This work was supported by National Institutes of Health grants HL44485 and HL28607.

Ethics Statement: All procedures were reviewed and approved by the Institutional Animal Care and Use Committee.
1. Preliminary Fabrication of Micropipettes, Restrainers, and Blockers
<ol>
	<li>Pull several clean borosilicate glass capillary tubes in half using an electronic puller adjusted so that, when pulled, the stretched portion of the tube is about 1 cm in length and the two halves are somewhat symmetric. Ensure that the taper is consistent with the dimensions in Figure 1. Use both halves for micropipettes, restrainers and blockers.
	<ol>
		<li>Bevel the micropipettes using an air driven grinding wheel30 with 0.5 &#181;m abrasive paper bathed with water. Set the angle of the micropipette holder so that it is about 30 degrees from horizontal.</li>
		<li>Thoroughly wash and dry the micropipettes by using suction to pull clean acetone through the tip and up the shaft. Inspect the micropipettes (Figure 1A) using a small upright microscope with 4&#215; and 40&#215; objectives fitted with an alligator clip micropipette holder and an eyepiece reticle.</li>
		<li>Select micropipettes with tip opening about 50 &#181;m long with a bevel whose length/width ratio is between 3.1 and 3.5 and with a sharply tapered point which helps penetrate the tough collagen fibers in the mesentery.</li>
		<li>Store 15-20 micropipettes of slightly varying sizes in a dust-free box.</li>
	</ol>
	</li>
	<li>Make restrainers using pulled capillary tubes prepared in step 1.1). Hold a pulled glass capillary tube briefly near a microflame to form a blunt end (Figure 1B). Store several in a dust-free box.</li>
	<li>Make microoccluders using pulled capillary tubes prepared in step 1.1). Hold a pulled capillary tube under a microflame and gently bend (using a tubing adapter, e.g., 23G) the fine tip (about 4 mm from tip) to make an angle of close to 120 degrees to the shaft (Figure 1C). Make and store several.</li>
</ol>
2. Animal Preparation and Surgery
<ol>
	<li>Anesthetize male (350-450 g) or female Sprague-Dawley rats (200-300 g) with pentobarbital sodium (80-100 mg/kg, Sigma-Aldrich, P3761) via subcutaneous injection; protect the mesentery by not using intraperitoneal injection.</li>
	<li>Throughout surgical procedures and experimental protocols, maintain anesthesia by supplemental injections (30 mg/kg) as needed. Determine depth of anesthesia by toe pinch or corneal reflex. After completion of the experimental procedure, euthanize the rat via pentobarbital overdose or intra-cardiac injection of saturated potassium chloride under anesthesia.</li>
</ol>
3. Prepare Solutions and Erythrocytes for use as Flow Markers
<ol>
	<li>Prepare mammalian Ringer&#39;s solution for superfusate and control perfusate solution (typically mammalian Ringer&#39;s solution&#160;containing 10 mg/ml fatty acid free bovine serum albumin, BSA).
	<ol>
		<li>Filter perfusate through 0.2 &#181;m syringe filters to remove tiny particles that might block the micropipette tip; filter into small clean container.</li>
	</ol>
	</li>
	<li>Prepare erythrocytes by drawing about 0.2 ml blood from the tail vein of the anesthetized rat into a 20G needle on a small syringe containing 0.05 ml heparin (1,000 U/ml) and transfer to a 15 ml centrifuge tube containing about 14 mL mammalian Ringer&#39;s solution&#160;solution.
	<ol>
		<li>Centrifuge to gently pack red cells (about max 200 &#215; g for 7 min). Draw off the supernatant and re-suspend the red cells in Ringer&#39;s solution.</li>
		<li>After centrifuging and removing supernatant three times leave the packed red cells in the bottom of the tube for later use. Note that this procedure reduces platelets in the final perfusates to less than 0.1% of normal levels31.</li>
	</ol>
	</li>
</ol>
4. Arrange the Tissue on the Animal Tray
<ol>
	<li>Shave the abdomen of the anesthetized rat from below navel to xiphoid process. Ensure that the shaved area extends around the right side (for rat placed on right side) so that hair does not form a wick to draw the superfusate out of the tissue well. Remove cut hairs with a wetted gauze pad or tissue.
	<ol>
		<li>Hold the skin with a blunt serrated forceps and make a center-line incision (2-3 cm long) through the skin using a sturdy scissors. Using a fine (iris) scissors make a similar incision through the linea alba, lifting the abdominal wall with serrated forceps to avoid damaging the gut.</li>
		<li>With the animal on its side on the animal tray, gently pull out the gut from the abdominal cavity using cotton-tipped applicators (wood stick) soaked in Ringer&#39;s solution and blunt serrated forceps. Arrange the gut in the tissue well so that the mesentery is draped over the pillar for viewing through the microscope taking care to avoid touching the mesentery (potentially damaging microvessels) with either forceps or cotton. 
		Note: The animal tray (Figure 2A) can be constructed from Plexiglas and requires a tissue well (about 3 mm deep), an optically clear pillar (e.g., polished quartz about 2 cm dia. and 5 mm high), and a warming pad adjusted to maintain the animal&#8217;s temperature. The thickness of the pillar must not exceed the working distance of the objective.</li>
	</ol>
	</li>
	<li>Position the animal tray on the microscope stage so that the mesentery is visible through the eyepieces.</li>
	<li>Position a gravity-fed drip line to continuously superfuse the mesentery with mammalian Ringer&#39;s solution maintained at 35&#8211;37 &#176;C. Use gauze pads to hold the gut in place, help retain moisture, and wick excess Ringer&#39;s solution&#160;off the surface. Adjust the flow and aspirate the effluent to maintain a consistent superfusate thickness.</li>
	<li>Identify target venular microvessels, which are unbranched segments downstream of convergent flow, one or two branches distal to true capillaries. Find an unbranched vessel (ideally, 600 to 1,000 &#181;m in length) having brisk blood flow and free of white cells sticking on the vessel wall.</li>
	<li>Position the test microvessel in the center of the microscope field and move the restrainer into position near the chosen cannulation site.</li>
</ol>
5. Fill Micropipette and Mount in Holder
<ol>
	<li>Just before cannulating, suspend the red cells in the perfusate. Add 7 &#181;l of packed red cells to 0.5 ml perfusate in a clean borosilicate test tube. Note: Hematocrits of 1-3% can be used, but consistent hematocrit will lead to consistent perfusate concentrations of any substances released from the red cells.</li>
	<li>Fit a 1 ml syringe with a 23G tubing adapter and PE 50 tubing (5-15 cm length). Draw the perfusate/red cell mixture into the syringe. Invert syringe to mix and expel all bubbles from tubing and adapter. For increased efficiency, fit tubing to several syringes before the experiment starts and keep them in a dust free box or plastic bag.</li>
	<li>Fill the micropipette by advancing the tubing into the large end of the micropipette until it abuts the tapered region. Apply a gentle quick push on the syringe plunger to fill the micropipette tip. Note: A tiny stream or droplet should be visible at the tip to evidence complete filling.</li>
	<li>Withdraw the tubing while gently pushing on the plunger to fill the wider part of the micropipette shaft. Remove small bubbles in the wide shaft by carefully flicking the micropipette shaft. Note: A similar procedure has been illustrated32.</li>
	<li>Place the micropipette into a pipette holder with a side port which allows a continuous fluid connection to a water manometer. Ensure that the holder, which is attached to a hydraulic drive used to control very fine advancement of the micropipette, is at a slight angle (15-25&#176;) to the horizontal so that the edge of the micropipette taper does not hit the gut or tray.</li>
	<li>Mount the hydraulic drive on a moveable micromanipulator, which has x, y, and z drives to enable close alignment to the test vessel (Figure 2).</li>
	<li>Adjust the hydrostatic pressure applied to the fluid in the micropipette using a syringe or water-filled rubber bulb to change the height of fluid in the water column of the manometer to about 40 cmH2O above the mesentery.</li>
	<li>Cannulate the microvessel as soon as possible after filling the pipette; red cells settle to the bottom of the pipette, so that after about 40 min very few red cells will flow into the vessel.</li>
</ol>
6. Microcannulation and Microvascular Pressure Measurement
<ol>
	<li>Before positioning the filled micropipette under the microscope, gently press the restrainer onto the tissue near the microvessel applying sufficient force to grip the tissue. Draw the restrainer back, slightly stretching the tissue in line with the microvessel so that the stresses in the mesenteric collagen fibers are aligned with the vessel.
	<ol>
		<li>Align the micropipette with the vessel and lower it into view through the eyepieces. Place the tip just upstream of the chosen cannulation site and lower it onto the tissue so that it partially obstructs flow within the vessel, but does not occlude it.</li>
	</ol>
	</li>
	<li>Cannulate the vessel by driving the pipette tip slowly into the vessel lumen using the hydraulic drive. Be careful not to push through the lower side of the vessel. 
	Note: As the micropipette enters the lumen, the perfusate rapidly washes the blood in the vessel out of the lumen and perfusate freely flows into the animal&#8217;s circulation distal to the test microvessel, displacing some of the normal blood perfusion in the downstream vessels.</li>
	<li>Lower the perfusion pressure by adjusting the manometer until the blood (as indicated by the animal&#8217;s red cells) is drawn back into the perfused segment; this determines the balance pressure where the red cells gently oscillate in the vessel lumen and measures the hydrostatic microvessel pressure (Pc) at the distal end of the microvessel segment. Maintain vessel perfusion at all pressures above this balance pressure.</li>
</ol>
7. Microocclusion and Collection of Data
<ol>
	<li>Optional step: Before using the micro-occluder to block the vessel, press it into the tissue in a non-used area of mesentery far from any vessel so that the tip accumulates a fine pad of tissue that protects the experimental vessel during repeated micro-occlusions.</li>
	<li>Place the blocker above the perfused vessel near the lower edge of the microscope field.</li>
	<li>Record the following with video and audio.
	<ol>
		<li>Verbally record the location of the block site and the lower screen edge as seen on the eyepiece reticle.</li>
		<li>With the tip of the blocker placed just above the microvessel, use the fine z control of the micromanipulator to gently lower the blocker until the flow in the vessel is occluded. Note the manometer pressure (Pc) on audio. Apply occlusion typically for 3-10 sec, and then release, restoring free perfusion. Move the block site up the vessel toward the pipette tip in 5-10 &#181;m steps based on time (&#62;5 min after initial block at a site), number of occlusions (3-5), to prevent vessel wall damage at the block site.</li>
	</ol>
	</li>
</ol>
8. Analysis of Data and Measurement of Lp (Water Permeability)
<ol>
	<li>During video playback, determine the initial distance (&#8467;0) from a marker red cell to the occlusion site by use of an image of a stage micrometer and the known positions on the images. Determine the initial velocity (d&#8467;/dt) of the marker cell from its position in several frames during the 3-10 sec of recorded images. Determine the vessel radius (r) from the images taken during occlusion (Figure 3).</li>
	<li>Calculate Jv/S for each occlusion as (d&#8467;/dt)&#215;(1/&#8467;0)&#215; (r/2). Calculate Lp at a constant pressure as (Jv/S) divided by the driving pressure (microvessel pressure &#8211; effective colloid osmotic pressure; see Discussion).</li>
</ol>

The authors have nothing to disclose.

Details of Lp calculations. Although transvascular fluid movement occurs while the vessel is freely perfused, such exchange is far too small to be measured during free perfusion because it is typically less than 0.01% of the vessel perfusion rate. However, when perfusion is transiently stopped by occluding the microvessel, transvascular flow (i.e., filtration) is measured from the movement of marker red cells in the lumen as the column of fluid between a marker red cell and ...

Microperfusion in the vasculature entails establishing controlled flow of an artificial perfusate of known composition via a micropipette in a blood vessel usually less than 40 &#181;m in diameter. The perfused vessel remains within its normal tissue environment and is perfused with the animal&#8217;s blood up to the time of cannulation. When used in conjunction with a range of video imaging or fluorometric techniques, in situ microperfusion enables measurement of water and solute flows across the walls of microvessels under conditions where the driving forces for these flows are known and the permeability properties of the vascular wall can be directly evaluated. Further, by controlling the composition of the fluid surrounding the microvessel in the tissue (perfusate and superfusate), the regulation of microvessel permeability and exchange can be investigated by enabling the endothelial cells forming the microvessel wall to be exposed to a variety of experimental conditions (agonists, modified perfusion conditions, fluorescent indicators to measure intracellular composition and signaling) for precisely measured periods of time (sec to hr). In addition, ultrastructural or cytochemical evaluations of key cellular molecular structures regulating the barrier can be investigated in the same microvessels in which permeability is directly measured. The approach thereby forms a bridge between investigation of the cellular and molecular mechanisms to modify endothelial barrier function in cultured endothelial cell monolayers and investigation in intact microvessels. See the following reviews for further evaluation1-6.
A limitation of microperfusion is that it can be used only in microvascular beds that are thin, transparent and have sufficient structural integrity to enable cannulation with a glass micropipette. While early investigations used frog microvessels in mesentery and thin cutaneous pectoris muscle7,8, by far the most commonly used preparation in mammalian models is the rat mesentery9-15. Most investigations have focused on acute changes in vascular permeability studied over periods of 1-4 hr, but more recent investigations have been extended to measurements on individual vessels 24-72 hr after an initial perfusion12,16. The recently developed CRISPR technology, which promises to make more genetically modified rat models available for studying vascular permeability regulation17 should enable the methods described in this communication to be applied in venular microvessels of the mesentery in these important new rat models.
The method requires an inverted microscope equipped with a custom built microscope stage large enough to hold both the animal preparation and at least three micromanipulators used to position microtools close to the perfused vessel and to align a perfusion micropipette with the vessel lumen. For example a custom platform for an x-y microscope stage (about 90 &#215; 60 cm) can be fabricated from a 1 cm thick steel plate with a rust-proof coating. The stage is attached to an engineering index table or two dove-tail slides mounted at right angles and supported on Teflon pillars or ball transfers for movement in the horizontal plane. A typical rig (see Figure 2) has much in common with the microscope and micropositioning equipment used for a range of intravital microcirculation experiments such as those to measure single vessel blood flow and hematocrit, local oxygen delivery by blood perfused microvessels, regulation of vascular smooth muscle tone, and the local microvascular accumulation of fluorescent tracers injected into the whole circulation.18-26
The fundamental aspect of the technique is the measurement of volume flow (Jv) across a defined surface area (S) of the microvessel wall. To accomplish this via the modified Landis technique described herein a simple inverted microscope is adequate. A small video camera is mounted on the image port and the video signal, with an added time base, is displayed on a video monitor and recorded either in digital form on a computer or as a digital or analog signal on a video recorder. Once the microvessel is cannulated the portion of the microvessel visible to the camera can be changed by moving the stage and manipulators as a unit without disrupting the cannulation.
Measurement of transvascular flows may also be combined with more detailed investigations using a sophisticated fluorescence microscope with appropriate filters such as rigs used for measurements of solute permeability, fluorescent ratio monitoring of cytoplasmic calcium or other cellular mechanisms, and confocal imaging 6,12,13,27. A key advantage of all microperfusion approaches is the ability to make repeated measures, on the same vessel, under controlled change of driving force such as hydrostatic and oncotic pressures, or induced change in vessel responses to inflammatory conditions. The most common design is a paired comparison of measured hydraulic conductivity (Lp) on the same vessel with the vessel first perfused via a micropipette filled with a control perfusate and the red cell suspension to establish a baseline permeability state, then with a second pipette with the test agent added to the perfusate. Multiple cannulations are possible with the cycle repeated after reperfusion with the control pipette.
The present protocol demonstrates the cannulation and microperfusion of a venular vessel in rat mesentery to record water fluxes across the microvessel wall and measure the Lp of the vessel wall, a useful index of the permeability of the common pathway for water and solutes across the intact endothelial barrier. The procedure is called the modified Landis technique because the original Landis principle of using the relative movement of red cells as a measure of transvascular fluid exchange after perfusion is blocked is preserved28, but the range of experimental conditions (e.g., the hydrostatic and albumin oncotic pressure differences across the microvessel wall) available after microperfusion is far greater than in uncannulated blood perfused microvessels8,29.

<table><tbody><tr><td>MICROSCOPE, TABLE AND STAGE</td><td></td><td></td><td></td></tr><tr><td>inverted microscope (metallurgical type) with trinocular head for video: example</td><td>Olympus</td><td>CK-40</td><td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td></tr><tr><td>inverted microscope (metallurgical type) with trinocular head for video: example</td><td>Leica</td><td>DMIL</td><td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td></tr><tr><td>narrow diameter, long working distance objective: example</td><td>Nikon</td><td>Nikon E Plan 10&amp;times;/0.25 LWD</td><td></td></tr><tr><td>stage platform--1/2 inch or 1 cm sheet steel</td><td>welding shop</td><td></td><td>this should be heavy to reduce vibration</td></tr><tr><td>Unislide x-y table: dove tail slides</td><td>Velmex</td><td>AXY4006W1</td><td></td></tr><tr><td>VIDEO</td><td></td><td></td><td></td></tr><tr><td>CCD video camera: example</td><td>Pulnix</td><td>TM-7CN (no longer available)</td><td>no color needed</td></tr><tr><td>video capture system with audio--generic</td><td></td><td></td><td></td></tr><tr><td>video playback system (completely still frame, single frame motion)</td><td></td><td></td><td></td></tr><tr><td>small microphone</td><td></td><td></td><td></td></tr><tr><td>MICROMANIPULATORS, HOLDERS</td><td></td><td></td><td></td></tr><tr><td>micromanipulator, XYZ (3)</td><td>Prior/Stoelting (no longer available)</td><td></td><td>look for fine Z, and larger range of travel in coarse drives for ease of positioning</td></tr><tr><td>hydraulic probe drive, one way</td><td>FHC</td><td>50-12-1C</td><td>need to buy either manual drive or electronic drive</td></tr><tr><td>manual drum drive&amp;nbsp;</td><td>FHC</td><td>50-12-9-02</td><td></td></tr><tr><td>or hydraulic drive, 3 way</td><td>Siskiyou Corporation</td><td>MX610 (1-way) or MX630 (3-way)</td><td>great for short arms, water filled and must be sent back for refill ~every 2 years</td></tr><tr><td>connectors/rods/holders</td><td>Siskiyou Corporation</td><td>MXC-2.5, MXB etc.</td><td></td></tr><tr><td>pin vise</td><td>Starrett</td><td>162C</td><td>to hold restrainer</td></tr><tr><td>pipette holder</td><td>World Prescision Instruments</td><td>MPH3</td><td></td></tr><tr><td>water manometer ~120 cm</td><td></td><td></td><td></td></tr><tr><td>MICROSCOPE TRAY</td><td></td><td></td><td></td></tr><tr><td>clear Plexiglas for microscope tray for animal</td><td></td><td></td><td></td></tr><tr><td>3/4 inch polished quartz disc ~1/4 inch tall</td><td>Quartz Scientific Inc.</td><td>custom</td><td>&amp;nbsp;(or polished plexiglass, glass); make sure the height is less than working distance of objective</td></tr><tr><td>Plexiglas glue (Weld-on 4: CAUTION CARCINOGEN)</td><td></td><td></td><td></td></tr><tr><td>medical adhesive for tissue well</td><td>NuSil</td><td>MED-1037</td><td></td></tr><tr><td>All-purpose silicone rubber heat mat, 5&quot; L x 2&quot; W</td><td>Cole Parmer</td><td>EW-03125-20</td><td>heater for microscope tray--needs cord and controller--240V version available</td></tr><tr><td>Power Cord Adapter for Kapton Heaters and Kits, 6 ft, 120 VAC</td><td>Cole Parmer</td><td>EW-03122-75</td><td></td></tr><tr><td>STACO 3PN1010B Variable-Voltage Controller, 10 A; 120 V In, 0-140 V Out</td><td>Cole Parmer</td><td>EW-01575-00</td><td></td></tr><tr><td>PIPET MANUFACTURE</td><td></td><td></td><td></td></tr><tr><td>vertical pipette puller</td><td>Sutter Instrument Company</td><td>P-30 with nichrome filament</td><td></td></tr><tr><td>1.5 mm OD thin wall capillary tubing</td><td>Sutter Instrument Company</td><td>B150-110-10</td><td></td></tr><tr><td>pipette grinder air stone and dissection microscope--see reference in text</td><td></td><td></td><td>or purchase a package from Sutter Instruments or World Precision Instruments</td></tr><tr><td>RX Honing Machine, System II</td><td>RX Honing Machine Corporation</td><td>MAC-10700 Rx System II Machine</td><td>alternative for air stone, use with a dissecting microscope mounted at an angle</td></tr><tr><td>&amp;nbsp;&amp;nbsp; with ceramic sharpening disc</td><td>RX Honing Machine Corporation</td><td></td><td>use &quot;as is&quot; or attach lapping film</td></tr><tr><td>lapping film sheets, 0.3 or 0.5 um</td><td>3M</td><td>part no. 051144 80827</td><td>268X Imperial lapping film sheets with adhesive back--can be purchased from Amazon</td></tr></tbody></table>

microperfusion technique to investigate regulation of microvessel permeability in rat mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

Figure 4 shows the results from measuring the time course of changes in Lp in a rat venular microvessel cannulated successively with four perfusates.33 The magnitude of Lp calculated at a constant pressure was used as a measure of changes in microvessel wall permeability, first in the control state with a perfusate containing 1% bovine serum albumin then when the vessel was exposed to the inflammatory agent bradykinin (Bk) using a second micropipette containing 10 nM Bk. ...

Watch this Scientific Journal Video about Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery at JoVE.com

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

microperfusion-technique-to-investigate-regulation-microvessel

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.7K Views.  University of California Davis. The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions. 

Video: Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Developmental Biology

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Medicine

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Immunology and Infection

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Endothelial PAR1 Role in Leukocyte Adhesion and NET Formation During I/R Injury

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric vessels, leading to both local and systemic pathologies with a poor prognosis. Both ischemia and reperfusion trigger a series of cellular and molecular responses, with inflammatory cells serving as key regulators of the pathology. These interactions with the ischemic endothelium are mediated by multiple adhesion receptors. Several animal models have been established to mimic this pathology and investigate the involved molecular pathways. In this study, a microsurgical model of I/R injury is combined with intravital microscopy to visualize leukocyte rolling, adhesion, and neutrophil extracellular trap (NET) formation. This model is applied to transgenic mice deficient in endothelial PAR1 (F2r) to assess the impact of PAR1 on leukocyte rolling and NET formation 1 h after ischemia and immediately following reperfusion. In vivo, Acridine Orange leukocyte staining was employed, and NETs were visualized using a nucleic acid stain. Interestingly, reduced leukocyte adhesion and NET formation were observed in mice lacking the endothelial PAR1 receptor. This model enables the in vivo analysis of key regulators involved in I/R injury.

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Summary

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Chapters in this video

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Rat Mesentery Angiogenesis Assay

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy