Authors thank Ms. Risa Otsuka for technical assistance and helping experiments. This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Young Scientists (B), 2010, 22700476, and Suzuken Memorial Foundation 2010 for K.T. And this study was supported by Research and Development of the Next-Generation Integrated Simulation of Living Matter, a part of the Development and Use of the Next-Generation Supercomputer Project of MEXT, and in part by JST ERATO Suematsu Gas Biology Project.

1. Labeling Erythrocytes with Fluorescein Isothiocyanate Isomer I (FITC)
All the experimental protocols we used were approved by the Animal Care Committee of Keio University School of Medicine.
<ol>
 <li> Anesthetize a donor mice, and after making a celiotomy incision withdraw whole blood from the inferior vena cava with a 1-ml syringe containing small amount of heparin. The mouse was immediately euthanized by sodium pentobarbital injection (100 mg/kg i.p.). </li>
 <li> Wash the whole blood twice with PBS (pH 7.4) by centrifugation for 5 min at 400 g in a swinging-bucket rotor. </li>
 <li> Dissolve 10 mg of FITC in 5 ml of 100 mM Na2HPO4 and filter it with a membrane having 0.22-&mu;m pores. </li>
 <li> In a test tube mix 1 ml of the FITC solution with 0.15 ml of 3 mM glucose, 0.25 ml of 180 mM NaH2PO4, and 1.5 ml of 100 mM Na2HPO4. Add 0.2 ml of the RBC pellet, and tap the tube for mixing. </li>
 <li> Keep the tube for 2 hours at 4 &deg;C and then wash the stained RBCs in PBS twice. </li>
 <li> Put a small amount of the RBC suspension on a slide grass and see if the RBCs look healthy (i.e., that their shape and size are normal) and that the fluorescence intensity is sufficient. </li>
 <li> Suspend the 0.1 ml of RBCs in 0.9 ml of PBS at pH 7.4, and keep the suspension at 4 &deg;C until injection. </li>
</ol>
2. Preparation of Oxygen-sensitive Dye
<ol>
 <li> Dissolve 500 mg of BSA in 10 ml of PBS at pH 7.4. </li>
 <li> Add 30 mg of Pd(II)-meso-tetra(4-carboxyphenyl)porphine (Pd-TCPP) to the BSA solution and stir overnight. </li>
 <li> (Optional) Extract BSA-bound Pd-TCPP by using gravity-flow chromatography or a spin column to separate it from free Pd-TCPP. </li>
 <li> Centrifuge the solution to remove undissolved Pd-TCPP, and filter the supernatant with a membrane filter having 0.22-&mu;m pores. </li>
 <li> Store 1-ml aliquots in tubes at -20 &deg;C. Avoid repeated freeze-thaw cycles. </li>
</ol>
3. Animal Preparation 
<ol>
 <li> For microscopic observation of the microcirculation prepare a plastic plate with a hole 20 mm in diameter, and tape a 30-mm-square square cover glass over the hole. </li>
 <li> Anesthetize a mouse, remove the fur, and prep the skin. Place a catheter in the tail vain for drug injection by using a 30 G needle connected to a 10-cm polyethylene (PE 10) catheter filled with heparinized PBS. </li>
 <li> After median incision, extend a main lobule of the liver on the plastic plate, and place the mouse in sternal recumbency. </li>
 <li> Prepare small slips with kitchen wrap (3 mm x 8 mm) and tile them around the edge of the hepatic lobe to inhibit moving of the liver with respiration and keeping it from drying. </li>
 <li> Observe the hepatic microcirculation under a microscope (with transmitted light), and confirm that the blood flow has no stasis in the field of view for at least 15 min. </li>
 <li> Slowly inject 0.2 ml of fluorescently labeled RBCs for blood flow observation or 0.2 ml of Pd-TCPP solution for pO2 measurement. This amount of FITC-labeled RBCs will account for 1/50 of all the RBCs in circulation in the visualized region. </li>
</ol>
4. Blood Flow Visualization
<ol>
 <li> Excite the FITC by irradiating it with mercury lamp light that has passed through a bandpass filter (450&#x2013;490 nm)1. </li>
 <li> Record the fluorescent image with a CCD camera. </li>
</ol>
5. pO2 Measurement
Pd-TCPP phosphorescence is relatively weak and should be detected with a high&#x2013;sensitivity detector. All experiments need to be performed in a dark room.
<ol>
 <li> The absorption peaks of Pd-TCPP are at 410 nm and 532 nm, so the second-harmonic wavelength 532 nm is recommended for excitation. This wavelength can be generated by a Nd:YAG pulse laser2. </li>
 <li> Feed the beam from the Nd:YAG laser into the appropriate port on the inverted microscope, and adjust the beam spot to a central position in the focal plane. The spatial resolution depends on the laser spot size, which is changed by passing the beam through a pinhole. </li>
 <li> To detect the phosphorescence, attach a long-pass filter (&gt;620 nm) and a photomultiplier tube (PMT) to the other port of the microscope. The PMT should be sensitive to red wavelengths, especially around 700 nm. </li>
 <li> Sample the phosphorescence signal (500 points sampled at 200 kHz) and calculate the time constant of the decay by fitting an exponential function to the decay. </li>
 <li> For calibration prepare two set of samples of Pd-TCPP solution with pO2 150 mmHg and 0 mmHg. Add dithionite sodium 1% in volume to sample to produce absence of oxygen. Keep the pH of the samples at 7.4 and the temperature at 37 &deg;C during calibration. </li>
 <li> Fix the Stern-Volmer constant kq and oxygen-free luminescence decay time &tau;0 in the equation (1)3. In our case, with Pd-TCPP solutions at 37 &deg;C and a pH of 7.4, kq is 374 mmHg-1 s-1 and &tau;0 is 0.74 ms. These measurement parameters depend on the equipment (i.e. laser, detector, other optical devices) and the signal processing method, so calibration is needed in each measurement system4. 
 &tau;0/&tau; = 1 + kq &bull; &tau;0 &bull; pO2&nbsp;&nbsp;&nbsp;&nbsp; (1) </li>
 <li> Set the mouse on the plate on the microscope stage, observe the hepatic microcirculation, and adjust the position of the target microvessel to the point of the laser spot. </li>
</ol>
6. Hepatitis Model
<ol>
 <li> Fast mice with free access to water overnight. </li>
 <li>Prepare a 20 mg per ml solution of acetaminophen (APAP) in DMSO. </li>
 <li> Inject it intraperitonealy at 1 ml/100 g bw (i.e., 200 mg/kg bw). After the injection, the mouse can be given free access to food and water 5. </li>
 <li> 24 hours after injection, anesthetize the mouse and expose the liver by making a median incision. If hepatitis is achieved, necrosis in the pericentral region will be easily visible to the naked eye. </li>
</ol>
7. Representative Results
Example images of hepatic microcirculation are shown in Figure 1, where (a) is a transmitted&#x2013;light image and (b) is a fluorescence image. Individual FITC-labeled erythrocytes are observed in the video movie, and portal venules, sinusoids, and central venules are recognized by blood flow directions.
As shown in Figure 2, the laser spot irradiating a central venule through a x100 objective lens was approximately 10 &mu;m in diameter. Intravascular pO2 in the hepatic microcirculation was measured in male C57BL/6 mice, and Figure 3(a) shows the relations between pO2 and vessel diameter in portal and central venules. The pO2 gradient from portal to central venules indicates that RBCs release oxygen efficiently while passing through sinusoids. As shown in Figure 3(b), the average pO2 was 59.8 mmHg in portal vessels, 48.2 mmHg in sinusoids, and 38.9 mmHg in central venules. 
When we produced acute hepatitis by injecting APAP intraperitonealy, intravascular pO2 was significantly elevated in each part of the hepatic microcirculation: p&lt;0.05 in portal veins and p&lt;0.01 in sinusoids and central veins (Figure 3(c)). 
<img src="/files/ftp_upload/3996/3996fig1.jpg" alt="Figure 1" /> 
Figure 1. Low-magnification images showing hepatic microcirculation with (a) transmitted light and (b) fluorescence. 
<img src="/files/ftp_upload/3996/3996fig2.jpg" alt="Figure 2" /> 
Figure 2. Image of laser spot irradiating a targeted central venule for pO2 measurement.
<img src="/files/ftp_upload/3996/3996fig3.jpg" alt="Figure 3" /> 
 Figure 3. pO2 gradient in hepatic microcirculation of healthy control mice and of mice with acetaminophen-induced hepatic injury. (a) shows pO2 versus vessel diameter for portal venules (PV) and central venules (CV), and (b) shows the oxygen gradient from PV to CV via sinusoids (S). The pO2 difference between PV and CV reflects the oxygen consumption of hepatocytes or other parenchymal cells. (c) shows that in hepatitis conditions induced by APAP administration, pO2 was significantly elevated in PV, S, and CV and the pO2 gradient decreased, indicating that hepatic oxygen consumption was compromised.

<ol>
	<li>Ishikawa, M., Sekizuka, E., Shimizu, K., Yamaguchi, N., Kawase, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+RBC+velocities+in+the+rat+pial+arteries+with+an+image-intensified+high-speed+video+camera+system.">Measurement of RBC velocities in the rat pial arteries with an image-intensified high-speed video camera system.</a> Microvasc. Res. 56, 166-172 (1998).</li><li>Tsukada, K., Sekizuka, E., Oshio, C., Tsujioka, K., Minamitani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+velocity+and+oxygen+tension+measurement+in+cerebral+microvessels+by+double-wavelength+photoexcitation.">Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.</a> J. Appl. Physiol. 96, 1561-1568 (2004).</li><li>Stern, O., Volmer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Abklingzeit+der+Fluoreszenz.">&#220;ber die Abklingzeit der Fluoreszenz.</a> Physik. Zeitschr. 20, 183-188 (1919).</li><li>Rumsey, W. L., Vanderkooi, J. M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+phosphorescence:+a+novel+method+for+measuring+oxygen+distribution+in+perfused+tissue.">Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue.</a> Science. 241, 1649-1651 (1988).</li><li>Soga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+metabolomics+reveals+ophthalmic+acid+as+an+oxidative+stress+biomarker+indicating+hepatic+glutathione+consumption.">Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption.</a> J. Biol. 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Invest. 101, 604-612 (1998).</li><li>Grote, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiologie der Menschen. , 560 (1980).</li><li>Suganuma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytes+with+T-state-stabilized+hemoglobin+as+a+therapeutic+tool+for+postischemic+liver+dysfunction.">Erythrocytes with T-state-stabilized hemoglobin as a therapeutic tool for postischemic liver dysfunction.</a> Antioxid Redox Signal. 8, 1847-1855 (2006).</li><li>Vanderkooi, J. M., Maniara, G., Green, T. J., Wilson, D. 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Pharmacol. 169, 369-405 (2010).</li><li>Hinson, J. A., Reid, A. B., McCullough, S. S., James, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetaminophen-induced+hepatotoxicity:+role+of+metabolic+activation,+reactive+oxygen/nitrogen+species,+and+mitochondrial+permeability+transition.">Acetaminophen-induced hepatotoxicity: role of metabolic activation, reactive oxygen/nitrogen species, and mitochondrial permeability transition.</a> Drug Metab. Rev. 36, 805-822 (2004).</li><li>Lieber, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+disorders+of+alcoholism.">Medical disorders of alcoholism.</a> N. Engl. J. Med. 333, 1058-1065 (1995).</li><li>Zimmerman, H. J., Maddrey, W. 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We have nothing to disclose.

Delivering oxygen to tissues is an essential role of the microcirculation, and a lot of papers describe diseases related to microvessel anatomy and rheology6. Liver blood flow is a combination of arterial and venous blood. About 30% of the blood flow to the liver is well-oxygenated blood provided by the hepatic artery and the other 70%, provided by the portal vein, is the poorly oxygenated blood venous outflow from the spleen and gastrointestinal tract7. Liver diseases such as fatty liver, shock, or...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Pd(II) 			meso-Tetra(4-carboxyphenyl)porphine</td>
 <td>Frontier Scientific, Inc.</td>
 <td>PdT790</td>
 </tr>
 <tr>
 <td>Fluorescein isothiocyanate 			isomer I</td>
 <td>Sigma-Aldrich Co.</td>
 <td>F7250</td>
 </tr>
 <tr>
 <td>Acetaminophen</td>
 <td>Sigma-Aldrich Co.</td>
 <td>A7085</td>
 </tr>
 </tbody>
</table> 
<table border="1">
<tbody>
 <tr>
 <td>Name 			of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 	<td>&nbsp;</td>
	<td>&nbsp;</td>
	<td>&nbsp;</td>
 <td>Equipment</td>
</tr> <tr>
 	<td>photomultiplier tube</td>
	<td>hamamatsu photonics k.k</td>
	<td>H10722-20 </td>
</tr>
</tbody>
</table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

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

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

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

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

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

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12.6K Aufrufe.  Keio University. Ein optisches System wurde entwickelt, um hepatischen Mikrozirkulation mit FITC-markierten Erythrozyten visualisieren und die Sauerstoff-Partialdruck in der Mikrogefäße laserunterstützte phosphorimetry messen. Diese Methode kann verwendet werden, um physiologische und pathologische Mechanismen durch die Analyse von mikrovaskulären Struktur, Durchmesser, Blutflussgeschwindigkeit, und Sauerstoff-Partialdruck zu untersuchen.

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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

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 ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

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 ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.