Limits...
P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

Lu YM, Tao RR, Huang JY, Li LT, Liao MH, Li XM, Fukunaga K, Hong ZH, Han F - J Neuroinflammation (2012)

Bottom Line: Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility.Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7).Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT

Background: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia.

Methods: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction.

Results: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

Conclusion: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

Show MeSH

Related in: MedlinePlus

Changes in FasL expression after microsphere embolism induction in the brain. (A) Representative immunoblots showing Fas and FasL expression at the indicated times following microsphere embolism in rats. Immunoblotting with an anti-β-actin antibody showed equal total protein loading in each lane. S, sham. (B) Quantitative analysis of Fas and FasL levels was performed by densitometric analysis of the immunoblots. The data are expressed as percentages of values obtained from sham animals (mean ± SEM,  n  = 6 rats). * P  <0.05; **P <0.01 vs. sham. (C) Fluorescent staining for FasL (green) and NeuN (red) was performed in ipsilateral brain regions 168 h after embolic injury in rats. (D) FasL (green, a, d) and GFAP (red, b, e) double staining was performed 168 h after embolic injury in rats. (E) Immunohistochemical localization of FasL (green) and ED1 (red) was examined 168 h after ME ischemia. Scale bar = 40 μm. (F) Quantification of colocalized FasL-NeuN, FasL-GFAP, and FasL-ED1 fluorescent signals in rats following ME injury. The mean overlapping fraction, the mean Pearson’s r and the mean ICQ are presented for each condition (the mean ± SEM, n = 6 rats). ME, microsphere embolism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3420259&req=5

Figure 2: Changes in FasL expression after microsphere embolism induction in the brain. (A) Representative immunoblots showing Fas and FasL expression at the indicated times following microsphere embolism in rats. Immunoblotting with an anti-β-actin antibody showed equal total protein loading in each lane. S, sham. (B) Quantitative analysis of Fas and FasL levels was performed by densitometric analysis of the immunoblots. The data are expressed as percentages of values obtained from sham animals (mean ± SEM, n  = 6 rats). * P <0.05; **P <0.01 vs. sham. (C) Fluorescent staining for FasL (green) and NeuN (red) was performed in ipsilateral brain regions 168 h after embolic injury in rats. (D) FasL (green, a, d) and GFAP (red, b, e) double staining was performed 168 h after embolic injury in rats. (E) Immunohistochemical localization of FasL (green) and ED1 (red) was examined 168 h after ME ischemia. Scale bar = 40 μm. (F) Quantification of colocalized FasL-NeuN, FasL-GFAP, and FasL-ED1 fluorescent signals in rats following ME injury. The mean overlapping fraction, the mean Pearson’s r and the mean ICQ are presented for each condition (the mean ± SEM, n = 6 rats). ME, microsphere embolism.

Mentions: FasL-Fas system is key initiators of the inflammatory response to injury [11]. To determine the roles of the Fas and FasL in microsphere-induced embolism, we initially used immunoblotting to investigate the temporal profile of FasL/Fas protein expression levels following microsphere embolism. A 40-kDa FasL immunoreactive protein band was detected by SDS-PAGE (Figure 2A). Representative blots are presented in Figure 2 and show a significant increase in FasL protein after 12 h in ME rats compared with sham rats (222.1 ± 15.6% vs. sham, P <0.05; Figure 2A and 2B). Further increases in FasL were evident after 168 h in the rats with microsphere embolism compared with the sham rats (348.1 ± 35.4% vs. sham, P <0.01). However, no significant differences in Fas expression were found (48 kDa) throughout the experiments (Figure 2A and 2B).


P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

Lu YM, Tao RR, Huang JY, Li LT, Liao MH, Li XM, Fukunaga K, Hong ZH, Han F - J Neuroinflammation (2012)

Changes in FasL expression after microsphere embolism induction in the brain. (A) Representative immunoblots showing Fas and FasL expression at the indicated times following microsphere embolism in rats. Immunoblotting with an anti-β-actin antibody showed equal total protein loading in each lane. S, sham. (B) Quantitative analysis of Fas and FasL levels was performed by densitometric analysis of the immunoblots. The data are expressed as percentages of values obtained from sham animals (mean ± SEM,  n  = 6 rats). * P  <0.05; **P <0.01 vs. sham. (C) Fluorescent staining for FasL (green) and NeuN (red) was performed in ipsilateral brain regions 168 h after embolic injury in rats. (D) FasL (green, a, d) and GFAP (red, b, e) double staining was performed 168 h after embolic injury in rats. (E) Immunohistochemical localization of FasL (green) and ED1 (red) was examined 168 h after ME ischemia. Scale bar = 40 μm. (F) Quantification of colocalized FasL-NeuN, FasL-GFAP, and FasL-ED1 fluorescent signals in rats following ME injury. The mean overlapping fraction, the mean Pearson’s r and the mean ICQ are presented for each condition (the mean ± SEM, n = 6 rats). ME, microsphere embolism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3420259&req=5

Figure 2: Changes in FasL expression after microsphere embolism induction in the brain. (A) Representative immunoblots showing Fas and FasL expression at the indicated times following microsphere embolism in rats. Immunoblotting with an anti-β-actin antibody showed equal total protein loading in each lane. S, sham. (B) Quantitative analysis of Fas and FasL levels was performed by densitometric analysis of the immunoblots. The data are expressed as percentages of values obtained from sham animals (mean ± SEM, n  = 6 rats). * P <0.05; **P <0.01 vs. sham. (C) Fluorescent staining for FasL (green) and NeuN (red) was performed in ipsilateral brain regions 168 h after embolic injury in rats. (D) FasL (green, a, d) and GFAP (red, b, e) double staining was performed 168 h after embolic injury in rats. (E) Immunohistochemical localization of FasL (green) and ED1 (red) was examined 168 h after ME ischemia. Scale bar = 40 μm. (F) Quantification of colocalized FasL-NeuN, FasL-GFAP, and FasL-ED1 fluorescent signals in rats following ME injury. The mean overlapping fraction, the mean Pearson’s r and the mean ICQ are presented for each condition (the mean ± SEM, n = 6 rats). ME, microsphere embolism.
Mentions: FasL-Fas system is key initiators of the inflammatory response to injury [11]. To determine the roles of the Fas and FasL in microsphere-induced embolism, we initially used immunoblotting to investigate the temporal profile of FasL/Fas protein expression levels following microsphere embolism. A 40-kDa FasL immunoreactive protein band was detected by SDS-PAGE (Figure 2A). Representative blots are presented in Figure 2 and show a significant increase in FasL protein after 12 h in ME rats compared with sham rats (222.1 ± 15.6% vs. sham, P <0.05; Figure 2A and 2B). Further increases in FasL were evident after 168 h in the rats with microsphere embolism compared with the sham rats (348.1 ± 35.4% vs. sham, P <0.01). However, no significant differences in Fas expression were found (48 kDa) throughout the experiments (Figure 2A and 2B).

Bottom Line: Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility.Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7).Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT

Background: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia.

Methods: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction.

Results: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

Conclusion: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

Show MeSH
Related in: MedlinePlus