This research was funded by the Netherlands Military Healthcare Insurance Foundation (Stichting Ziektekosten Verzekering Krijgsmacht) under grant number 20-0232 and the Dutch Heart Foundation 2021 E. Dekker Grant (03-006-2021-T019 to IAM). We also thank Lindy Alles, Paul Bloemen, and Ed van Bavel for their outstanding assistance.&#160;

Methods for Inducing Cerebral Arterial Gas Embolism in Rats: ECA vs CCA Approach

<ol>
	<li>Mitchell, S. J., Bennett, M. H., Moon, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decompression+sickness+and+arterial+gas+embolism.">Decompression sickness and arterial gas embolism.</a> N Engl J Med. 386 (13), 1254-1264 (2022).</li><li>Bessereau, J., 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=Long-term+outcome+of+iatrogenic+gas+embolism.">Long-term outcome of iatrogenic gas embolism.</a> Intensive Care Med. 36 (7), 1180-1187 (2010).</li><li>Brown, A. E., Rabinstein, A. A., Braksick, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics,+imaging+findings,+and+outcomes+of+cerebral+air+embolism.">Clinical characteristics, imaging findings, and outcomes of cerebral air embolism.</a> Neurocrit Care. 38 (1), 158-164 (2023).</li><li>Weenink, R. P., Hollmann, M. W., Van Hulst, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+cerebral+arterial+gas+embolism.">Animal models of cerebral arterial gas embolism.</a> J Neurosci Methods. 205 (2), 233-245 (2012).</li><li>Rosengren, L., Persson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+blood-brain+barrier+leakage+to+Evans+blue-labelled+albumin+after+air+embolism+in+ethanol-intoxicated+rats.">Enhanced blood-brain barrier leakage to Evans blue-labelled albumin after air embolism in ethanol-intoxicated rats.</a> Acta neuropathologica. 38 (2), 149-152 (1977).</li><li>Furlow, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+air+embolism+of+the+brain:+An+analysis+of+the+technique+in+the+rat.">Experimental air embolism of the brain: An analysis of the technique in the rat.</a> Stroke. 13 (6), 847-852 (1982).</li><li>Gerriets, 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+rat+model+for+cerebral+air+microembolisation.">A rat model for cerebral air microembolisation.</a> J Neurosci Methods. 190 (1), 10-13 (2010).</li><li>Juenemann, M., 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=Impact+of+bubble+size+in+a+rat+model+of+cerebral+air+microembolization.">Impact of bubble size in a rat model of cerebral air microembolization.</a> J Cardiothorac Surg. 8, 198 (2013).</li><li>C Schaefer, 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=Investigation+of+experimental+endovascular+air+embolisms+using+a+new+model+for+the+generation+and+detection+of+highly+calibrated+micro+air+bubbles.">Investigation of experimental endovascular air embolisms using a new model for the generation and detection of highly calibrated micro air bubbles.</a> J Endovasc Ther. 30 (3), 461-470 (2022).</li><li>Bederson, J. B., 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=Rat+middle+cerebral+artery+occlusion:+Evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination.</a> Stroke. 17 (3), 472-476 (1986).</li><li>Schulte, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+biological+dyes+and+stains:+Pitfalls+and+possibilities.">Standardization of biological dyes and stains: Pitfalls and possibilities.</a> Histochemistry. 95 (4), 319-328 (1991).</li><li>Wayman, C., 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=Performing+permanent+distal+middle+cerebral+with+common+carotid+artery+occlusion+in+aged+rats+to+study+cortical+ischemia+with+sustained+disability.">Performing permanent distal middle cerebral with common carotid artery occlusion in aged rats to study cortical ischemia with sustained disability.</a> J Vis Exp. (108), e53106 (2016).</li><li>Themistoklis, K. 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We have described how to introduce air emboli in the rat cerebral arteries using two methods and have shown that introduction through a needle inserted in the CCA has multiple advantages over a method that involves embolization through a catheter into the ECA. Specifically, we observed fewer complications with the CCA-entry method, as well as a more consistent bubble diameter and reduced surgical time. The CCA-entry method results in dose-dependent CND, and abnormalities on MRI indicative of cerebral infarction as confirmed with histology.
The initial choice of the ECA-entry method was inspired by Gerriets et al.7. However, we identified several difficulties with this approach, including substantial variations in bubble size and a higher surgical complication rate compared to the CCA-entry method. A primary source of these complications is related to the catheter length. In our model, using a short catheter (125 mm) helped maintain the stability of the bubbles because the longer the catheter, the higher the probability that bubbles merge while flowing through the catheter9. However, in the ECA-entry method, a longer catheter facilitates easier placement and leverage for movement. Using a short catheter in the ECA-entry method results in frequent dislocation and deterioration of the ECA stump due to excessive manipulation.
A second difficulty encountered in the ECA-entry method regarded the creation of consistently sized bubbles (Figure 6). In the ECA-entry method, the flow of saline through the catheter has to be temporarily halted while the catheter is inserted into the ECA. When the arterial flow is restored and the embolization can start, the catheter is suddenly subjected to the blood pressure of the rat. As a result, this leads to blood retrogradely entering the catheter and bubble generator. The effect of the fluctuating blood pressure on the pressure inside the bubble generator leads to a higher variation in bubble size, sometimes leading to cylindrical-shaped bubbles filling up the channel (Figure 6B). This can be avoided by increasing the pressure within the system before positioning the catheter in the ECA. This is best done by a second person for precise timing. Furthermore, since this method is more time-consuming, it leads to a larger amount of saline being infused into the rat than the CCA-entry method. In the CCA-entry method, saline is continuously flowing through the catheter, and the needle is inserted in the direction of the blood flow through the CCA, thereby solving the pressure gradient issue described above. This results in the absence of backflow into the catheter and a more uniform bubble size.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67397/67397fig06.jpg" /> 
Figure 6: Example recording and analysis of bubble production. Images display screen grabs showing the real-time analysis of the total number of bubbles, total volume, and average diameter in the measurement area (green, upper left corner). The measurement area is highlighted between the orange lines on the right. The bubble diameter and volume are calculated based on the horizontal diameter. Images include the formation of (A) successfully generated air bubbles and (B) unsuccessfully generated cylindrical bubbles. <a href="https://www.jove.com/files/ftp_upload/67397/67397fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Despite the CCA-entry method being a favorite, we still encountered various technical difficulties. Firstly, preparation of the PPA is challenging, since accidental compression of the vagus nerve can result in depression of breathing and subsequent death of the animal12. To reduce this risk, one should tilt the rat slightly sideways and approach the PPA from the cranial direction. In addition, due to the challenging anatomy of the ICA and PPA bifurcation, there is a risk of vascular damage and uncontrollable bleeding. This can only be circumvented by improving surgical skills. These challenges highlight that mastering the CAGE model in rats is complex and requires significant practice and precision13.
The proposed technical setup of the air bubble generator has its limitations, particularly related to the custom-made glass capillaries, because of their fragility. The tubing that connects the pneumatic regulator to the capillary is prone to breakage when adjusting the three-way valve for pressure release after embolization. Furthermore, replacing a capillary requires destroying the existing capillary due to the heat-shrink rubber being permanently attached to it. Additionally, each capillary has unique bubble characteristics due to minor differences in tip diameter and shape. Lastly, the manual operation of the bubble generation via the pneumatic regulator required significant experience. Inexperienced handling can result in the production of excessively large bubbles. Automated pressure regulation with a feedback loop from the Python code could enhance automated precision in future studies.
Our thorough documentation, encompassing both the technical specification of the bubble generator, a detailed surgical protocol, and providing the software, makes an important contribution to this field of research. Our CCA technique ensures minimal disruption to physiological cerebral perfusion, by maintaining CCA flow throughout the procedure and abolishing the need to sacrifice the ECA. Our study provides a reliable and reproducible experimental model to investigate CAGE and its potential treatments.

Cerebral arterial gas embolism (CAGE) is characterized by the lodging of gas bubbles within the cerebral arterial circulation, leading to a spectrum of neurological impairments. This condition is predominantly known as a complication in diving, where overexpansion of the lungs during ascent results in barotrauma and air entry into the pulmonary veins, which then flow to the cerebral arteries1. In addition to this well-known occurrence in diving, medical professionals are increasingly recognizing CAGE as a complication of invasive medical procedures. Iatrogenic air embolism can occur during the placement, handling, or removal of central venous catheters and chest drains, as well as throughout the course of open and endovascular procedures, including heart valve interventions, thoracic endovascular aortic repairs and endovascular thrombectomy in ischemic stroke2,3. Despite its clinical significance, the research on CAGE, particularly using animal models, remains sparse and fragmented4.
Since the pioneering study by Rosengren et al. in 1977 with rats, animal models for CAGE have undergone significant refinement5. The approach employed by Rosengren involved cannulation of the common carotid artery (CCA) to introduce a total volume of 10 &#181;L of air. This technique was not without limitations, including altered hemodynamics due to arterial ligation and the uncontrolled size and excessive volume of the air embolus6. Furlow&#39;s method, described in 1982, improved the precision of air embolization by advancing a catheter into the internal carotid artery and administering a total air volume of 5 &#181;L. However, although its importance was recognized early, the concept of uniform bubble size was only implemented decades later. Gerriets et al. were able to produce a consistent number of bubbles with a uniform diameter, initially 160 &#181;m, later reduced to 45 &#181;m7,8. The surgical method used here required sacrificing the external carotid artery (ECA). Recently, Schaefer et al. introduced a less invasive method by inserting a microcatheter into the CCA through the femoral artery, more accurately mimicking air embolism scenarios seen during endovascular procedures9. Their method had the limitation of not ligating the arterial branches of the CCA (for example, ECA and pterygopalatine artery (PPA)), thereby allowing bubbles to not only flow to the desired cerebral arteries but also to non-cerebral territories. This may result in inconsistent cerebral ischemic damage, complicating the reproducibility of experiments.
Despite the advancements in preclinical CAGE models, challenges remain in replicating bubble generation techniques, standardizing surgical methods, and acquiring consistent cerebral lesions. The current study introduces both a conventional surgical approach that requires sacrificing the ECA and an alternative method in which air bubbles are injected directly into the CCA. We report detailed procedures, challenges, and open-source software for real-time bubble analysis. We also include the technical details needed to build a bubble generator.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum Crossed Roller XYZ Stage Center Drive Metric Threads with Fine Pitch Screw</td>
			<td>Optosigma&#160;</td>
			<td>TAM-405CLFP</td>
			<td>Part of frame (Step 1.1.)&#160;</td>
		</tr>
		<tr>
			<td>Basler Ace - acA2440-35um</td>
			<td>Basler AG</td>
			<td>107208</td>
			<td>High speed camera (Step 1.4.)</td>
		</tr>
		<tr>
			<td>Bupivacaine 2.5 mg/ml</td>
			<td>Aurobindo Pharma B.V.</td>
			<td>RVG20949</td>
			<td>Medication perioperative (Step 3.8.3.)</td>
		</tr>
		<tr>
			<td>Buprenorfine 0.3 mg/ml</td>
			<td>Indivior</td>
			<td>112515</td>
			<td>Medication perioperative (Step 3.2.1.)</td>
		</tr>
		<tr>
			<td>Custom glass chamber</td>
			<td>Technoglas Lab. App. B.V.</td>
			<td>-</td>
			<td>Custom made (Step 1.3.1.)</td>
		</tr>
		<tr>
			<td>Duratears</td>
			<td>Alcon</td>
			<td>-</td>
			<td>Artificial tears (Step 3.2.5.)</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Aesculap</td>
			<td>GT416-VR</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-Pneumatic Regulator - ITV0010-3L</td>
			<td>SMC</td>
			<td>ITV0010-3L</td>
			<td>Pneumatic regulator of bubble generator (Step 1.2.)</td>
		</tr>
		<tr>
			<td>GC100T-15 thin wall W/O filament 1.0mmOD</td>
			<td>Multi Channel Systems</td>
			<td>300036</td>
			<td>Borosilicate glass capillaries (Step 1.5.)</td>
		</tr>
		<tr>
			<td>Graphpad Tool: www.graphpad.com/quickscalcs/randomize1/</td>
			<td>Dotmatics</td>
			<td>-</td>
			<td>Randomly assign subjects to treatment groups&#160;</td>
		</tr>
		<tr>
			<td>Heatshrink rubber</td>
			<td>Pro-POWER</td>
			<td>1190988</td>
			<td>Holds capillary and pneumatic tubing in place (Step 1.2.)</td>
		</tr>
		<tr>
			<td>Isoflurane 1000 mg/g</td>
			<td>Laboratorios Karizoo S.A.</td>
			<td>118938</td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Dell</td>
			<td>-</td>
			<td>12th Gen Intel&#174; Core&#8482; i5-1235U 1.30 GHz, 16.0 GB ram, Windows 10</td>
		</tr>
		<tr>
			<td>Light source station with two dual white LED and goosenecks</td>
			<td>Euromex Microscopen B.V.</td>
			<td>LE.5212</td>
			<td>Led light source (Step 1.4.)</td>
		</tr>
		<tr>
			<td>Micro forceps bent</td>
			<td>Aesculap</td>
			<td>BD329R</td>
			<td>(Step 3.3.2.)</td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>Silber</td>
			<td>GU1870</td>
			<td>For inserting needle in CCA (Step 3.7.3.)</td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>HEBU medical</td>
			<td>HB7384</td>
			<td>Vascular scissor (Step 3.6.3)</td>
		</tr>
		<tr>
			<td>Micro vascular clip</td>
			<td>Biemer</td>
			<td>FD562R</td>
			<td>(Step 3.6.1.)&#160;</td>
		</tr>
		<tr>
			<td>Microlance 3 (21G, 27G and 30G)</td>
			<td>BD Medical</td>
			<td>304000</td>
			<td>(Step 1.3.2.)</td>
		</tr>
		<tr>
			<td>Mosquito artery clamp</td>
			<td>Aesculap</td>
			<td>BH105R</td>
			<td>(Step 3.4.3.)</td>
		</tr>
		<tr>
			<td>NexiusZoom</td>
			<td>Euromex Microscopen B.V.</td>
			<td>NZ.1903-B</td>
			<td>Microscope for surgery (Step 3.3.)</td>
		</tr>
		<tr>
			<td>Narishige PB-7</td>
			<td>Narishige Group</td>
			<td>-</td>
			<td>Micropipette puller (Step 1.5.1.)</td>
		</tr>
		<tr>
			<td>Optomechanical mounts, adapter and post assemblies&#160;</td>
			<td>Thor Labs</td>
			<td>-</td>
			<td>Various parts to hold the bubble generator body in static position (Step 1.1.)</td>
		</tr>
		<tr>
			<td>PE-10 tubing</td>
			<td>Intramedic</td>
			<td>427401</td>
			<td>Catheter (Step 1.3.2.)</td>
		</tr>
		<tr>
			<td>Perfusor Space</td>
			<td>B.Braun</td>
			<td>8713030</td>
			<td>Syringe pump (Step 1.6.1.)</td>
		</tr>
		<tr>
			<td>Plan Achromat Objective, 0.10 NA, 18.5 mm WD 4X&#160;</td>
			<td>Olympus</td>
			<td>RMS4X</td>
			<td>Magnification lens (Step 1.4.)&#160;</td>
		</tr>
		<tr>
			<td>Python</td>
			<td>Python Software Foundation</td>
			<td>-</td>
			<td>Version 3.11.2 (Step 2.2.1.)</td>
		</tr>
		<tr>
			<td>Pylon viewer</td>
			<td>Basler AG</td>
			<td>-</td>
			<td>Version 7.4.0 (Step 2.1.1.)</td>
		</tr>
		<tr>
			<td>Rubber O-RING 1 x 1 mm silicone</td>
			<td>Op den Velde Industrie B.V.</td>
			<td>99002887</td>
			<td>Prevents leakage of saline (Step 1.3.3.)</td>
		</tr>
		<tr>
			<td>Rubber O-RING 6 x 1 mm silicone</td>
			<td>Op den Velde Industrie B.V.</td>
			<td>99002886</td>
			<td>Holds glass chamber in place (Figure 2.)</td>
		</tr>
		<tr>
			<td>Rodent Warmer X1 with Rat Heating Pad and Rectal Probe</td>
			<td>Stoelting</td>
			<td>53800R</td>
			<td>Heating pad (Step 3.1.2.)</td>
		</tr>
		<tr>
			<td>Skeleton Fine Forceps</td>
			<td>Hoskins</td>
			<td>2710-B-2074</td>
			<td>(Step 3.3.2.)</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Charles River Laboratory</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of cerebral arterial gas embolism in rat

We present a methodological approach for preclinical research of cerebral arterial gas embolism (CAGE), a condition characterized by gas bubbles within the cerebral circulation causing multifocal ischemia. The current work describes two surgical methods to induce CAGE in the rat: one with injection of air via the external carotid artery (ECA), thereby sacrificing the vessel, and one via direct injection into the common carotid artery (CCA). Male Wistar rats were used and divided into groups (n=5) to undergo either the ECA- or CCA-entry method with injection of different air emboli volumes (6000, 7000 and 8000 nL) or sham surgery. A custom-made bubble generator was used to produce gas emboli with consistent size, and open-source software was developed for real-time bubble analysis. The comparison between the two methods revealed the CCA approach to be superior in terms of consistent bubble production within the bubble generator, reduced embolization time and fewer complications.

Neurological outcome 
Table 1 gives an overview of all inclusions and exclusions across the different experimental groups. None of the sham-operated rats showed any CND. In the ECA-CAGE group (8000 nL), two out of five rats did not exhibit CND, while the three remaining rats experienced CND, of which two died within 24 h. In the CCA-CAGE group (8000 nL) all animals showed CND, three of five did not survive up to 48 h. In the CCA-CAGE groups with lower volumes of air, all rats survived postoperatively. All five rats in the 7000 nL group demonstrated CND, whereas in the 6000 nL group, two out of five rats showed CND.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3"></td>
			<td colspan="2">ECA</td>
			<td colspan="4">CCA</td>
		</tr>
		<tr>
			<td colspan="3"></td>
			<td>sham</td>
			<td>8000 nL</td>
			<td>sham</td>
			<td>8000 nL</td>
			<td>7000 nL</td>
			<td>6000 nL</td>
		</tr>
		<tr>
			<td colspan="3">Included</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td colspan="3">Exhibited clinical neurological deficits</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>5</td>
			<td>5</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="3">Mortality &#60; 24 h</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="3">Mortality 24-48 h</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="3">Excluded total</td>
			<td>3</td>
			<td>4</td>
			<td>1</td>
			<td>0</td>
			<td>2</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Excluded due to bleeding complication</td>
			<td>2</td>
			<td>3</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="3">Excluded due to thrombotic complication</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Intraoperative death due to vagal nerve compression</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Animal group inclusions and exclusion. The number of rats in each group, inclusions and exclusions, deaths, and survival with clinical neurological deficits.
MRI 
Figure 4&#160;shows a representative T2-weighted image of a rat that received 7000 nL air bubbles through the CCA-entry method, showing cortical hyperintensities. Similar abnormalities were seen in all animals in the CCA-CAGE group that received 7000 or 8000 nL, and to a lesser extent in the 6000 nL group and the ECA-CAGE group. Notably, while none of the animals in the sham groups showed any CND, one ECA-sham rat exhibited an area of hyperintensity on MRI; in the CCA-sham group, no rats displayed abnormalities on MRI.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67397/67397fig04.jpg" /> 
Figure 4: MRI example images. Representative T2-weighted MRI images (3 days post CAGE surgery) showing cortical hyperintensities due to CAGE in a rat of the CCA-CAGE 7000 nL group. <a href="https://www.jove.com/files/ftp_upload/67397/67397fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histology 
Figure 5&#160;shows a representative H&#38;E-stained brain section of the 7000 nL CCA-CAGE rat from Figure 4, demonstrating cortical ischemic brain damage with neuronal cell loss and reactive gliosis, including reactive astrogliosis and microglial activation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67397/67397fig05.jpg" /> 
Figure 5: Post-mortem histology. Representative H&#38;E staining of the rat from Figure 4 showing cortical tissue of the contra-lesional side with (A) intact neurons and (B) the ipsi-lesional side with ischemic cortical tissue with neuronal cell loss and reactive gliosis (arrows). <a href="https://www.jove.com/files/ftp_upload/67397/67397fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Technical challenges 
Due to technical challenges, the ECA-entry method had a substantially lower success rate compared to the CCA-entry method (Table 1). This predominantly stemmed from the short catheter length required in the ECA-entry method, which frequently resulted in catheter dislocation and bleeding. Additionally, the ECA-entry method also resulted in an approximately 20 min longer surgery time, as well as a larger variation in bubble diameter.
Supplementary Figure 1: Details of the air bubble generator. (A) Images of the body of the bubble generator and the capillary puller highlight their assembly and functional aspects. (B) Lateral view of the bubble generator, showing its design and structural features from a side perspective. (C) Frontal view of the bubble generator, illustrating the key frontal aspects and features. <a href="https://www.jove.com/files/ftp_upload/67397/JoVE_Supplementary figure1_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Steps for running the software. This file provides a detailed guide on the procedures and steps to be followed for effectively running and utilizing the software associated with the air bubble generator. <a href="https://www.jove.com/files/ftp_upload/67397/JoVE_Supplementary figure 2_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Python code. Code consists of the two scripts (A and B) that should be saved within the same folder. <a href="https://www.jove.com/files/ftp_upload/67397/JoVE_Supplementaryfile1_Revised.zip" target="_blank">Please click here to download this File.</a>

Induction of Cerebral Arterial Gas Embolism in Rat

This protocol provides a detailed description of inducing cerebral air emboli in rats. It compares direct injection into the common carotid artery and introduction through the external carotid artery. It provides a technical description of the air bubble generator, the effect of different air volumes, and procedural challenges.

We present a methodological approach for preclinical research of cerebral arterial gas embolism (CAGE), a condition characterized by gas bubbles within ...

induction-of-cerebral-arterial-gas-embolism-in-rat

Research

JoVE Journal

Medicine

333 Views.  Amsterdam UMC. This protocol provides a detailed description of inducing cerebral air emboli in rats. It compares direct injection into the common carotid artery and introduction through the external carotid artery. It provides a technical description of the air bubble generator, the effect of different air volumes, and procedural challenges.

Video: Induction of Cerebral Arterial Gas Embolism in Rat

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 &mu;m &plusmn; 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 &plusmn; 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.