Name: a saline/bipolar radiofrequency energy device as an adjunct for hemostasis in solid organ injury/trauma
Uploaded: 2020-07-28T12:30:00
Description: Watch this Scientific Journal Video about A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma at JoVE.com

The authors have no acknowledgments.

Procedures involving animal subjects have been approved by the Animal Care Committee at the University of Calgary and follow the guidelines set by the Canadian Council of Animal Care. The committee ensures the study is ethical and that the animals are treated humanely.
1. Model Preparation
<ol>
	<li>House the 50 kg adult male pig in an animal care facility for 1 week prior to the surgery to acclimatize the animal to the housing conditions and the handlers. Fast the model for a minimum of 6 h...

<ol>
	<li>Kauvar, D. S., Lefering, R., Wade, C. E. <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+hemorrhage+on+trauma+outcome:+an+overview+of+epidemiology,+clinical+presentations,+and+therapeutic+considerations.">Impact of hemorrhage on trauma outcome: an overview of epidemiology, clinical presentations, and therapeutic considerations.</a> Journal of Trauma and Acute Care Surgery. 60 (6), S3-S11 (2006).</li><li>Shrestha, 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=Damage-control+resuscitation+increases+successful+nonoperative+management+rates+and+survival+after+severe+blunt+liver+injury.">Damage-control resuscitation increases successful nonoperative management rates and survival after severe blunt liver injury.</a> Journal of Trauma and Acute Care Surgery. 78 (2), 336-341 (2015).</li><li>Kozar, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trauma+Association/critical+decisions+in+trauma:+operative+management+of+adult+blunt+hepatic+trauma.">Trauma Association/critical decisions in trauma: operative management of adult blunt hepatic trauma.</a> Journal of Trauma and Acute Care Surgery. 71 (1), 1-5 (2011).</li><li>Peitzman, A. B., Richardson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+of+injuries+to+the+solid+abdominal+organs:+a+50-year+perspective+from+the+Journal+of+Trauma.">Surgical treatment of injuries to the solid abdominal organs: a 50-year perspective from the Journal of Trauma.</a> Journal of Trauma and Acute Care Surgery. 69 (5), 1011-1021 (2010).</li><li>Aloia, T. A., Zorzi, D., Abdalla, E. K., Vauthey, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-surgeon+technique+for+hepatic+parenchymal+transection+of+the+noncirrhotic+liver+using+saline-linked+cautery+and+ultrasonic+dissection.">Two-surgeon technique for hepatic parenchymal transection of the noncirrhotic liver using saline-linked cautery and ultrasonic dissection.</a> Annals of surgery. 242 (2), 172-177 (2005).</li><li>Ball, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+novel+energy+technology+for+arresting+ongoing+liver+surface+and+laceration+hemorrhage.">Use of a novel energy technology for arresting ongoing liver surface and laceration hemorrhage.</a> Canadian Journal of Surgery. 57 (4), E146 (2014).</li><li>Ball, C. G., 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=Use+of+a+novel+saline/bipolar+radiofrequency+energy+instrument+as+an+adjunct+for+arresting+ongoing+solid+organ+surface+and+laceration+bleeding+in+critically+injured+patients.">Use of a novel saline/bipolar radiofrequency energy instrument as an adjunct for arresting ongoing solid organ surface and laceration bleeding in critically injured patients.</a> Injury. 47 (9), 1996-1999 (2016).</li><li>Ball, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+a+novel+saline/bipolar+radiofrequency+energy+instrument+for+arresting+ongoing+solid+and+non-solid+organ+hemorrhage+in+a+swine+model.">The efficacy of a novel saline/bipolar radiofrequency energy instrument for arresting ongoing solid and non-solid organ hemorrhage in a swine model.</a> Injury. 47 (12), 2706-2708 (2016).</li><li>Swindle, M. M., Smith, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+for+performing+experimental+surgery+in+swine.">Best practices for performing experimental surgery in swine.</a> Journal of Investigative Surgery. 26 (2), 63-71 (2013).</li><li>Cantle, P. M., Roberts, D. J., Holcomb, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+Control+Resuscitation+Across+the+Phases+of+Major+Injury+Care.">Damage Control Resuscitation Across the Phases of Major Injury Care.</a> Current Trauma Reports. 3 (3), 238-248 (2017).</li><li>Gaarder, C., Naess, P. A., Buanes, T., Pillgram-Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+surgical+trauma+care+training+with+a+live+porcine+model.">Advanced surgical trauma care training with a live porcine model.</a> Injury. 36 (6), 718-724 (2005).</li><li>Harvin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+the+damage:+morbidity+and+mortality+after+emergent+trauma+laparotomy.">Control the damage: morbidity and mortality after emergent trauma laparotomy.</a> The American Journal of Surgery. 212 (1), 34-39 (2016).</li></ol>

The rapid and effective control of hemorrhage is an essential component of modern damage control resuscitation10. A variety of operative and adjunctive techniques are available to arrest hemorrhage in a solid organ injury3. None of these techniques has proven to be universally applicable or successful in achieving hemostasis. The initial experience with the SBRF device described here has been positive6,7,...

Uncontrolled hemorrhage due to solid organ injury remains a leading cause of morbidity and mortality in both blunt and penetrating trauma1. With the advent of effective damage control resuscitation strategies, the rate of non-operative management for abdominal trauma continues to increase2. As a result, patients requiring operative management have increasingly complex injuries and associated physiologic derangement. In these patients, early control of hemorrhage is an essential component of effective damage control resuscitation and desirable outcomes.
The surgical management of solid organ injuries remains a key competency for trauma, acute care, and general surgeons. A wide variety of surgical techniques and hemostatic adjuncts for these injuries have been described3. Traditional techniques for treating solid organ bleeding include coagulation by high-voltage electrocautery, application of topical hemostatic agents, sutured repairs, and partial or total organ excision. Argon beam coagulation has also been described4. While each of these techniques has a role in achieving hemostasis, none is universally applicable or successful.
Many novel tools and hemostatic therapies have been described in the elective surgical setting. This is especially true in the realm of hepatobiliary surgery5. As familiarity with these tools increases, many of them have also shown promise in the surgical management of traumatic injuries. One such device utilizes a combination of ignited saline and bipolar radiofrequency energy to arrest hemorrhage. Additionally, it has the ability to simultaneously seal small- to medium-sized bile ducts within the liver6. The positive experience with this tool in the management of solid organ injuries has been described previously6,7,8.
The goal of this publication is to demonstrate the potential application of this novel device using simulated solid organ injuries in a porcine model.

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			<td height="21" style="height:21px;width:256px;">Aquamantys pump generator</td>
			<td style="width:173px;">Medtronic</td>
			<td style="width:173px;">40-402-1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aquamantys 6.0 bipolar sealer</td>
			<td>Medtronic</td>
			<td>23-112-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrosurgical pencil with tip</td>
			<td>Megadyne</td>
			<td>0039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Porcine animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Porcine ventilator/induction and anesthetic medications</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 x 1 liter bags of 0.9% normal saline</td>
			<td></td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 x scalpels (#10)</td>
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			<td height="21" style="height:21px;">Belfour abdominal retractor</td>
			<td></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction tubing</td>
			<td></td>
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		</tr>
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			<td height="21" style="height:21px;">Suction tip</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction device/wall connector</td>
			<td></td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction canister</td>
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		</tr>
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			<td height="21" style="height:21px;">Debakey forceps</td>
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			<td height="21" style="height:21px;">Metz scissors</td>
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		</tr>
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			<td height="21" style="height:21px;">Curved Mayo scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Closing suture (1-0 Nylon)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 x Laparotomy sponges</td>
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		</tr>
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			<td height="21" style="height:21px;">2 x Kelley clamps</td>
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		</tr>
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			<td height="21" style="height:21px;">2 x snap clamps</td>
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		</tr>
	</tbody>
</table>

a saline/bipolar radiofrequency energy device as an adjunct for hemostasis in solid organ injury/trauma

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional techniques for arresting this ongoing bleeding include coagulation by high voltage electrocautery, topical hemostatic application, and the delivery of ignited argon gas. The goal of this study/video was to demonstrate the efficacy of a new energy device for arresting persistent solid organ hemorrhage.A novel instrument utilizing bipolar radiofrequency (RF) energy which acts to ignite/boil dripping saline from a simple handpiece is employed to arrest ongoing bleeding from solid organ injuries in a porcine model. This instrument is extrapolated from experience within elective hepatic resections. An escalating series of injuries to solid organs within a porcine model will be created. This will be followed by arresting hemorrhage with this novel energy device in sequence. A standard suction device will also be employed. This simple saline/RF energy instrument has the potential to arrest ongoing solid organ surface/capsular bleeding, as well as moderate hemorrhage associated with deep lacerations.

The SBRF device described herein provides effective hemostasis for a variety of solid organ injuries. The efficacy of the SBRF device in a porcine model has been described previously8. The results of this study are republished here with permission from the authors.
Using a porcine model, injuries of increasing severity were applied to four separate models. The injuries were described as surface decapsulat...

Watch this Scientific Journal Video about A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma at JoVE.com

A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma

The goal of this publication is to demonstrate the potential application of a novel device using simulated solid organ injuries in a porcine model.

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional ...

Life threatening solid organ hemorrhage can be extremely difficult to control. The application of a saline bipolar radio frequency instrument allows the rapid and effective regulation of significant solid organ hemorrhage. This technique is effective for the control of both bleeding, as well as biliary leakage.It's simple to learn, and can be rapidly and effectively performed with minimal formal training. The use of this technique is intuitive. It can be rapidly and effectively applied, even if the practitioner lacks extensive previous experience with the device or extensive knowledge of intro organ anatomy.When the animal is ready, open the hand piece of an ignited SBRF device prepared according to the manufacturer's specifications and connect the handpiece to the generator. Then set the saline flow rate to low and the radio frequency power setting to 160 watts. Establish an adequate exposure of the solid organs of interest, mobilizing other structures and inserting a retractor as necessary.Using a number 10 scalpel blade, apply an abrasive back and forth, one to two millimeter deep, two square centimeter area, superficial force to the liver capsule to induce capsular bleeding. Continue to create one centimeter deep, solid organ lacerations of increasing severity with the scalpel, from five centimeters to the entire length of the liver tissue. Then, using a blunt device such as a Kelly clamp, use a stabbing motion to create penetrating injuries of a partial to full thickness.After simulating all of the appropriate experimental injuries, depress the button of the handpiece to initiate the simultaneous flow of 0.9%saline and the delivery of bipolar radio frequency energy. The saline will boil at the site of application. Position the tip of the device directly onto the raw of the liver surface to superficial areas of bleeding or within defects in the liver itself, and apply concurrent suctioning from a standard surgical aspirator as needed to deliver the heated saline and energy directly to the areas of ongoing hemorrhage.Using a gentle back and forth motion, heat the tissues to approximately 100 degrees Celsius. An auditory pop will occur after three to five seconds. Signifying that the burn is complete.Then move the instrument in an organized manner to the next targeted site, applying precisely directed high voltage electrocautery in conjunction with the application of the SBRF and suction devices to obtain hemostasis as necessary. When all of the sites of the hemorrhage have been cauterized, apply the tip of the device across the injured edge of the liver parenchyma to seal the small to medium bile ducts. In this representative experiment, the depth of the tissue penetration by the SBRF device varied by target organ.Notably, no tissue coagulation was observed when the inferior vena cava was targeted, likely due to the heat sink effect from significant blood flow, which further supports the safety of the device usage around large vascular structures. Saline bipolar radio frequency devices are superb instruments for surgeons who must stop ongoing hemorrhage in challenging areas. They also facilitate primary hemostasis in solid organs without requiring any extensive solid organ anatomy knowledge.

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Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...