Toll-like receptor-3 activation increases the vulnerability of the neonatal brain to hypoxia-ischemia - PubMed (original) (raw)
Toll-like receptor-3 activation increases the vulnerability of the neonatal brain to hypoxia-ischemia
Linnea Stridh et al. J Neurosci. 2013.
Abstract
Susceptibility and progression of brain injury in the newborn is closely associated with an exacerbated innate immune response, but the underlying mechanisms are often unclear. Toll-like receptors (TLRs) are important innate immune sensors that may influence the vulnerability of the developing brain. In the current study, we provide novel data to show that activation of the viral innate immune receptor TLR-3 sensitizes the neonatal brain to subsequent hypoxic-ischemic (HI) damage. Poly inosinic:poly cytidylic acid (Poly I:C), a synthetic ligand for TLR-3, was administered to neonatal mice 14 h before cerebral HI. Activation of TLR-3 before HI increased infarct volume from 3.0 ± 0.5 to 15.4 ± 2.1 mm³ and augmented loss of myelin basic protein from 13.4 ± 6.0 to 70.6 ± 5.3%. The sensitizing effect of Poly I:C was specific for the TLR-3 pathway because mice deficient in the TLR-3 adaptor protein Toll/IL-1R domain-containing adaptor molecule-1 (TRIF) did not develop larger brain damage. The increased vulnerability was associated with a TRIF-dependent heightened inflammatory response, including proinflammatory cytokines, chemokines, and the apoptosis-associated mediator Fas, whereas there was a decrease in reparative M2-like CD11b⁺ microglia and phosphorylation of Akt. Because TLR-3 is activated via double-stranded RNA during most viral infections, the present study provides evidence that viral infections during pregnancy or in the neonate could have great impact on subsequent HI brain injury.
Figures
Figure 1.
Experimental design. Poly I:C or saline was injected intraperitoneally to P8 mice. Animals were killed at 1 and 6 h for protein analysis and at 6 and 14 h for gene expression analysis. In separate animals, HI was induced at 14 h after Poly I:C/saline injection. Animals were killed 24 h after HI for flow cytometry analysis and at 5 d after HI for immunohistochemistry (IHC).
Figure 2.
TLR-3 activation sensitize the brain to HI. Neuropathological analysis at 5 d after HI showed a significant increase in both infarct volume (A) and tissue loss (C) in Poly I:C-treated WT mice compared with saline/HI mice. In WT mice, the infarct area at 6 levels of the brain (B) and tissue loss (D) were significantly increased throughout the brain in Poly I:C/HI compared with saline/HI animals. In TRIF KO mice, no differences were found between poly I:C/HI and saline/HI animals. E, Representative images of MAP-2-stained sections in WT and TRIF KO mice 5 d after saline/HI and Poly I:C/HI. Anteroposterior levels, from ∼1 to −2 mm of bregma, are indicated by numbers in B and D, where level 1 indicates the most posterior level. Data are presented as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001; n = 20–21 per group. Scale bar: E, 1 mm.
Figure 3.
Effects of TLR-3 activation on white matter injury. In WT mice, white matter injury was increased in Poly I:C/HI WT mice compared with saline/HI at both the hippocampal (A) and striatum (B) levels. No differences were seen between treatment groups in the TRIF KO mice. C, Representative images of MBP-stained sections in WT and TRIF KO mice after Poly I:C/HI or saline/HI at the hippocampal level. Data are shown as mean ±SEM percentage loss of MBP stain. ***p ≤ 0.001; n = 20–21 per group. Insets represent the area that is depicted at higher magnification. Scale bars: top, 20 μm; bottom, 5 μm.
Figure 4.
Inflammatory response after HI. The inflammatory response, as indicated by Iba-1 immunostaining, showed a pattern of amoeboid-like cells in injured brain regions. The degree and distribution of Iba-1 expression appeared to correlate to the degree of brain damage in all experimental groups. The morphology of the Iba-1-positive cells in injured areas did not differ between Poly I:C/HI-treated (B, D) or saline/HI-treated (A, C) animals or between genotypes. Insets represent the area that is depicted at higher magnification. Scale bars: left, 1 mm; right, 5 μm.
Figure 5.
Gene expression analysis after Poly I:C administration. In WT mice, Poly I:C (10 mg/kg) increased mRNA expression of IL-6 at 6 h (A), IL-1β at 14 h (B), TNF-α at both 6 and 14 h (C), IFN-β at both 6 and 14 h (D), IP-10 at 6 h (E), MCP-1 at both 6 and 14 h (F), and Fas at 6 h (G) compared with controls (saline). In TRIF KO animals, there were no differences in expression of any of the genes between Poly I:C- and saline-injected mice. Data are presented as mean ± SEM; *p ≤ 0.05, ***p ≤ 0.001, n = 7–10 per group.
Figure 6.
Poly I:C effects on MAPK and IκB phosphorylation. In WT mice, Poly I:C (10 mg/kg) reduced the phosphorylation of Akt at 1 h (A), whereas phosphorylation of IκB was increased at 1 h (B). There were no changes in the phosphorylation of ERK, JNK, or p38 (C–E). Data are presented as mean ± SEM; *p ≤ 0.05, **p ≤ 0.01; n = 8 per group.
Figure 7.
Poly I:C reduces reparative M2-like microglia after hypoxic–ischemic injury. A, Gating strategy for analysis of CD11b+ microglia cells. A P1 gate was defined in forward and side scatter. 7AAD was used to distinguish between live and dead cells, and microglia marker CD11b was then gated from the live cells. B, The overall population of CD11b+ microglia increased in the ipsilateral hemisphere compared with the contralateral hemisphere in Poly I:C/HI animals but not in saline/HI mice. Within the CD11b+ population, two different populations were distinguished: a CD11b+high-expressing and a CD11b+low-expressing population, which were both affected by HI. The CD11b+high population increased in the injured hemisphere in both saline/HI-and Poly I:C/HI-treated animals. In contrast, the CD11b+low population was decreased in the injured hemisphere in both saline/HI- and Poly I:C/HI-treated animals. The CD11b+high population in the injured hemisphere was lower in Poly I:C/HI compared with saline/HI animals, whereas the CD11b+low population was higher in the injured hemisphere after Poly I:C/HI compared with saline/HI. C, There were no significant changes in M1-like (proinflammatory) CD11b+CD86+ cells in the ipsilateral compared with the contralateral hemisphere after either Poly I:C/HI or saline/HI. In contrast, there was a decrease in M2-like (reparative) CD11b+CD206+cells in the injured hemisphere in both saline/HI- and Poly I:C/HI-treated animals. In the contralateral hemisphere, the percentage of CD11b+CD206+cells was lower in Poly I:C/HI-treated animals compared with saline/HI-treated animals. Data are presented as mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n = 8–9 per group.
References
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