Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul;42(7):2033-44.
doi: 10.1161/STROKEAHA.110.601369. Epub 2011 May 5.

Phosphatidylinositol-3-kinase gamma plays a central role in blood-brain barrier dysfunction in acute experimental stroke

Rong Jin  1 , Zifang Song, Shiyong Yu, Abigail Piazza, Anil Nanda, Josef M Penninger, D Neil Granger, Guohong Li
Affiliations

Phosphatidylinositol-3-kinase gamma plays a central role in blood-brain barrier dysfunction in acute experimental stroke

Rong Jin et al. Stroke. 2011 Jul.

Abstract

Background and purpose: Phosphoinositide 3-kinase (PI3K)-γ is linked to inflammation and oxidative stress. This study was conducted to investigate the role of the PI3Kγ in the blood-brain barrier dysfunction and brain damage induced by focal cerebral ischemia/reperfusion.

Methods: Wild-type and PI3Kγ knockout mice were subjected to middle cerebral artery occlusion (60 minutes) followed by reperfusion. Evans blue leakage, brain edema, infarct volumes, and neurological deficits were examined. Oxidative stress, neutrophil infiltration, and matrix metallopeptidase-9 were assessed. Activation of nuclear factor-κB and expression of proinflammatory and pro-oxidative genes were studied.

Results: PI3Kγ deficiency significantly reduced blood-brain barrier permeability and brain edema formation, which were time-dependently correlated with preventing the degradation of the tight junction protein, claudin-5, and the basal lamina protein, collagen IV, and the phosphorylation of myosin light chain in brain microvessels. PI3Kγ deficiency suppressed ischemia/reperfusion-induced nuclear factor-κB p65 (Ser536) phosphorylation and the expression of the pro-oxidant enzyme NADPH oxidase (Nox1, Nox2, and Nox4) and proinflammatory adhesion molecules (E- and P-selectin, intercellular adhesion molecule-1) at different time points. These molecular changes were associated with significant inhibition of oxidative stress (superoxide production and malondialdehyde content), neutrophil infiltration, and matrix metallopeptidase-9 expression/activity in PI3Kγ knockout mice. Eventually, PI3Kγ deficiency significantly reduced infarct volumes and neurological scores at 24 hours after ischemia/reperfusion.

Conclusions: Our results provide the first direct demonstration that PI3Kγ plays a significant role in ischemia/reperfusion-induced blood-brain barrier disruption and brain damage. Future studies need to explore PI3Kγ as a potential target for stroke therapy.

PubMed Disclaimer

Figures

Figure 1
Figure 1. PI3Kγ deficiency reduces BBB leakage, edema formation, infarct volume, and improves neurological outcome
A, Upper panel shows representative pictures of Evans blue leakage in the brains and coronal sections (bregma +0.70 mm) in wildtype (WT) and PI3Kγ knockout (KO) mice at 4 and 48 h after tMCAO. Lower panel shows the quantitative analysis of Evans blue leakage. L represents the ischemic (left) side, and R represents the contralateral (right) side. n= 5 mice per time point per group, **P<0.01 versus WT controls. B, Brain tissue water content and edema volume in WT and KO mice at 24 and 48 h after tMCAO. n=8 mice per group, *P<0.05 versus WT controls. C, Infarct volumes. Representative photographs of TTC staining (upper panel) and infarct volumes (lower panel) at 24h after tMCAO in WT and KO mice (n=12/group). D, Neurological outcomes. Neurological Bederson score (upper panel) and grip test score (lower panel) as assessed at day 1 after tMCAO in WT and KO mice (n=12/group). *P<0.05 versus WT mice.
Figure 1
Figure 1. PI3Kγ deficiency reduces BBB leakage, edema formation, infarct volume, and improves neurological outcome
A, Upper panel shows representative pictures of Evans blue leakage in the brains and coronal sections (bregma +0.70 mm) in wildtype (WT) and PI3Kγ knockout (KO) mice at 4 and 48 h after tMCAO. Lower panel shows the quantitative analysis of Evans blue leakage. L represents the ischemic (left) side, and R represents the contralateral (right) side. n= 5 mice per time point per group, **P<0.01 versus WT controls. B, Brain tissue water content and edema volume in WT and KO mice at 24 and 48 h after tMCAO. n=8 mice per group, *P<0.05 versus WT controls. C, Infarct volumes. Representative photographs of TTC staining (upper panel) and infarct volumes (lower panel) at 24h after tMCAO in WT and KO mice (n=12/group). D, Neurological outcomes. Neurological Bederson score (upper panel) and grip test score (lower panel) as assessed at day 1 after tMCAO in WT and KO mice (n=12/group). *P<0.05 versus WT mice.
Figure 1
Figure 1. PI3Kγ deficiency reduces BBB leakage, edema formation, infarct volume, and improves neurological outcome
A, Upper panel shows representative pictures of Evans blue leakage in the brains and coronal sections (bregma +0.70 mm) in wildtype (WT) and PI3Kγ knockout (KO) mice at 4 and 48 h after tMCAO. Lower panel shows the quantitative analysis of Evans blue leakage. L represents the ischemic (left) side, and R represents the contralateral (right) side. n= 5 mice per time point per group, **P<0.01 versus WT controls. B, Brain tissue water content and edema volume in WT and KO mice at 24 and 48 h after tMCAO. n=8 mice per group, *P<0.05 versus WT controls. C, Infarct volumes. Representative photographs of TTC staining (upper panel) and infarct volumes (lower panel) at 24h after tMCAO in WT and KO mice (n=12/group). D, Neurological outcomes. Neurological Bederson score (upper panel) and grip test score (lower panel) as assessed at day 1 after tMCAO in WT and KO mice (n=12/group). *P<0.05 versus WT mice.
Figure 2
Figure 2. PI3Kγ deficiency reduces cerebrovascular claudin-5 and collagen IV degradation and myosin light chain (MLC) phosphorylation
A through C, Immunostaining of claudin-5 (A), collagen IV (B), and phospho-MLC (Ser19) (C) in WT and PI3Kγ KO mice at 4, 24, and 48 h after tMCAO. The region of interest is shown in adjoining coronal section. Ctrl and Ipsi. represents contralateral and ipsilateral hemispheres, respectively. n=5 per time point per group. Scale bar: 50 μm. D, Western blot analysis of the phospho-MLC(ser19) (18 kDa) in the ischemic cortex (region of interest shown in adjoining coronal section). Samples from sham-operated animals served as controls. Quantified band intensities are presented as fold-changes of control (Ctrl). n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs Ctrl.
Figure 2
Figure 2. PI3Kγ deficiency reduces cerebrovascular claudin-5 and collagen IV degradation and myosin light chain (MLC) phosphorylation
A through C, Immunostaining of claudin-5 (A), collagen IV (B), and phospho-MLC (Ser19) (C) in WT and PI3Kγ KO mice at 4, 24, and 48 h after tMCAO. The region of interest is shown in adjoining coronal section. Ctrl and Ipsi. represents contralateral and ipsilateral hemispheres, respectively. n=5 per time point per group. Scale bar: 50 μm. D, Western blot analysis of the phospho-MLC(ser19) (18 kDa) in the ischemic cortex (region of interest shown in adjoining coronal section). Samples from sham-operated animals served as controls. Quantified band intensities are presented as fold-changes of control (Ctrl). n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs Ctrl.
Figure 2
Figure 2. PI3Kγ deficiency reduces cerebrovascular claudin-5 and collagen IV degradation and myosin light chain (MLC) phosphorylation
A through C, Immunostaining of claudin-5 (A), collagen IV (B), and phospho-MLC (Ser19) (C) in WT and PI3Kγ KO mice at 4, 24, and 48 h after tMCAO. The region of interest is shown in adjoining coronal section. Ctrl and Ipsi. represents contralateral and ipsilateral hemispheres, respectively. n=5 per time point per group. Scale bar: 50 μm. D, Western blot analysis of the phospho-MLC(ser19) (18 kDa) in the ischemic cortex (region of interest shown in adjoining coronal section). Samples from sham-operated animals served as controls. Quantified band intensities are presented as fold-changes of control (Ctrl). n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs Ctrl.
Figure 2
Figure 2. PI3Kγ deficiency reduces cerebrovascular claudin-5 and collagen IV degradation and myosin light chain (MLC) phosphorylation
A through C, Immunostaining of claudin-5 (A), collagen IV (B), and phospho-MLC (Ser19) (C) in WT and PI3Kγ KO mice at 4, 24, and 48 h after tMCAO. The region of interest is shown in adjoining coronal section. Ctrl and Ipsi. represents contralateral and ipsilateral hemispheres, respectively. n=5 per time point per group. Scale bar: 50 μm. D, Western blot analysis of the phospho-MLC(ser19) (18 kDa) in the ischemic cortex (region of interest shown in adjoining coronal section). Samples from sham-operated animals served as controls. Quantified band intensities are presented as fold-changes of control (Ctrl). n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs Ctrl.
Figure 3
Figure 3. PI3Kγ deficiency reduces NF-kB p65 phosphorylation and proinflammatory gene expression
A, Western blot analysis of the phospho-p65 (ser536) and total p65 in the ischemic cortex at 4 and 24 h after tMCAO. The region of interest is shown in adjoining coronal section. Samples from sham-operated animals served as controls. Similar results were obtained in three separate experiments. B & C, Real-time RT-PCR analysis of mRNA expression of the NADPH oxidase subunits (Nox-1, Nox-2, Nox-4) (B) and the leukocyte-endothelial adhesion molecules (E-selectin, P-selectin, ICAM-1) (C) in the ischemic cortex (region of interest shown in adjoining coronal section) at indicated times after tMCAO. n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs sham control.
Figure 3
Figure 3. PI3Kγ deficiency reduces NF-kB p65 phosphorylation and proinflammatory gene expression
A, Western blot analysis of the phospho-p65 (ser536) and total p65 in the ischemic cortex at 4 and 24 h after tMCAO. The region of interest is shown in adjoining coronal section. Samples from sham-operated animals served as controls. Similar results were obtained in three separate experiments. B & C, Real-time RT-PCR analysis of mRNA expression of the NADPH oxidase subunits (Nox-1, Nox-2, Nox-4) (B) and the leukocyte-endothelial adhesion molecules (E-selectin, P-selectin, ICAM-1) (C) in the ischemic cortex (region of interest shown in adjoining coronal section) at indicated times after tMCAO. n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs sham control.
Figure 3
Figure 3. PI3Kγ deficiency reduces NF-kB p65 phosphorylation and proinflammatory gene expression
A, Western blot analysis of the phospho-p65 (ser536) and total p65 in the ischemic cortex at 4 and 24 h after tMCAO. The region of interest is shown in adjoining coronal section. Samples from sham-operated animals served as controls. Similar results were obtained in three separate experiments. B & C, Real-time RT-PCR analysis of mRNA expression of the NADPH oxidase subunits (Nox-1, Nox-2, Nox-4) (B) and the leukocyte-endothelial adhesion molecules (E-selectin, P-selectin, ICAM-1) (C) in the ischemic cortex (region of interest shown in adjoining coronal section) at indicated times after tMCAO. n= 4 per time point per group. *P<0.05 vs WT, #P<0.05 vs sham control.
Figure 4
Figure 4. PI3Kγ deficiency reduces ROS and MDA production
A, (Left panel) Representative DHE fluorescence images showing the superoxide signal taken from the ischemic cortex and striatum 4h after tMCAO. The region of interest is shown in adjoining coronal section indicated by asterisks. B, Semiquantitative analysis of ROS production in cortex and striatum as described in Methods at 4 and 24 h after tMCAO. Data represent mean ± S.E. n = 5 per time point per group, *P<0.05, **P<0.01 versus WT. C, ELISA assay for MDA protein in the ischemic cortex (region of interest shown in adjoining coronal section) at 4 and 24 h after tMCAO in WT and KO mice. MDA level is also expressed as ratio of ipsilateral/contralateral (ipsi/ctrl). n=5 per time point per group, **P<0.01 versus WT.
Figure 4
Figure 4. PI3Kγ deficiency reduces ROS and MDA production
A, (Left panel) Representative DHE fluorescence images showing the superoxide signal taken from the ischemic cortex and striatum 4h after tMCAO. The region of interest is shown in adjoining coronal section indicated by asterisks. B, Semiquantitative analysis of ROS production in cortex and striatum as described in Methods at 4 and 24 h after tMCAO. Data represent mean ± S.E. n = 5 per time point per group, *P<0.05, **P<0.01 versus WT. C, ELISA assay for MDA protein in the ischemic cortex (region of interest shown in adjoining coronal section) at 4 and 24 h after tMCAO in WT and KO mice. MDA level is also expressed as ratio of ipsilateral/contralateral (ipsi/ctrl). n=5 per time point per group, **P<0.01 versus WT.
Figure 5
Figure 5. PI3Kγ deficiency reduces neutrophil infiltration and neutrophil-associated MMP-9
A&B, Immunostaining for neutrophils (A) and MMP-9 (B) in the ischemic cortex in WT and PI3Kγ KO mice at the indicated times after tMCAO. The number of the cells positively stained for neutrophils and MMP-9 was calculated in the 3 predefined regions of interest shown in adjoining coronal section. Bar = 50μm. n = 5 per time point per group, *P<0.05 versus WT control. C, Neutrophil infiltration was quantified by MPO assay. ELISA assay of MPO protein in ischemic cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. *P<0.05 versus WT control. In A–C, Samples from sham-operated animals served as controls (Ctrl). D, Double immunostaining showing the co-localization of MMP-9 with the specific neutrophil marker (PMN), but not with microglial (Iba1), astrocytic (GFAP), or neutronal (neuN) markers in the ischemic cortex at 24 h after tMCAO. Bar = 50μm.
Figure 5
Figure 5. PI3Kγ deficiency reduces neutrophil infiltration and neutrophil-associated MMP-9
A&B, Immunostaining for neutrophils (A) and MMP-9 (B) in the ischemic cortex in WT and PI3Kγ KO mice at the indicated times after tMCAO. The number of the cells positively stained for neutrophils and MMP-9 was calculated in the 3 predefined regions of interest shown in adjoining coronal section. Bar = 50μm. n = 5 per time point per group, *P<0.05 versus WT control. C, Neutrophil infiltration was quantified by MPO assay. ELISA assay of MPO protein in ischemic cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. *P<0.05 versus WT control. In A–C, Samples from sham-operated animals served as controls (Ctrl). D, Double immunostaining showing the co-localization of MMP-9 with the specific neutrophil marker (PMN), but not with microglial (Iba1), astrocytic (GFAP), or neutronal (neuN) markers in the ischemic cortex at 24 h after tMCAO. Bar = 50μm.
Figure 5
Figure 5. PI3Kγ deficiency reduces neutrophil infiltration and neutrophil-associated MMP-9
A&B, Immunostaining for neutrophils (A) and MMP-9 (B) in the ischemic cortex in WT and PI3Kγ KO mice at the indicated times after tMCAO. The number of the cells positively stained for neutrophils and MMP-9 was calculated in the 3 predefined regions of interest shown in adjoining coronal section. Bar = 50μm. n = 5 per time point per group, *P<0.05 versus WT control. C, Neutrophil infiltration was quantified by MPO assay. ELISA assay of MPO protein in ischemic cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. *P<0.05 versus WT control. In A–C, Samples from sham-operated animals served as controls (Ctrl). D, Double immunostaining showing the co-localization of MMP-9 with the specific neutrophil marker (PMN), but not with microglial (Iba1), astrocytic (GFAP), or neutronal (neuN) markers in the ischemic cortex at 24 h after tMCAO. Bar = 50μm.
Figure 5
Figure 5. PI3Kγ deficiency reduces neutrophil infiltration and neutrophil-associated MMP-9
A&B, Immunostaining for neutrophils (A) and MMP-9 (B) in the ischemic cortex in WT and PI3Kγ KO mice at the indicated times after tMCAO. The number of the cells positively stained for neutrophils and MMP-9 was calculated in the 3 predefined regions of interest shown in adjoining coronal section. Bar = 50μm. n = 5 per time point per group, *P<0.05 versus WT control. C, Neutrophil infiltration was quantified by MPO assay. ELISA assay of MPO protein in ischemic cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. *P<0.05 versus WT control. In A–C, Samples from sham-operated animals served as controls (Ctrl). D, Double immunostaining showing the co-localization of MMP-9 with the specific neutrophil marker (PMN), but not with microglial (Iba1), astrocytic (GFAP), or neutronal (neuN) markers in the ischemic cortex at 24 h after tMCAO. Bar = 50μm.
Figure 6
Figure 6. PI3Kγ deficiency reduces MMP-9 activity and protein content
A, Gelatin zymogram. Note that MMP-9 (92 and 82 kDa) and MMP-2 (63 kDa) bands were detected in the homogenates of ischemic (left, L) and contralateral (right, R) cortex 24 h after tMCAO. The region of interest is shown in adjoining coronal section. B, Densitometric analysis of the bands shown in A. Data represent mean ± s.d. from 3 separate experiments. *P<0.05 versus WT (saline-treated) control. C. ELISA assay of MMP-9 protein in ischemic (left) and contralateral (right) cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. **P<0.01 versus WT control.
Figure 6
Figure 6. PI3Kγ deficiency reduces MMP-9 activity and protein content
A, Gelatin zymogram. Note that MMP-9 (92 and 82 kDa) and MMP-2 (63 kDa) bands were detected in the homogenates of ischemic (left, L) and contralateral (right, R) cortex 24 h after tMCAO. The region of interest is shown in adjoining coronal section. B, Densitometric analysis of the bands shown in A. Data represent mean ± s.d. from 3 separate experiments. *P<0.05 versus WT (saline-treated) control. C. ELISA assay of MMP-9 protein in ischemic (left) and contralateral (right) cortex (region of interest shown in adjoining coronal section) 24 h after tMCAO. n=5 for each group. **P<0.01 versus WT control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jung JE, Kim GS, Chen H, Maier CM, Narasimhan P, Song YS, Niizuma K, Katsu M, Okami N, Yoshioka H, Sakata H, Goeders CE, Chan PH. Reperfusion and neurovascular dysfunction in stroke: from basic mechanisms to potential strategies for neuroprotection. Mol Neurobiol. 2010;41:172–179. - PMC - PubMed
    1. Jin R, Yang G, Li G. Molecular insights and therapeutic targets for blood-brain barrier disruption in ischemic stroke: critical role of matrix metalloproteinases and tissue-type plasminogen activator. Neurobiol Dis. 2010;38:376–385. - PMC - PubMed
    1. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25. - PubMed
    1. Sandoval KE, Witt KA. Blood–brain barrier tight junction permeability and ischemic stroke. Neurobiol Dis. 2008;32:200–219. - PubMed
    1. Barr TL, Latour LL, Lee KY, Schaewe TJ, Luby M, Chang GS, El-Zammar Z, Alam S, Hallenbeck JM, Kidwell CS, Warach S. Blood-brain barrier disruption in humans is independently associated with increased matrix metalloproteinase-9. Stroke. 2010;41:e123–128. - PMC - PubMed

Publication types

Substances