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, 27 (4), 309-319

Interleukin-4 Contributes to Degeneration of Dopamine Neurons in the Lipopolysaccharide-treated Substantia Nigra in vivo

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Interleukin-4 Contributes to Degeneration of Dopamine Neurons in the Lipopolysaccharide-treated Substantia Nigra in vivo

Eugene Bok et al. Exp Neurobiol.

Abstract

The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN) in vivo of lipopolysaccharide (LPS)-treated rat. Tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral DA neurons at 3 and 7 day post-LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 day and remained elevated at 7 day post-LPS. IL-4 immunoreactivity was detected exclusively in microglia. IL-4 neutralizing antibody (NA) significantly increased survival of DA neurons in LPS-treated SN in vivo by inhibiting microglial activation and production of proinflammatory mediator such as IL-1β as assessed by immunihistochemical, RT-PCR and ELISA analysis, respectively. Accompanying neuroprotection are IL-4NA effects on decreased disruption of blood-brain barrier and astrocytes. The present data suggest that endogenously expressed IL-4 from reactive microglia may be involved in the neuropathological processes of degeneration of DA neurons occurring in Parkinson's disease.

Keywords: Dopaminergic neurons; Interleukin-4; Lipopolysaccharides; Parkinson disease; Substantia nigra.

Figures

Fig. 1
Fig. 1. LPS induces degeneration of DA neurons and microglial activation in the SN in vivo. PBS (3 µl) or LPS (5 µg/3 µl) was unilaterally injected into the SN. At 7 d post-injection, the coronal sections (40 µm) were selected and processed for TH (A, B, E, F), NeuN (C, G) immunohistochemical staining or Nissl staining (D, H). (B, F) Higher magnifications of (A, E), respectively. (I) Stereological counting results of TH+ and Nissl+ neurons in the SN at 7 d post-injection. The SN tissues were immunostained with CD11b (J, K, P, Q), Iba-1 (L, M R, S), and CD68 (N, O, T, U) antibodies for microglia in the SN. (K, M, O, Q, S, U) Higher magnifications of (J, L, N, P, R, T), respectively. Dotted lines indicate SNpc where DA neurons were degenerated. Scale bars: A, E, 250 µm; B, F, 25 µm; C, D, G, H, 50 µm. J, L, N, P, R, T, 500 µm; K, M, O, Q, S, U, 50 µm. SNpc, substantia nigra pars compacta; SNr, substantia nigra reticulata. Data are presented as the means±SEM of four to seven animals per group. *p<0.001, significantly different from PBS (ANOVA and Bonferroni analyses).
Fig. 2
Fig. 2. LPS induces IL-4 expression in the microglia in the SN in vivo. (A~D) Immunofluorescence staining of IL-4 in the SN at 1 d (B), 3 d (C), and 7 d (D) after intranigral injection of LPS or PBS as a control (A, 1d). (E~L) At 3 d post injection, the SN tissues were immunostained simultaneously with IL-4 (Red) and TH (E, I; green), CD11b (F, J; green), Iba-1 (G, K; green) or CD68 (H, L; green). (E~H) PBS injected SN. (I~L) LPS injected SN. Arrows indicate IL-4+ cells merged with CD11b+, Iba-1+, and CD68+ cells. (M) Quantification of IL-4 expression in TH+, CD11b+, Iba-1+ or CD68+cells in the SN. Scale bars: A, E, 250 µm; B, F, 25 µm; C, D, G, H, 50 µm. J, L, N, P, R, T, 500 µm; K, M, O, Q, S, U, 50 µm. SNpc, substantia nigra pars compacta; SNr, substantia nigra reticulata. Data are presented as the means±SEM of four to eight animals per group. *p<0.05, **p<0.01, significantly different from PBS (ANOVA and Bonferroni analyses). Scale bar: A~D, 100 mm; E~L, 25 mm.
Fig. 3
Fig. 3. LPS induces degeneration of DA neurons and microglial activation in the SN in vivo. LPS (5 µg/3 µl) was unilaterally injected into the SN in the presence of gIgG (1 µg) or IL-4NA (1 µg). At 7 d post-injection, the coronal sections were selected and processed for TH (A, B, E, F), NeuN (C, G) immunohistochemical staining or Nissl staining (D, H). (B, F) Higher magnifications of (A, E), respectively. (I) Stereological counting results of TH+ and Nissl+ neurons in the SN at 7 d post-injection. The SN tissues were immunostained with CD11b (J, K, P, Q), Iba-1 (L, M R, S), and CD68 (N, O, T, U) antibodies for microglia in the SN. (K, M, O, Q, S, U) higher magnifications of (J, L, N, P, R, T), respectively. Dotted lines indicate SNpc where DA neurons were degenerated. Scale bars: A, E, 250 µm; B, F, 25 µm; C, D, G, H, 50 µm. J, L, N, P, R, T, 500 µm; K, M, O, Q, S, U, 50 µm. Data are presented as the means±SEM of four to five animals per group. *p<0.001, significantly different from LPS with gIgG (ANOVA and Bonferroni analyses).
Fig. 4
Fig. 4. Neutralization of IL-4 downregulates IL-1β expression in the SN in vivo. Animals were intranigrally infused with LPS (5 µg/3 µl) or PBS as a control in the presence or absence of gIgG or IL-4NA and sacrificed 1 d later for immunohistochemical analysis (A~C), RT-PCR (D, E), and ELISA (F) of the SN. Photomicrographs (A) and the quantification (B) of CD68+ cells in the SN. (C) Colocalization of IL-1b (red) within CD11b+ cells (green). Arrows indicate merged cells. Representative photograph (D) and histogram for the quantification (E) of RT-PCR shows that IL-4NA decrease mRNA expression of IL-1β in the SN. (F) The amounts of IL-1b in the SN were measured using a ELISA technique. Data are presented as the means±SEM of 3 to 5 animals per group. *p<0.01, **p<0.001, compared with PBS; #p<0.01, compared with LPS or LPS with gIgG (ANOVA and Student-Newman-Keuls analysis). Scale bars: A, C: 50 µm.
Fig. 5
Fig. 5. Neutralization of IL-4 prevents disruption of BBB and astrocytes in the SN in vivo. Animals were administered LPS or PBS in the presence or absence of gIgG or IL-4NA and sacrificed at indicated time point. (A~E) Focal leakage of the BBB was demonstrated by FITC-albumin microangiography. The dashed lines indicate the SNpc. (F~J) The SN tissues were immunostained with anti-GFAP antibody for astrocyte. Dotted lines indicate the lacking area of GFAP staining in the SN. Arrows indicate syringe track. (K) Quantification of BBB leakage density and GFAP immuno negative (i.n.) area at 3 day. Data are presented as the means±SEM of Three to five animals per group. *p<0.01, significantly different from PBS; #p<0.01, significantly different from LPS or LPS with gIgG (ANOVA and Student-Newman-Keuls analysis). Scale bars: A~E, 500 µm; F~J, 200 µm.

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