Objective

2

To investigate the effect of

Xingnao Kaiqiao

acupuncture therapy (for regaining the consciousness and opening the orifices) on cerebral mitochondrial dynamics homeostasis in middle cerebral artery occlusion/reperfusion (MCAO/R) model rats

and to explore the mechanism of this therapy on cerebral ischemia-reperfusion injury (CIRI).

Methods

2

A total of 64 healthy male SD rats were randomly divided into a sham-operation group

a model group

an acupuncture group

and an inhibitor group

with 16 rats in each group. Except for the sham-operation group

the MCAO/R model was established in the remaining groups using the modified Longaʹs method. In the acupuncture group

acupuncture was delivered at bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26)

for 30 min per intervention

once daily for 5 consecutive days. In the inhibitor group

the mitochondrial division inhibitor 1 (Mdivi-1) was injected intraperitoneally (2 mg/kg)

once daily for 5 consecutive days. Separately

before cerebral ischemia

after ischemia

after ischemia-reperfusion and after intervention completion

using a laser speckle contrast imaging (LSCI) system

the cerebral blood perfusion in the affected cerebral cortex region was monitored. After intervention completion

the neurological deficits were evaluated using the Bederson score; the grip strength was measured with a small animal grip meter; the cerebral infarction volume percentage was quantified via 2

3

5-triphenyltetrazolium chloride (TTC) staining; the mitochondrial ultrastructure in endothelial cells within the ischemic penumbra (IP) region of the cerebral cortex was observed using transmission electron microscopy; the fluorescence co-localization area of optic atrophy 1 (OPA1) and the endothelial cell marker CD31 in the cortical IP region was analyzed by immunofluorescence; the protein expression of mitochondrial fusion protein 2 (MFN2)

OPA1

and dynamin-related protein 1 (DRP1) in the cortical IP region was detected using Western blot.

Results

2

Except in the sham-operation group

cerebral blood perfusion in the affected cerebral cortex decreased after cerebral ischemia in the rest groups compared with that before cerebral ischemia (

P

<

0.001). When compared with the time point after cerebral ischemia

cerebral blood perfusion in the affected cerebral cortex was elevated after cerebral ischemia-reperfusion in each group (

P

<

0.001). After intervention completion

in comparison with the model group

the ratios of cerebral blood perfusion were elevated in both the acupuncture group and the inhibitor group (

P

<

0.001); and the ratio of cerebral blood perfusion rose in the acupuncture group compared with that in the inhibitor group (

P

<

0.001). Compared with the sham-operation group

the model group exhibited the increase of neurological deficit score and cerebral infarction volume percentage (

P

<

0.001)

and the decline of grip strength (

P

<

0.001). When compared with the model group

both the acupuncture group and the inhibitor group showed the decrease of neurological deficit scores and cerebral infarction volume percentage (

P

<

0.01

P

<

0.05

P

<

0.001)

and the increase of grip strength (

P

<

0.001). Compared with the inhibitor group

cerebral infarction volume percentage was lower and grip strength was higher in the acupuncture group (

P

<

0.001). In the model group

the endothelial cell mitochondria exhibited severe swelling

exhibiting a spherical morphology predominantly

with extensive cristae fragmentation or even disappearance

and excessive vacuolization. In both the acupuncture group and the inhibitor group

the ultrastructural damage to endothelial cell mitochondria was partially ameliorated; the acupunct

ure group demonstrated a superior restoration of mitochondrial ultrastructure in comparison with the inhibitor group. When compared with the sham-operation group

the area ratio of co-localization of OPA1 and CD31 decreased in the model group (

P

<

0.001). The area ratio of co-localization of OPA1 and CD31 increased in either the acupuncture group or the inhibitor group in comparison with the model group

respectively (

P

<

0.001). The area ratio of co-localization of OPA1 and CD31 in the acupuncture group was higher than that of the inhibitor group (

P

<

0.001). Compared with the sham-operation group

the model group exhibited the lower protein expression of MFN2 and OPA1 (

P

<

0.001) and protein higher expression of DRP1 (

P

<

0.001). When compared with the model group

the acupuncture group showed the increase of protein expression of MFN2 and OPA1 and the decrease of DRP1 protein expression (

P

<

0.001

P

<

0.05)

while in the inhibitor group

the protein expression of MFN2 was elevated and that of DRP1 declined (

P

<

0.05

P

<

0.01). When compared with the inhibitor group

the acupuncture group displayed higher MFN2 expression (

P

<

0.01) and lower DRP1 expression (

P

<

0.01).

Conclusion

2

Xingnao Kaiqiao

acupuncture therapy mitigates neurological deficits and enhances cerebral blood supply of ischemic region in MCAO/R rats

and the underlying mechanism may be related to regulating mitochondrial dynamics homeostasis of endothelial cells in the IP of cerebral cortex

and attenuating mitochondrial damage.