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. 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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 injection enables dependable, spatially limited, highly concentrated staining, and requires small dye dosages13,14. Additionally, venous injection allows a quick clearance of fluorescein14,15. An alternative method is to inject contrast agent directly into the chosen artery. This method was not used in these experiments as the investigators wanted to prevent contaminating the surgical field with blood and fluorescein. In order to reduce this risk, peripheral venous contrast agent injection is recommended13.
Advantages of &#160;FVA are high contrast (easily detectable with the human eye), high sensitivity as shown above (Table 2), low cost and easy handling16. Fluorescein sodium was the chosen contrast agent to examine perfusion. Visible light alone can be used for excitation of the dye and emission of the typical green light. Nevertheless, this contrast agent works best with blue light (approximately 480 nm) and emits a strong green light (wavelength approximately 530 nm)15. According to Yoshioka et al., fluorescein colors the artery extremely quickly14. Furthermore, the flow of fluorescein-enriched blood can be observed in real time15,17. The short time needed for FVA presents another advantage; in this series it took an average of 2 min (&#177; 1min) to conduct one FVA.
The disadvantage of using fluorescein as a contrast agent is that it works well with only thin artery walls which demand very careful dissection. Ichikawa et al. showed the extinction of dye due to the thwarted emission of light through thicker walls by calcification or undissected arteries15. After injection, fluorescein is metabolised to fluorescent fluorescein glucuronide in the liver. Within 30 min after injection, the concentration of fluorescein glucuronide exceeds the concentration of fluorescein18. Fluorescein requires a long clearance time. An immediate reinjection after intravenous injection of fluorescein is not recommended as the artery and aneurysm are already fluorescent from the first injection17.
The molecular weight of fluorescein is only 376 kDa which allows leakage of the dye. The vascular wall also becomes fluorescent which could lead to false positive flow evaluations (increasing with time after application). A patchy coloration of the vessel wall was observed starting approximately 5 min after injection of fluorescein14. The spotty coloration, however, was only observed in larger arteries. Small and medium arteries did not show this staining structure17. It is recommended to evaluate the aneurysm immediately in order to detect residual filling.
Although there is a very low risk of toxicity, some cases of fluorescein leading to cardiac and respiratory reactions have been described14. In this study no severe adverse event occurred; the only complications were 2 cases of ear vein perforation. According to Lane et al., sodium fluorescein is not harmful even when used in humans17. On the other hand, fluorescein is quite unstable and should not be exposed to white light16&#160;- a red light source can be used instead.
In order to choose the concentration of fluorescein for rabbits, the investigators started with the lowest known working dose in rats (0.2 mL of 100 mg/mL fluorescein sodium) and increased it gradually to 1 mL. A strong fluorescence signal was registered at that dose. The dosage was increased gradually to test if the fluorescence improves - which was not the case. The authors decided to continue with 1 mL of 100 mg/mL fluorescein sodium13.
Another dye available to examine vessels intraoperatively is ICG. Its size is 775 kDa and such barely penetrates the surrounding tissues14. Because of its longer emission wavelengths, tissue is penetrated more easily because tissues are more transparent at 800 nm19 and deeper structures become visible14,16. Excitation wavelength within the 750-800 nm are required16,20 and the emission wavelength from the contrast agent is approximately 800 nm16, making both invisible to the human eye. Due to its short half-life time in blood plasma, the dye can be injected and reused repeatedly16. Limitations to using this dye include problems with thick-walled arteries20 and the necessity of light outside the visible spectrum13. As a consequence, ICG is dependent on expensive equipment and not readily applicable in every laboratory.
In conclusion, FVA is a fast, inexpensive and reliable method with high sensitivity to screen patency of aneurysms and parent arteries in rodent aneurysm models. It is associated with virtually no morbidity and mortality. It allows real-time blood flow monitoring during surgery and follow-up. To improve its efficacy, the injection should be carried out in the dark and is best performed on meticulously dissected vessels. This method can easily and safely be implemented in a cerebrovascular laboratory, and can minimize experiment costs.

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>
		<col />
		<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 performed in one rat due to difficulties puncturing the femoral vein.
Of 16 aneurysms in eight rabbits, two aneurysms showed persistent perfusion of the parent artery (confirmed macroscopically) (see Table&#160;1) while FVA identified five cases with residual perfusion. 14 rabbit aneurysms showed no residual perfusion macroscopically, however 11 (79%) were subsequently detected using FVA. Residual perfusion was observed macroscopically in 25 of 48 rats (Table&#160;1), and the other 23 rats showed no macroscopic signs of residual perfusion (Figure 2). 22 of those 23 aneurysms were then confirmed using FVA (96%). Altogether, 25 of 27 cases could be confirmed, resulting in a positive predictive value of 92.6%, a sensitivity rate of 100% and specificity of 94.1%. (Table&#160;2).
In summary, 25 aneurysms showed residual perfusion, 53 parent arteries were patent and 11 were occluded as confirmed macroscopically and on video angiography. There were only minor complications associated with FVA in rabbits; such as perforation of marginal ear vein during catheterization. No further adverse events were experienced. No mortality and no morbidity due to FVA was reported.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59782/59782fig1.jpg" /> 
Figure 1: Visualizing patency in a rabbit. (A) Patency of the parent artery is clearly visible on the fluorescence image (green emission from fluorescein are seen). (B) This artery is occluded (fluorescence image). Both arteries were inspected macroscopically (C-D). Panel (C) shows in the artery from panel A; the lumen is open. Panel (D) shows artery from panel B where occlusion can be seen macroscopically. Orange dotted lines mark the borders of the parent artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59782/59782fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59782/59782fig2.jpg" /> 
Figure 2: Visualizing perfusion in a rat. (A) This panel shows a residually perfused aneurysm (red dotted line marks the residual perfusion). (B) No perfusion can be detected. Panel (C) shows the artery from panel A during macroscopic scrutiny; the aneurysm orifice is open. (D) Macroscopic view of the neointima on an occluded aneurysm. Orange dotted lines mark the parent artery and the aneurysm&#8217; dome. Panels (A) and (B) are fluorescence-only images and the green color shows fluorescein emission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59782/59782fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td></td>
			<td>Patency/Residual Perfusion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Macroscopical +</td>
			<td>Macroscopical -</td>
			<td>Fluorescein +</td>
			<td>Fluorescein -</td>
		</tr>
		<tr>
			<td>Rabbits</td>
			<td>2</td>
			<td>14</td>
			<td>5</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Rats</td>
			<td>23</td>
			<td>25</td>
			<td>22</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>25</td>
			<td>39</td>
			<td>27</td>
			<td>32</td>
		</tr>
	</tbody>
</table>
Table 1: Patency testing. Patency of parent artery was only tested in rabbits and is illustrated here. Fluorescein detected more patencies of parent arteries than macroscopical evaluation. (All rats in this setting had an open parent artery, as aneurysms were sutured on the abdominal aorta.) The patency of aneurysms was tested in rats only. Twenty-two of 23 macroscopically detected patencies were confirmed using FVA. Twenty-one of 25 showed no patency on FVA.
<table>
	<tbody>
		<tr>
			<td></td>
			<td>Macroscopic positive</td>
			<td>Macroscopic negative</td>
			<td>total</td>
		</tr>
		<tr>
			<td>Fluorescein positive</td>
			<td>25</td>
			<td>2</td>
			<td>27</td>
		</tr>
		<tr>
			<td>Fluorescein negative</td>
			<td>0</td>
			<td>32</td>
			<td>32</td>
		</tr>
		<tr>
			<td>total</td>
			<td>25</td>
			<td>34</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Two-by-two table used to calculate specificity and sensitivity of FVA.

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K Views.  Kantonsspital Aarau. 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.

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations 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 ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...