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. 2021 Jan;9(1):19.
doi: 10.21037/atm-20-2936.

The trend of indirect anastomosis formation in a 2-vessel occlusion plus encephalo-myo-synangiosis rat model

Wensheng Li  1 , Lei Wei  2 , Bocheng Wang  3 , Shuangqi Gao  1 , Tengchao Huang  1 , Zhangyu Li  1 , Robin Bhattarai  4 , Hui Wang  1 , Ying Guo  1 , Chuan Chen  1
Affiliations

The trend of indirect anastomosis formation in a 2-vessel occlusion plus encephalo-myo-synangiosis rat model

Wensheng Li et al. Ann Transl Med. 2021 Jan.

Abstract

Background: Basic research on the factors influencing indirect anastomosis formation in a 2-vessel occlusion plus encephalo-myo-synangiosis (2VO + EMS) rat model is conducive to improving the efficacy of indirect revascularization surgery in the clinic. However, the time point at which anastomosis between the rat temporal muscle (TM) and brain naturally has the greatest effect after encephalo-myo-synangiosis (EMS) remains unknown. Therefore, we conducted this study to explore the peak time of indirect anastomosis formation in the 2VO + EMS rat model.

Methods: Forty 2VO + EMS rats were randomly divided into five groups (n=8) according to the length of time (by week) after EMS, and 2VO rats were used as the control group (n=8). The expression of vascular endothelial growth factor (VEGF) and CD31 on the EMS side of the brain, perfusion ratio [improvement of cerebral blood perfusion (CBP) on the EMS side] and Morris water maze (MWM) results were compared between groups. Furthermore, the trends of the above variables were explored over weeks.

Results: Overall, the expression of VEGF and CD31, the perfusion ratio and the cognitive improvement in the 2VO + EMS rat model gradually increased over weeks after EMS. The VEGF and CD31 expression (as detected by immunofluorescence), perfusion ratio and number of times crossing the platform area peaked at 4 weeks after EMS. In addition, both the escape latency and the time spent in the target quadrant peaked in the fifth week after EMS.

Conclusions: After establishing the 2VO + EMS rat model, the degree of endothelial cell (EC) proliferation and CBP improvement on the EMS side of the brain peaked at 4 weeks after EMS, whereas the cognitive improvement peaked in the fifth week.

Keywords: Angiogenesis; cognitive function; encephalo-myo-synangiosis (EMS); endothelial cell proliferation; indirect anastomosis formation.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-2936). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The experimental schedule. CCA, common carotid artery; LDF, laser Doppler flowmetry; 2VO, 2-vessel occlusion; EMS, encephalo-myo-synangiosis; MRI-ASL, magnetic resonance imaging arterial spin labeling; IF, immunofluorescence; MWM, Morris water maze.
Figure 2
Figure 2
Procedures for modeling the 2VO + EMS rat. (A,B) Procedures for bilateral CCA ligation. The black arrow indicates the left CCA. Representative images showing the EMS procedures, including (C) reflection of the skin, (D) reflection of the TM tissue, (E) opening of the DM (the black asterisk indicates the TM and the yellow arrow indicates the DM margin) and (F) stitching of the TM and DM tissue (the black arrow indicates the DM). (G) Harvested brain samples from 2VO + EMS rats (the black arrow indicates the TM). (H) HE-stained brain slide from a 2VO + EMS rat. The yellow frame indicates the brain cortex involved in EMS. Bar =1 mm. EMS, encephalo-myo-synangiosis; 2VO, 2-vessel occlusion; DM, dura mater; TM, temporal muscle; CCA, common carotid artery.
Figure 3
Figure 3
Schematic drawing showing the EMS procedure. EMS, encephalo-myo-synangiosis.
Figure 4
Figure 4
The change in CBF after 2VO. (A) Representative graph showing the CBF changes before and after 2VO; (B) Doppler flowmetry results showing that the CBF values in all the rats dropped to 22.55%±4.26% of the baseline levels, and the decrease rate in the 2VO group was significantly lower than that in the 2VO sham group; (C) the column chart showing that the operation time was not significantly different between groups. ***, P<0.001. 2VO, 2-vessel occlusion; EMS, encephalo-myo-synangiosis; CBF, cerebral blood flow.
Figure 5
Figure 5
Representative immunofluorescence staining showing CD31 expression on both sides of the 2VO + EMS rat brain. (A) HE-stained brain slides from 2VO + EMS rats. The two black frames indicate the part of the brain in close contact with the TM tissue and the brain on the symmetrical part of the non-EMS side. Bar =1 mm. (B) HE and immunofluorescence results showing that there were significantly more CD31 (+) cells in the brain on the EMS side than on the non-EMS side. Bar =200 µm. The yellow curve indicates the range of the brain surface involved in EMS. HE, hematoxylin and eosin; 2VO, 2-vessel occlusion; EMS, encephalo-myo-synangiosis.
Figure 6
Figure 6
IF results showing both VEGF and CD31 expression in the EMS side brains from each group. (A) Dual IF staining showing VEGF (+) and CD31 (+) cells in the EMS-side brains in each group. Bar =20 µm; (B) counts of VEGF (+) cells in each group; (C) counts of CD31 (+) cells in each group; (D) counts of VEGF & CD31 (+) cells in each group; (E) representative IF results showing both VEGF (+) and CD31 (+) cells. Bar =5 µm. The yellow arrow indicates a VEGF (+) cell, the white arrow indicates a CD31 (+) cell and the red arrow indicates the overlap of VEGF (+) and CD31 (+) cells. The error bars represent the ± SD. *, P<0.05; **, P<0.01; #, P>0.05. IF, immunofluorescence; TM, temporal muscle; VEGF, vascular endothelial growth factor; EMS, encephalo-myo-synangiosis.
Figure 7
Figure 7
The results of MRI-ASL in each group. (A) Representative MRI-T2 and MRI-ASL films showing the CBP differences between each group. The black arrow indicates the ischemic brain tissue covered by TM. The gray circles indicate the ROIs for CBP detection. (B) A chart showing the CBP values of the ROIs in each group. (C) The results of perfusion ratios in each group. The error bars represent the ± SD. *, P<0.05; ***, P<0.001; #, P>0.05. CBP, cerebral blood perfusion; ASL, arterial spin labeling; ROI, region of interest; TM, temporal muscle.
Figure 8
Figure 8
MWM test results showing the cognitive improvement in each group. (A) Representative swimming path of each group. (B) Average escape latency of each group. #, P>0.05, 2VO + EMS6 vs. 2VO + EMS5; *, P<0.05, 2VO + EMS5 vs. 2VO + EMS4; ***, P<0.001, 2VO sham vs. 2VO + EMS5. (C) Time spent in the target quadrant of each group. *, P<0.05; #, P>0.05. (D) Average swimming speed of each group. (E) Times of crossing the platform area of each group. *, P<0.05; #, P>0.05. The error bars represent the ± SD. MWM, Morris water maze; 2VO, 2-vessel occlusion; EMS, encephalo-myo-synangiosis.
Figure 9
Figure 9
Diagrams of curves showing the trends of VEGF expression, CD31 expression, perfusion ratio improvement and cognitive improvement over weeks.
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