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Recently, the latest research result "DL-3-n-butylphthalide Increases Collateriogenesis and Functional Recovery after Focal Ischemic Stroke in Mice" by the team of Professor Ling Wei from Emory University in the United States was published in the journal Aging and Disease (IF=5.
402) This study aims to evaluate the effect of butylphthalide (NBP) on collateral angiogenesis in in vitro cell culture and in vivo ischemic stroke models
.
The results of in vitro studies proved that: NBP significantly increased the expression of αSMA/CD-31 co-labeled cells and the angiogenesis marker PDGFRα
.
In vivo studies have shown that NBP treatment can increase the expression of VEGF and Ang-1 in the tissues around the infarct, reduce the level of nNOS, increase the αSMA/BrdU co-labeled cells in the area around ischemia and infarction, and restore the ipsilateral collateral vessels.
It can improve the local cerebral blood flow and promote the recovery of nerve function
.
Research background Stroke is the main cause of human death and disability, including long-term dysfunction
.
The current clinical treatment is mainly thrombolytic therapy using tissue plasminogen activator (tPA) or thrombectomy by mechanical means, but the number of patients benefiting is limited
.
On the other hand, the improvement of collateral circulation after stroke has become a promising therapy for tissue repair and functional recovery.
At present, most studies are focused on promoting angiogenesis, neurogenesis and neuroplasticity around the infarct
.
A number of clinical trials of thrombectomy have shown that the collateral circulation around the ischemic core area is an important factor in determining the prognosis of stroke patients
.
The collateral circulation of acute ischemic stroke is a mechanism to save ischemic penumbra and improve ischemia
.
Therefore, improving blood flow and promoting new collateral circulation has become a new and effective stroke therapy
.
Collateral branch refers to the branched blood vessel structure that connects adjacent large blood vessels and exists in most tissues
.
The main function of collateral vessels is to change the blood flow path and provide blood perfusion to the blood supply area of the occluded blood vessel
.
Cerebral collateral circulation means that when the blood supply artery of the brain is severely narrowed or occluded, the blood flows through other blood vessels (collateral or newly formed vascular anastomosis) to reach the ischemic area, so that the ischemic tissue can be compensated by perfusion to varying degrees
.
Artery-artery or vein-venous anastomosis can form collateral circulation, so that the ischemic tissue can be perfused to different degrees
.
Previous studies have shown that NBP can promote post-stroke angiogenesis and reduce neurovascular inflammation by increasing vascular-related factors bFGF, HIF-1α and VEGF.
Therefore, in this study, we used the focal cerebral ischemia model to explore the administration of nasal drops of NBP.
Influence on the formation of collateral arteries and functional recovery after stroke
.
Research methods This research is divided into two parts: in vitro and in vivo experiments
.
In vitro experiments differentiated mouse-induced pluripotent stem cell-derived vascular progenitor cells (iPSC-VPC) from iPSC originally generated from embryonic fibroblasts
.
Immunofluorescence was used to detect the expression of neovascularization marker PDGFRα; immunocytochemical staining was used to detect the expression of CD-31, PDGFR, ɑSMA and pVEGFR2 that promote angiogenesis
.
In the in vivo experiment, C57BL/6 mice were divided into three groups: sham operation group, placebo control group and NBP treatment group
.
The occlusion of the common carotid artery induced ischemic stroke by permanent ligation of the right middle cerebral artery for 7 minutes
.
NBP (80 mg/kg) or placebo was administered intranasally 1 hour after stroke, repeated once a day until death
.
He was given bromodeoxyuridine (BrdU, 50 mg/kg/day) from the third day until death
.
Measure the diameter of arteries under a fluorescence microscope, Western blot analysis to detect the expression of VEGF, Ang-1, Tie-2 and nNOS protein levels, laser scanning imaging to measure local cerebral blood flow (LCBF), cylinder test and corner test to test mice Functional and behavioral changes
.
Research results ➤ NBP promotes differentiation of vascular endothelial cells.
In vascular endothelial progenitor cells cultured from mouse iPSCs, immunocytochemical staining showed that NBP (10μM) treatment for 48 hours significantly increased the expression of angiogenesis marker PDGFRα, while phosphorylated VEGFR2 ( p-VEGFR2) remains unchanged (Figures: 1A-1D)
.
NBP treatment for 48 hours can increase the percentage of αSMA/CD-31 double-positive cells in the total cells from 20-25% to more than 70% (Figure 1E and 1F)
.
About 80% of αSMA-positive cells expressed Glut-1, another vascular endothelial marker (Figure 1G)
.
In addition, muscle/endoplasmic reticulum Ca2+-ATPase (SERCA2) was observed in all these cultured cells, indicating the ability of these differentiated vascular cells to regulate Ca2+ homeostasis (Figure 1H)
.
➤ NBP promotes the growth of collaterals after stroke.
The research team observed the effect of NBP on collateral circulation in a focal ischemia model.
14 days after the stroke, immunohistochemical staining was used to observe the pre-existing and new blood vessels, which can be seen Large and small arteries/arterioles on the surface of the cortex (Figure 1A)
.
The research team took advantage of the αSMA-GFP marker in transgenic mice to identify collateral vessels based on the distribution pattern of GFP fluorescence
.
Image the GFP-positive arterioles on the surface of the brain and grid them (Figures 2B and 2C)
.
Three days after the stroke, MCAO branches were measured according to different branch levels (Figure 3D and 3E)
.
On the 14th day after stroke, the diameters of all collateral vessels around the infarction in each group were analyzed (Figure 2F)
.
On day 3 after stroke, the number of MCAO branches measured at levels II and V increased significantly after NBP treatment (Figure 3E)
.
On the 14th day after stroke, the diameters of the sham operation group and the infarct group were 11.
8±0.
89μm and 16.
9±0.
82μm, respectively
.
NBP treatment further increased the diameter of the collateral vessels, and the ipsilateral cortex was 20.
23±0.
81μm (Figure 2F)
.
Using αSMA-GFP and BrdU combined labeling, calculate the newly generated smooth muscle cells in the area around ischemia and infarction to determine the arterialization after stroke (Figure 3A-3C)
.
In the NBP group (Figures 3C and 3D), more GFP/BrdU dual-labeled cells were observed.
Studies have shown that NBP treatment significantly increased the blood vessel density in the area around ischemia and infarction
.
There was no difference in the contralateral cortex between the control group and the NBP group (Figure 3E and 3F)
.
➤ NBP increases the expression of VEGF after stroke In order to explore the possible mechanism of NBP in promoting angiogenesis, the study evaluated the vascular regulators in the area around ischemia and infarction
.
The protein expression of VEGF, Ang-1 and nNOS was detected 14 days after stroke (Figure 4A)
.
Western blot analysis showed that NBP treatment for 14 days significantly increased the expression of VEGF in the area around the infarction, showing that NBP can promote angiogenesis (Figure 4A and 4B)
.
The expression of nNOS increased after stroke, and NBP treatment significantly reduced the expression of nNOS compared with the control (Figure 4A and 4D)
.
Compared with the stroke control group, the level of Tie-2 in mice treated with NBP was significantly higher (Figure 4E and 4F)
.
➤ NBP increases local cerebral blood flow after stroke.
Oxygen and nutrients in the blood are essential for cell survival and tissue repair.
Therefore, laser Doppler imaging technology is used to measure LCBF (Figure 5A)
.
14 days after stroke, the LCBF recovery rate of the control group in the area around ischemia and infarction was about 80% (Figure 5B), while the recovery rate of LCBF in the similar area of the NBP group was 90%, and there was no difference from the sham operation group
.
➤ NBP promotes the improvement of neurological function in the early and long-term stroke.
Past data show that the survival rate of neurons is significantly improved after NBP administration
.
Studies have shown that the NBP group observed significant behavioral improvement 3 days after stroke
.
Early NBP treatment can increase the use of the left limb in the cylinder experiment and correct the sensory-related steering shift in the corner experiment (Figure 6A and 6B)
.
Several experiments were conducted to evaluate the effect of NBP on long-term sensory and motor dysfunction after stroke on 14 days
.
The sticky label test showed that NBP treatment significantly shortened the time for mice to remove sticky spots (Figure 6C and 6D); von Frey fiber stimulation showed that NBP treatment can alleviate the enhancement of mechanical stimulation sensation in stroke mice (Figure 6E and Figure 6E and Figure 6D).
6F); In addition, the study also found that NBP treatment can improve social cognitive deficits after stroke and significantly increase the total social time after stroke (Figure 6J)
.
The conclusions of the study indicate that NBP promotes angiogenesis in the ischemia and peri-infarct area by increasing the expression of angiogenesis factors after ischemic stroke, and restores the local cerebral blood flow and motor function in the ischemic area
.
This study provides new evidence for butylphthalide to improve the acute and long-term functional prognosis of stroke through collateral circulation, and provides important clinical guidance
.
Literature index: Zheng Zachory Wei, Dongdong Chen, Ling Wei et al.
DL-3-n-butylphthalide Increases Collateriogenesis and Functional Recovery after Focal Ischemic Strokein Mice.
[J].
Aging and disease.
DOI: 10.
14336/AD.
2020.
1226.
402) This study aims to evaluate the effect of butylphthalide (NBP) on collateral angiogenesis in in vitro cell culture and in vivo ischemic stroke models
.
The results of in vitro studies proved that: NBP significantly increased the expression of αSMA/CD-31 co-labeled cells and the angiogenesis marker PDGFRα
.
In vivo studies have shown that NBP treatment can increase the expression of VEGF and Ang-1 in the tissues around the infarct, reduce the level of nNOS, increase the αSMA/BrdU co-labeled cells in the area around ischemia and infarction, and restore the ipsilateral collateral vessels.
It can improve the local cerebral blood flow and promote the recovery of nerve function
.
Research background Stroke is the main cause of human death and disability, including long-term dysfunction
.
The current clinical treatment is mainly thrombolytic therapy using tissue plasminogen activator (tPA) or thrombectomy by mechanical means, but the number of patients benefiting is limited
.
On the other hand, the improvement of collateral circulation after stroke has become a promising therapy for tissue repair and functional recovery.
At present, most studies are focused on promoting angiogenesis, neurogenesis and neuroplasticity around the infarct
.
A number of clinical trials of thrombectomy have shown that the collateral circulation around the ischemic core area is an important factor in determining the prognosis of stroke patients
.
The collateral circulation of acute ischemic stroke is a mechanism to save ischemic penumbra and improve ischemia
.
Therefore, improving blood flow and promoting new collateral circulation has become a new and effective stroke therapy
.
Collateral branch refers to the branched blood vessel structure that connects adjacent large blood vessels and exists in most tissues
.
The main function of collateral vessels is to change the blood flow path and provide blood perfusion to the blood supply area of the occluded blood vessel
.
Cerebral collateral circulation means that when the blood supply artery of the brain is severely narrowed or occluded, the blood flows through other blood vessels (collateral or newly formed vascular anastomosis) to reach the ischemic area, so that the ischemic tissue can be compensated by perfusion to varying degrees
.
Artery-artery or vein-venous anastomosis can form collateral circulation, so that the ischemic tissue can be perfused to different degrees
.
Previous studies have shown that NBP can promote post-stroke angiogenesis and reduce neurovascular inflammation by increasing vascular-related factors bFGF, HIF-1α and VEGF.
Therefore, in this study, we used the focal cerebral ischemia model to explore the administration of nasal drops of NBP.
Influence on the formation of collateral arteries and functional recovery after stroke
.
Research methods This research is divided into two parts: in vitro and in vivo experiments
.
In vitro experiments differentiated mouse-induced pluripotent stem cell-derived vascular progenitor cells (iPSC-VPC) from iPSC originally generated from embryonic fibroblasts
.
Immunofluorescence was used to detect the expression of neovascularization marker PDGFRα; immunocytochemical staining was used to detect the expression of CD-31, PDGFR, ɑSMA and pVEGFR2 that promote angiogenesis
.
In the in vivo experiment, C57BL/6 mice were divided into three groups: sham operation group, placebo control group and NBP treatment group
.
The occlusion of the common carotid artery induced ischemic stroke by permanent ligation of the right middle cerebral artery for 7 minutes
.
NBP (80 mg/kg) or placebo was administered intranasally 1 hour after stroke, repeated once a day until death
.
He was given bromodeoxyuridine (BrdU, 50 mg/kg/day) from the third day until death
.
Measure the diameter of arteries under a fluorescence microscope, Western blot analysis to detect the expression of VEGF, Ang-1, Tie-2 and nNOS protein levels, laser scanning imaging to measure local cerebral blood flow (LCBF), cylinder test and corner test to test mice Functional and behavioral changes
.
Research results ➤ NBP promotes differentiation of vascular endothelial cells.
In vascular endothelial progenitor cells cultured from mouse iPSCs, immunocytochemical staining showed that NBP (10μM) treatment for 48 hours significantly increased the expression of angiogenesis marker PDGFRα, while phosphorylated VEGFR2 ( p-VEGFR2) remains unchanged (Figures: 1A-1D)
.
NBP treatment for 48 hours can increase the percentage of αSMA/CD-31 double-positive cells in the total cells from 20-25% to more than 70% (Figure 1E and 1F)
.
About 80% of αSMA-positive cells expressed Glut-1, another vascular endothelial marker (Figure 1G)
.
In addition, muscle/endoplasmic reticulum Ca2+-ATPase (SERCA2) was observed in all these cultured cells, indicating the ability of these differentiated vascular cells to regulate Ca2+ homeostasis (Figure 1H)
.
➤ NBP promotes the growth of collaterals after stroke.
The research team observed the effect of NBP on collateral circulation in a focal ischemia model.
14 days after the stroke, immunohistochemical staining was used to observe the pre-existing and new blood vessels, which can be seen Large and small arteries/arterioles on the surface of the cortex (Figure 1A)
.
The research team took advantage of the αSMA-GFP marker in transgenic mice to identify collateral vessels based on the distribution pattern of GFP fluorescence
.
Image the GFP-positive arterioles on the surface of the brain and grid them (Figures 2B and 2C)
.
Three days after the stroke, MCAO branches were measured according to different branch levels (Figure 3D and 3E)
.
On the 14th day after stroke, the diameters of all collateral vessels around the infarction in each group were analyzed (Figure 2F)
.
On day 3 after stroke, the number of MCAO branches measured at levels II and V increased significantly after NBP treatment (Figure 3E)
.
On the 14th day after stroke, the diameters of the sham operation group and the infarct group were 11.
8±0.
89μm and 16.
9±0.
82μm, respectively
.
NBP treatment further increased the diameter of the collateral vessels, and the ipsilateral cortex was 20.
23±0.
81μm (Figure 2F)
.
Using αSMA-GFP and BrdU combined labeling, calculate the newly generated smooth muscle cells in the area around ischemia and infarction to determine the arterialization after stroke (Figure 3A-3C)
.
In the NBP group (Figures 3C and 3D), more GFP/BrdU dual-labeled cells were observed.
Studies have shown that NBP treatment significantly increased the blood vessel density in the area around ischemia and infarction
.
There was no difference in the contralateral cortex between the control group and the NBP group (Figure 3E and 3F)
.
➤ NBP increases the expression of VEGF after stroke In order to explore the possible mechanism of NBP in promoting angiogenesis, the study evaluated the vascular regulators in the area around ischemia and infarction
.
The protein expression of VEGF, Ang-1 and nNOS was detected 14 days after stroke (Figure 4A)
.
Western blot analysis showed that NBP treatment for 14 days significantly increased the expression of VEGF in the area around the infarction, showing that NBP can promote angiogenesis (Figure 4A and 4B)
.
The expression of nNOS increased after stroke, and NBP treatment significantly reduced the expression of nNOS compared with the control (Figure 4A and 4D)
.
Compared with the stroke control group, the level of Tie-2 in mice treated with NBP was significantly higher (Figure 4E and 4F)
.
➤ NBP increases local cerebral blood flow after stroke.
Oxygen and nutrients in the blood are essential for cell survival and tissue repair.
Therefore, laser Doppler imaging technology is used to measure LCBF (Figure 5A)
.
14 days after stroke, the LCBF recovery rate of the control group in the area around ischemia and infarction was about 80% (Figure 5B), while the recovery rate of LCBF in the similar area of the NBP group was 90%, and there was no difference from the sham operation group
.
➤ NBP promotes the improvement of neurological function in the early and long-term stroke.
Past data show that the survival rate of neurons is significantly improved after NBP administration
.
Studies have shown that the NBP group observed significant behavioral improvement 3 days after stroke
.
Early NBP treatment can increase the use of the left limb in the cylinder experiment and correct the sensory-related steering shift in the corner experiment (Figure 6A and 6B)
.
Several experiments were conducted to evaluate the effect of NBP on long-term sensory and motor dysfunction after stroke on 14 days
.
The sticky label test showed that NBP treatment significantly shortened the time for mice to remove sticky spots (Figure 6C and 6D); von Frey fiber stimulation showed that NBP treatment can alleviate the enhancement of mechanical stimulation sensation in stroke mice (Figure 6E and Figure 6E and Figure 6D).
6F); In addition, the study also found that NBP treatment can improve social cognitive deficits after stroke and significantly increase the total social time after stroke (Figure 6J)
.
The conclusions of the study indicate that NBP promotes angiogenesis in the ischemia and peri-infarct area by increasing the expression of angiogenesis factors after ischemic stroke, and restores the local cerebral blood flow and motor function in the ischemic area
.
This study provides new evidence for butylphthalide to improve the acute and long-term functional prognosis of stroke through collateral circulation, and provides important clinical guidance
.
Literature index: Zheng Zachory Wei, Dongdong Chen, Ling Wei et al.
DL-3-n-butylphthalide Increases Collateriogenesis and Functional Recovery after Focal Ischemic Strokein Mice.
[J].
Aging and disease.
DOI: 10.
14336/AD.
2020.
1226.
