Activation of microglia induces symptoms of Parkinson's disease in wild-type, but not in IL-1 knockout mice

Abstract

Background: Parkinson's disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice.

Methods: Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection.

Results: LPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice.

Conclusions: The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment.

Figure 1

Expression of inflammatory cytokine in an acute LPS-treated mouse. (A) Immunohistochemical analysis of the effect of LPS treatment on the microglia in the SNC. PBS (2 μL/injection) or LPS (5 μg/2 μL/injection) was injected into the SNR bilaterally. These representative photomicrographs are coronal sections of the SNC at 6 hr after PBS (upper row) or acute LPS treatment (lower row). Triple immunofluorescence staining was performed with CD11b (green), IL-1β (red) and TH (blue) antibodies. Immunoreactivity for IL-1β was co-localized with that for CD11b after LPS treatment (yellow in the merged photomicrograph). Scale bars: 20 μm (B) Effect of LPS treatment on the gene expression of inflammatory cytokine in the midbrain. The midbrain was obtained 6 hr after the acute LPS treatment. Values are expressed as percentages of those in the PBS-treated group and represented as mean ± SEM. *P < 0.05 compared with the PBS-treated group. IL, interleukin; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNC, substantia nigra pars compact; SNR, substantia nigra pars reticulata; Τ Η , tyrosine hydroxylase.

Figure 2

Immunohistochemical analysis of the effect of the subacute LPS treatment on CD11b-positive microglia. PBS (2 μL/injection, bilaterally) or LPS (5 μg/2 μL/injection, bilaterally) was injected into the SNR daily for 5 consecutive days. (A) Representative photomicrographs are coronal sections of the SNC 6 hr after the final PBS (upper rows) or LPS treatment (lower rows). Double immunofluorescence staining was performed with a CD11b antibody (green) and DAPI (blue). Scale bars: 50 μm (B) Number of CD11b immunopositive cells in the SNC. The number of microglial cells was counted between 200 μm and 300 μm from the injection site. The results are expressed as the mean ± SEM for four mice in each group. DAPI, 4',6-diamidino-2-phenylindole; IL, interleukin; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNC, substantia nigra pars compact; SNR, substantia nigra pars reticulata.

Figure 3

Immunohistochemical analysis of the effect of subacute LPS treatment on oxidative stress markers. PBS (2 μL/injection, bilaterally) or LPS (5 μg/2 μL/injection, bilaterally) was injected into the SNR daily for 5 consecutive days. (A) Representative photomicrographs are coronal sections of the SNC region 6 hr after the final PBS (upper row) or LPS treatment (lower row). Double immunofluorescence staining was performed with 4HNE (red, upper row) or NT (red, lower row) antibody and DAPI (blue). Scale bars: 20 μm (B) Numbers of 4HNE and NT immunopositive cells in the SNC. The results are expressed as the mean ± SEM for four mice in each group. 4HNE, 4-hydroxynonenal; DAPI, 4',6-diamidino-2-phenylindole; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNC, substantia nigra pars compact; SNR, substantia nigra pars reticulata.

Figure 4

Detection of cell death using Fluoro-Jade B and TH immunostaining. PBS (2 μL/injection, bilaterally) or LPS (5 μg/2 μL/injection, bilaterally) was injected into the SNR daily for 5 consecutive days. (A) The representative photomicrographs of Fluoro-Jade B staining are coronal sections of the SNC 6 hr, 21 days and 30 days after the final PBS (left) or LPS treatment (right). The positive control was prepared after a traumatic brain injury (TBI) in the cortex. (B) The representative photographs of TH immunostaining using diaminobenzidine, are coronal sections of the SN 6 hr after the final PBS (left) or LPS treatment (right). LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SN, substantia nigra; SNC, substantia nigra pars compact; SNR, substantia nigra pars reticulata; Τ Η , tyrosine hydroxylase.

Figure 5

Immunohistochemical analysis of the effect of subacute LPS treatment on α-synuclein. PBS (2 μL/injection, bilaterally) or LPS (5 μg/2 μL/injection, bilaterally) was injected into the SNR daily for 5 consecutive days. (A) Representative photomicrographs are coronal sections of the SNC 30 days after the final PBS (upper) or LPS treatment (lower). Triple immunofluorescence staining was performed with α-synuclein (red) antibody, TH (green) antibody and DAPI (blue). Scale bars: 20 μm (B) Effect of LPS treatment on the gene expression of α-synuclein in the midbrain 30 days after the final LPS treatment. Values are expressed as percentages of those in the PBS-treated group and represented as mean ± SEM. *P < 0.05 compared with the PBS-treated group. DAPI, 4',6-diamidino-2-phenylindole; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNC, substantia nigra pars compact; SNR, substantia nigra pars reticulata; Τ Η , tyrosine hydroxylase.

Figure 6

Effect of subacute LPS treatment on performance in the rotarod test for wild-type, IL-1 KO and TNFα KO mice. PBS (2 μL/injection, bilaterally, open column) or LPS (5 μg/2 μL/injection, bilaterally, closed column) was injected into the SNR daily for 5 consecutive days. The rotarod test was conducted at 2 hr and 8, 15 and 22 days after the final PBS or LPS treatment. The test was performed three times and latency to fall off was recorded for each trial. The median latency time was calculated for each mouse. The results are expressed as the mean ± SEM in each group. *P < 0.05 compared with the PBS-treated group. IL, interleukin; KO, knockout; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; TNF, tumor necrosis factor.

Figure 7

Effect of subacute LPS treatment on the gene expression of tyrosine hydroxylase in the midbrain. PBS (2 μL/injection, bilaterally) or LPS (5 μg/2 μL/injection, bilaterally) was injected into the SNR daily for 5 consecutive days. The midbrain was obtained 6 hr after the final PBS or LPS treatment. Values are expressed as percentages of those in the PBS-treated group and represented as mean ± SEM. *P < 0.05 compared with the PBS-treated group. IL, interleukin; KO, knockout; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SEM, standard error of the mean; SNR, substantia nigra pars reticulata; TNF, tumor necrosis factor.