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

Summary

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.

Abstract

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.

Introduction

The fact that an increasing number of patients with coronary heart diseases have benefited from revascularization therapies for decades does not negate the fatal consequences of MIRI. As described by Yellon and Hausenloy, approximately 30% of the final infarct size can be attributed to MIRI¹. Cardiac microvascular endothelial dysfunction is the core mechanism underlying MIRI and the "no-reflow" phenomenon and is the target of many cardioprotective agents and classical cardioprotective pathways². Moreover, endothelial hyperpermeability of microvessels can trigger myocardial edema³ and the over-infiltration of pro-inflammatory cells, including neutrophils⁴, macrophages, and erthrocytes⁵.

MIRI-induced oxidative stress and overexpressed pro-inflammatory cytokines, such as the members of the tumor necrosis factor family⁶ and the interleukin family⁷, activate signaling pathways of cell apoptosis to accelerate the death of ischemic myocardium. Erythrocytes penetrate the vulnerable endothelial barrier and accumulate in the extracellular matrix (ECM) to form intramyocardial hematoma⁸. Macrophage- and neutrophil-derived matrix metalloproteinases disassemble collagen in the ECM, which increases the risk of mechanical complication and post-infarction ventricular aneurysm⁹. Thus, the permeability of the endothelial barrier is a crucial sign for the prognostic evaluation of acute myocardial infarction and marks the severity of MIRI to some degree.

FITC-dextran is an array of polysaccharides comprised of different lengths of branched glucose molecules of molecular weight ranging from 4,000 Da to 70,000 Da. It is widely used as the standard tracer to demonstrate the permeability of cell membranes, functional physiological barriers such as the blood-brain barrier¹⁰, and mucosal membranes¹¹ under specific treatments and pathological conditions. The permeability of one specific membrane varies for different substances according to their molecular weights. Usually, albumin is restricted to cardiac microvessels; however, it can penetrate the endothelium in the early stage of IR, causing myocardial edema. As the molecular weight of albumin is approximately 68,000 Da, 70,000 Da FITC-dextran would be a suitable agent to demonstrate endothelial hyperpermeability after IR. Moreover, dextran has been widely adopted to maintain the blood volume and colloid osmotic pressure of shock patients, and FITC-labeled tracers are used in numerous in vivo or in vitro studies with few adverse effects.

In addition to FITC-dextran, endothelial permeability has been measured with Evans Blue (EB) staining. After injection into the blood circulation, EB binds to serum albumin tightly and forms a complex¹². Thus, this EB-albumin complex, not EB alone, can be used for the measurement of permeability, which means that the accuracy of such an EB-based method can be influenced by different concentrations of serum albumin in experimental subjects. Additionally, EB has been reported to poison the organs of experimental animals, especially the heart, liver, and lungs, via cellular degeneration and increased vascular permeability¹³. Endothelium undergoing ischemia/reperfusion would be more sensitive to such detrimental effects than normal endothelium, which can confound the significance of experimental results.

Compared to EB dye, FITC-dextran is exogenous, and the binding affinity to albumin is negligible. Hence, FITC-dextran remains in the free state and is not bound by macromolecules, such as albumin and tissue proteins. Thus, if administered according to body weight, FITC-dextran can work at equal blood concentration and is not affected by individual differences in serum albumin concentration and blood volume. Tongxinluo (TXL), a traditional Chinese medicine, was used to test the diagnostic sensitivity of this method. TXL reduces MIRI by maintaining the endothelial cellular ultrastructure and endothelial barrier integrity¹⁴. TXL is also used to cure angina pectoris and improve myocardial no-reflow after infarction clinically¹⁵. It contains Radix ginseng, Buthus martensi, Hirudo, Eupolyphaga seu steleophaga, Scolopendra subspinipes, Periostracum cicadae, Radix paeoniae rubra, Semen ziziphi spinosae, Lignum dalbergiae odoriferae, Lignum santali albi, and Borneolum syntheticum. TXL has been shown to protect the microvascular endothelium via the MEK/ERK pathway¹⁶ and endothelial nitric oxide synthase-mediated mechanisms^5,17 and alleviate ischemia/reperfusion injury (IRI) of organs¹⁸, especially the heart, in a dose-dependent manner. Here, rats were fed with 0.5 g/kg TXL powder (an equivalent dose to that used clinically in humans) as described¹⁹. This paper provides a method to functionally assess the endothelial barrier of cardiac microvessels after IR via the measurement of the total fluorescence intensity of extravasated 70,000 Da FITC-dextran compared to EB.

Protocol

Animal experiments were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" issued by the US National Institutes of Health (Bethesda, MD, USA, NIH Publication No. 85-23, revised 1996) and the "Regulation to the Care and Use of Experimental Animals" of the Beijing Council on Animal Care (1996). The study protocol was approved by the Care of Experimental Animals Committee of Fuwai Hospital. Male SPF-class Sprague-Dawley rats (SD rats) weighing 150-180 g were used in this study.

1. Preparation of reagents, materials, and equipment

1. Prepare reagents and solutions including 70% ethanol, FITC-dextran (70,000 Da, dissolved in ice-cold saline to make 10% w/v solution for use), hair removal cream, mounting medium with 4',6-diamidino-2-phenylindole (DAPI, 0.5-10 µg/mL), 1% w/v EB/saline solution, and 5% w/v pentobarbital sodium/saline solution (stored in 4 °C in the dark).
2. Sterilize all surgical tools and materials in advance, including surgical tape; gauze pads; surgical gloves; 16 G endotracheal tube; insulin syringe; operating scissors; large, curved dissecting forceps; straight dissecting forceps; straight mosquito forceps; curved mosquito forceps; retractors (30 mm Spread); 5 cm microneedle holders; 3-0 polypropylene suture (taper needle); and 6-0 polypropylene suture (round needle).
3. Check and ensure that all equipment, including the constant temperature warming system, illumination system, ventilator, automated quantitative pathology imaging system, and ultraviolet freezing slicer, are in good condition.

2. Preparing the rats for the surgical procedures

1. Divide all SD rats into Sham group, IR group, and TXL group (IR+TXL group) randomly.
   NOTE: In this protocol, six rats were randomly assigned to each group, n=6.
2. Weigh each SD rat, record the weight, and calculate the dose of TXL to be administered for each rat.
3. Administer ibuprofen at a dose of 30 mg/kg by gavage to each rat at least 12 h prior to thoracotomy to alleviate preoperative pain and distress.
4. Administer a superfine powder of TXL at a dose of 0.5 g/kg by gavage 12 h and 1 h to each SD rat in the IR+TXL group before anesthetization, as described¹⁹.
5. Disinfect the operating table with 70% ethanol and place the sterilized surgical tools and materials on the disinfected surface.
6. Adjust the temperature of the constant temperature warming system to maintain the body temperature of the rats at 37 ± 1 °C.

3. Establish a rat model of cardiac ischemia/reperfusion

1. Inject 5% w/v pentobarbital sodium/phosphate-buffered saline (PBS) solution at a dose of 50 mg/kg (0.1 mL/100 g body weight) into the abdominal cavity to anesthetize the rat. Prepare the surgical site by carefully removing the fur from the neck to the chest by shaving or using a depilatory cream.
2. Gently adjust the position of the tongue as needed with sterilized tweezers and wipe respiratory secretion with sterilized cotton balls. Keep the glottis clearly in view and insert an endotracheal tube into the trachea gently.
3. Connect the endotracheal tube to a ventilator circling at 65-80 breaths per minute and adjust the tidal volume at 4-5 mL by fixing the rat's head and limbs.
4. Place a sterile drape over the rat, disinfect the operating area with tincture of iodine followed by 70% ethanol, and repeat this procedure three times.
5. Make a left parasternal incision at the level of the fourth intercostal space to expose the left musculus pectoralis major and pectoralis minor.
6. Dissect the fascia between the musculus pectoralis major and pectoralis minor using a blunt dissection technique to expose the left fourth intercostal space.
   NOTE: Avoid excessive tissue damage and vascular injury that will cause severe bleeding and postoperative death.
7. Dissect the intercostal muscles, position a chest retractor within the fourth intercostal space, and then spread the ribs.
8. Tear the pericardium and remove the attached adipose tissue.
   NOTE: Avoid damaging other organs with sharp surgical instruments, especially the lungs. Damage to the lungs can result in the development of pneumothorax and hemopneumothorax during the operation, causing cacoethic consequences ranging from hemodynamic disorders to reduced animal survival.
9. Manipulate the position of the thymus and expose the ascending aorta and left atria gently. Identify and ligate the proximal left anterior descending (LAD) artery transiently for 45 min with a 6-0 polypropylene suture (see Figure 1 A-D).
   NOTE: This procedure is identical for sham control rats except that the LAD is not ligated.
10. Remove the chest retractor and draw the ribs together in an interrupted suture pattern with a 3-0 polypropylene suture.
11. Restore negative intrathoracic pressure to ensure effective breathing.
    NOTE: Two methods can be used to restore negative intrathoracic pressure: i) before closing the chest cavity, researchers can plug up the air outlet of the ventilator for 2 cycles to inflate the lungs. ii) The air remaining in the chest cavity can be drawn out with a syringe after the intercostal space has been closed.
12. After hair removal and disinfection, make an incision in the right lower limb and dissect the superficial fascia from the right femoral vein.
13. Loosen the ligation and check the respiration and circulation conditions after inducing myocardial ischemia for 45 min.
14. Inject 10% (w/v) FITC-dextran/saline solution into the femoral vein at 0.15 mL/100 g body weight, and press the femoral vein for 1 min with sterile cotton balls to stanch the bleeding (Figure 2A,B).
    NOTE: Alternatively, inject 1% w/v EB/saline solution into the femoral vein at a dose of 0.5 mL/100 g body weight.
15. Sew up the incisions with a 3-0 polypropylene suture and disinfect the incisions, followed by postoperative analgesia with ibuprofen, as described above.

4. Preparation of frozen tissue slices for fluorescence measurement

1. Inject 5% w/v pentobarbital sodium/PBS solution at a dose of 150 mg/kg (0.3 mL/100 g body weight) intraperitoneally to anesthetize the rats deeply after 3 h of myocardial reperfusion.
2. Slit the abdominal cavity and expose the aorta abdominalis and vena cava (see in Figure 3A).
3. Inject 50 mL of ice-cold PBS into the vena cava steadily, cut the aorta abdominalis to flush blood from the myocardium, and harvest the heart.
   NOTE: Observe changes in the lungs and liver. A sweating liver suggests that saline is accurately injected into the vena cava (see in Figure 3C), and a pale lung is considered a sign of a well-perfused myocardium (see in Figure 3B).
4. In a dark room, rinse the heart in ice-cold saline or PBS gently to remove any remaining blood and FITC-dextran (or EB) from the chambers of the heart.
5. Cut off the heart from the ligation and discard the myocardium above the ligation.
6. Embed the heart in molds with embedding compound followed by storage at -80 °C.
7. Unload the frozen tissues from the embedding molds and load them on sample holders in a prechilled -20 °C chamber of a freezing slicer.
8. Adjust the thickness of a single slice to 5-8 µm and slice the tissue at approximately the midpoint of the base-apex axis (e.g., the middle of the ischemia).
9. Load the sliced tissue on adhesive microscope slides and seal each slice with one drop (approximately 40 µL) of fluorescent mounting medium containing DAPI.
10. Keep the slides in a dry and dark box.
    ​NOTE: If the slides cannot be scanned immediately, store them in a dry, cool, and dark place to prevent the formation of condensate water and fluorescence quenching.

5. Fluorescence measurement and data analysis

1. Set up the imaging system and check the software connected to the scanner.
2. Load the slides in the slide shelves and insert them into the scanner.
3. Open the slide scanner software (see the Table of Materials) and set up a new exposure protocol by clicking on Edit Protocol | New….
   NOTE: If the scanner or its affiliated software is not available, a laser confocal microscope (see the Table of Materials) or other ordinary slide scanners with the fluorescence mode (see the Table of Materials) may be an alternative to the automated quantitative fluorescence scanner.
4. Rename the protocol and study, and select the fluorescence mode.
5. Define Whole Slide Scan | Pixel Resolution and Multispectral Regions | Pixel Resolution as 0.25 µm (40x) respectively.
6. Click on Edit Filters and Bands... to select the Whole Slide Scan Filters column and the Multispectral Region Bands column.
   NOTE: If EB is used, select Whole Slide Scan Filters | DAPI and Texas Red or AF495. If FITC-dextran is used, select Multispectral Region Bands | DAPI and FITC or AF488.
7. Click on Edit Exposures... and load the carrier.
8. Select the target slot and click on Take overview.
   NOTE: This takes a brightfield scan of each heart section. Select the myocardium for focusing or select the area blindly.
9. Select the target slide, adjust Stage Height to approximately -50 µm, and click on Auto Focus.
10. Click on Whole Scan Exposure(ms)-DAPI | Autoexpose.
    NOTE: The exposure time of DAPI for each slide does not have to be the same because images with DAPI are used to illustrate cross-sections of the heart, which are convenient for outlining the area of the myocardium but not to calculate fluorescence density.
11. Click on Whole Scan Exposure(ms)-FITC | Autoexpose.
    NOTE: Click on Whole Scan Exposure(ms)-Texas Red | Autoexpose if using EB. It is important to remember and ensure that the exposure time of FITC or Texas Red is the same in all slide scans.
12. Click on Auto Focus again and ensure that the slide is in focus.
13. Click on Back and save the protocol.
14. Repeat steps 5.7-5.14 for each slide.
15. Return to the home page and click on Scan Slides.
16. Select a slot, rename all the slides, and select the corresponding exposure protocol for each slide.
17. Scan the slides and capture the images.
18. Open the images with slide viewer software for the scans (see the Table of Materials).
19. Magnify all the images to a relatively equal extent.
20. Snapshot and save images as .tiff.
    NOTE: It is better to save the images with and without scale bars at the same time. Images with scale bars can be used for publication, whereas those without scale bars can be used for statistical analysis.
21. Open ImageJ software, click on Analyze, and select Set Measurement.
22. Select Area | Integrated density.
23. Open the image in the FITC channel with Image J.
24. Click on Analyze again and record the numerical value of integrated density (IntDen) that appears.
    NOTE: As scale bars in the images can be mistaken for grayscale of fluorescence by computers, use images without scale bars to measure IntDen.
25. Close the FITC image and open the image in the DAPI channel.
26. Outline a cross-section of the myocardium.
27. Click on Analyze and collect Area data.
28. Calculate the ratios of IntDen/Area (mean of fluorescence) for each heart.
29. Input all ratios in statistical analysis software.

Discussion

This paper presents a protocol to assess the dysfunction of the cardiac endothelial barrier after being exposed to MIRI. It should be noted that there are several crucial steps in this protocol that determine the reliability and accuracy of the results. All steps involving FITC-dextran are to be performed in a dark room. The FITC-dextran/saline solutions must be stored at 4 °C and wrapped with tin foil. Additionally, coronary arteries must be ligated, as illustrated in Figure 1, so that the myocardium can be reperfused immediately after the loop is loosened. Researchers can choose 5-0 or 6-0 polyethylene sutures because thinner strings under 7-0 are more likely to cut myocardial fibers and vessels. Importantly, the tracer solution should be injected into the femoral veins and not the tail veins. Each heart should be well-perfused and well-rinsed with ice-cold saline before and after being sacrificed, respectively.

Many researchers specializing in MIRI are inclined to study the disrupted subcellular structure of the endothelium in the earlier stage of myocardial reperfusion-the formation of apoptotic bodies, swelling mitochondria, and abnormal intercellular junctions-to prove the hyperpermeability of the endothelium via electron microscopic examination. Immunoblotting has also been used to measure microvascular permeability because the decrease in levels of tight conjunction proteins is regarded as the core mechanism of endothelial injury and malfunction of endothelial barrier over time. For instance, VE-cadherin²⁰ and β-catenin²¹ are key elements of endothelial cell-to-cell adhesion junctions, and these connections contribute to the maintenance of vascular integrity²². Tight junctions, such as JAM-A^23,24 and occludin²⁵, also cooperatively participate in the construction of the endothelial barrier in the circulatory system.

Currently, an increasing number of studies suggest that mitochondrial function and mitochondrial homeostasis could be key targets of protective pathways against reperfusion injury. As reported in pathophysiological and pharmacological studies, mitophagy can play an important role in the cellular death of the endothelium and myocardium induced by IR²⁶. In addition, mitochondrial fission, mitochondrial proteostasis, and mitochondrial quality control may protect high-risk myocardium in reperfused areas^27,28. The above methods focus more on demonstrating different mechanisms or causes of hyperpermeability than on displaying endothelial permeability directly and visually. Moreover, sample preparation for electron microscopic examination is complicated and dangerous. Glutaraldehyde, acetone, and osmic acid solutions used in electron microscopy probably volatilize and erode the mucosa, conjunctiva, and skin, even threatening lives if used in uncontrolled environments.

Compared to these indirect and life-threatening methods, this method, which measures the fluorescence intensity emitted by FITC-dextran extravasation, is ideal for assessing cardiac microvascular permeability. First, 70,000 Da FITC-dextran cannot penetrate through the endothelium under normal physiological conditions but can enter the myocardial interstitial space under IR, which allows a clear differentiation between rats under different treatments. Additionally, the FITC-dextran solution is injected into the blood via the femoral veins, which guarantees that all of the fluid is injected into the rat's circulation. In contrast, tail vein injection is more difficult in rats because the tail vein is indistinct and covered with a thick stratum. Further, unlike regular paraformaldehyde fixation and paraffin embedding, myocardial tissue treatment is simplified and optimized using the quick-freeze pathological section technique used in this study. Short storage time, light-proof environment, and cryopreservation may together alleviate fluorescence quenching of FITC.

More importantly, this method can minimize selection bias during statistical analysis. We perform sufficient heart perfusion with saline to ensure that nearly all the blood mixed with FITC-dextran is douched out from the myocardium and the heart chambers, which can eliminate the fluorescence emitted by any FITC remaining in the vessels. If this is not the case, fluorescence merged with capillaries should be excluded and trimmed artificially. While collecting tissue from areas of interest, no visual boundary was observed between the ischemic myocardium and the remote myocardium, especially after EB dye staining. This hindered the collection of samples from these representative areas. Using the method described herein, researchers can obtain images of whole sections and analyze the fluorescence intensity using a quantitative slider scanner.

Interestingly, EB dye can be an alternative, albeit suboptimal, to FITC-dextran. Compared with the relatively harsh storage conditions of FITC-dextran, EB powder or its solution can remain stable at room temperature for a long time. Additionally, EB is relatively inexpensive for most institutions. Nonetheless, certain disadvantages could limit its application. First, EB staining shows lower sensitivity than FITC-dextran, which may underestimate the differences among groups and increase the sample size of each group. Besides, EB shows stronger adhesion to proteins. The endocardium and epicardium are more easily stained by EB, which can influence the results of fluorescence density. Further, obvious filling defects in the reperfusion area remarkedly reduce the image quality, which can limit the use of EB.

To extend the application of this method in an MI model, the method was tested in an MI model. First, FITC-dextran was injected into blood vessels after LAD ligation. As expected, a low level of fluorescence was emitted from the slice because FITC-dextran in blood flow cannot pass through the ligated site, and no FITC-dextran entered the ischemic area. FITC-dextran was also applied 5 min before ligation to ensure the passage of FITC-dextran into all organs. Consequently, FITC-dextran failed to penetrate the microvascular endothelium probably because no perfusion pressure in ligated blood vessels can drive FITC-dextran through the endothelium. Generally, this method probably requires significantly more modification to satisfy its application in the MI model.

Despite the above advantages, there are limitations to this method. First, this protocol was not tested in IR models of other animals, which may limit its application in preclinical and clinical studies. Second, FITC-labeled dyes are more expensive than other common dyes such as EB. However, unlike EB, as 10% w/v FITC-dextran solution, diluted with serum, rarely changes the color of the myocardium, FITC-dextran staining and EB/2,3,5-triphenyltetrazole chloride (TTC) dual staining may be conducted in the same heart, which can trim the budget with respect to the number of animals and the dyes used. Last, the slide scanner used in this study-the Quantitative Slide Scanner and similar instruments-may not be available in some laboratories. In summary, this protocol presents a simplified, reliable, and visual method for the detection of cardiac microvascular endothelial permeability in an IR model in rats using a fluorescence assay based on an automated quantitative pathology imaging system. This protocol demonstrates that 70,000 Da FITC-dextran performs better than EB in the functional assessment of the endothelial barrier of cardiac microvessels after IR injury.

Materials

Name	Company	Catalog Number	Comments
16 G Angiocather	Becton Dickinson, USA	2089312
3-0 polypropylene suture, taper needle	Ethicon, USA	KGP693
4’,6-diamidino-2-phenylindole (DAPI)	Invitrogen, USA	D3571	Alternative to Mounting medium with DAPI
6-0 polypropylene suture, round needle	Ethicon, USA	KGJ656
Adhesive Microscope Slides	CITOTEST, China	188105
ALC-V8S Ventilator	Shanghai Alcott Biotech, China	ALC-V8S
Constant Temperature Warming System	Shanghai Alcott Biotech, China	ALC-HTP203
Dissecting Forceps-Large Cvd	RWD life Science, China	F12007-10
Dissecting Forceps-Str	RWD life Science, China	F12005-10
FITC-dextran, 70,000Da	Sigma-Aldrich, USA	53471
Hair Removal Cream	Nair, USA	N/A
Insulin Syringe,1ml	Becton Dickinson, USA	8155672
Microneedle holders	RWD life Science, China	F31047-12
Mosquito Forceps-Cvd	RWD life Science, China	F22007-12
Mounting medium with DAPI	ZSGB-BIO, China	ZLI-9557
Multi-position Fiber Optic Illuminator System	RWD life Science, China	76301
Operating Scissors	RWD life Science, China	S14014-12
Pannoramic SCAN	3DHISTECH, Budapest, Hungary	SC150-211305	Alternative slide scan to Vectra Polaris
Phenochart 1.0.8	PerkinElmer, USA	N/A	Slide viewer software
Phosphate buffered saline	HyClone,USA	SH30256.01
Retractors,30mm Spread,5cm	RWD life Science, China	F22005-45
SP8 Confocal microscope	Leica, German	TCS-SP8 SR	Alternative to Vectra Polaris
Surgical tape	3M, USA	1527C-0
Tissue-Tek O.C.T. Compound	SAKURA,Japan	0822-00
Tongxinluo superfine powder	Yiling, China	Z19980015
Ultraviolet Freezing Slicer	Leica, German	CM1950
Vectra Polaris 1.0.7 software	PerkinElmer, USA	N/A	Slide scan software
Vectra Polaris Quantitative Slide Scanner	PerkinElmer, USA	VP1814N1068