Neuroprotective effect of L-DOPA-induced interleukin-13 on striatonigral degeneration in cerebral ischemia

Abstract

Levodopa (L-DOPA) treatment is a clinically effective strategy for improving motor function in patients with ischemic stroke. However, the mechanisms by which modulating the dopamine system relieves the pathology of the ischemic brain remain unclear. Emerging evidence from an experimental mouse model of ischemic stroke, established by middle cerebral artery occlusion (MCAO), suggested that L-DOPA has the potential to modulate the inflammatory and immune response that occurs during a stroke. Here, we aimed to demonstrate the therapeutic effect of L-DOPA in regulating the systemic immune response and improving functional deficits in mice with ischemia. Transient MCAO led to progressive degeneration of nigrostriatal dopamine neurons and significant rotational behavior in mice. Exogenous L-DOPA treatment attenuated the striatonigral degeneration and reversed motor behavioral impairment. Notably, treatment with L-DOPA significantly increased IL-13 but reduced IFN-γ in infarct lesions. To investigate the role of IL-13 in motor behavior, we stereotaxically injected anti-IL-13 antibodies into the infarct area of the mouse brain one week after MCAO, followed by L-DOPA treatment. The intervention reduced dopamine, IL-13, and IL-10 levels and exacerbated motor function. IL-13 is potentially expressed on CD4 T cells, while IL-10 is mainly expressed on microglia rather than astrocytes. Finally, IL-13 activates the phagocytosis of microglia, which may contribute to neuroprotection by eliminating degenerating neurons. Our study provides evidence that the L-DOPA-activated dopamine system modulates peripheral immune cells, resulting in the expression of anti-inflammatory and neuroprotective cytokines in mice with ischemic stroke.

Fig. 1. tMCAO induces degeneration of nigrostriatal dopamine neurons in vivo.

A Representative cresyl violet-stained sections of sham and tMCAO mouse brains at 1 and 2 weeks after tMCAO. B. The graph shows the estimated infarct area (EI). The EI was calculated by subtracting the total ipsilateral volume from the total contralateral volume (black line) (namely, differences between the hemispheres, green). NI (noninjured tissue, yellow); IS (ischemic scar tissue, red); T (total ipsilateral tissue = NI + IS); Numbers of tMCAO mice at 1 week (n = 6) and 2 weeks (n = 6). C–F Photomicrographs of TH+ fibers in the striatum (STR) and TH+ cells in the substantia nigra (SN) at 1 week and 2 weeks after tMCAO are shown. The scale bars are 1000 μm (STR) and 400 μm (SN). The optical density of the TH+ fibers in the STR is presented. The number of TH+ cells in the SN is displayed. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a significant difference compared to the sham group. The mean ± SD is calculated by six pictures of each animal obtained from the sham (n = 4) and tMCAO (n = 3) groups. The difference in the p-value between the two groups was evaluated using the Student’s unpaired t-test. The staining intensity or the number of TH+ cells was quantified on seven slides per mouse. C contralateral, I ipsilateral.

Fig. 2. The amphetamine-induced rotation test and dopamine levels in the striatum.

A Scheme illustrating the rotational testing in the striatum following amphetamine administration at 1 day, 1 week, and 2 weeks after tMCAO. B The results of the amphetamine-induced rotation test were recorded for 1 day (n = 10), 1 week (n = 14), and 2 weeks (n = 10). C The dopamine levels of the ipsilateral striatum were measured using ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences compared to the sham group. Mean ± SD: Sham (1 d, n = 5; 1w, n = 4; 2w, n = 11); tMCAO (1 d, n = 3; 1w, n = 7; 2w, n = 13). The statistical p-value between the two groups was evaluated using the Student’s unpaired t-test.

Fig. 3. Effect of L-DOPA on tMCAO.

A Scheme illustrating the administration of L-DOPA/benserazide and the amphetamine-induced rotation test. B Results of the amphetamine-induced rotation test at 1 and 2 weeks after treatment with L-DOPA/benserazide in tMCAO mice (n = 9 per group). C Measurement of dopamine levels in the ipsilateral striatum tissues by ELISA (n = 9–10 per group). D Photomicrographs of TH+ cells in the SN and TH+ fibers in the STR are shown for the vehicle and L-DOPA/benserazide groups 2 weeks after tMCAO. E The optical density of TH+ fibers in the STR is presented. F The number of TH+ cells in the SN is displayed. Each bar represents the mean ± SD. *p < 0.05 and ***p < 0.001 indicate statistically significant differences compared to the sham group. The statistical significance between the two groups was evaluated using the Student’s unpaired t-test.

Fig. 4. Expression of cytokines in the brain tissues after tMCAO.

A, B. IL-13 and IFN-γ levels were measured in the ipsilateral striatum tissues by ELISA. IL-13 (Sham: 1 d, n = 6; 1w, n = 6; 2w, n = 6; tMCAO: 1 d, n = 5; 1w, n = 4; 2w, n = 10), IFN-γ (Sham: 1 d, n = 6; 1w, n = 8; 2w, n = 3; tMCAO: 1 d, n = 6; 1w, n = 6; 2w, n = 5). C, D Slides from frozen tissues were stained with anti-CD4 (green) and anti-IL-13 (red) antibodies. Fluorescence images of IL-13 in CD4 T cells were merged (yellow). IL-13-producing cells were counted in CD4 T cells in the striatum. Mean ± SD: IL-13 (tMCAO: 1w, n = 8; 2w, n = 7). E, F Slides from paraffin tissues were stained with anti-CD4 (green) and anti-IFN-γ (red) antibodies. Fluorescence images of IFN-γ in CD4 T cells were merged (yellow). IFN-γ-producing cells were counted in CD4 T cells in the striatum. Mean ± SD: IFN-γ (tMCAO: 1w, n = 4; 2w, n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a significant difference compared to the sham group. The statistical p-value between the two groups was evaluated using the Student’s unpaired t-test.

Fig. 5. Effect of anti-IL-13 antibody on tMCAO.

A–C Measurement of IL-13, IL-10, and IFN-γ levels in the ipsilateral striatum tissues of stroke mice treated with L-DOPA/benserazide was performed using ELISA (n = 3–6 per group). Each bar represents the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistically significant difference compared to the vehicle control group. The statistical significance between the two groups was evaluated using the Student’s unpaired t-test. D Scheme of anti-IL-13 Ab and L-DOPA/benserazide injection and amphetamine-induced rotation. E The amphetamine-induced rotation test was conducted at 1 and 2 weeks after treatment with L-DOPA/benserazide and anti-IL-13 neutralizing Ab (1 μg/μL) in tMCAO (n = 7–10 per group). F Dopamine levels in ipsilateral brain tissues were measured using ELISA (n = 4 per group). G–I Levels of IL-13, IL-10, and IFN-γ in the ipsilateral striatum tissues were measured using ELISA (n = 4 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a significant difference compared to the control IgG group. The result was expressed as the mean ± SD, and the statistical p-value between the two groups was evaluated using the Student’s unpaired t-test.

Fig. 6. Effects of dopamine on IL-13 levels in vitro.

A, B. CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibodies (1 μg/mL) in the presence of 5 μg/mL of dopamine. After 48 h, IL-13 and IFN-γ levels in the culture media were measured using ELISA (n = 3 per group), and the level of Foxp3 expression was measured by flow cytometry. MFI: mean fluorescence intensity. C Scheme of a lymphocyte coculture system with astrocytes. D–F Lymphocytes were cocultured with astrocytes (including microglia) and stimulated with anti-CD3 and anti-CD28 antibodies (1 μg/mL) in the presence of dopamine and an anti-IL-13 neutralizing antibody (0.5 μg/mL). After 48 h, levels of IL-13, IL-10, and IFN-γ were measured using ELISA (n = 4–12 per group). G–I. The astrocyte culture contained ca. 20~30% of CD11b+ microglia. Therefore, microglia and astrocytes were purified with over 94% purity using MACS. The purified cell populations were then stimulated with recombinant IL-13 cytokines (20 ng/mL). After 24 h, IL-10 expression was measured in the culture media using ELISA (n = 3–4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate a significant difference compared to the control group. The result was expressed as the mean ± SD, and the statistical p-value between the two groups was evaluated using the Student’s unpaired t-test. The comparison of multiple groups was evaluated using a one-way ANOVA with Tukey’s multiple comparison test. These experiments have been repeated at least two times.

Fig. 7. IL-13-induced phagocytic activity of microglia.

A Schematic diagram illustrating the testing of microglia’s efficacy in phagocytosing apoptotic neurons. B–F Purified microglia were cocultured with CFSE-stained neurons. Primary dopaminergic (C, D) and cortical (E, F) neurons were stained with CFSE. Purified microglia were cocultured with neurons in the presence of rIL-13 (20 ng/mL). After 15 min (dopaminergic neurons) and 30 min (cortical neurons), microglia were stained with anti-CD11b antibodies, and the CFSE-positive cells indicating phagocytosis were analyzed using flow cytometry. (n = 3 per group). *p < 0.01 indicates a significant difference compared to the control group. The result was expressed as the mean ± SD, and the statistical p-value between the two groups was evaluated using the Student’s unpaired t-test. These experiments have been repeated at least two times. All schematic graphics were created using a subscription to BioRender.com.