Amoeboid microglia in the periventricular white matter induce oligodendrocyte damage through expression of proinflammatory cytokines via MAP kinase signaling pathway in hypoxic neonatal rats

Abstract

Hypoxic injury in the perinatal period results in periventricular white matter (PWM) lesions with axonal damage and oligodendroglial loss. It also alters macrophage function by perpetuating expression of inflammatory mediators. Relevant to this is the preponderance of amoeboid microglial cells (AMC) characterized as active macrophages in the developing PWM. This study aimed to determine if AMC produce proinflammatory cytokines that may be linked to the oligodendroglial loss observed in hypoxic PWM damage (PWMD). Wistar rats (1 day old) were subjected to hypoxia, following which upregulated expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), TNF receptor 1 (TNF-R(1)) and IL-1 receptor 1 (IL-1R(1)) was observed. This was coupled with apoptosis and expression of TNF-R(1) and IL-1R(1) in oligodendrocytes. Primary cultured microglial cells subjected to hypoxia (3% oxygen, 5% CO(2) and 92% nitrogen) showed enhanced expression of TNF-alpha and IL-1beta. Furthermore, mitogen-activated protein (MAP) kinase signaling pathway was involved in the expression of TNF-alpha and IL-1beta in microglia subjected to hypoxia. Our results suggest that following a hypoxic insult, microglial cells in the neonatal rats produce inflammatory cytokines such as TNF-alpha and IL-1beta via MAP kinase signaling pathway. These cytokines are detrimental to oligodendrocytes resulting in PWM lesion.

Figure 1

Real‐time RT‐PCR analysis of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), TNF receptor 1 (TNF‐R₁) and IL‐1 receptor 1 (IL‐1R₁) mRNA expression in the corpus callosum of postnatal rats at 3, 24 h, 3, 7 and 14 days after the hypoxic exposure and their corresponding controls. A–D. Graphical representations of the fold changes in TNF‐α, IL‐1β, TNF‐R₁ and IL‐1R₁, respectively, quantified by normalization to the β‐actin as an internal control. Each bar represents the mean ± SD. There is a significant difference in mRNA levels after the hypoxic exposure when compared with the corresponding controls. (*P < 0.05.)

Figure 2

Western blotting of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), TNF receptor 1 (TNF‐R₁) and IL‐1 receptor1 (IL‐1R₁) protein expression in the corpus callosum tissue supernatants of rats at 3 and 24 h, and at 3, 7 and 14 days after the hypoxic exposure and their corresponding controls (Cn). A. TNF‐α (30 kDa), IL‐1β (17 kDa), TNF‐R₁ (55 kDa) and IL‐1R₁ (80 kDa) immunoreactive bands, respectively. B–E. Bar graphs showing significant changes in the optical density of TNF‐α, IL‐1β, TNF‐R₁ and IL‐1R₁, respectively, following hypoxic exposure when compared with their corresponding controls (mean ± SD). (*P < 0.05.)

Figure 3

Confocal images showing the distribution of lectin‐labeled (A,D,G,J, green), tumor necrosis factor‐α (TNF‐α) (B,E, red) and interleukin‐1β (IL‐1β) (H,K, red) immunoreactive amoeboid microglial cells (AMC; arrows) in the corpus callosum at 24 h after the hypoxic exposure and the corresponding control rat. The colocalized expression of lectin and TNF‐α as well as lectin and IL‐1β AMC can be seen in C and F, and I and L, respectively. The expression of TNF‐α (E) and IL‐1β (K) in AMC (arrows) is markedly enhanced after the hypoxic exposure. The bar graph in M shows a significant increase in the percentage of TNF‐α and IL‐1β immunoreactive microglia after the hypoxic exposure when compared with their corresponding controls (*P < 0.01). Scale bars: A–L, 50 µm.

Figure 4

Confocal images showing the distribution of CC₁ (A,D,G,J, green), tumor necrosis factor receptor 1 (TNF‐R₁) (B,E, red) and interleukin‐1 receptor 1 (IL‐1R₁) (H,K, red) in oligodendrocytes (arrows) and their processes in the corpus callosum at 7 days after the hypoxic exposure and the corresponding control. The colocalized expression of CC₁ with TNF‐R₁ and IL‐1R₁ is depicted in C and F, I and L, respectively. Note the expression of TNF‐R₁ and IL‐1R₁ is upregulated after the hypoxic exposure. The percentage of TNF‐R₁ and IL‐1R₁ immunoreactive positive oligodendrocytes is significantly increased as shown in bar graph M (*P < 0.01). Scale bars: A–L, 20 µm.

Figure 5

Electron micrographs showing necrotic cells in the corpus callosum at 24 h (A) and at 7 days (C) after the hypoxic exposure, Apoptotic cells (B,D) and amoeboid microglial cells (AMC) with internalized necrotic (E) and apoptotic (F) cells (arrows) in the corpus callosum at 7 days after the hypoxic exposure. Scale bars A, C, D = 0.5 μm; B = 1 μm; E, F = 2 μm.

Figure 6

Confocal images showing CC₁‐immunolabeled oligodendrocytes (arrows) (A,D, red) and apoptotic cells (B,E; green) (arrows) as detected by terminal deoxynucleotidyl transferase (Tdt)‐mediated dUTP nick end labeling (TUNEL) in the corpus callosum of a control and a hypoxic rat at 7 days after the hypoxic exposure. The colocalized expression of CC₁ and TUNEL labeling can be seen in C and F. Bar graphs in G and H show the significant increase in percentage of apoptotic nuclei (G) and decrease in the number of CC₁‐positive oligodendrocytes/mm² (H) in the corpus callosum after the hypoxic exposure (*P < 0.01). Scale bars: A–F, 20 µm.

Figure 7

mRNA and protein expression of tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in cultured control microglia and at 1, 2, 4 and 6 h after hypoxic exposure. A,B. Graphical representations of the fold changes in TNF‐α mRNA and IL‐1β mRNA, respectively, quantified by normalization to the β‐actin as an internal control. C. TNF‐α (30 kDa) and IL‐1β (17 kDa) immunoreactive protein bands. D,E. Bar graphs showing changes in the optical density of TNF‐α and IL‐1β, respectively, following hypoxic exposure. Significant differences in mRNA and protein levels in microglial cells after the hypoxic exposure are evident when compared with controls. (*P < 0.05.)

Figure 8

Confocal images of cultured control microglia showing the expression of lectin (A,G, green), tumor necrosis factor‐α (TNF‐α) (B, red), interleukin‐1β (IL‐1β) (H, red) and colocalized expression of lectin and TNF‐α (C) as well as lectin and IL‐1β (I). D–F show the expression of lectin (D, green), TNF‐α (E, red) and colocalized expression of lectin and TNF‐α (F) after treatment with 3% oxygen for 4 h. Note the elevated expression of TNF‐α following treatment with 3% oxygen for 4 h (E) as compared with the control cells (B). J–L show the expression of lectin (J, green), IL‐1β (K, red) and colocalized expression of lectin and IL‐1β (L) after treatment with 3% oxygen for 4 h. The expression of IL‐1β is greatly increased in the microglial cells after hypoxic exposure for 4 h. The bar graph (M) shows a significant increase in the percentage of TNF‐α and IL‐1β immunoreactive microglia after the hypoxic exposure when compared with the control cells (*P < 0.01). Scale bars: A–L, 50 µm.

Figure 9

Western blot analysis showing hypoxia‐induced tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) production via activation of mitogen‐activated protein kinase pathway in microglia. A–C. c‐Jun N‐terminal kinase (JNK), p38 and ERK phosphorylation, and total JNK, p38 and ERK immunoreactive bands. D–G. Immunoreactive bands that indicate that SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) suppress the expression TNF‐α and IL‐1β, respectively, in the microglial cells after hypoxic exposure for 4 h. H–J. Bar graphs showing significant changes in the optical density of JNK, p38 and ERK phosphorylation, respectively, following hypoxic exposure. K–N. Bar graphs showing significant suppression in expression of TNF‐α and IL‐1β by SP600125 and SB203580, respectively. (*P < 0.05.)

Figure 10

Confocal images of cultured microglia showing the expression of the N‐terminal phosphorylated c‐Jun (phos‐c‐Jun) (B, red), counter‐staining with DAPI (A, blue) and colocalized expression of DAPI and phos‐c‐Jun (C). D–F show the expression of phos‐c‐Jun (E, red), counter‐staining with DAPI (D, blue), and colocalized expression of DAPI and phos‐c‐Jun (F) after treatment of cells with 3% oxygen for 45 minutes. The expression of phos‐c‐Jun in the nucleus is greatly increased after hypoxia for 45 minutes. G represents the percentage of phospho‐c‐Jun‐positive control microglial cells and cells treated with 3% oxygen (P < 0.01). Scale bars: A–F, 50 µm.