Pha-543613 preserves blood–brain barrier integrity after intracerebral hemorrhage in mice anoxic brain injury recovery

Vasogenic brain edema develops after intracerebral hemorrhage (ICH) as a consequence of increased blood–brain barrier (BBB) permeability. 1 the hematoma-induced disintegration of endothelial tight junctions facilitates capillary leakage, and the mass effect of extravasated fluid contributes to the patient’s delayed neurological deterioration. 2 tight junction proteins, such as claudins, occludin, and junctional adhesion molecule-1, span the membrane of 2 adjacent endothelial cells, thus, forming the initial barrier between blood and brain. 1 moreover, decreased expression of these trans-membrane proteins indicates reduced barrier integrity and increased paracellular permeability. 3, 4

Previous studies demonstrated the regulatory role of β-catenin in claudin-3 and claudin-5 gene expression, both being essential BBB constituents. 3, 5 unphosphorylated, cytosolic β-catenin translocates into the nucleus, and, by binding to the T cell factor/lymphoid enhancer factor, it promotes claudin-3 transcription. 3 contrary to this barrier-maintaining effect, β-catenin has also been reported to form a complex with the forkhead box O1 transcription factor, which subsequently represses claudin-5 transcription. 5

anoxic brain injury recovery

After hemorrhagic stroke, cytotoxic events activate the ubiquitously expressed glycogen synthase kinase-3β (GSK-3β), 6 which phosphorylates serine and threonine residues of β-catenin, initiating its ubiquitin-dependent proteasomal degradation. 7 however, pharmacological stimulation of the α7 nicotinic acetylcholine receptor (α7nachr) activates the phosphatidylinositol 3-kinase (PI3K)-akt signaling pathway, and akt has been shown to inhibit GSK-3β, thereby stabilizing β-catenin. 6

Our earlier work demonstrated that selective α7nachr stimulation reduced brain edema in rodents subjected to experimental hemorrhagic stroke, 6, 8 but the mechanism remains poorly understood. Because functional α7nachrs are located in cerebral microvascular endothelial cells, 9 we hypothesized that PHA-543613, a selective α7nachr agonist, would stabilize β-catenin through PI3K-akt–induced inhibition of GSK-3β, therefore, increasing claudin-3 expression and improving BBB functional integrity after experimental ICH in mice.Anoxic brain injury recovery

PHA-543613 reduced GSK-3β activation, thereby stabilizing β-catenin and tight junction proteins

Western blot analyses of the ipsilateral brain hemisphere were conducted at 24 hours after surgery (n=5 per group). Changes in protein expression of phosphorylated and, therefore, activated GSK-3β (p–GSK-3β, tyr216) was quantified as a ratio to total GSK-3β ( figure 3A). GSK-3 phosphorylation was significantly increased in the ipsilateral brain hemisphere of mice subjected to ICH ( P0.05, compared with sham). However, PHA-543613 treatment significantly reduced the p–GSK-3β/GSK-3β ratio ( P0.05, compared with vehicle), which was reversed by MLA and wortmannin ( P0.05, compared with PHA-12 mg). Β-catenin phosphorylation enhances its degradation.Anoxic brain injury recovery increased phosphorylated β-catenin (p–β-catenin; ser33/37 and thr41) levels were found in vehicle animal brains ( P0.05, compared with sham; figure 3B). PHA-543613 significantly reduced the p–β-catenin/β-catenin ratio ( P0.05, compared with vehicle), which was tendentially abolished by MLA ( P=0.05) and reversed by wort-mannin ( P0.05). Experimental ICH reduced claudin-3 and claudin-5 levels ( P0.05, compared with sham; figure 3C and 3D), but PHA-543613 treatment increased their expression significantly ( P0.05, compared with vehicle). Mice in the PHA+MLA group presented significantly reduced claudin-3 levels ( P0.05) and tended to reduce claudin-5 levels ( P=0.09) when compared with vehicle animals.Anoxic brain injury recovery wortmannin reversed the initial increase of claudin-3 and claudin-5 by PHA-543613 ( P0.05).


The present study aimed to explain how α7nachr stimulation minimized cerebral edema formation in mice subjected to ICH. Consistent with our previous work, 6 PHA-543613 administration reduced total perihematomal brain edema, which we evaluated via the wet weight/dry weight method. Because this technique does not distinguish between vasogenic and cytotoxic brain edema, 10 we incorporated evans blue assays to determine the functional integrity of the BBB. As expected, PHA-543613 reduced BBB permeability after ICH, which was associated with less albumin-bound dye extravasation into the affected brain hemisphere.Anoxic brain injury recovery therefore, we propose that PHA-543613 reduced brain edema formation after ICH, at least to some extent, by preserving BBB functional integrity.

Although primarily expressed by neurons and microglia, functional α7nachrs have been located in BBB-comprising microvascular endothelial cells. 9 in addition to their function as ligand-gated ion channels, α7nachr stimulation activates PI3K-akt signaling through an unidentified mechanism. 11 moreover, akt has been reported to antagonize hemorrhage-induced GSK-3β activation, 6 resulting in dephosphorylation of GSK-3β substrates, including β-catenin. 7 in accordance with this theory, we found quantitatively more phosphorylated GSK-3β and β-catenin in the brains of ICH animals, but PHA-543613 administration reversed these hemorrhage-evoked molecular consequences.Anoxic brain injury recovery

Unphosphorylated β-catenin associates with sequence-specific DNA-binding factors of the T cell factor/lymphoid enhancer factor family, thus, promoting the transcription of claudin-3, 3 which we found highly expressed in PHA-543613–treated ICH mouse brains. Contrary to this barrier-maintaining effect, β-catenin, when in complex with forkhead box O1, represses endothelial-specific claudin-5 expression. 5 surprisingly, the present study found that PHA-543613–induced β-catenin stabilization was associated with increased claudin-5 expression. This discrepancy may be attributed to PI3K-akt–induced forkhead box O1 inhibition, as previously reported. 5 consistent with our findings, pharmacological β-catenin stabilization increased claudin-5 expression in a rat model of focal cerebral ischemia. 12

anoxic brain injury recovery

The observed functional and morphological changes, evoked by PHA-543613, were ameliorated or reversed by coadministration of MLA or wortmannin with the treatment. MLA is a competitive inhibitor of α7nachr; and wortmannin irreversibly inhibits PI3K, thus preventing akt activation. 8 yet, both interventions failed to induce BBB disruption, brain edema, and neurofunctional deficits significantly after experimental ICH in mice, when administered alone. 6 based on these findings, we suggest that functional α7nachrs and PI3K are required for PHA-543613–induced BBB protection.

Claudin-3 and claudin-5 are transmembrane proteins essential for maintaining the diffusion barrier provided by tight junctions. 3, 13 the selective loss of claudin-3 in cerebral microvascular endothelial cells has been linked to pathological conditions, including autoimmune encephalomyelitis and human glioblastoma multiforme. 13 additionally, claudin-5 knockout mice presented increased BBB permeability and died shortly after birth. 4 therefore, prospective treatments that effectively increase claudin-3 and claudin-5 expressions may ameliorate BBB breakdown in various central nervous system diseases.Anoxic brain injury recovery

The present study has several limitations. First, we did not implement the collagenase-induced ICH mouse model. Collagenases are proteolytic enzymes that may impair the BBB to a greater extent than blood toxicity alone. 14 in fact, hijioka et al 15 observed that α7nachr stimulation failed to significantly reduce brain edema in a collagenase-induced ICH model. Furthermore, pathophysiological mechanisms of edema formation may differ between these models. Second, β-catenin is a structural adaptor protein of endothelial adherens junctions and may, therefore, directly generate BBB stabilization. 7 third, anti-inflammatory 11 and antiapoptotic 6, 8 properties of α7nachr agonism were not evaluated in this study.Anoxic brain injury recovery

In summary, α7nachr agonist PHA-543613 reversed ICH-induced brain edema, possibly by stabilizing β-catenin and, therefore, increasing claudin-3 and claudin-5 expressions. These molecular changes were paralleled by preserved BBB integrity and improved sensorimotor deficits in ICH animals. However, α7nachr antagonist MLA and PI3K inhibitor wortmannin reversed the treatment effects. Based on these findings, we conclude that α7nachr-induced activation of the PI3K-akt pathway stabilized β-catenin through GSK-3β inhibition, thereby increasing subsequent claudin-3 and claudin-5 expression.