Name: fluorescence angiography for evaluation of aneurysm perfusion and parent artery patency in rat and rabbit aneurysm models
Uploaded: 2019-07-24T14:00:00
Description: 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

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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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. 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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">
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	<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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Research

JoVE Journal

Neuroscience

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 ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

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 ...