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The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage.

Hanafy KA - J Neuroinflammation (2013)

Bottom Line: Our results suggest that SAH pathology could have different phases.These results could explain why therapies tailored to aSAH patients have failed for the most part.Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of NeuroCritical Care, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, The Center for Life Science, 3 Blackfan Circle, Boston, MA 02215, USA. khanafy@bidmc.harvard.edu

ABSTRACT

Background: Although microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively.

Methods: Subarachnoid hemorrhage was induced in TLR4-/-, TRIF-/-, MyD88-/- and wild type C57BL/6 mice by injecting 60 μl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes.

Results: On post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model.

Conclusion: Our results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

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Immunohistochemistry of TLR4 co-localization among the different cell types of the murine brain. Representative images from four mice per group and four different fields of view on post-operative day (POD) 7 A. Immunohistochemistry across shows staining with Toll-like receptor 4 (TLR4) in the first panel, Iba1 (for microglia) in the second panel, and the merge in the third panel. Arrows delineate co-localization. B. Immunohistochemistry showing individual panels and merge for astrocytes with Glial Fibrillary acidic protein. C. Immunohistochemistry for β III Tubulin reflecting co-localization of TLR4 and neurons. Scale Bars: 15 μm. D. Quantification based on four mice and four different fields of view at POD 7 and POD 15 showing microglia express the most Toll-like receptor 4 (TLR4) by ANOVA at both time points (P <0.001).
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Figure 4: Immunohistochemistry of TLR4 co-localization among the different cell types of the murine brain. Representative images from four mice per group and four different fields of view on post-operative day (POD) 7 A. Immunohistochemistry across shows staining with Toll-like receptor 4 (TLR4) in the first panel, Iba1 (for microglia) in the second panel, and the merge in the third panel. Arrows delineate co-localization. B. Immunohistochemistry showing individual panels and merge for astrocytes with Glial Fibrillary acidic protein. C. Immunohistochemistry for β III Tubulin reflecting co-localization of TLR4 and neurons. Scale Bars: 15 μm. D. Quantification based on four mice and four different fields of view at POD 7 and POD 15 showing microglia express the most Toll-like receptor 4 (TLR4) by ANOVA at both time points (P <0.001).

Mentions: Additionally, we measured neuronal apoptosis in these groups, as described in Figure 2. Interestingly, TRIF−/− SAH had a statistically equivalent neural apoptotic burden to WT SAH on POD 7, while on POD 15, there was no difference between MyD88−/− SAH and WT SAH. In terms of minimal apoptotic burden, there was no difference between the number of apoptotic neurons quantified in sham, TLR4−/− SAH, and MyD88 −/− SAH on POD 7. At POD 15, there was no difference between the number of apoptotic neurons quantified in sham, TLR4−/− SAH and TRIF−/− SAH (Figure 3B). Of note, the LPS injected mice, demonstrated significantly less neuronal apoptosis at POD 7, compared to WT SAH; however at POD 15 there was no difference between these groups. With the understanding that the TLR4 pathway may play a role in vasospasm and neuronal apoptosis, we wanted to identify what type of cell was expressing TLR4. Microglia, astrocytes and neurons were examined for TLR4 expression with representative images shown in Figure 4A-C and quantification of TLR4 co-localization in Figure 4D. Based on these results, we found that the majority of TLR4 is expressed in microglia at both POD 7 and 15 after SAH.


The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage.

Hanafy KA - J Neuroinflammation (2013)

Immunohistochemistry of TLR4 co-localization among the different cell types of the murine brain. Representative images from four mice per group and four different fields of view on post-operative day (POD) 7 A. Immunohistochemistry across shows staining with Toll-like receptor 4 (TLR4) in the first panel, Iba1 (for microglia) in the second panel, and the merge in the third panel. Arrows delineate co-localization. B. Immunohistochemistry showing individual panels and merge for astrocytes with Glial Fibrillary acidic protein. C. Immunohistochemistry for β III Tubulin reflecting co-localization of TLR4 and neurons. Scale Bars: 15 μm. D. Quantification based on four mice and four different fields of view at POD 7 and POD 15 showing microglia express the most Toll-like receptor 4 (TLR4) by ANOVA at both time points (P <0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750560&req=5

Figure 4: Immunohistochemistry of TLR4 co-localization among the different cell types of the murine brain. Representative images from four mice per group and four different fields of view on post-operative day (POD) 7 A. Immunohistochemistry across shows staining with Toll-like receptor 4 (TLR4) in the first panel, Iba1 (for microglia) in the second panel, and the merge in the third panel. Arrows delineate co-localization. B. Immunohistochemistry showing individual panels and merge for astrocytes with Glial Fibrillary acidic protein. C. Immunohistochemistry for β III Tubulin reflecting co-localization of TLR4 and neurons. Scale Bars: 15 μm. D. Quantification based on four mice and four different fields of view at POD 7 and POD 15 showing microglia express the most Toll-like receptor 4 (TLR4) by ANOVA at both time points (P <0.001).
Mentions: Additionally, we measured neuronal apoptosis in these groups, as described in Figure 2. Interestingly, TRIF−/− SAH had a statistically equivalent neural apoptotic burden to WT SAH on POD 7, while on POD 15, there was no difference between MyD88−/− SAH and WT SAH. In terms of minimal apoptotic burden, there was no difference between the number of apoptotic neurons quantified in sham, TLR4−/− SAH, and MyD88 −/− SAH on POD 7. At POD 15, there was no difference between the number of apoptotic neurons quantified in sham, TLR4−/− SAH and TRIF−/− SAH (Figure 3B). Of note, the LPS injected mice, demonstrated significantly less neuronal apoptosis at POD 7, compared to WT SAH; however at POD 15 there was no difference between these groups. With the understanding that the TLR4 pathway may play a role in vasospasm and neuronal apoptosis, we wanted to identify what type of cell was expressing TLR4. Microglia, astrocytes and neurons were examined for TLR4 expression with representative images shown in Figure 4A-C and quantification of TLR4 co-localization in Figure 4D. Based on these results, we found that the majority of TLR4 is expressed in microglia at both POD 7 and 15 after SAH.

Bottom Line: Our results suggest that SAH pathology could have different phases.These results could explain why therapies tailored to aSAH patients have failed for the most part.Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of NeuroCritical Care, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, The Center for Life Science, 3 Blackfan Circle, Boston, MA 02215, USA. khanafy@bidmc.harvard.edu

ABSTRACT

Background: Although microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively.

Methods: Subarachnoid hemorrhage was induced in TLR4-/-, TRIF-/-, MyD88-/- and wild type C57BL/6 mice by injecting 60 μl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes.

Results: On post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model.

Conclusion: Our results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

Show MeSH
Related in: MedlinePlus