated mice, numbers of the activated microglia were increased with their accumulation in the SNc. P8 refers to postnatal day 8; and 60w, to 60-day-old mice (Sawada H. et al., 2007, J Neurosci

Neonatal microglia are activated macrophage colony-stimulating factor (M-CSF) dependently from late gestation up to two weeks after birth, and are very proliferative and easily activated under normal circumstances (Sawada M. et al., 1990; Thery et al., 1990). Sawada H. et al. (2007) showed that in MPTP-administered neonatal mice their microglia activated by treatment with systemic LPS showed neurotrophic potential toward dopaminergic neurons. Neonatal (postnatal day 7) mice treated with MPTP showed decreases in the number of dopaminergic (A9) neurons in the SN (Fig. 1 and 2), TH activity, and the levels of DA and the metabolite 3,4-dihydroxyphenylacetic acid in the midbrain. However, cell viability of dopaminergic (A9) neurons and these markers increased in mice treated with MPTP and LPS, along with marked LPS-induced activation of microglia (Fig. 1 and 2). A modest activation of microglia and a significant decrease in the number of

Res, Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and Sons).

**4. Nuroprotective role of microgliae** 

Rabchevsky and Streit, 1997; Suzuki et al., 2001).

dopaminergic (A9) neurons were observed in the MPTP-treated mice, whereas mice treated with the MPTP and LPS demonstrated marked microglial activation and a tendency toward recovery against cell toxicity, as compared with the MPTP-treated mice (Fig. 3A). These MPTP-LPS-treated mice showed increased levels of pro-inflammatory cytokines of IL-1β and IL-6. LPS-activated microglia in neonatal and aged mice had different phenotypic effects on dopaminergic neurons exposed to MPTP. In contrast, the number of dopaminergic neurons in the SN in aged mice (60 weeks) treated with MPTP was significantly decreased, and an increase in the number of microglia treated with MPTP and LPS produced a further decrease in the number of dopaminergic neurons (Fig. 1 and 2). The relationship between microglial activation and viability of dopaminergic (A9) neurons for the three groups (saline control, MPTP treated, and MPTP-LPS treated mice) of aged mice showed an inverse correlation (Fig. 3B). These results suggest that LPS-activated microglia in aged mice may be neurotoxic, whereas in neonatal mice they may have neurotrophic potential.

Fig. 2. Analysis of effects of LPS treatment on numbers of dopaminergic (A9) neurons and CD 11b-positive activated microglia in MPTP-treated neonatal and aged mice. **A**: Number of dopaminergic (A9) neurons of the SN for the saline, MPTP, and LPS-MPTP groups in P8 mice. The number of dopaminergic (A9) neurons in the MPTP group was significantly decreased, whereas that for the LPS-MPTP group was recovered. **B**: Number of CD11bimmunopositive microglial cells in the SN in P8 mice. The LPS-MPTP group demonstrated marked microglial activation. **C**: Number of dopaminergic (A9) neurons of the three groups in the SN of 60w mice. The number in the MPTP and LPS-MPTP groups was significantly decreased. **D**: Number of CD11b-immunopositive microglia for the three groups in the SN of 60w mice. Severe microglial activation was observed in the LPS-MPTP group. Values represent the mean±SD. \**p* < 0.05; \*\**p* < 0.01 versus saline group, ##*p* < 0.01 versus MPTP group, by the use of the unpaired Student's *t* test (Sawada H. et al., 2007, J Neurosci Res, Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and Sons).

Role of Microglia in Inflammatory Process in Parkinson's Disease 337

been reported to be relevant to the pathogenesis of PD in relation to microglia. The infiltration of peripheral immune cells into the brain and its relevance to PD have been reported. Infiltrates of CD4+ T cells were found in the SN of PD patients, and CD4+ T cells were neurotoxic in MPTP-treated mice (Brochard et al., 2009). In contrast, mice lacking CD4+ T cells showed attenuated nigro-striatal degeneration induced by MPTP. Another report indicated that nitrated α-synuclein, which is abundant in Lewy bodies, was detected in peripheral lymphocytes in cervical lymph nodes from MPTP-treated mice (Benner et al., 2008). The transfer of T cells from mice immunized with nitrated α-synuclein into MPTPtreated mice caused significant infiltration into the brain and a neuroinflammatory response with accelerated dopaminergic neuron loss (Benner et al., 2008). The consequence of two subsets of CD4+ lymphocytes that acted on microglia was reported to be different (Reynolds et al., 2009). In the presence of nitrated α-synuclein, one subset, i.e., CD4+ CD25- (effector) T cells, enhanced the microglial activation and neurotoxic responses by secreting TNF-α and IFN-γ; and the other, CD4+ CD25+ (regulatory) T cells, suppressed the microglia activation and induced microglia apoptosis by secreting IL-10 and TGF-β. On the other hand, the Th1 and Th17 cells, other subsets of CD4+ T cells, increased the production of NO, superoxide, TNF-α and IL-1β from microglia, and decreased the production of neurotrophic factors such as insulin-like growth factor (IGF)-1 (Appel, 2009). Consequently, neuronal injury, which may trigger the release of increased levels of nitrated α-synuclein, may enhance the microglia-mediated neurotoxicity. CD4+ CD25+ T cells suppressed the inflammatory effects of Th17 cells. Another subset of CD4+ T cells, Th2 cells, produced IL-4, increased the release of IGF-1 from microglia, and decreased the release of free radicals, resulting in enhanced

Many reports on postmortem PD brains and PD animal models indicate that activated microglia have neurotoxic effects and may play a significant role in progression of the disease (Block et al., 2007). Microglial activation was also reported to be neurotoxic in experimental PD models produced by MPTP (Furuya et al., 2004; Wu et al., 2002, 2003). ROS production from microglia adversely affects the neurons. Previous studies demonstrated that NADPH oxidase-mediated microglial superoxide production is important to MPTP- or rotenone-induced dopaminergic toxicity (Gao et al., 2003; Wu et al., 2002, 2003). NADPH oxidase subunit (gp91)-deficient mice showed attenuated microglial

There have been many reports indicating the neurotoxic effects of activated microglia, especially in aged animals (Sawada H. et al., 2007; Sawada M. et al., 2008; Sugama et al., 2003). Aging is thought to be an important factor in idiopathic PD. Aging is speculated to promote a change from the protective to the toxic phenotype of activated microglia, as in the toxic change in microglia hypothesized by Sawada M. et al. (2006). Cultures of amyloid βpeptide (Aβ)-stimulated microglia from aged rats were reported to show more evidence of toxicity than those from middle-aged or embryonic mice (Viel et al., 2001). Furthermore, MPTP neurotoxicity is greater in aged mice than in young mice, and is accompanied by age-

As described above, activated microglia acted in neuroprotection in MPTP-treated neonatal mice. However, microglia in neonatal or young animals might also act as neurotoxicity, depending on the condition of microglial activation. Sawada H. et al. (2010) reported that

production of superoxide and dopaminergic cell death (Wu et al., 2003).

related microglial activation (Sugama et al., 2003).

neuronal protection.

**5. Neurotoxic role of microglia** 

Using an ischemic gerbil model, Imai et al. (2007) also showed neuroprotective effects of exogenously administered microglia. Microglia cells were isolated from neonatal gerbils by labeling with a fluorescent dye. When the isolated microglia were systemically injected into the subclavian artery in experimental ischemic gerbils, the cells migrated to ischemic hippocampal regions (CA1 pyramidal neurons); and the number of surviving hippocampal neurons was greater in the host gerbils than in the control ischemic animals. This neuroprotective effect was enhanced when the isolated microglia were stimulated by interferon-γ. Administration of exogenous microglia to the ischemic gerbils improved the performance of the animals in a passive-avoidance learning task. The ischemic animals revealed increased expression of neurotrophic factors BDNF and GDNF in their hippocampal regions. Thus, administration of isolated neonatal microglia may potentially have neurotrophic effects on injured brain regions.

Recently, Saijo et al. (2009) reported that an orphan nuclear receptor, Nurr1, protected dopaminergic neurons against impairment by inhibiting the expression of pro-inflammatory mediators produced by microglia and astrocytes, such as TNF-α, IL-1β, and inducible NOsynthesizing (iNOS) enzyme. Nurr1 is known to play an essential role in the generation and maintenance of dopaminergic neurons. When Nurr1 expression was reduced by shRNA, inflammatory substances were increased in microglia; and with further amplification by astrocytes, these substances caused dopaminergic neuron death in the SN.

Fig. 3. Relationship between activated microglia and dopaminergic (A9) neurons in saline, MPTP, and LPS-MPTP groups of individual neonatal or aged mice. **A**: In P8 mice, only slight activation of microglia and decrease in number of dopaminergic (A9) neurons were found for the MPTP group, whereas the LPS-MPTP group demonstrated marked microglial activation and a tendency toward protection against loss of dopaminergic (A9) neurons compared with the MPTP group. **B**: In 60w mice, an inverse correlation (R=0.81) was observed between the 2 parameters when data for all 3 groups was plotted (Sawada H. et al., 2007, J Neurosci Res, Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and Sons).

The question as to whether or not peripheral macrophages or lymphocytes can cross the BBB into the PD brain remains still controversial. However, peripheral T lymphocytes have

Using an ischemic gerbil model, Imai et al. (2007) also showed neuroprotective effects of exogenously administered microglia. Microglia cells were isolated from neonatal gerbils by labeling with a fluorescent dye. When the isolated microglia were systemically injected into the subclavian artery in experimental ischemic gerbils, the cells migrated to ischemic hippocampal regions (CA1 pyramidal neurons); and the number of surviving hippocampal neurons was greater in the host gerbils than in the control ischemic animals. This neuroprotective effect was enhanced when the isolated microglia were stimulated by interferon-γ. Administration of exogenous microglia to the ischemic gerbils improved the performance of the animals in a passive-avoidance learning task. The ischemic animals revealed increased expression of neurotrophic factors BDNF and GDNF in their hippocampal regions. Thus, administration of isolated neonatal microglia may potentially

Recently, Saijo et al. (2009) reported that an orphan nuclear receptor, Nurr1, protected dopaminergic neurons against impairment by inhibiting the expression of pro-inflammatory mediators produced by microglia and astrocytes, such as TNF-α, IL-1β, and inducible NOsynthesizing (iNOS) enzyme. Nurr1 is known to play an essential role in the generation and maintenance of dopaminergic neurons. When Nurr1 expression was reduced by shRNA, inflammatory substances were increased in microglia; and with further amplification by

Fig. 3. Relationship between activated microglia and dopaminergic (A9) neurons in saline, MPTP, and LPS-MPTP groups of individual neonatal or aged mice. **A**: In P8 mice, only slight activation of microglia and decrease in number of dopaminergic (A9) neurons were found for the MPTP group, whereas the LPS-MPTP group demonstrated marked microglial activation and a tendency toward protection against loss of dopaminergic (A9) neurons compared with the MPTP group. **B**: In 60w mice, an inverse correlation (R=0.81) was observed between the 2 parameters when data for all 3 groups was plotted (Sawada H. et al., 2007, J Neurosci Res, Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and

The question as to whether or not peripheral macrophages or lymphocytes can cross the BBB into the PD brain remains still controversial. However, peripheral T lymphocytes have

astrocytes, these substances caused dopaminergic neuron death in the SN.

have neurotrophic effects on injured brain regions.

Sons).

been reported to be relevant to the pathogenesis of PD in relation to microglia. The infiltration of peripheral immune cells into the brain and its relevance to PD have been reported. Infiltrates of CD4+ T cells were found in the SN of PD patients, and CD4+ T cells were neurotoxic in MPTP-treated mice (Brochard et al., 2009). In contrast, mice lacking CD4+ T cells showed attenuated nigro-striatal degeneration induced by MPTP. Another report indicated that nitrated α-synuclein, which is abundant in Lewy bodies, was detected in peripheral lymphocytes in cervical lymph nodes from MPTP-treated mice (Benner et al., 2008). The transfer of T cells from mice immunized with nitrated α-synuclein into MPTPtreated mice caused significant infiltration into the brain and a neuroinflammatory response with accelerated dopaminergic neuron loss (Benner et al., 2008). The consequence of two subsets of CD4+ lymphocytes that acted on microglia was reported to be different (Reynolds et al., 2009). In the presence of nitrated α-synuclein, one subset, i.e., CD4+ CD25- (effector) T cells, enhanced the microglial activation and neurotoxic responses by secreting TNF-α and IFN-γ; and the other, CD4+ CD25+ (regulatory) T cells, suppressed the microglia activation and induced microglia apoptosis by secreting IL-10 and TGF-β. On the other hand, the Th1 and Th17 cells, other subsets of CD4+ T cells, increased the production of NO, superoxide, TNF-α and IL-1β from microglia, and decreased the production of neurotrophic factors such as insulin-like growth factor (IGF)-1 (Appel, 2009). Consequently, neuronal injury, which may trigger the release of increased levels of nitrated α-synuclein, may enhance the microglia-mediated neurotoxicity. CD4+ CD25+ T cells suppressed the inflammatory effects of Th17 cells. Another subset of CD4+ T cells, Th2 cells, produced IL-4, increased the release of IGF-1 from microglia, and decreased the release of free radicals, resulting in enhanced neuronal protection.

#### **5. Neurotoxic role of microglia**

Many reports on postmortem PD brains and PD animal models indicate that activated microglia have neurotoxic effects and may play a significant role in progression of the disease (Block et al., 2007). Microglial activation was also reported to be neurotoxic in experimental PD models produced by MPTP (Furuya et al., 2004; Wu et al., 2002, 2003).

ROS production from microglia adversely affects the neurons. Previous studies demonstrated that NADPH oxidase-mediated microglial superoxide production is important to MPTP- or rotenone-induced dopaminergic toxicity (Gao et al., 2003; Wu et al., 2002, 2003). NADPH oxidase subunit (gp91)-deficient mice showed attenuated microglial production of superoxide and dopaminergic cell death (Wu et al., 2003).

There have been many reports indicating the neurotoxic effects of activated microglia, especially in aged animals (Sawada H. et al., 2007; Sawada M. et al., 2008; Sugama et al., 2003). Aging is thought to be an important factor in idiopathic PD. Aging is speculated to promote a change from the protective to the toxic phenotype of activated microglia, as in the toxic change in microglia hypothesized by Sawada M. et al. (2006). Cultures of amyloid βpeptide (Aβ)-stimulated microglia from aged rats were reported to show more evidence of toxicity than those from middle-aged or embryonic mice (Viel et al., 2001). Furthermore, MPTP neurotoxicity is greater in aged mice than in young mice, and is accompanied by agerelated microglial activation (Sugama et al., 2003).

As described above, activated microglia acted in neuroprotection in MPTP-treated neonatal mice. However, microglia in neonatal or young animals might also act as neurotoxicity, depending on the condition of microglial activation. Sawada H. et al. (2010) reported that

Role of Microglia in Inflammatory Process in Parkinson's Disease 339

Table 1. Volumes of the necrotic or FJB positive areas and number of the CD11b positive microglia in ethanol-injured striatum. These data are obtained from 4-5 of neonatal P8 mice. Values represent the mean±SD. ★ *p* < 0.05; ★★ *p* < 0.01, by use of the unpaired Student's

Sawada M. et al. (1998, 1999, 2006) proposed a hypothesis of toxic change of microglia. He with collaborators separated two subsets of microglia with neuroprotective or neurotoxic phenotypes from mouse brain by cell sorting and established a cell line for each. The 6-3 cell line of one subset produced a greater amount of ROS stimulated by phorbol myristate acetate (PMA) than did the other, the Ra2 cell line. Both clones were dependent on granulocyte macrophage colony-stimulating factor (GM-CSF). When both microglia cell lines were co-cultured with N18 neuronal cells, which are sensitive to oxidative stress by hydrogen peroxide to produce dose-dependent cell death, and are stimulated with PMA, the viability of the N18 cells was increased when the cell were co-cultured with Ra2 cells and decreased in the presence of 6-3 cells. These results indicate that 6-3 cells were of the

A toxic change in microglia phenotypes from neuroprotection to neurotoxicity was observed by transfecting the cells with cDNA encoding HIV-1 Nef protein, indicating the conversion of microglia from a neurotrophic to a neurotoxic subtype (Vilhardt et al., 2002). When Ra2 cells were transfected with Nef protein by using lenti virus, these normally neuroprotective cells produced ROS with activation of NADPH oxidase, in contrast to the non-transfected Ra2 cells, which did not produce ROS. When Nef-Ra2 cells were co-cultured with N-18 neuronal cells, the viability of N-18 cells was decreased; whereas that of N-18 cells cocultured with Ra2 cells was not lowered. These results suggest a toxic change in nef-

According to a report on conditional ALS transgenic mice, the disease progression was determined by the expression of mutant superoxide dismutase (SOD)1 protein not in motor neurons but in microglia (Boillée et al., 2006). Microglial activation was observed from the disease onset to the progression in the spinal cord in these animals. The disease progression accelerated in the transgenic mice that expressed mutant SOD1 in all systemic cells including microglia, whereas the viability was improved in the transgenic mice that expressed normal SOD1 only in their microglia. However, the early phase of disease progression showed no difference between above the two types of transgenic mice. This report concluded that microglia had little effect on the early disease phase but participated on the later disease progression in ALS. According to the hypothesis of toxic change in microglia by Sawada M., microglial toxic changes might appear during the progression

neurotoxic phenotype, and that Ra2 cells were of neuroprotective one.

transfected microglia accompanied by ROS production.

*t* test.

phase of ALS.

**6. Toxic change in microglia** 

LPS-activated neonatal microglia showed the neurotoxic phenotype in an ethanol-induced brain injury model produced by the stereotaxic injection of ethanol into the mouse striatum (Takeuchi et al., 1998; Toyama et al., 2008; Fig. 4). In this ethanol-injected model produced a large and round or oval shaped brain lesion without hemorrhage, infection or other unexpected effects that might affect the cytokine networks in the brain. Neonatal mice were pretreated with systemic LPS or saline injection (i.p.) daily for 5 days from postnatal day 3 (P3) to P7. Local injection of 100% ethanol (2.0 μl) produced more severe neuronal damage than seen in the MPTP-induced PD model. A large lesion with a necrotic core was observed in the ethanol-injected striatum; and activated microglia had migrated to the outside of this necrotic mass, where Fluoro-Jade B (FJB)-positive degenerative neurons were observed (Fig. 4). After the ethanol-induced damage, activated microglia accumulated in the FJB-positive regions and eliminated damaged neurons by causing delayed neuronal death (Fig. 4). By previous treatment with systemic LPS or saline treatment as a control, the volumes of necrotic and degenerative areas in the striatum were further increased along with an increase in the number of activated microglia by LPS (Table 1). The number of iNOSpositive microglia also tended to be increased by the LPS treatment.

Fig. 4. Morphological changes due to ethanol injection into the striatum of neonatal (P8) mice; detection of neuronal injury by Fluoro-Jade B (FJB) staining and activated microglia by CD11b immunostaining. A large diameter of lesion was observed in ethanol-injected striatum 24 hr after the injection with the core of necrotic mass. Degenerative cells were observed outside the necrotic mass. **A:** In FJB staining, many FJB-positive cells were seen in the degenerative region. **B:** In CD11b immunostaining, the large number of activated microglia was found in the degenerative region. n; necrotic region, d; degenerative region. FJB-positive degenerative neurons are shown in saline-treated (**C**), and LPS-treated (**D**) mice. The number of FJB-positive degenerative cells was increased in LPS-treated mice. CD11b-positive microglia in ethanol-injected ipsilateral striatum are shown in saline-treated (**E**), and LPS-treated (**F**) mice. Increase in the number of activated microglia was observed most markedly in LPS-treated mice.


Table 1. Volumes of the necrotic or FJB positive areas and number of the CD11b positive microglia in ethanol-injured striatum. These data are obtained from 4-5 of neonatal P8 mice. Values represent the mean±SD. ★ *p* < 0.05; ★★ *p* < 0.01, by use of the unpaired Student's *t* test.
