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.remotexs.ntu.edu.sg/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><tbody><tr><td>Aluminum Crossed Roller XYZ Stage Center Drive Metric Threads with Fine Pitch Screw</td><td>Optosigma&amp;nbsp;</td><td>TAM-405CLFP</td><td>Part of frame (Step 1.1.)&amp;nbsp;</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&amp;nbsp;</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&amp;reg; Core&amp;trade; 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.)&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Olympus</td><td>RMS4X</td><td>Magnification lens (Step 1.4.)&amp;nbsp;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Medicine

335 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

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Neuroscience

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model for disease research and drug development. However, the commonly used four-vessel occlusion method mainly involves inserting an electrocoagulation pen into the bilateral pterygoid foramen of the first cervical vertebra behind the neck to electrocoagulate the vertebral arteries. This process carries the risk of incomplete electrocoagulation, bleeding, and damage to the brainstem and spinal cord. Twenty-four hours after surgery, re-anesthetized rats undergo carotid artery ligation in front of the neck. Two surgeries expose the rats to a higher risk of infection and increase the experimental period. In this study, during a single surgical procedure, an anterior cervical incision was used to locate the key site where the vertebral artery penetrates the first cervical vertebra. The bilateral vertebral arteries were electrocauterized under visual conditions, while the bilateral common carotid arteries were separated to place loose knots. When the limbs of the rats began twitching, the bilateral common carotid arteries were quickly ligated to induce ischemic coma. This method can avoid the risk of infection caused by two surgical operations and is easy to perform with a high success rate, providing a useful reference for relevant practitioners.

Author Spotlight: Enhancing Cerebral Ischemia Research with a Simplified Rat Model

This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model ...

Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

Source: Luo, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60783">Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation.</a> J. Vis. Exp. (2020)
This video demonstrates a procedure to induce tissue damage or infarction in the pons of an anesthetized rat using electrical stimulation.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

Source: B&#246;mers, J. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62567">Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

Source: B&#246;mers, J. et al. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. ...

Induction of Cerebral Arterial Gas Embolism in Rat

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Chapters in this video

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with ¹⁸F-FDG

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection