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 strictly<s...

<ol>
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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. 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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>

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 border="0" cellpadding="0" cellspacing="0" style="width:651px;" width="651">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">For rabbits</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aluminium foil</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Animal shaver</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black tape</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blue filter</td>
			<td style="width:149px;">Thorlabs MF475-35</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Body warm plate</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:149px;">Sony NEX-5R</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catheter</td>
			<td style="width:149px;">22G Vasofix Safety</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfictant</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescein sodium</td>
			<td style="width:149px;">Fluorescein Faure 10%</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glas plate</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Green filter</td>
			<td style="width:149px;">Thorlabs MF539-43</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incontinence pad</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infusion pump</td>
			<td style="width:149px;">Perfusor Secura</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine hydrochloride</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>any generic products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:149px;">25G</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ringer&#39;s Solution</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile sheets</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical instruments</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>micro forceps, micro scissor, blunt surgical scissor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical microscope</td>
			<td colspan="3">OPMI, Carl Zeiss AG, Oberkochen, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe 2ml, 5ml, 50ml</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tape</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Three-way-stopcock</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Torch light</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazin</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>any generic products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">For rats</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aluminium foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Animal shaver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blue filter</td>
			<td style="width:149px;">Thorlabs MF475-35</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Body warm plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:149px;">Sony NEX-5R</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfictant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescein sodium</td>
			<td style="width:149px;">Fluorescein Faure 10%</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Green filter</td>
			<td style="width:149px;">Thorlabs MF539-43</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incontinence pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine hydrochloride</td>
			<td></td>
			<td></td>
			<td>any generic products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medetomidine hydrochloride</td>
			<td></td>
			<td></td>
			<td>any generic products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:149px;">25G</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ringer&#39;s Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical instruments</td>
			<td></td>
			<td></td>
			<td>micro forceps, micro scissor, blunt surgical scissor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical microscope</td>
			<td colspan="3">OPMI, Carl Zeiss AG, Oberkochen, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe 2ml, 5ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">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 ...

This method is a reliable and easy technique for visualizing real-time blood flow in aneurysms and vessels and can be used in different animal models. The main advantage of this technique is that it facilitates visualization of blood flow during surgery without requiring any expensive devices or extensive surgery. Before beginning the procedure use black tape to secure a green bandpass filter to act as an excitation filter onto a flashlight.And check that the flashlight only emits blue light. Equip the camera with a green bandpass filter to function as an emission light filter. Only green light should now be able to pass through.Then, disinfect the work space with a disinfectant solution and cover the table with sterile drapes to prevent contamination. To perform fluorescein video angiography or FVA in a rabbit confirm that the anesthetized animal does not respond to pain stimulus before positioning the rabbit in the supine position. Shave the neck around the sternocleidomastoic muscle.Focus the camera on the dissected artery and use aluminum foil to cover a five milliliter syringe containing 100 milligrams/milliliter of fluorescein sodium. Turn off the lights as much as possible and inject 0.3 milliliters/kilogram of fluorescein sodium intravenously into the injection port of the catheter. Next, flush the syringe with 0.5 milliliters of saline solution to ensure that all of the dye has been injected.And immediately illuminate the surgical field with the modified flashlight. As soon as the light is turned on begin filming with the modified camera. Blood flow should be visible within a few seconds of the injection.For macroscopic analysis of the vessel after angiography resect the aneurysms and the parent artery complex in follow-up animals, or perform visual confirmation of flow by observing the filling of the aneurysm and/or bypass after removal of the temporary clips. In this representative study, FVA was able to be performed in eight out of 10 rabbits. Of the 16 aneurysms identified in the eight rabbits two aneurysms demonstrated persistent perfusion of the parent artery, while FVA identified five cases with residual perfusion.14 rabbit aneurysms exhibited no residual perfusion macroscopically, while 11 were subsequently detected using FVA. Residual perfusion was observed macroscopically in 23 of 48 rats, and the other 25 rats showed no macroscopic signs of residual perfusion. 22 of the 23 aneurysms without macroscopic perfusion were then confirmed using FVA.Overall, 25 of 27 rabbit and rat combined cases could be confirmed, resulting in a positive predictive value of 92.6%a sensitivity rate of 100%and a specificity of 94.1%The most important things when attempting this procedure are to dissect the vessels properly and to work in a darkened room when administering and visualizing fluorescein. This procedure can be used in any animal model to evaluate blood flow in any visible vessel.

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JoVE Journal

Neuroscience

6.9K vistas.  Kantonsspital Aarau. Este método es una técnica fiable y fácil para visualizar el flujo sanguíneo en tiempo real en aneurismas y vasos y se puede utilizar en diferentes modelos animales. La principal ventaja de esta técnica es que facilita la visualización del flujo sanguíneo durante la cirugía sin necesidad de ningún dispositivo caro o cirugía extensa. Antes de comenzar el procedimiento, utilice cinta negra para fijar un filtro de paso de banda verde para que actúe como un filtro de excitación en una...