Indomethacin promotes survival of new neurons in the adult murine hippocampus accompanied by anti-inflammatory effects following MPTP-induced dopamine depletion

Abstract

Background: Parkinson's disease (PD) is characterized by dopaminergic cell loss and inflammation in the substantia nigra (SN) leading to motor deficits but also to hippocampus-associated non-motor symptoms such as spatial learning and memory deficits. The cognitive decline is correlated with impaired adult hippocampal neurogenesis resulting from dopamine deficit and inflammation, represented in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) mouse model of PD. In the inflammatory tissue, cyclooxygenase (COX) is upregulated leading to an ongoing inflammatory process such as prostaglandin-mediated increased cytokine levels. Therefore, inhibition of COX by indomethacin may prevent the inflammatory response and the impairment of adult hippocampal neurogenesis.

Methods: Wildtype C57Bl/6 and transgenic Nestin-GFP mice were treated with MPTP followed by short-term or long-term indomethacin treatment. Then, aspects of inflammation and neurogenesis were evaluated by cell counts using immunofluorescence and immunohistochemical stainings in the SN and dentate gyrus (DG). Furthermore, hippocampal mRNA expression of neurogenesis-related genes of the Notch, Wnt, and sonic hedgehog signaling pathways and neurogenic factors were assessed, and protein levels of serum cytokines were measured.

Results: Indomethacin restored the reduction of the survival rate of new mature neurons and reduced the amount of amoeboid CD68+ cells in the DG after MPTP treatment. Indomethacin downregulated genes of the Wnt and Notch signaling pathways and increased neuroD6 expression. In the SN, indomethacin reduced the pro-inflammatory cellular response without reversing dopaminergic cell loss.

Conclusion: Indomethacin has a pro-neurogenic and thereby restorative effect and an anti-inflammatory effect on the cellular level in the DG following MPTP treatment. Therefore, COX inhibitors such as indomethacin may represent a therapeutic option to restore adult neurogenesis in PD.

Keywords: Hippocampus; Indomethacin; Inflammation; MPTP; Neurodegeneration; Neurogenesis; Parkinson’s disease; Substantia nigra.

Fig. 1

Timeline of the experimental procedure. MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride, BrdU: 5-bromo-2′-deoxyuridine

Fig. 2

Results of histological cell counts of proliferating cells in the dentate gyrus. Absolute numbers of newborn mature neurons (a) and all newborn cells (b), and subtypes of newborn progenitor cells in short-term- (c) and long-term-treated (d) mice in the dentate gyrus revealed by immunohistological and immunofluorescent analysis. N = 6–8/group. A two-way ANOVA with factors neurotoxin, drug, and their interaction was performed. A significant interaction was followed by a Bonferroni post hoc test with *p ≤ 0.05, **p ≤ 0.01. CTR: control; MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride

Fig. 3

Representative confocal images of the subtypes. Proliferating subtypes of neurogenesis in the dentate gyrus: progenitor cell type 1 (a), progenitor cell type 2a (b), progenitor cell type 2b (c), progenitor cell type 3 (d), and mature neuron (e). Scale bars indicate 10 μm. BrdU: 5-bromo-2′-deoxyuridine; DCX: doublecortin; GFP: green fluorescent protein; NeuN: neuronal nuclei

Fig. 4

Hippocampal gene expression analysis. Quantitative real-time PCR was performed for the effector genes gli1, hes5, and lef1 of the sonic hedgehog, Notch, and Wnt signaling pathways, respectively, and for the neurogenic factors neuroD6 and ngn1 in short-term- (a) and long-term-treated (b) mice. Gene expression is displayed as fold changes of mRNA levels in relation to CTR + vehicle. N = 5/group. A two-way ANOVA with factors neurotoxin, drug, and their interaction was performed. A significant interaction was followed by a Bonferroni post hoc test: *p ≤ 0.05, **p ≤ 0.01. CTR: control; MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride

Fig. 5

Results of histological cell counts of cellular inflammation in the dentate gyrus. Absolute numbers of Iba-1-positive cells (a), CD68-positive cells (b), amoeboid CD68-positive cells (c), and ramified CD68-positive cells (d) and representative images of CD68-positive cells in CTR mice (e) and MPTP-treated mice (f). Black arrows indicate amoeboid CD68-positive cells, and blue arrows indicate ramified CD68-positive cells. In the short-term treatment group, indomethacin reduces the increased number of amoeboid CD68-positive cells after dopamine depletion. Scale bars indicate 100 and 10 μm in the higher magnification. N = 6–8/group. A two-way ANOVA with main factors neurotoxin, drug, and their interaction was performed. A significant interaction was followed by Bonferroni post hoc test with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. CTR: control; MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride

Fig. 6

Results of histological cell counts and representative images of dopaminergic neurons in the substantia nigra. Absolute numbers of TH-positive cells (a) and representative images, displaying the dopaminergic cell loss after neurotoxic treatment in the substantia nigra in mice of the CTR + vehicle (b) and MPTP + vehicle (c) group, representatively. Scale bars indicate 200 μm. N = 6–11/group. CTR: control; MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride

Fig. 7

Results of histological cell counts and representative images of cellular inflammation in the substantia nigra. Absolute numbers of Iba-1-positive cells (a), CD68-positive cells (b), amoeboid CD68-positive cells (c), and ramified CD68-positive cells (d) and representative images of Iba-1-positive cells in CTR mice (e) and MPTP-treated mice (f), displaying the higher amount of Iba-1-positive cells after dopamine depletion in the substantia nigra. Scale bars indicate 100 μm. N = 5–8/group. A two-way ANOVA with main factors neurotoxin, drug, and their interaction was performed. A significant interaction was followed by Bonferroni post hoc test with *p ≤ 0.05, **p ≤ 0.01, ***p  ≤ 0.001. CTR: control; MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride