Name: integrated compensatory responses in a human model of hemorrhage
Uploaded: 2016-11-20T15:00:00
Description: Watch this Scientific Journal Video about Integrated Compensatory Responses in a Human Model of Hemorrhage at JoVE.com

This work is supported by funding from the United States Army, Medical Research and Materiel Command, Combat Casualty Care Program.&#160;We thank LTC Kevin S. Akers, MD and Ms. Kristen R. Lye for their assistance in making the video.

Prior to any human procedure, the institutional&#160;review board (IRB) must approve the protocol. The protocol used in this study was approved by the US Army Medical Research and Materiel Command IRB. The protocol is designed to demonstrate the physiological responses of compensation to a progressive reduction in central blood volume similar to that experienced by individuals during ongoing hemorrhage in a controlled and reproducible laboratory setting. The laboratory room temperature is controlled at 23 - 25 &#730;C.
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<ol>
	<li>Convertino, V. A., Wirt, M. D., Glenn, J. P., Lein, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+compensatory+reserve+for+early+and+accurate+prediction+of+hemodynamic+compromise:+a+review+of+the+underlying+physiology.">The compensatory reserve for early and accurate prediction of hemodynamic compromise: a review of the underlying physiology.</a> Shock. 45 (6), 580-590 (2016).</li><li>Eastridge, B. 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=Death+on+the+battlefield+(2001-2011):+Implications+for+the+future+of+combat+casualty+care.">Death on the battlefield (2001-2011): Implications for the future of combat casualty care.</a> Journal of Trauma and Acute Care Surgery. 73 (6), S431-S437 (2012).</li><li>Orlinsky, M., Shoemaker, W., Reis, E. D., Kerstein, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+body+negative+pressure+as+a+model+to+study+progression+to+acute+hemorrhagic+shock+in+humans.">Lower body negative pressure as a model to study progression to acute hemorrhagic shock in humans.</a> J. Appl. Physiol. 96, 1249-1261 (2004).</li><li>Convertino, V. 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=Inspiratory+resistance+maintains+arterial+pressure+during+central+hypovolemia:+implications+for+treatment+of+patients+with+severe+hemorrhage.">Inspiratory resistance maintains arterial pressure during central hypovolemia: implications for treatment of patients with severe hemorrhage.</a> Crit Care Med. 35 (4), 1145-1152 (2007).</li><li>Carter, R., Hinojosa-Laborde, C., Convertino, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+integration+of+mechanisms+associated+with+high+tolerance+to+progressive+reductions+in+central+blood+volume:+the+compensatory+reserve.">Variability in integration of mechanisms associated with high tolerance to progressive reductions in central blood volume: the compensatory reserve.</a> Physiol Reports. 4 (1), (2016).</li><li>Convertino, V. A., Sather, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasoactive+neuroendocrine+responses+associated+with+tolerance+to+lower+body+negative+pressure+in+humans.">Vasoactive neuroendocrine responses associated with tolerance to lower body negative pressure in humans.</a> Clin. Physiol. 20, 177-184 (2000).</li><li>Convertino, V. 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=Use+of+advanced+machine-learning+techniques+for+noninvasive+monitoring+of+hemorrhage.">Use of advanced machine-learning techniques for noninvasive monitoring of hemorrhage.</a> J. Trauma. 71 (1 Suppl), S25-S32 (2011).</li><li>Convertino, V. A., Rickards, C. A., Ryan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+mechanisms+associated+with+heart+rate+and+vasoconstrictor+reserves.">Autonomic mechanisms associated with heart rate and vasoconstrictor reserves.</a> Clin. Auton. Res. 22, 123-130 (2012).</li><li>Rickards, C. A., Ryan, K. L., Cooke, W. H., Convertino, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+to+central+hypovolemia:+the+influence+of+oscillations+in+arterial+pressure+and+cerebral+blood+velocity.">Tolerance to central hypovolemia: the influence of oscillations in arterial pressure and cerebral blood velocity.</a> J. Appl. Physiol. 111 (4), 1048-1058 (2011).</li><li>Johnson, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductions+in+central+venous+pressure+by+lower+body+negative+pressure+of+blood+loss+elicit+similar+hemodynamic+responses.">Reductions in central venous pressure by lower body negative pressure of blood loss elicit similar hemodynamic responses.</a> J. Appl. Physiol. 117, 131-141 (2014).</li><li>van Helmond, N., 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=Coagulation+Changes+during+Lower+Body+Negative+Pressure+and+Blood+Loss+in+Humans.">Coagulation Changes during Lower Body Negative Pressure and Blood Loss in Humans.</a> Am. J. Physiol. Heart Circ. Physiol. 309, H1591-H1597 (2015).</li><li>Gerhardt, R., Berry, J., Blackbourne, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+life-saving+interventions+performed+by+out-of-hospital+combat+medical+personnel.">Analysis of life-saving interventions performed by out-of-hospital combat medical personnel.</a> J. Trauma. 71, S109-S113 (2011).</li><li>Pinsky, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+evaluation+and+monitoring+in+the+ICU.">Hemodynamic evaluation and monitoring in the ICU.</a> Chest. 132 (6), 2020-2029 (2007).</li><li>Rivers, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+goal-directed+therapy+in+the+treatment+of+severe+sepsis+and+septic+shock.">Early goal-directed therapy in the treatment of severe sepsis and septic shock.</a> N.Engl.J.Med. , 1368-1377 (2001).</li><li>Rivers, E. 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Using LBNP to cause progressive and continuous reductions in central blood volume, we were able to induce a typical response of hemodynamic decompensation in the subject, characterized by a sudden onset of hypotension and bradycardia (Figure 7). It is important to understand that the integrated compensatory response to hemorrhage is very complex,19 resulting in significant individual variability in the tolerance to blood loss.1 As such, some individuals have relatively responsive co...

The most important function of the cardiovascular system is the control of adequate perfusion (blood flow and oxygen delivery) to all tissues of the body through homeostatic regulation of arterial blood pressure. Various mechanisms of compensation (e.g., autonomic nervous system activity, cardiac rate and contractility, venous return, vasoconstriction, respiration) contribute to maintain normal physiological levels of oxygen in the tissues.1 Reductions in circulating blood volume such as those caused by hemorrhage can compromise the ability of cardiovascular compensatory mechanisms and ultimately lead to low arterial blood pressure, serious tissue hypoxia, and circulatory shock that can be fatal.
Circulatory shock caused by severe bleeding (i.e., hemorrhagic shock) is a leading cause of death due to trauma.2 One of the most challenging aspects of preventing a patient from developing shock is our inability to recognize its early onset. Early and accurate assessment of the progression toward the development of shock is currently limited in the clinical setting by technologies (i.e., medical monitors) that provide measurements of vital signs that change very little in the early stages of blood loss because of the body&#39;s numerous compensatory mechanisms for regulating blood pressure.3-6 As such, the capability to measure the sum total of the body&#39;s reserve to compensate for blood loss represents the most accurate reflection of tissue perfusion state and the risk of developing shock.1 This reserve is called the compensatory reserve which can be accurately assessed by real-time measurements of changes in features of the arterial waveform.1 Depletion of the compensatory reserve replicates the terminal cardiovascular instability observed in critically ill patients with sudden onset of hypotension; a condition known as hemodynamic decompensation.7
The relationship between the utilization of the compensatory reserve and regulation of blood pressure during ongoing blood loss in humans can be demonstrated in the laboratory using a comprehensive set of physiological measurements (e.g., blood pressures, heart rate, arterial blood oxygen saturation, stroke volume, cardiac output, vascular resistance, respiration rate, pulse character, mental status, end-tidal CO2, tissue oxygen) provided by standard physiological monitoring during continuous progressive reductions in central blood volume similar to those that occur during hemorrhage. Lowered central blood volume can be induced noninvasively with progressive increases in Lower Body Negative Pressure (LBNP).8 Using this combination of physiological measurements and LBNP, the conceptual understanding of how to assess the body&#39;s ability to compensate for reduced central blood volume can easily be demonstrated. This study depicts the prelab preparation, the demonstration of compensatory response in relation to other physiological responses during simulated hemorrhage, and the postlab evaluation of results. The experimental techniques necessary for making measurements of compensatory reserve are demonstrated in a human volunteer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynamic Research Evaluation Workstation (DREW) data acquisition syetem</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom Built by ISR personnel.&#160; The DREW allows for time synchronization of both digital and analog signal data collection from up to 16 independent instruments with a sampling rate of 1000 Hz.</td>
		</tr>
		<tr>
			<td>Finometer</td>
			<td>Finapress Medical Systems (FMS)</td>
			<td>Model 1</td>
			<td>Device that provides non-invasive, continuous measurements of brachial artery blood pressure and arterial oxygen saturation (SpO2) using two separate infrared finger photophlethymography cuff sensors.</td>
		</tr>
		<tr>
			<td>BCI Capnocheck Plus</td>
			<td>Smith Medical PM Inc.</td>
			<td>9004</td>
			<td>Capnograph used to measure&#160; end tidal CO2 and respiration rate</td>
		</tr>
		<tr>
			<td>CipherOX&#160;</td>
			<td>Flashback Technologies Inc.</td>
			<td>R200</td>
			<td>Investigational device used to calculate Compensatory Reserve Index (CRI)</td>
		</tr>
		<tr>
			<td>Nonin 9560 Pulse Oximeter</td>
			<td>Nonin</td>
			<td>9560</td>
			<td>finger pulse oximeter</td>
		</tr>
		<tr>
			<td>Lower Body Negative Pressure Chamber (LBNP)</td>
			<td>NASA</td>
			<td>79K32632-1</td>
			<td>Custom Chamber built by NASA</td>
		</tr>
		<tr>
			<td>ECG Biotach</td>
			<td>Gould</td>
			<td>13-6615-65</td>
			<td>Electrocardiograph for measuring ECG</td>
		</tr>
		<tr>
			<td>Nasal CO2 Sample Line</td>
			<td>Salter Labs</td>
			<td>REF 4000</td>
			<td>Latex free nasal cannula for sampling expired air</td>
		</tr>
	</tbody>
</table>

integrated compensatory responses in a human model of hemorrhage

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of hemorrhaging patients is difficult because current clinical tools provide measures of vital signs that remain stable during the early stages of bleeding due to compensatory mechanisms. Consequently, there is a need to understand and measure the total integration of mechanisms that compensate for reduced circulating blood volume and how they change during ongoing progressive hemorrhage. The body&#39;s reserve to compensate for reduced circulating blood volume is called the &#39;compensatory reserve&#39;. The compensatory reserve can be accurately evaluated with real-time measurements of changes in the features of the arterial waveform measured with the use of a high-powered computer. Lower Body Negative Pressure (LBNP) has been shown to simulate many of the physiological responses in humans associated with hemorrhage, and is used to study the compensatory response to hemorrhage. The purpose of this study is to demonstrate how compensatory reserve is assessed during progressive reductions in central blood volume with LBNP as a simulation of hemorrhage.

The LBNP procedure causes a reduction in air pressure around the lower torso and legs. As this vacuum is progressively increased, blood volume shifts from the head and upper torso to the lower body to create a state of central hypovolemia. The progressive reduction in central blood volume (i.e., LBNP) produces significant alterations in the features of the arterial waveform measured with the infrared finger photoplethysmograph (Figure 5). The Compensatory Reserve...

Watch this Scientific Journal Video about Integrated Compensatory Responses in a Human Model of Hemorrhage at JoVE.com

Integrated Compensatory Responses in a Human Model of Hemorrhage

The purpose of this protocol is to demonstrate the techniques for measuring compensatory responses to reduced central blood volume using lower body negative pressure as a noninvasive experimental model of human hemorrhage which can be used to quantify the total integration of compensatory mechanisms to blood volume deficit in humans.

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of ...

The overall goal of this procedure is to simulate central hypovolimia associated with hemorrhage in humans using lower-body negative pressure and to assess the individual's capacity to compensate during this challenge. Lower-body negative pressure is a valid model of human hemorrhage, which has been used to identify early predictors of hemorrhagic shock. The main advantages of this procedure are that all measurements are acquired by non-invasive methods and that tolerance to hemorrhage can be safely assessed in the subjects.Using lower-body negative pressure, we developed the measurement of compensatory reserve which can provide an early assessment of the progression towards shock during hemorrhage and is more accurate than the current tools to monitor vital signs. Compensatory reserve reflects the integration of all physiological compensatory mechanisms involved in the response to hemorrhage in an individual patient and therefore tracks the progression to shock with a high degree of specificity. Prior to the study visit, instruct the subject to avoid caffeine, alcohol, and strenuous exercise 24 hours prior to testing and to avoid eating at least two hours prior in the even that hemodynamic decompensation induces nausea.On the study day, begin by turning on all the equipment that requires warm-up and calibration:a data acquisition system, two infrared finger sensors, a capnograph, and a finger pulse oximeter. Synchronize all of the instruments with internal clocks by adjusting the timestamp on each instrument to match a lab master-clock that should be used to mark time during the experiment. Next, escort the subject into an exam room and perform a medical screening exam to ensure that the subject meets minimal health requirements as well as all inclusion criteria.Then, bring the subject into the testing room, inform them about the experimental procedure, and obtain written consent to participate in the study. Next, place the neoprene lower-body negative pressure, or LBNP, skirt on the subject. Insure that the skirt is snug around the waist and torso in order to create an airtight seal.Instruct the subject to lay supine on the bed of the LBNP chamber while straddling a stationary post to secure the torso in place. Then, ask the subject to relax their lower body. Slide the bed into the chamber and attach the neoprene skirt to the chamber opening in order to create an airtight seal.Next, place electrocardiogram, or ECG, electrodes on the right and left humeral clavicular joints and on the right and left lower ribs in a modified lead to configuration for continuous measurement of heart rate. Position the subject's arms on the arm rests, adjusted so that the hands are supported at heart level. Place an infrared finger photoplethysmography device on the left and right middle fingers for continuous non-invasive beat to beat measurement of blood pressure.Enter subject information to enable the appropriate assumptions for calculation of stroke volume, cardiac output, and peripheral vascular resistance by the model flow algorithm. Then, attach the finger cuffs to the pressure monitors. Calibrate the devices and record blood pressure according to the manufacturer instructions.Then, place the finger pulse oximeter on the right index finger for continuous measurement of compensatory reserve. Finally, place an nasal cannula on the subject and instruct them to breathe through their nose to assure sensitive reflections in inspiration and expiration. Connect the nasal cannula to the capnograph for the continuous measurement of respiration and end tidal CO2.Begin data recording by clicking the start button on the data acquisition system and record baseline data for five minutes. Initiate the first level of central hypovolimia by turning on the vacuum motor and setting negative pressure to negative 15 millimeters of mercury and hold this pressure for five minutes. Then, increase the LBNP to negative 30 millimeters of mercury, negative 45 millimeters of mercury, negative 60 millimeters of mercury, and to negative 70 millimeters of mercury and hold each pressure for five minutes.Continue to increase LBNP levels by negative 10 millimeters of mercury every five minutes until negative 100 millimeters of mercury LBNP or the point of hemodynamic decompensation where the compensatory reserve index, or CRI value, is zero. During the experiment, observe the progressive decrease in the reserve to compensate which is represented by a gradual reduction in the colored bar on the screen. As the bar changes colors from green to yellow to red, notice that the measurement of the CRI moves from a value of one to zero.After the experiment is complete, terminate the LBNP by pressing the pressure release button on the LBNP chamber. Continue recording data on the data acquisition system for 10 minutes after the cessation of LBNP. Stop recording data at the end of the 10 minute recovery period by clicking the stop button on the data acquisition system.Detach all instrumentation from the subject and remove the subject from the LBNP chamber. Ask the subject to sit after stepping down from the LBNP platform to ensure they are symptom-free before leaving the laboratory. Finally, download data files from the acquisition system for extraction of the CRI, mean arterial pressure, heart rate, and SpO2 values.The progressive reduction in central blood volume, or LBNP, produces significant alterations in the features of the arterial wave form. Here, the values for mean arterial pressure, MAP, heart rate, SpO2, CRI, and LBNP are plotted against time. CRI decreases early and progressively throughout the multiple steps of LBNP while changes in heart rate, mean arterial pressure, and SpO2 occur during later phases of hemorrhage.The LBNP technique will continue to be used as a valid model of human hemorrhage to provide data for creating, testing, and refining future algorithms and devices to measure compensatory reserve. The ability to measure the compensatory changes associated with blood loss is critical to providing acute care in emergency situations in both military and civilian settings.

integrated-compensatory-responses-in-a-human-model-of-hemorrhage

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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) ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

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 ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...