Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

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 mortality^1,2,3,4. 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, depending on cell migration from the adjacent vessel and the aneurysm wall.⁵ To date, the origin of endothelial cells in neointima formation of endovascular treated aneurysms remains unclear. There is an ongoing debate in the literature about the source from which neointima-forming cells are recruited.

By using a cell-tracer injection of CM-Dil dye (see the Table of Materials) in the abdominal aorta of rats, we aimed to analyze the role of endothelial cells, originating in the parent artery, in neointima formation at two different FU time points (day 7 and day 21) (Figure 1). An advantage of the model is the direct local cell-tracer incubation in vivo in a parent artery prior to aneurysm suture, allowing FU at later time points. In vivo injection techniques, such as cell-tracer incubation, have not been described in the literature. An advantage of this technique is the direct, one-point, intraoperative, in vivo injection, which makes the model robust and reproducible.

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 followed^6,7. 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 performed using the nonparametric Wilcoxon-Mann-Whitney U test. Probability values (p) of ≤ 0.05 and/or ≤ 0.01 were considered significant.

1. Preoperative phase-general preparation and anesthesiological aspects

1. Randomize rats into either coil or stent treatment groups (Figure 2) via a web-based randomization system. Now, perform a preoperative clinical examination of all animals planned for surgery next to a quiet, aseptic operating room maintaining a room temperature of 23 ± 3 °C. Analyze the animals' behavior and inspect the mucous membranes and turgor as part of the preoperative clinical examination.
2. Record the weight of each animal.
3. Prior to surgery, incubate the arterial pouches from donor rats in 0.1% sodium dodecyl sulfate for 10 h at 37 °C to obtain decellularized aneurysms⁸. Collect these pouches from donor animals a few days before the surgery.
   1. Prepare the full length of the abdominal aorta with microscissors and forceps and apply 6-0 nonabsorbable ligatures at an interval of 3-4 mm.
   2. Directly generate vital aneurysms intraoperatively by a previously ligated arterial vessel pouch from the thoracic part of a donor animal⁹. Perform thoracotomy with scissors and surgical forceps at the indicated FU time point and ligate the vessel pouch at the desired length.
4. Directly implant the pouch into the recipient and harvest the aneurysm from the donor animal for further macroscopic analysis and histological processing.
5. For anesthesia induction, place all rats in a clean box provided with oxygen (O₂) until loss of consciousness after 5-10 min. Anesthetize rats with a subcutaneous (SC) injection of a mixture of fentanyl 0.005 mg/kg, medetomidine 0.15 mg/kg, and midazolam 2 mg/kg.
   NOTE: This ensures a surgical plane of at least 45 min.
6. Check the depth of anesthesia by the absence of the pedal withdrawal reflex.
7. Place the rats in a supine position and shave the thoracoabdominal part with an electric shaver.
8. Fixate the 4 paws of the rats with tape on a board, covered by a heating pad connected to an autoregulating rectal probe. Insert the rectal probe in the rat's anus to maintain the desired temperature of 37 °C with the help of the heating pad.
9. Now, install a sensor on the right hind leg connected to a computerized system for checking vital signs intraoperatively.
10. Cover the rat's nose and mouth with a face mask. If requiring prolonged anesthesia, start isoflurane (1.0-2.0% titrated to effect in 100% O₂).
11. Disinfect the surgical field with povidone-iodine or alternating disinfectants and drape the surgical field in a sterile fashion.
12. For perianesthetic care, apply a sterile ophthalmic lubricant to the eyes and cover them with an opaque foil mask to prevent drying and damage from the surgical lamp.
13. Throughout surgery, supply oxygen continuously via the face mask, monitor the body temperature, and provide heat using a heating pad, maintaining normothermia.
14. Monitor other vital signs continuously (pulse and breath distension, heart and breath rate, and oxygen saturation).

2. Operative phase - cell-tracer injection

NOTE: The detailed surgical approach in the Helsinki rat microsurgical sidewall aneurysm model⁹ and techniques for coil- and stent-implantation are described elsewhere^8,10,11.

1. Store the fluorescent lipophilic cell-tracer at ≤ -20 °C all the time, protected from light.
2. Perform the surgery by preparing the rat aorta and caval vein, followed by the separation of both, as well as proximal and distal temporary clamping of the aorta.
   NOTE: This technique has been described previously⁹.
   1. Clamp the proximal and distal parts of the aorta with two temporary titan clips.
3. Put one microswab with purple padding each under the proximal and distal parts of the aorta for better visualization of the artery.
4. Now, protect the abdomen with wet gauze.
5. On the day of the operation, dissolve 2 µL of the cell-tracer by pipetting in 1 mL of phosphate-buffered saline (PBS).
6. Transfer the mixture to a 1 mL syringe fitted with a 27-1/2 G (0.4 x 13 mm) sterile cannula.
   NOTE: Be careful to avoid light exposure while performing steps 2.5 and 2.6.
7. Turn off the light in the operating room. While looking under a microscope, perform the one-point injection in the middle ventral part of the aorta using micro forceps and carefully inject 1 mL of heparinized 0.9% saline solution.
8. Inject the cell-tracer carefully (Video 1) and immediately turn off the operating microscope as well. Again, protect the abdomen with wet gauze.
9. Let the dye incubate for at least 15 min. After the incubation period, turn on the microscope and operating room lights.
10. Perform the longitudinal arteriotomy and suturing of the aneurysm, as described elsewhere¹¹.
    1. Use microforceps and microscissors to perform the arteriotomy so that its length averages the diameter of the harvested aneurysm (step 1.3). To ensure the correct length, place the aneurysm beside the aorta before performing arteriotomy. Suture the aneurysm with 8-10 single-stitches using a nonabsorbable 10-0 suture, and carefully remove the temporary clamps-starting distally-under continuous irrigation with heparinized saline. Close the wound in a layered fashion. Of note, use a coil packing density of 1 cm.
       NOTE: The technique of coil- or stent-implantation has been described elsewhere^8,10.

3. Postoperative phase-monitoring and analgetic care

1. At the end of the surgery, reverse the anesthesia with an SC injection mixture of buprenorphine 0.05 mg/kg, atipamezol 0.75 mg/kg, and flumazenil 0.2 mg/kg. Let each operated animal recover in a clean cage until fully awake and warm, as needed, with a heating lamp.
2. For 3 days, administer 1 mg/kg meloxicam (one injection or oral application per day) and buprenorphine (0.05 mg/kg four times each day) SC. Overnight, provide buprenorphine continuously in the drinking water with the same dosing: 6 mL buprenorphine 0.3 mg/mL, 360 mL drinking water, 10 mL of 5% glucose.
3. In the immediate postoperative phase, house each animal in a single cage for protection. Regroup the animals after 24 h.
4. If any rat shows distressed or aggressive behavior after SC injection, administer buprenorphine in the drinking water during the day.
5. Provide soft feed on the cage floor to support feeding and recovery postoperatively.
6. Observe and take care of all the animals according to the wellbeing and pain score sheet.
7. Administer rescue analgesia SC (meloxicam 1 mg/kg and 0.05 mg/kg buprenorphine) when needed.

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 controversial^12,13. Overall, 31 male Lewis rats were included in this investigation; only 4 died prematurely (12.9% mortality).

Materials

Name	Company	Catalog Number	Comments
3-0 resorbable suture	Ethicon Inc., USA	VCP428G
4-0 non-absorbable suture	B. Braun, Germany	G0762563
6-0 non-absorbable suture	B. Braun, Germany	C0766070
9-0 non-absorbable suture	B. Braun, Germany	G1111140
Atipamezol	Arovet AG, Switzerland
Bandpass filter blue	Thorlabs	FD1B	any other
Bandpass filter green	Thorlabs	FGV9	any other
Bipolar forceps			any other
Bicycle spotlight			any other
Board (20 x 10 cm)			any other
Buprenorphine	Indivior, Switzerland	1014197
Camera	Sony NEX-5R, Sony, Tokyo, Japan
Cannula (27-1/2 G)			any other
Cell count software	Image-J version 1.52n, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/
CellTracker CM-Dil dye	ThermoFisher SCIENTIFIC, USA	C7000
Coil-Device	Styker, Kalamazoo, MI, USA		2 cm of Target 360 TM Ultra, 2-mm diameter
Desinfection			any other
Eye-lubricant			any other
Fentanyl	Sintetica, S.A., Switzerland	98683	any generic
Flumazenil	Labatec-Pharma, Switerzland
Fluoresceine	Curatis AG	5030376	any generic
Fluorescence microscope	Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8
Foil mask			any other
Glucose (5%)			any other
Heating pad	Homeothermic Control Unit, Harvard, Edenbridge, England		any other
Isotonic sodium chloride solution (0.9%)	Fresenius KABI	336769	any generic
Isoflurane			any generic
Longuettes			any other
Meloxicam	Boehringer Ingelheim	P7626406	any generic
Medetomidine	Virbac, Switzerland	QN05CM91
Micro needle holder			any other
Midazolam	Roche, Switzerland
Monitoring-system	Starr Life Sciences Corp., 333 Allegheny Ave, Oakmont, PA 15139, United States
Needle holder			any other
O2-Face mask			any other
Operation microscope	OPMI, Carl Zeiss AG, Oberkochen, Germany		any other
Oxygen			any other
Rectal temperature probe			any other
Scalpell	Swann-Morton	210	any other
Small animal shaver			any other
Smartphone			any other
Sodium dodecyl sulfate (0.1%)	Sigma-Aldrich	11667289001
Soft feed	Emeraid Omnivore		any generic
Soft tissue forceps			any other
Soft tissue spreader			any other
Stainless steel sponge bowls			any other
Stent-Device	Biotroni, Bülach, Switzerland		modified magmaris device, AMS with polymer coating, 6-mm length, 2-mm diameter
Sterile micro swabs			any other
Straight and curved microforceps			any other
Straight and curved microscissors			any other
Straight and curved forceps			any other
Surgery drape			any other
Surgical scissors			any other
Syringes 1 mL, 2 mL, and 5 mL			any other
Tape			any other
Vascular clip applicator	B. Braun, Germany	FT495T
Yasargil titan standard clip (2x)	B. Braun Medical AG, Aesculap, Switzerland	FT242T	temporary