Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Summary

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Abstract

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.

Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.

Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

Introduction

Evidence of complete aneurysm obliteration and parent artery integrity is of utmost importance in aneurysm surgery. There are several options to confirm parent artery patency and aneurysm occlusion, such as Doppler sonography, conventional cerebral angiography (DSA), computed tomography angiography (CTA) or magnetic resonance angiography (MRA)^1,2. However, these are expensive and time-consuming methods which are often not available in a laboratory setting. Furthermore, they may have relevant side effects such as radiation exposure or need for additional sedation of experimental animals to avoid movement artefact.

With an increasing number of new endovascular devices emerging, there is a consecutive need for preclinical testing of such devices. However, these studies often rely on post-mortem analysis (e.g., macro pathology and histology) and lack information on dynamic perfusion. Furthermore, for the researcher it may be crucial to obtain immediate and reliable information during an experimental surgical procedure. Fluorescence angiography is a cost-effective and easy to perform visualization technique^1,3,4.

As such, indocyanine green (ICG) video angiography is often used in clinical neurosurgical procedures and has extensively been studied^5,6. Fluorescein video angiography (FVA) is an alternative technique, with the additional advantage of creating a fluorescence signal that is within the wavelength range of human vision, and can thus be seen by the naked eye without an extended spectrum infrared camera⁷. Fluorescein video angiography is less often used in clinical cerebrovascular surgery and reports on FVA in experimental settings are scarce^1,4.

The aim of this report is to demonstrate the feasibility and scope of applications of FVA in rat and rabbit preclinical cerebrovascular research.

Protocol

The rodents were housed in an animal care facility and experiments were reviewed and approved by the Committee for Animal Welfare at the University of Bern, Switzerland (BE 108/16) and (BE65/16). All animals were maintained on a standard laboratory diet with free access to food and water. All animal experiments were conducted under careful consideration of the 3Rs (replacement, reduction and refinement). Ten female New Zealand White rabbits and 48 male Wistar rats were included. ARRIVE guidelines were followed strictly⁸.

NOTE: Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats as described before^4,9. The imaging procedure and macroscopic analysis was performed 4 weeks after aneurysm creation.

1. Preparation of material needed for fluorescein video angiography

1. Modify the flashlight by taping on a blue bandpass filter (see the Table of Materials), which will function as an excitation filter. The torch should then only emit blue light. Use black tape to avoid any leakage of unfiltered light.
2. Equip the camera (e.g., attached to the microscope) with a green bandpass filter (see the Table of Materials), which will function as a emission light filter. Only green light should now be able to pass through.

2. Preparation of workplace and materials

1. Disinfectant the workspace with disinfectant solution.
2. Cover the table with sterile drapes to prevent contamination.
3. Use sterile instruments for the surgery.

3. Preparation of animals for the surgery

1. Weigh the animals.
2. Induce anaesthesia and adjust the dose according to the weight.
   1. For rabbits, start balanced anesthesia. Shield their eyes with one hand during injection to reduce their fright reaction. Cover the cage with a sheet to help sedate the animals.
   2. Anesthetize rats in a gas chamber with 4% isoflurane and 96% oxygen prior to the injection.
3. Monitor the depth of anaesthesia. Pinch between their toes to make sure the animals are asleep.
   1. Reposition the rabbits onto their backs. They should not react.
   2. For rats, pinch their tails and ensure that no reaction is observed.
4. Apply ointment on the rodents' eyes to prevent dryness. Pull out the rats' tongues to avoid any chance of swallowing.
5. Start with preservation of anaesthesia.
   1. For rabbits, catheterize (22 G shielded IV catheter with injection port, see the Table of Materials) the ear vein. Maintain balanced anaesthesia. Use a three-way stopcock to enable multiple simultaneous injections.
   2. For rats, inject 50 mg/kg ketamine hydrochloride and 0.5 mg/kg medetomidine hydrochloride intraperitoneally. Monitor anaesthesia with a noxious toe pinch during surgery. In the case of reaction, administer additional anaesthetic.
6. Tape the animals onto the board in a supine position and closely shave the incision location. Disinfect the area with Betaseptic. 
   1. For rabbits, disinfect the neck, especially around sternocleidomastoid muscle.
   2. For rats, disinfect the area from bladder to transvers colon.
7. Administer oxygen through a mask throughout the surgery and maintain body temperature with a heating pad.

4. Preparation of the artery

1. For best results, thoroughly dissect the chosen vessel from the surrounding tissue^9,10.
   1. For rats, identify the tail vein (less invasive, preferably used for surviving animals) or dissect a femoral vein for fluorescein injection^4,11.
      NOTE: For rabbits, no further dissection of vessels is needed for fluorescein injection as the ear vein is already being used for anaesthesia.
2. Position a white pad under chosen vessel to increase contrast with the surrounding tissue.
3. Focus the camera mounted to the microscope on the dissected artery.

5. Fluorescein video angiography

1. Cover the 5 mL syringe filled with fluorescein sodium (100 mg/mL, see the Table of Materials) with aluminium foil to protect from exposure of light. Turn off the lights (as much as possible) and inject fluorescein sodium intravenously. Inject under darkness to prevent photobleaching.
   1. For rabbits, inject 0.3 mL/kg fluorescein sodium through the three-way-stopcock into the catheterized ear vein.
   2. For rats, inject 0.4 mL/kg fluorescein sodium into the femoral vein via a catheter or a 25 G needle.
2. Flush the needle or the catheter with 0.5 mL saline solution to ensure that all dye is injected.
3. Illuminate the surgical field with the modified flashlight.
4. Commence filming with the modified camera. Blood flow should be visible a few seconds after injection (Figure 1).
   NOTE: Here, we used frame rate = 50 frames/s, focal length = 70 mm, and F3.4.

6. Macroscopic analysis

1. Resect the aneurysms and parent artery complex, and evaluate the patency macroscopically by opening the parent artery with micro-scissors and evaluate the lumen of the parent artery and the anerysm’s orifice (see Figure 1, 2)⁹. Measure the sizes of the aneurysms. Aneurysm-parent-artery-complex can then be stored for further analysis (e.g., histology).

Results

Heart rate and blood pressure were monitored during surgery. Mean heart rate was 193/min in rabbits and 196/min in rats. The rabbits' body weight ranged 3.05-4.18 kg, and the rats weighed 335-690 g.

We were able to perform FVA in eight out of ten rabbits (Figure 1). Four aneurysm examinations in two rabbits were not recorded with the camera due to technical difficulties. No technical difficulties involving FVA in rats were reported. However, FVA could not be p...

Discussion

FVA is a promising and uncomplicated method to examine vessels in rodents and can be performed with commercial devices and off-the-shelf equipment. FVA can be implemented during any surgery where intraoperative evaluation of vessel integrity is needed as the vessels need proper dissection first.

The authors preferred venous injection to arterial injection due to the lower risk of inadvertent events such as infection, ischemia and compartment syndrome¹². Intravenous inje...

Materials

Name	Company	Catalog Number	Comments
For rabbits
Aluminium foil
Animal shaver
Black tape
Blue filter	Thorlabs MF475-35
Body warm plate
Camera	Sony NEX-5R
Catheter	22G Vasofix Safety
Disinfictant
Fluorescein sodium	Fluorescein Faure 10%
Glas plate
Green filter	Thorlabs MF539-43
Incontinence pad
Infusion pump	Perfusor Secura
Ketamine hydrochloride			any generic products
Needle	25G
Oxygen
Ringer's Solution
Sterile sheets
Surgical instruments			micro forceps, micro scissor, blunt surgical scissor
Surgical microscope	OPMI, Carl Zeiss AG, Oberkochen, Germany
Syringe 2ml, 5ml, 50ml
Tape
Three-way-stopcock
Torch light
Xylazin			any generic products
For rats
Aluminium foil
Animal shaver
Black tape
Blue filter	Thorlabs MF475-35
Body warm plate
Camera	Sony NEX-5R
Disinfictant
Fluorescein sodium	Fluorescein Faure 10%
Green filter	Thorlabs MF539-43
Incontinence pad
Isoflurane
Ketamine hydrochloride			any generic products
Medetomidine hydrochloride			any generic products
Needle	25G
Oxygen
Plate
Ringer's Solution
Sterile sheets
Surgical instruments			micro forceps, micro scissor, blunt surgical scissor
Surgical microscope	OPMI, Carl Zeiss AG, Oberkochen, Germany
Syringe 2ml, 5ml
Tape
Torch light