Name: microperfusion technique to investigate regulation of microvessel permeability in rat mesentery
Uploaded: 2015-09-12T14:00:00
Description: Watch this Scientific Journal Video about Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery at JoVE.com

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</stron...

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 border="0" cellpadding="0" cellspacing="0">
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		<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&#215;/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></td>
			<td></td>
			<td></td>
			<td></td>
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			<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>
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			<td></td>
			<td></td>
			<td></td>
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		</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&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></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>&#160;(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&#34; L x 2&#34; 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></td>
			<td></td>
			<td></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>&#160;&#160; with ceramic sharpening disc</td>
			<td>RX Honing Machine Corporation</td>
			<td></td>
			<td>use &#34;as is&#34; 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 ...

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