Disruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia

Abstract

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (-/-) , TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88-/-,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88-/-, 23.9±9.9 mm(3), TRIF mutant, 26.7±5.8 mm(3) and WT, 17.9±8.4mm(3). These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

Figure 1

Figure 1a: Representative Western blot of 7 independent transfections of total PC12 cell lysates incubated with anti-MyD88 antibody showing decreased expression of MyD88 protein in PC12 cells transfected with MyD88 siRNA for 96h compared to PC12 cells transfected with negative control siRNA. β-actin loading controls are shown below each band. Figure 1b: Average quantitation of MyD88 protein expression in PC12 cells transfected with negative control siRNA compared to PC12 cells transfected with MyD88 siRNA from 7 independent experiments. Quantitation of MyD88 protein levels 96h after transfection with respective siRNAs was done by densitometry using Alpha Ease software, version 4.0 (Alpha Innotech). MyD88 protein expression was significantly decreased in PC12 cells transfected with MyD88 siRNA compared to PC12 cells transfected with negative control siRNA (p=0.0004). Figure 1c: Average viability of PC12 cells following OGD after knock-down of MyD88 expression. PC12 cells were transfected with MyD88 or negative control siRNA for 96h, and cells exposed to OGD for 15h. Data represent averages from 3 independent experiments. Percent cell viability was determined after OGD by means of flow cytometric analysis using Hoechst 33342 and PI staining. Percent cell viability following OGD was not statistically different in PC12 cells transfected with MyD88 siRNA compared to cells transfected with negative control siRNA as shown above.

Figure 1

Figure 1a: Representative Western blot of 7 independent transfections of total PC12 cell lysates incubated with anti-MyD88 antibody showing decreased expression of MyD88 protein in PC12 cells transfected with MyD88 siRNA for 96h compared to PC12 cells transfected with negative control siRNA. β-actin loading controls are shown below each band. Figure 1b: Average quantitation of MyD88 protein expression in PC12 cells transfected with negative control siRNA compared to PC12 cells transfected with MyD88 siRNA from 7 independent experiments. Quantitation of MyD88 protein levels 96h after transfection with respective siRNAs was done by densitometry using Alpha Ease software, version 4.0 (Alpha Innotech). MyD88 protein expression was significantly decreased in PC12 cells transfected with MyD88 siRNA compared to PC12 cells transfected with negative control siRNA (p=0.0004). Figure 1c: Average viability of PC12 cells following OGD after knock-down of MyD88 expression. PC12 cells were transfected with MyD88 or negative control siRNA for 96h, and cells exposed to OGD for 15h. Data represent averages from 3 independent experiments. Percent cell viability was determined after OGD by means of flow cytometric analysis using Hoechst 33342 and PI staining. Percent cell viability following OGD was not statistically different in PC12 cells transfected with MyD88 siRNA compared to cells transfected with negative control siRNA as shown above.

Figure 1

Figure 1a: Representative Western blot of 7 independent transfections of total PC12 cell lysates incubated with anti-MyD88 antibody showing decreased expression of MyD88 protein in PC12 cells transfected with MyD88 siRNA for 96h compared to PC12 cells transfected with negative control siRNA. β-actin loading controls are shown below each band. Figure 1b: Average quantitation of MyD88 protein expression in PC12 cells transfected with negative control siRNA compared to PC12 cells transfected with MyD88 siRNA from 7 independent experiments. Quantitation of MyD88 protein levels 96h after transfection with respective siRNAs was done by densitometry using Alpha Ease software, version 4.0 (Alpha Innotech). MyD88 protein expression was significantly decreased in PC12 cells transfected with MyD88 siRNA compared to PC12 cells transfected with negative control siRNA (p=0.0004). Figure 1c: Average viability of PC12 cells following OGD after knock-down of MyD88 expression. PC12 cells were transfected with MyD88 or negative control siRNA for 96h, and cells exposed to OGD for 15h. Data represent averages from 3 independent experiments. Percent cell viability was determined after OGD by means of flow cytometric analysis using Hoechst 33342 and PI staining. Percent cell viability following OGD was not statistically different in PC12 cells transfected with MyD88 siRNA compared to cells transfected with negative control siRNA as shown above.

Figure 2

Figure 2a: Embryonic cortical neurons were isolated from WT, MyD88-/- and TRIF mutant mice and subjected to 5h OGD. Cell viability following OGD was determined by flow cytometric analysis. Percent neuron survival for each group was determined by comparing cell viability after OGD to cell viability of control cortical neurons that did not undergo OGD within each group. Data shown represent average percent neuron survival from 4 independent experiments for each group. Average percent neuron survival following 5h OGD was similar for cortical neurons from all 3 groups of mice. Figure 2b: The percent of cell death due to apoptosis (A) and necrosis (N) was determined for each experimental group by flow cytometric analysis using Hoechst 33342 and Propidium Iodide staining. Results represent data from 4 independent experiments. Percentages of apoptotic and necrotic cells were not significantly different for cortical neurons from all 3 groups of mice following 5h OGD.

Figure 2

Figure 2a: Embryonic cortical neurons were isolated from WT, MyD88-/- and TRIF mutant mice and subjected to 5h OGD. Cell viability following OGD was determined by flow cytometric analysis. Percent neuron survival for each group was determined by comparing cell viability after OGD to cell viability of control cortical neurons that did not undergo OGD within each group. Data shown represent average percent neuron survival from 4 independent experiments for each group. Average percent neuron survival following 5h OGD was similar for cortical neurons from all 3 groups of mice. Figure 2b: The percent of cell death due to apoptosis (A) and necrosis (N) was determined for each experimental group by flow cytometric analysis using Hoechst 33342 and Propidium Iodide staining. Results represent data from 4 independent experiments. Percentages of apoptotic and necrotic cells were not significantly different for cortical neurons from all 3 groups of mice following 5h OGD.

Figure 3

Figure 3a: Mice were subjected to global forebrain ischemia via 6-minute bilateral common carotid occlusion and hippocampal CA1 neuron survival was determined 7 days later (total N= 30; n= 12, 9 and 9 per group for WT, MyD88-/- and TRIF mutant mice respectively). Representative hippocampal sections (5X) from all 3 groups of mice are shown in the upper panels and in the lower panels are representative CA1 sections (40 X) from the same hippocampi. Figure 3b: The percentage of CA1 neurons that survived 7 days following 6-minute BCCAO, as determined by light microscopy, from WT, MyD88-/- and TRIF mutant mice are shown. There were no differences in percent CA1 neuronal survival following BCCAO among the 3 groups of mice.

Figure 3

Figure 3a: Mice were subjected to global forebrain ischemia via 6-minute bilateral common carotid occlusion and hippocampal CA1 neuron survival was determined 7 days later (total N= 30; n= 12, 9 and 9 per group for WT, MyD88-/- and TRIF mutant mice respectively). Representative hippocampal sections (5X) from all 3 groups of mice are shown in the upper panels and in the lower panels are representative CA1 sections (40 X) from the same hippocampi. Figure 3b: The percentage of CA1 neurons that survived 7 days following 6-minute BCCAO, as determined by light microscopy, from WT, MyD88-/- and TRIF mutant mice are shown. There were no differences in percent CA1 neuronal survival following BCCAO among the 3 groups of mice.

Figure 4

Figure 4a: Mice were subjected to permanent MCAO) and infarct volumes determined 24 h later (total=21; n=7 per group). Representative coronal brain sections, stained with Cresyl violet show infarct sizes from WT, MyD88-/- and TRIF mutant mice. Infarct sizes were similar in all 3 groups of mice. Figure 4b: Comparison of infarct volumes from WT, MyD88-/- and TRIF mutant mice following permanent MCAO (n=7 per group). Infarct areas from sections as shown in Fig 4a were determined using NIH image J software and absolute infarct volumes were calculated as described in the methods section. There was a trend towards increased infarct volumes in MyD88-/- and TRIF mutant mice compared to WT mice, but these differences were not statistically significant, (p=0.16).

Figure 4

Figure 4a: Mice were subjected to permanent MCAO) and infarct volumes determined 24 h later (total=21; n=7 per group). Representative coronal brain sections, stained with Cresyl violet show infarct sizes from WT, MyD88-/- and TRIF mutant mice. Infarct sizes were similar in all 3 groups of mice. Figure 4b: Comparison of infarct volumes from WT, MyD88-/- and TRIF mutant mice following permanent MCAO (n=7 per group). Infarct areas from sections as shown in Fig 4a were determined using NIH image J software and absolute infarct volumes were calculated as described in the methods section. There was a trend towards increased infarct volumes in MyD88-/- and TRIF mutant mice compared to WT mice, but these differences were not statistically significant, (p=0.16).