The authors wish to thank Andy Poczobutt and Dr. Jori Leszczynski for their valuable help and edits to this manuscript.&#160;Supported by Grants received from the National Heart, Lung and Blood Institute (NHLBI RO1 HL078929, PPG HL014985 and RO3 HL095439) and the Department of Veterans&#39; Affairs (Merit Review).

All applicable international, national, and/or institutional guidelines for the care and use of animals (mice) were followed in the experiments described in this manuscript.
This method uses FVBN adult mice, aged 16 - 20 weeks, found to be optimal for the purposes of this study. Day 1 includes steps 1 - 5 and Day 2 includes steps 6 - 7 (Figure 1).
1. Equipment Preparation
<ol>
	<li>Secure an adequate supply of sterile, disposable syringes and needles, if ketamine/xylazine is used as the anesthetic (as recommended). If isoflurane is used as the anesthetic, check the oxygen tank and the fluid level of isoflurane to make sure there are adequate supplies for the experiment before starting. Also, assemble the nosecone breathing circuits and attach them to the induction box; attach new charcoal canisters to the breathing circuits. Prepare the induction box by turning on the oxygen and ascertaining that the second stage reads approximately 50 psi.</li>
	<li>Turn on the heating pad to 37 &#176;C.</li>
	<li>Prepare the rectal temperature probe for the surgery.</li>
</ol>
2. Mouse Preparation
This step includes anesthesia, hair removal, and positioning (adult FVBN mice-age 16 - 20 weeks).
<ol>
	<li>Weigh the mice and record the weights.</li>
	<li>Anesthetize the mice.
	<ol>
		<li>Administer ketamine and xylazine IP (80 - 100 mg/kg and 7.5 - 16 mg/kg, respectively). However, it is recommended to begin with lower doses of ketamine and xylazine (about 30 mg/kg and 6 mg/kg, respectively).</li>
		<li>Maintain the anesthesia with about 0.1 - 0.25 times initial doses of ketamine/xylazine throughout the surgery.&#160;Ketamine and xylazine were the anesthetic agent(s) of choice in this particular study, as more reproducibility and survivability were observed. The anesthetic agent(s) of choice in other studies may be found to be different.&#160; If isoflurane is used, put the mouse in an induction chamber and turn on isoflurane to 5% until the mouse loses full consciousness. Then use a nasal nosecone set at 1.5 - 2.5% isoflurane throughout the remainder of the procedure.</li>
	</ol>
	</li>
	<li>Monitor the mice every 2 - 3 min by toe pinch to check for appropriate depth of anesthesia.</li>
	<li>Shave the ventral neck area of the mouse.</li>
	<li>Place the mouse in a supine position on the preheated pad. Secure the paws and feet of the mouse to the surgical surface with tape.</li>
	<li>Place artificial tear ointment onto the eyes to prevent drying out during surgery.</li>
</ol>
3. Surgical Details
<ol>
	<li>Make a 1 cm incision in the right ventral neck over the jugular vein.</li>
	<li>Apply one or two drops of lidocaine (1 - 2%) into the incision area for pain management and to promote vasodilation. Wait 2 min. for the lidocaine to take effect.&#160;</li>
	<li>Expose and isolate the right internal jugular vein via blunt dissection. Tie the vein off with 4-0 suture and gently retract the rostral end of the vessel with a hemostat. Cut a hole, using fine scissors, about 3 mm below or caudal to the tie, approximately halfway through the diameter of the vein.</li>
	<li>Mark a&#160;PV-1 polyvinyl catheter 1.5 cm from the end and insert it into the jugular vein through the hole using a vessel dilator, and thread the catheter towards the caudal end of the vessel, approximately 1.5 cm.</li>
	<li>Tie the catheter securely within the caudal part of the vessel (below the cut), with 4-0 silk. Tie the rostral part of the vessel to the outside of the catheter with the loose ends of the suture which was used to tie off the rostral jugular vein, in step 3.3.</li>
	<li>Tack the skin loosely back together around the catheter with 4-0 silk to help prevent loss of body heat and desiccation of tissues.</li>
	<li>Connect the catheter to a syringe containing heparinized saline (10 U/mL) and flush the catheter.</li>
</ol>
4. Injections
<ol>
	<li>Inject the Evans blue solution (50 &#956;L of a 30 mg/mL solution in 0.9% normal, unheparinized saline, or approximately 50 mg/kg) into the jugular vein catheter, followed by a small volume of heparinized saline to flush the line.</li>
	<li>2 min later, inject substance P (100 &#956;L of a 0.3 &#956;M solution in 0.9% normal, unheparinized saline, or 1 nmol/kg), followed by a small volume of heparinized saline to flush the line. Substance P augments the extravasation of plasma proteins through the endothelial layer; in this protocol, it routinely induces an augmentation of plasma extravasation values of approximately 1.5-fold, making plasma extravasation values easier to measure.</li>
	<li>Wait 18 min after the substance P is injected. During this time, the Evans blue dye will equilibrate and circulate.</li>
	<li>Terminate the experiment 18 min after the injection of substance P (20 min after Evans blue injection) by sacrificing the mouse with cervical dislocation. It is likely that the mouse can be directly cervically dislocated, without first giving an overdose of anesthetic, as the mouse is probably still well-anesthetized from the surgery. However, if it is necessary, an overdose of the anesthetics ketamine/xylazine, isoflurane, or pentobarbital may be given, followed by cervical dislocation.</li>
</ol>
5. Isolation of Organs
<ol>
	<li>Cut open the chest cavity of the mouse and gravity-perfuse (from a height of about 51 cm or 20&#34;) the heart and blood vessels with 50 mL of 50 mM sodium citrate, pH 3.5. pH 3.5 presumably preserves Evans blue binding to albumin.</li>
	<li>Excise 1 - 5 relevant organs (tissues) with a dissecting scalpel (e.g. urinary bladder, kidney, stomach, liver, pancreas, proximal or distal colon, ileum, duodenum, flank skin, ears, tail, heart, and/or lungs) and remove any residual contents, if present.</li>
	<li>Rinse the organs in room temperature (RT) phosphate-buffered saline (PBS; 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 8.0 g of NaCl, and 0.20 g of KCl in 1 L, pH 7.4).</li>
	<li>Blot the organs with tissue, cut each organ in half, and weigh each half (wet weights, in g).</li>
	<li>Dry one half of the tissue in a drying oven at 150 &#176;C, on foil, for 48 h.</li>
	<li>Place the other tissue half in a consistent volume (up to 200 &#181;L) of formamide in a microfuge tube for 48 h (and up to 72 h) to extract the Evans blue.</li>
</ol>
6. Measurement of Tissue OD
<ol>
	<li>Remove 50 &#181;L of Evans blue-infused formamide (after 48 - 72 h RT incubation) from the microfuge tube and place into one well of a 96-well polystyrene plate. Be careful not to transfer tissue pieces along with the formamide.</li>
	<li>Place 50 &#181;L of new, pure formamide into each of two empty wells of the 96-well plate for the blanks.</li>
	<li>Measure and record the OD620 of each well of the 96-well plate on an absorbance plate reader.&#160;620 nm is the absorbance max of Evans blue.</li>
</ol>
7. Calculation of Plasma Extravasation
<ol>
	<li>Weigh the dry tissue half which has been in the oven for 48 h.</li>
	<li>Calculate the wet weight/dry weight ratio for the specific organ of interest from each individual mouse, starting with the wet weight of this tissue half (obtained in step 5.4), divided by the dry weight of this same tissue half (obtained in step 7.1).</li>
	<li>Calculate the dry weight (in g) of the tissue half in formamide by dividing the wet weight of the tissue half before it was placed in formamide (obtained in step 5.4) by the wet: dry weight ratio for the specific organ of interest (calculated in step 7.2).</li>
	<li>Calculate the corrected OD620 values. Starting with the OD620 value from each experimental well of the 96-well plate (containing Evans blue-infused formamide from each tissue, obtained in step 6.3), subtract the blank well OD620 value (the mean OD620 value of the two wells containing pure formamide, prepared in step 6.2, OD620 values obtained in step 6.3) from each experimental value.</li>
	<li>Calculate the plasma extravasation value by dividing the corrected OD620 (calculated in step 7.4) by the dry weight of the tissue half in formamide (calculated in step 7.3). The units of plasma extravasation will be OD620/g dry weight.</li>
	<li>Analyze data and express as the mean &#177; SEM. Statistically compare groups by t test or one-way analysis of variance and Scheff&#233;&#39;s multiple-comparison&#160;test (lycofs01.lycoming.edu), as appropriate.</li>
</ol>

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The authors have nothing to disclose.

As discussed above, the study of plasma extravasation may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. The successful use of the plasma extravasation protocol (above), using Evans blue dye, has been demonstrated in the current manuscript. Although the data shown back the hypothesis that NEP may protect the vasculature against plasma extravasation, this is a secondary goal presently, with the primary goal being to present an optimized protocol which may be...

Vascular leak in the organs refers to extravasation, or leakage of blood plasma through gaps produced in the endothelium of post capillary venules in the organs. This plasma extravasation or increased vascular permeability, which may arise from some type of an inflammatory response, may have grave consequences. Thus, it is important that this phenomenon, its causes, modulators, and consequences, are studied and understood, and likewise, that investigators have good tools and protocols with which to study them. The endothelial gaps may be produced via a number of stimuli, but usually are produced by the action of peptide neurotransmitters and/or tachykinins on the endothelia. One of the major naturally occurring mediators of this process, which results in increased plasma extravasation, is the undecapeptide tachykinin neuropeptide, substance P1.
Methods to investigate and measure vascular permeability or plasma extravasation, which use the albumin-binding property of Evans blue dye, have been developed, and are usually known for their accuracy, simplicity, economy, safety, and ability to allow the determination of plasma extravasation from several tissues at once, if so desired2,3,4,5,6,7,8,9. This Evans blue protocol for assessing plasma extravasation in the organs of FVBN mice uses all these, but adds some important modifications that make it generally useful and adaptable for future studies, involving the average laboratory that conducts or will conduct important studies of factors associated with plasma extravasation or vascular permeability. In this protocol, substance P is introduced to the mice at 1 nmol/kg, which augments the extravasation of plasma by 1.5-fold. This increases the sensitivity of the protocol, resulting in more easily observable and obtainable results. Other factors that impact permeability, such as various other peptides, chemicals, or some forms of toxic injury, may be used or studied by other laboratories, as desired. Jugular vein injections are used in this protocol to introduce Evans blue and substance P systemically, which requires terminal surgery. However, jugular vein injections5,7,10, even after consideration of the necessary terminal surgical techniques, are easier to master and lead to the production of more consistent results than other venous injections, including tail vein injections4,9. Although it may be possible for Evans blue to be delivered by retro-orbital venous sinus injections, no references in the literature have been found that use this method of delivery of Evans blue. However, as for tail vein injections, the high degree of expertise and practice to reproducibly master this technique greatly limits its use for successful Evans blue injections. In contrast, the alternative jugular vein injection method as described in our protocol, offers a technically obtainable solution. A crucial procedure for perfusion of the mouse&#39;s veins, performed just after the sacrifice of the Evans blue-perfused mouse, removes excess Evans blue dye, and has been standardized in this protocol. Previously described methods of perfusion have been carefully examined and modified to obtain the present procedure.Other modifications described here are all optimized, straightforward, and inexpensive.
There are some important limitations of the Evans blue dye method. For example, low sensitivity sometimes associated with this method may prevent some additional gross pathological and histological examination of tissues from Evans blue-injected animals. However, these and other limitations have led to the development of alternative methods and models that, nonetheless, still use Evans blue. The measurement of Evans blue by fluorescence (rather than by visual-range) spectroscopy may increase the sensitivity of the method. Additionally, fluorescence microscopy of Evans blue-stained tissues was developed to allow for observation of vascular leak in more distinct locations11. Also, whole-body imaging and scanning of a live animal previously injected with Evans blue12&#160;allows for investigation of Evans blue concentrations in a continuous manner, rather than at one specific chosen time point of the experiment. However, this method requires the availability of appropriate imaging facilities, and may be very expensive. Modifications involving Evans blue and performed in an in vitro type of model, such as in a cell culture or chick chorioallantoic model13 (CAM) have also been described. These models are monitored by fluorescence and intravital14&#160;microscopy, and allow the quantification of vascular permeability changes over time, but may raise questions regarding accurate modeling of in vivo conditions and may also be expensive.
There have been other methods developed to determine and quantify vascular leak or permeability, which do not involve the administration of Evans blue. These methods may employ an appropriate fluorescent molecule (such as albumin or fluorescein), or an isotopically labeled or otherwise tagged molecule, to live animals (or to cell culture or chorioallantoic (CAM) models13, followed by non-invasive imaging (PET scanning, MRI, intravital microscopy, whole body scanning) or by invasive imaging (fluorescent microscopy)3,12,15. Although these techniques may offer a number of advantages over other Evans blue methods, they also have disadvantages, which may include their considerable complexities, requisite expertise, resources, and high monetary costs.
Neprilysin16 (the peptidase enzyme NEP, also known as CD10, MME, or Enkephalinase) has been suggested to be involved in inhibiting plasma extravasation, at least in part, through the enzymatic metabolism and inactivation of endogenous substance P. Thus, in tissues in which the cell surface peptidase NEP occurs, there may be an attenuation of the effect of substance P, presumably by the peptidase activity of NEP.
Initially, we tested for substance P-induced plasma extravasation utilizing this modified Evans blue protocol, with FVBN wild type (WT) and NEP knockout (KO) mice. NEP involvement in substance P-augmented plasma extravasation was suspected from these initial studies, and we describe these and further experiments involving NEP&#39;s role in plasma extravasation. However, the focus of this manuscript is not NEP or its role in plasma extravasation, but rather the plasma extravasation experiments themselves. The NEP results are representative of the kind of results that may be obtained through use of this modified protocol. The Evans blue method to measure plasma extravasation has been optimized and modified, as described in detail below for FVBN mice.

<table border="0" cellpadding="0" cellspacing="0" style="width:687px;" width="686">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:167px;">Vet One</td>
			<td style="width:119px;">200-070</td>
			<td style="width:185px;">inhaled anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ketamine</td>
			<td style="width:167px;">Vet One</td>
			<td style="width:119px;">200-055</td>
			<td>injectable anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine</td>
			<td style="width:167px;">Lloyd Laboratories</td>
			<td style="width:119px;">139-236</td>
			<td>injectable anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">syringes (10,3 &#38; 1 cc)</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">309604, 309657, 309659</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">needles (20G1,23G1 &#38; 26G1/2)</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">305178, 305193, 305111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane induction chamber</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:119px;">941443</td>
			<td>1 Liter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nosecone breathing circuits&#160;</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:119px;">RC2</td>
			<td>Rodent Circuit Controller 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">oxygen tank</td>
			<td style="width:167px;">Airgas</td>
			<td style="width:119px;">UN 1072</td>
			<td>100% medical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heating pad</td>
			<td style="width:167px;">CWE Inc.</td>
			<td style="width:119px;">TC-1000</td>
			<td>temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rectal temperature probe</td>
			<td style="width:167px;">CWE Inc.</td>
			<td>10-09012</td>
			<td>mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">balance (for rodents)</td>
			<td style="width:167px;">Ohaus</td>
			<td style="width:119px;">CS 2000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>15000-00</td>
			<td style="width:185px;">Vannas Spring scissors 3mm straight blade (cutting vessels)&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11151-10</td>
			<td style="width:185px;">Graefe extra fine forceps (isolating mouse vessels)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-hemostats</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>13009-12</td>
			<td style="width:185px;">Halstead-mosquito hemostats (blunt dissect, hold tissue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools -suture drivers</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>12502-12</td>
			<td style="width:185px;">Olsen-Hegar suture drivers (suturing)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11627-12</td>
			<td style="width:185px;">Adson-Brown alligator forceps (tissue grasping suturing, rat)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14110-15</td>
			<td style="width:185px;">Mayo tough cut scissors 15 cm (surgery, dissection, bones, rat)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>18025-10</td>
			<td style="width:185px;">suture tying forceps (used for Millar cath)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14078-10</td>
			<td style="width:185px;">Lexer Baby scissors straight (surgery, mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11254-20</td>
			<td style="width:185px;">Dumont #5 fine-tip forceps (rat vessels, dissection)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14082-09</td>
			<td style="width:185px;">Dissector scissors 12 mm (surgery, rat mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11051-10</td>
			<td style="width:185px;">10 cm Graefe forceps (tissue grasping, rat mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11251-35</td>
			<td style="width:185px;">Dumont 5/45 forceps (introducer for vessels)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-retractors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>17012-11</td>
			<td style="width:185px;">Weitlaner retractors 2/3 tooth (rat surgical)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11294-00</td>
			<td style="width:185px;">Dumont #4 forceps (vessel isolation rats, mice)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11297-00</td>
			<td style="width:185px;">Dumont #7 forceps (tissue grasping, dissection)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14058-11</td>
			<td style="width:185px;">tough cut iris scissors (mouse dissection, bones)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11009-13</td>
			<td style="width:185px;">serrated, curved Semken forceps (tissue grasping, mouse rat)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-hemostats</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>13003-10</td>
			<td style="width:185px;">Hartman curved hemostats (blunt dissect, hold tissue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11006-12</td>
			<td style="width:185px;">Adson serrated forceps (tissue grasping)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">clippers</td>
			<td style="width:167px;">Oster</td>
			<td style="width:119px;">A5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tape</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:119px;">159015G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">artificial tear ointment</td>
			<td style="width:167px;">Akorn Inc</td>
			<td style="width:119px;">13985-600-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lidocaine</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-4277-01</td>
			<td>2% injectable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinyl catheters</td>
			<td style="width:167px;">Tygon</td>
			<td style="width:119px;">PV-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Evans blue</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Substance P</td>
			<td style="width:167px;">Bachem&#160;</td>
			<td style="width:119px;">H-1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heparin</td>
			<td style="width:167px;">Sagent Pharmaceuticals</td>
			<td style="width:119px;">25201-400-10</td>
			<td>1000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saline solution</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-7138-09</td>
			<td>0.9% sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phenobarbital&#160;</td>
			<td style="width:167px;">Vortech</td>
			<td style="width:119px;">0298-9373-68</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium citrate</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">BP327-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">P4417-50TAB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes for blotting</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">06-666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">formamide</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">47670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microbalance</td>
			<td style="width:167px;">Denver Instrument</td>
			<td style="width:119px;">APX-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microfuge tubes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polystyrene 96 well plate</td>
			<td style="width:167px;">Becton Dickenson</td>
			<td style="width:119px;">351172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">absorbance plate reader</td>
			<td style="width:167px;">BioTek</td>
			<td style="width:119px;">Synergy 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gels</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>3450014&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">protein molecular weight standard</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>1610374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protran supported nitrocellulose</td>
			<td style="width:167px;">Amersham (GE)</td>
			<td style="width:119px;">10600015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gel box</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>1658005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween20</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">P-1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">S271-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary NEP polyclonal antibody&#160;</td>
			<td style="width:167px;">R &#38; D Systems</td>
			<td style="width:119px;">AF1182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">doxycycline chow</td>
			<td style="width:167px;">Teklad (HARLAN)</td>
			<td>TD.130750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FVB/NJ wild type mice</td>
			<td style="width:167px;">Jackson</td>
			<td style="width:119px;">001800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">secondary antibody (goat anti-rabbit)</td>
			<td style="width:167px;">ZyMed</td>
			<td style="width:119px;">81-6120</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL solution-Western Lightening Plus</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:119px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">film</td>
			<td style="width:167px;">Pierce</td>
			<td style="width:119px;">34091</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized evans blue protocol to assess vascular leak in the mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In Figure 1, a schematic of the procedure is shown, which has been found to result in the most reliable and consistent substance P-induced plasma extravasation values from organs of FVBN mice. This procedure usually takes two days of work, separated by at least 48 h of waiting time. It is possible to spread it out even more, if this is done consistently for all experiments to be compared. For example, after the organs are isolated on Day 1, the organs can be ...

Watch this Scientific Journal Video about An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse at JoVE.com

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

an-optimized-evans-blue-protocol-to-assess-vascular-leak-in-the-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.1K Views.  University of Colorado Denver. In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Video: An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.