This study was supported in part by a research grant from the Kantonsspital Aarau, Switzerland.

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 before4,9. The imaging procedure and macroscopic analysis was performed 4 weeks after aneurysm creation.
1. Preparation of material needed for fluorescein video angiography
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Preparation of workplace and materials
<ol>
	<li>Disinfectant the workspace with disinfectant solution.</li>
	<li>Cover the table with sterile drapes to prevent contamination.</li>
	<li>Use sterile instruments for the surgery.</li>
</ol>
3. Preparation of animals for the surgery
<ol>
	<li>Weigh the animals.</li>
	<li>Induce anaesthesia and adjust the dose according to the weight.
	<ol>
		<li>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.</li>
		<li>Anesthetize rats in a gas chamber with 4% isoflurane and 96% oxygen prior to the injection.</li>
	</ol>
	</li>
	<li>Monitor the depth of anaesthesia. Pinch between their toes to make sure the animals are asleep.
	<ol>
		<li>Reposition the rabbits onto their backs. They should not react.</li>
		<li>For rats, pinch their tails and ensure that no reaction is observed.</li>
	</ol>
	</li>
	<li>Apply ointment on the rodents&#39;&#160;eyes to prevent dryness. Pull out the rats&#39;&#160;tongues to avoid any chance of swallowing.</li>
	<li>Start with preservation of anaesthesia.
	<ol>
		<li>For rabbits, catheterize (22 G shielded IV catheter with injection port, see the Table of Materials) the ear vein. Maintain balanced&#160;anaesthesia. Use a three-way stopcock to enable multiple simultaneous injections.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tape the animals onto the board in a supine position and closely shave the incision location. Disinfect the area with Betaseptic.&#160;
	<ol>
		<li>For rabbits, disinfect the neck, especially around sternocleidomastoid muscle.</li>
		<li>For rats, disinfect the area from bladder to transvers colon.</li>
	</ol>
	</li>
	<li>Administer oxygen through a mask throughout the surgery and maintain body temperature with a heating pad.</li>
</ol>
4. Preparation of the artery
<ol>
	<li>For best results, thoroughly dissect the chosen vessel from the surrounding tissue9,10.
	<ol>
		<li>For rats, identify the tail vein (less invasive, preferably used for surviving animals) or dissect a femoral vein for fluorescein injection4,11. 
		NOTE: For rabbits, no further dissection of vessels is needed for fluorescein injection as the ear vein is already being used for anaesthesia.</li>
	</ol>
	</li>
	<li>Position a white pad under chosen vessel to increase contrast with the surrounding tissue.</li>
	<li>Focus the camera mounted to the microscope on the dissected artery.</li>
</ol>
5. Fluorescein video angiography
<ol>
	<li>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.
	<ol>
		<li>For rabbits, inject 0.3 mL/kg fluorescein sodium through the three-way-stopcock into the catheterized ear vein.</li>
		<li>For rats, inject 0.4 mL/kg fluorescein sodium into the femoral vein via a catheter or a 25 G needle.</li>
	</ol>
	</li>
	<li>Flush the needle or the catheter with 0.5 mL saline solution to ensure that all dye is injected.</li>
	<li>Illuminate the surgical field with the modified flashlight.</li>
	<li>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.</li>
</ol>
6. Macroscopic analysis
<ol>
	<li>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&#8217;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).</li>
</ol>

<ol>
	<li>Kakucs, C., Florian, I. A., Ungureanu, G., Florian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+Angiography+in+Intracranial+Aneurysm+Surgery:+A+Helpful+Method+to+Evaluate+the+Security+of+Clipping+and+Observe+Blood+Flow.">Fluorescein Angiography in Intracranial Aneurysm Surgery: A Helpful Method to Evaluate the Security of Clipping and Observe Blood Flow.</a> World Neurosurgery. 105, 406-411 (2017).</li><li>Ajiboye, N., Chalouhi, N., Starke, R. M., Zanaty, M., Bell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unruptured+Cerebral+Aneurysms:+Evaluation+and+Management.">Unruptured Cerebral Aneurysms: Evaluation and Management.</a> ScientificWorldJournal. 2015, 954954 (2015).</li><li>Suzuki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+blood+flow+in+perforating+arteries+using+fluorescein+cerebral+angiography+during+aneurysm+surgery.">Confirmation of blood flow in perforating arteries using fluorescein cerebral angiography during aneurysm surgery.</a> Journal of Neurosurgery. 107 (1), 68-73 (2007).</li><li>Gruter, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Video+Angiography+for+Evaluation+of+Dynamic+Perfusion+Status+in+an+Aneurysm+Preclinical+Experimental+Setting.">Fluorescence Video Angiography for Evaluation of Dynamic Perfusion Status in an Aneurysm Preclinical Experimental Setting.</a> Operative Neurosurgery. , (2019).</li><li>Raabe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+evaluation+of+surgical+microscope-integrated+intraoperative+near-infrared+indocyanine+green+videoangiography+during+aneurysm+surgery.">Prospective evaluation of surgical microscope-integrated intraoperative near-infrared indocyanine green videoangiography during aneurysm surgery.</a> Journal of Neurosurgery. 103 (6), 982-989 (2005).</li><li>Riva, M., Amin-Hanjani, S., Giussani, C., De Witte, O., Bruneau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+Green+Videoangiography+in+Aneurysm+Surgery:+Systematic+Review+and+Meta-Analysis.">Indocyanine Green Videoangiography in Aneurysm Surgery: Systematic Review and Meta-Analysis.</a> Neurosurgery. , (2017).</li><li>Kuroda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+injection+fluorescein+videoangiography+in+aneurysm+surgery.">Intra-arterial injection fluorescein videoangiography in aneurysm surgery.</a> Neurosurgery. 72, 141-150 (2013).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+Bioscience+Research+Reporting:+The+ARRIVE+Guidelines+for+Reporting+Animal+Research.">Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research.</a> PLOS Biology. 8 (6), 1000412 (2010).</li><li>Marbacher, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Helsinki+rat+microsurgical+sidewall+aneurysm+model.">The Helsinki rat microsurgical sidewall aneurysm model.</a> Journal of Visualized Experiments. (92), e51071 (2014).</li><li>Marbacher, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+bilobular,+bisaccular,+and+broad-neck+microsurgical+aneurysm+formation+in+the+rabbit+bifurcation+model+for+the+study+of+upcoming+endovascular+techniques.">Complex bilobular, bisaccular, and broad-neck microsurgical aneurysm formation in the rabbit bifurcation model for the study of upcoming endovascular techniques.</a> American Journal of Neuroradiology. 32 (4), 772-777 (2011).</li><li>Shurey, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+model+in+microsurgery+education:+classical+exercises+and+new+horizons.">The rat model in microsurgery education: classical exercises and new horizons.</a> Archives of Plastic Surgery. 41 (3), 201-208 (2014).</li><li>Foster, S. D., Lyons, M. S., Runyan, C. M., Otten, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mimic+of+soft+tissue+infection:+intra-arterial+injection+drug+use+producing+hand+swelling+and+digital+ischemia.">A mimic of soft tissue infection: intra-arterial injection drug use producing hand swelling and digital ischemia.</a> World Journal of Emergency Medicine. 6 (3), 233-236 (2015).</li><li>Flower, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+technique+for+indocyanine+green+and+sodium+fluorescein+dye+angiography+of+the+eye.">Injection technique for indocyanine green and sodium fluorescein dye angiography of the eye.</a> Investigative Ophthalmology &amp; Visual Science. 12 (12), 881-895 (1973).</li><li>Yoshioka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantage+of+microscope+integrated+for+both+indocyanine+green+and+fluorescein+videoangiography+on+aneurysmal+surgery:+case+report.">Advantage of microscope integrated for both indocyanine green and fluorescein videoangiography on aneurysmal surgery: case report.</a> Neurologia medico-chirurgica (Tokyo). 54 (3), 192-195 (2014).</li><li>Ichikawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+and+Clinical+Experience+with+a+Simple+Device+for+Performing+Intraoperative+Fluorescein+Fluorescence+Cerebral+Angiography:+Technical+Notes.">Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes.</a> Neurologia medico-chirurgica. 56 (3), 141-149 (2016).</li><li>Alander, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+indocyanine+green+fluorescent+imaging+in+surgery.">A review of indocyanine green fluorescent imaging in surgery.</a> International Journal of Biomedical Imaging. 2012, 940585 (2012).</li><li>Lane, B., Bohnstedt, B. N., Cohen-Gadol, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+comparative+study+of+microscope-integrated+intraoperative+fluorescein+and+indocyanine+videoangiography+for+clip+ligation+of+complex+cerebral+aneurysms.">A prospective comparative study of microscope-integrated intraoperative fluorescein and indocyanine videoangiography for clip ligation of complex cerebral aneurysms.</a> Journal of Neurosurgery. 122 (3), 618-626 (2015).</li><li>Blair, N. P., Evans, M. A., Lesar, T. S., Zeimer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+and+fluorescein+glucuronide+pharmacokinetics+after+intravenous+injection.">Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.</a> Investigative Ophthalmology &amp; Visual Science. 27 (7), 1107-1114 (1986).</li><li>Hillmann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+optical+imaging+of+physiological+responses+to+photostimulation+in+human+photoreceptors.">In vivo optical imaging of physiological responses to photostimulation in human photoreceptors.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (46), 13138-13143 (2016).</li><li>Golby, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Image-Guided Neurosurgery. , (2015).</li></ol>

All authors confirm no conflicts of interest.

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...

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 technique1,3,4.
As such, indocyanine green (ICG) video angiography is often used in clinical neurosurgical procedures and has extensively been studied5,6. 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 scarce1,4.
The aim of this report is to demonstrate the feasibility and scope of applications of FVA in rat and rabbit preclinical cerebrovascular research.

<table><tbody><tr><td>&lt;strong&gt;For rabbits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Animal shaver</td><td></td><td></td><td></td></tr><tr><td>Black tape</td><td></td><td></td><td></td></tr><tr><td>Blue filter</td><td>Thorlabs MF475-35</td><td></td><td></td></tr><tr><td>Body warm plate</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td>Sony NEX-5R</td><td></td><td></td></tr><tr><td>Catheter</td><td>22G Vasofix Safety</td><td></td><td></td></tr><tr><td>Disinfictant</td><td></td><td></td><td></td></tr><tr><td>Fluorescein sodium</td><td>Fluorescein Faure 10%</td><td></td><td></td></tr><tr><td>Glas plate</td><td></td><td></td><td></td></tr><tr><td>Green filter</td><td>Thorlabs MF539-43</td><td></td><td></td></tr><tr><td>Incontinence pad</td><td></td><td></td><td></td></tr><tr><td>Infusion pump</td><td>Perfusor Secura</td><td></td><td></td></tr><tr><td>Ketamine hydrochloride</td><td></td><td></td><td>any generic products</td></tr><tr><td>Needle</td><td>25G</td><td></td><td></td></tr><tr><td>Oxygen</td><td></td><td></td><td></td></tr><tr><td>Ringer&#039;s Solution</td><td></td><td></td><td></td></tr><tr><td>Sterile sheets</td><td></td><td></td><td></td></tr><tr><td>Surgical instruments</td><td></td><td></td><td>micro forceps, micro scissor, blunt surgical scissor</td></tr><tr><td>Surgical microscope</td><td>OPMI, Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Syringe 2ml, 5ml, 50ml</td><td></td><td></td><td></td></tr><tr><td>Tape</td><td></td><td></td><td></td></tr><tr><td>Three-way-stopcock</td><td></td><td></td><td></td></tr><tr><td>Torch light</td><td></td><td></td><td></td></tr><tr><td>Xylazin</td><td></td><td></td><td>any generic products</td></tr><tr><td>&lt;strong&gt;For rats&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Animal shaver</td><td></td><td></td><td></td></tr><tr><td>Black tape</td><td></td><td></td><td></td></tr><tr><td>Blue filter</td><td>Thorlabs MF475-35</td><td></td><td></td></tr><tr><td>Body warm plate</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td>Sony NEX-5R</td><td></td><td></td></tr><tr><td>Disinfictant</td><td></td><td></td><td></td></tr><tr><td>Fluorescein sodium</td><td>Fluorescein Faure 10%</td><td></td><td></td></tr><tr><td>Green filter</td><td>Thorlabs MF539-43</td><td></td><td></td></tr><tr><td>Incontinence pad</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Ketamine hydrochloride</td><td></td><td></td><td>any generic products</td></tr><tr><td>Medetomidine hydrochloride</td><td></td><td></td><td>any generic products</td></tr><tr><td>Needle</td><td>25G</td><td></td><td></td></tr><tr><td>Oxygen</td><td></td><td></td><td></td></tr><tr><td>Plate</td><td></td><td></td><td></td></tr><tr><td>Ringer&#039;s Solution</td><td></td><td></td><td></td></tr><tr><td>Sterile sheets</td><td></td><td></td><td></td></tr><tr><td>Surgical instruments</td><td></td><td></td><td>micro forceps, micro scissor, blunt surgical scissor</td></tr><tr><td>Surgical microscope</td><td>OPMI, Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Syringe 2ml, 5ml</td><td></td><td></td><td></td></tr><tr><td>Tape</td><td></td><td></td><td></td></tr><tr><td>Torch light</td><td></td><td></td><td></td></tr></tbody></table>

fluorescence angiography for evaluation of aneurysm perfusion and parent artery patency in rat and rabbit aneurysm models

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.

Heart rate and blood pressure were monitored during surgery. Mean heart rate was 193/min in rabbits and 196/min in rats. The rabbits&#39; 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...

Watch this Scientific Journal Video about Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models at JoVE.com

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

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.

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

fluorescence-angiography-for-evaluation-aneurysm-perfusion-parent

Research

JoVE Journal

Neuroscience

6.9K Views.  Kantonsspital Aarau. Deze methode is een betrouwbare en eenvoudige techniek voor het visualiseren van real-time bloedstroom in aneurysma's en bloedvaten en kan worden gebruikt in verschillende diermodellen. Het belangrijkste voordeel van deze techniek is dat het visualisatie van de bloedstroom tijdens de operatie vergemakkelijkt zonder dat er dure apparaten of uitgebreide chirurgie nodig zijn. Gebruik voor het begin van de procedure zwarte tape om een groene bandpassfilter te beveiligen om op een zaklamp te funge...

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

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Medicine

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat&#39;s descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.

Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular devices. Animal models respecting rheological, hemodynamic and aneurysm wall conditions are highly warranted. Therefore, the aim of the present study was to design a novel standardized and reproducible surgical technique to create autologous arterial pouch bifurcation aneurysms with non-modified and modified wall conditions in rabbits.
Bifurcation aneurysms were created by end-to-side anastomosis of the right on the left common carotid artery, both serving as parent arteries for the arterial pouch, which was microsurgically sewn on. Grafts were taken from the proximal right common carotid artery, either for the control (n = 7, immediate autologous re-implantation) or modified (n = 7, incubated with 100 international units elastase for 20 minutes before autologous re-implantation) group. Pouch and parent artery patency were controlled by fluorescence angiography immediately after creation. At follow-up (28 days), all rabbits underwent contrast enhanced magnetic resonance angiography and fluorescence angiography followed by aneurysm harvesting, macroscopic and histological evaluation.
A total of 16 female New Zealand White rabbits were operated upon. Two animals died prematurely. At follow-up, 85.72% of all aneurysms remained patent. Both groups revealed an increase in aneurysm size over time; this was more pronounced in the control group (6.48 &#177; 1.81 mm3 at time of creation vs. 19.85 &#177; 6.40 mm3 at follow-up, p = 0.037) than in the modified group (8.03 &#177; 1.08 mm3 at time of creation vs. 20.29 &#177; 6.16 mm3 at follow-up, p = 0.054).
Our findings demonstrate the adequacy of this new rabbit model which allows for the creation of bifurcation aneurysms with different wall conditions in a microsurgical approach. Given the excellent long-term patency and the property of aneurysm growth over time, this model may serve as an important tool for preclinical evaluation of novel endovascular therapies.

Developing and testing endovascular devices for intracranial aneurysm treatment is still of great importance. Most aneurysm models used today miss either the important characteristics of an arterial degenerated wall or the hemodynamics of a true bifurcation. Therefore, we aimed to design a novel arterial pouch bifurcation model in rabbits.

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular ...

Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the understanding of the pathophysiological processes and the development and testing of new therapeutic strategies. This study aims to describe a new rabbit aneurysm model that allows the creation of two elastase-digested saccular aneurysms with different hemodynamic conditions within the same animal.
Five female New Zealand white rabbits with a mean weight of 4.0 (&#177; 0.3) kg and mean age of 25 (&#177;5) weeks underwent microsurgical stump and bifurcation aneurysm creation. One aneurysm (stump) was created by right common carotid artery (CCA) exposure at its origin at the brachiocephalic trunk. A temporary clip was applied at the CCA origin and another, 2 cm above. This segment was treated with a local injection of 100 U of elastase for 20 min. A second aneurysm (bifurcation) was created by suturing an elastase-treated arterial pouch into the end-to-side anastomosis of the right CCA to left CCA. Patency was controlled by fluorescence angiography immediately after creation.
The average duration of surgery was 221 min. The creation of two aneurysms in the same animal was successful in all rabbits without complication. All aneurysms were patent immediately after surgery except for one bifurcation aneurysm, which showed an extreme tissue reaction due to elastase incubation and an immediate intraluminal thrombosis. No mortality was observed during surgery and up to one-month follow-up. Morbidity was limited to a transient vestibular syndrome (one rabbit), which recovered spontaneously within one day.
Demonstrated here for the first time is the feasibility of creating a two-aneurysm rabbit model with stump and bifurcation hemodynamic characteristics and highly degenerated wall conditions. This model allows the study of the natural course and potential treatment strategies on the basis of aneurysm biology under different flow conditions.

This protocol describes the steps for the creation of a rabbit model with two elastase-digested aneurysms with different hemodynamics (stump and bifurcation constellation). This allows the testing of novel endovascular devices in aneurysms with different angioarchitecture and hemodynamic conditions within a single animal.

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Microsurgical Creation of Giant Bifurcation Aneurysms in Rabbits for the Evaluation of Endovascular Devices

Giant aneurysms are dangerous lesions requiring endovascular treatment, with high rates of aneurysm recanalization and re-rupture. Reliable in vivo models are rare but are required for testing new endovascular devices.&#160;We demonstrate the technical aspects of the creation of giant bifurcation aneurysms in New Zealand white rabbits (2.5-5.5 kg). A 25-30 mm long venous pouch is taken from the external jugular vein, and a bifurcation between both carotid arteries is created microsurgically. The pouch is sutured in the bifurcation to mimic a giant aneurysm. This protocol summarizes our previously published standard technique for venous pouch true arterial bifurcation aneurysms and highlights its essential modification steps for giant aneurysms. Using this modified technique, we were able to create an animal model for giant aneurysms with high comparability to humans regarding the hemodynamics and coagulation systems. Furthermore, low morbidity and high aneurysm patency rates were achieved. The proposed giant aneurysm model offers an excellent possibility for testing new endovascular devices.

Here, we describe the technique for the microsurgical creation of giant bifurcation aneurysms in rabbits for the evaluation of endovascular devices.

Giant aneurysms are dangerous lesions requiring endovascular treatment, with high rates of aneurysm recanalization and re-rupture. Reliable in vivo models ...

Near-infrared Fluorescence Imaging of Abdominal Aortic Aneurysms

Source: Arvin H. Soepriatna1, Kelsey A. Bullens2, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>1
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
2 Department of Biochemistry, Purdue University, West Lafayette, Indiana
Near-infrared fluorescence (NIRF) imaging is an exciting optical technique that utilizes fluorescent probes to visualize complex biomolecular assemblies in tissues. NIRF imaging has many advantages over conventional imaging methods for noninvasive imaging of diseases. Unlike single photon emission computed tomography (SPECT) and positron emission tomography (PET), NIRF imaging is rapid, high-throughput, and does not involve ionizing radiation. Furthermore, recent developments in engineering target-specific and activatable fluorescent probes provide NIRF with high specificity and sensitivity, making it an attractive modality in studying cancer and cardiovascular disease. The presented procedure is designed to demonstrate the principles behind NIRF imaging and how to conduct in vivo and ex vivo experiments in small animals to study a variety of diseases. The specific example shown here employs an activatable fluorescent probe for matrix metalloproteinase-2 (MMP2) to study its uptake in two different rodent models of abdominal aortic aneurysms (AAAs).

Source: Arvin H. Soepriatna1, Kelsey A. Bullens2, and Craig J. Goergen1
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, ...

Education

JoVE Science Education

Engineering

Biomedical Engineering

Assessing Vessel Perfusion via Angiography: A Technique to Introduce Contrast Agent in Descending Aorta to Visualize Blood Vessel Perfusion in Rabbit Model

Source: Sligar, A. D et al., <a href="https://www.jove.com/v/58964">Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits.</a> J. Vis. Exp. (2019).
This video describes the technique for performing angiography by injecting a contrast agent into the descending aorta to visualize blood vessel perfusion in a rabbit model. This technique helps to understand the perfusion in blood vessels with blockages and after treatment during recovery.

Source: Sligar, A. D et al., Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits. J. Vis. Exp. (2019).
This video describes the technique for ...

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

Samenvatting

Meer video's verkennen

PLAYLIST