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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We performed a one-point, lipophilic cell-tracer injection to track endothelial cells, followed by an arteriotomy and suturing of sidewall aneurysms on the abdominal rat aorta. Neointima formation seemed dependent on the parent artery in decellularized aneurysms and was promoted by the recruitment from aneurysm wall cells in vital cell-rich walls.

Abstract

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and thrombus formation. The source of endothelial cells covering the endoluminal layer of the neointima remains unclear. Therefore, the aim of the present study was to investigate the origin of neointima-forming cells after cell-tracer injection in the already well-established Helsinki rat microsurgical sidewall aneurysm model.

Sidewall aneurysms were created by suturing decellularized or vital arterial pouches end-to-side to the aorta in male Lewis rats. Before arteriotomy with aneurysm suture, a cell-tracer injection containing CM-Dil dye was performed into the clamped aorta to label endothelial cells in the adjacent vessel and track their proliferation during follow-up (FU). Treatment followed by coiling (n = 16) or stenting (n = 15). At FU (7 days or 21 days), all rats underwent fluorescence angiography, followed by aneurysm harvesting and macroscopic and histological evaluation with immunohistological cell counts for specific regions of interest.

None of the 31 aneurysms had ruptured upon follow-up. Four animals died prematurely. Macroscopically residual perfusion was observed in 75.0% coiled and 7.0% of stented rats. The amount of cell-tracer-positive cells was significantly elevated in decellularized stented compared to coiled aneurysms with respect to thrombus on day 7 (p = 0.01) and neointima on day 21 (p = 0.04). No significant differences were found in thrombus or neointima in vital aneurysms.

These findings confirm worse healing patterns in coiled compared to stented aneurysms. Neointima formation seems particularly dependent on the parent artery in decellularized aneurysms, whereas it is supported by the recruitment from aneurysm wall cells in vital cell-rich walls. In terms of translation, stent treatment might be more appropriate for highly degenerated aneurysms, whereas coiling alone might be adequate for aneurysms with mostly healthy vessel walls.

Introduction

Subarachnoid hemorrhage caused by the rupture of an intracranial aneurysm (IA) is a devastating neurosurgical condition associated with high morbidity and mortality1,2,3,4. In addition to microsurgical clipping, which provides direct endothelium-to-endothelium contact, endovascular devices have gained increasing importance over the past decades for treating ruptured and incidentally discovered IAs. The healing response in endovascularly treated IAs mainly depends on neointima formation and thrombus organization. Both are synergic processes, ....

Protocol

Veterinary support was performed according to institutional guidelines. Experiments were approved by the Local Ethics Committee, Switzerland (BE 60/19). The ARRIVE guidelines and 3R principles have been strictly followed6,7. Thirty-one male Lewis rats, 12 weeks old and weighing 492 ± 8 g, were included. House all rats at a room temperature of 23 °C and a 12 h light/dark cycle. Provide free access to water and pellets. Statistical analyses have been.......

Representative Results

A total of 31 animals were included in the laboratory setting: 27 rats were included in the final statistical analysis; 4 rats died prematurely (12.9% mortality rate). Intraoperatively, breath distension was significantly (p = 0.03) reduced in stent- (12.9 μm ± 0.7) compared to coil-treated (13.5 μm ± 0.6) rats. Fluorescence angiography was performed for every rat at the end of the final FU. Reperfusion was indicated in all 6 coil-treated animals, whereas reperfusion was observed in only 12.5.......

Discussion

This study demonstrates that neointima formation is mediated via endothelial cells originating in the parent artery of the aneurysm complex but is supported by the recruitment of cells derived from the aneurysm wall in vital aneurysms. Nevertheless, the role of circulating progenitor cells in aneurysm healing remains controversial12,13. Overall, 31 male Lewis rats were included in this investigation; only 4 died prematurely (12.9% mortality).

Acknowledgements

The authors thank Alessandra Bergadano, DVM, PhD, for the dedicated supervision of long-term animal health. This work was supported by the research funds of the Research Council, Kantonsspital Aarau, Aarau, Switzerland, and the Swiss national science foundation SNF (310030_182450).

....

Materials

NameCompanyCatalog NumberComments
3-0 resorbable sutureEthicon Inc., USAVCP428G
4-0 non-absorbable sutureB. Braun, GermanyG0762563
6-0 non-absorbable sutureB. Braun, GermanyC0766070
9-0 non-absorbable sutureB. Braun, GermanyG1111140
AtipamezolArovet AG, Switzerland
Bandpass filter blueThorlabsFD1Bany other
Bandpass filter greenThorlabsFGV9any other
Bipolar forcepsany other
Bicycle spotlightany other
Board (20 x 10 cm)any other
BuprenorphineIndivior, Switzerland1014197
CameraSony NEX-5R, Sony, Tokyo, Japan
Cannula (27-1/2 G)any other
Cell count softwareImage-J version 1.52n, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/
CellTracker CM-Dil dyeThermoFisher SCIENTIFIC, USAC7000
Coil-DeviceStyker, Kalamazoo, MI, USA2 cm of Target 360 TM Ultra, 2-mm diameter
Desinfectionany other
Eye-lubricantany other
FentanylSintetica, S.A., Switzerland98683any generic
FlumazenilLabatec-Pharma, Switerzland
FluoresceineCuratis AG5030376any generic
Fluorescence microscopeOlympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8
Foil maskany other
Glucose (5%)any other
Heating padHomeothermic Control Unit, Harvard, Edenbridge, Englandany other
Isotonic sodium chloride solution (0.9%)Fresenius KABI336769any generic
Isofluraneany generic
Longuettesany other
MeloxicamBoehringer IngelheimP7626406any generic
MedetomidineVirbac, SwitzerlandQN05CM91
Micro needle holderany other
MidazolamRoche, Switzerland
Monitoring-systemStarr Life Sciences Corp., 333 Allegheny Ave, Oakmont, PA 15139, United States
Needle holderany other
O2-Face maskany other
Operation microscopeOPMI, Carl Zeiss AG, Oberkochen, Germanyany other
Oxygenany other
Rectal temperature probeany other
ScalpellSwann-Morton210any other
Small animal shaverany other
Smartphoneany other
Sodium dodecyl sulfate (0.1%)Sigma-Aldrich11667289001
Soft feedEmeraid Omnivoreany generic
Soft tissue forcepsany other
Soft tissue spreaderany other
Stainless steel sponge bowlsany other
Stent-DeviceBiotroni, Bülach, Switzerlandmodified magmaris device, AMS with polymer coating, 6-mm length, 2-mm diameter
Sterile micro swabsany other
Straight and curved microforcepsany other
Straight and curved microscissorsany other
Straight and curved forcepsany other
Surgery drapeany other
Surgical scissorsany other
Syringes 1 mL, 2 mL, and 5 mLany other
Tapeany other
Vascular clip applicatorB. Braun, GermanyFT495T
Yasargil titan standard clip (2x)B. Braun Medical AG, Aesculap, SwitzerlandFT242Ttemporary

References

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