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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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The effects of microglial ablation on neuronal apoptosis. A. Top panel: merge of DAPI and TUNEL staining from dentate gyrus of wild type subarachnoid hemorrhage (SAH) after intracerebroventricular (ICV) PBS liposome injection on POD 7 and 15. B. Bottom panel: merge of DAPI and TUNEL staining in wild type SAH after ICV clodronate injection on POD 7 and 15. Scale Bars: 10 μm in all panels. C. Quantification of apoptotic neurons on POD 7 displaying significant reduction, by Student’s t-test (P <0.03), after ICV clodronate injection and microglial ablation. D. Quantification of apoptotic neurons on POD 15 revealing no change between ICV PBS and clodronate injections. N = 3 for each group and error bars are standard deviations.
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Figure 7: The effects of microglial ablation on neuronal apoptosis. A. Top panel: merge of DAPI and TUNEL staining from dentate gyrus of wild type subarachnoid hemorrhage (SAH) after intracerebroventricular (ICV) PBS liposome injection on POD 7 and 15. B. Bottom panel: merge of DAPI and TUNEL staining in wild type SAH after ICV clodronate injection on POD 7 and 15. Scale Bars: 10 μm in all panels. C. Quantification of apoptotic neurons on POD 7 displaying significant reduction, by Student’s t-test (P <0.03), after ICV clodronate injection and microglial ablation. D. Quantification of apoptotic neurons on POD 15 revealing no change between ICV PBS and clodronate injections. N = 3 for each group and error bars are standard deviations.

Mentions: Finally, we determined whether microglial depletion effected neuronal apoptosis in Figure 7, and found a similar bimodal theme. At POD 7, the depletion of microglia resulted in a significant decrease in neuronal apoptosis (Figure 7A, C); while at POD 15, microglial depletion had no effect (Figure 7B, D).


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

Hanafy KA - J Neuroinflammation (2013)

The effects of microglial ablation on neuronal apoptosis. A. Top panel: merge of DAPI and TUNEL staining from dentate gyrus of wild type subarachnoid hemorrhage (SAH) after intracerebroventricular (ICV) PBS liposome injection on POD 7 and 15. B. Bottom panel: merge of DAPI and TUNEL staining in wild type SAH after ICV clodronate injection on POD 7 and 15. Scale Bars: 10 μm in all panels. C. Quantification of apoptotic neurons on POD 7 displaying significant reduction, by Student’s t-test (P <0.03), after ICV clodronate injection and microglial ablation. D. Quantification of apoptotic neurons on POD 15 revealing no change between ICV PBS and clodronate injections. N = 3 for each group and error bars are standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: The effects of microglial ablation on neuronal apoptosis. A. Top panel: merge of DAPI and TUNEL staining from dentate gyrus of wild type subarachnoid hemorrhage (SAH) after intracerebroventricular (ICV) PBS liposome injection on POD 7 and 15. B. Bottom panel: merge of DAPI and TUNEL staining in wild type SAH after ICV clodronate injection on POD 7 and 15. Scale Bars: 10 μm in all panels. C. Quantification of apoptotic neurons on POD 7 displaying significant reduction, by Student’s t-test (P <0.03), after ICV clodronate injection and microglial ablation. D. Quantification of apoptotic neurons on POD 15 revealing no change between ICV PBS and clodronate injections. N = 3 for each group and error bars are standard deviations.
Mentions: Finally, we determined whether microglial depletion effected neuronal apoptosis in Figure 7, and found a similar bimodal theme. At POD 7, the depletion of microglia resulted in a significant decrease in neuronal apoptosis (Figure 7A, C); while at POD 15, microglial depletion had no effect (Figure 7B, D).

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