This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

All animal experiments and procedures were approved by the Tulane University&#39;s Institutional Animal Care and Use Committee (IACUC).
1. Surgical Procedure Setup
<ol>
	<li>Autoclave instruments, surgical supplies, and culture supplies prior to surgery. Surgical supplies for each rat include: 1 drape, 1 drape with pre-cut hole (0.5 in x 1.5 in) in the center, gauze pads, and 1 absorbent underpad. Surgical instruments include: 1 scalpel with a number 10 blade, 2 pairs of tweezers, and a pair of fine scissors. Culture supplies include: 1 drape, 1 pair of tweezers, and prepared 6-well plate inserts with polycarbonate filters.</li>
	<li>Sterilize a plexiglass platform, a surgical stage and a surgical benchtop space with 70% ethanol. Keep the surgical stage in a sterile bowl until use.
	<ol>
		<li>Create a surgical stage by drilling an approximately 2 in by 1 in hole in the center of a 100 mm culture dish. Next, use sandpaper to smooth any sharp edges and add a layer of silicone glue to the hole&#39;s edges to create a raised surface for the tissues.</li>
		<li>Alternatively, design the surgical stage using CAD software and make by 3-D printing (Figure 1).</li>
	</ol>
	</li>
	<li>Place a sterile absorbent underpad down and lay a plexiglass platform on top of it. Place the drape, without a pre-cut hole, over a heated pad next to the absorbent underpad.</li>
	<li>Pre-warm sterile phosphate-buffered saline (PBS), media and saline to 37 &#176;C. Place media and PBS in separate culture dishes atop the heating pad and place saline in a 50 mL conical tube next to the surgical setup.</li>
	<li>Make sure all packages are opened prior to the beginning of the surgery to ensure sterile handling of all materials. A complete list of the common tools used in this procedure are listed in the Table of Specific Surgical Materials and Tools.</li>
</ol>
2. Mesentery Tissue Harvesting
<ol>
	<li>Use adult male Wistar rats (350 &#177; 25 g; 6 - 8 weeks of age). Other strains and ages of rats can be substituted.</li>
	<li>Anesthetize the rat via an intramuscular injection of ketamine (80 mg/kg body weight) and xylazine (8 mg/kg body weight). Confirm the rat is under anesthesia by pinching between the toes to check for a reflex response; there should be none.&#160;Pre-emptive analgesia for this terminal procedure is not necessary.</li>
	<li>Shave the abdominal region and remove remaining hair using hair removal cream. Wipe abdominal skin twice with 70% isopropyl alcohol followed by povidone-iodine. For the wipes the surgeon should start at the center of the surgical site and move to the outside of the prepared area in a circular manner as to not overlap areas that have been previously scrubbed with the same piece of sterile gauze or sterile cotton swab. Then transfer animal to the sterile surgical setup and place atop the plexiglass platform.</li>
	<li>Using a scalpel blade, make a 0.75 in - 1.25 in incision in the gut starting 1 in below the sternum. Be careful not to puncture the bowel or mesentery (1 layer of skin, 1 layer of connective tissue, and 1 layer of muscle).</li>
	<li>Place a drape with a pre-cut hole over the incision and place a sterile surgical stage atop the drape. Ensure the opening aligns with the incision. Use sterile cotton-tipped applicators to locate and pull out the ileum through the surgical stage opening.</li>
	<li>Pull 6 - 8 mesenteric windows through the stage using cotton-tipped applicators, and be careful not to touch the windows (Figure 1). Tissues are typically harvested from the ileum region of the small intestine starting near the cecum. Keep exposed tissues moist with warmed sterile saline as needed using a sterile syringe to drip the solution.</li>
	<li>Euthanize the rat via intracardiac injection of pentobarbital sodium (0.2 mL per rat). Before removing mesenteric windows, ensure the rat is euthanized by palpating the heart; there should be no pulse.</li>
	<li>Remove desired mesentery tissues by using tweezers to grab the fat pad and fine scissors to cut the window. Leave a border of fat (2 mm) around the window. Wash tissues once in warmed sterile PBS and once in media.</li>
	<li>Return exteriorized ileum to the abdominal cavity and dispose of animal according to institutional guidelines.</li>
</ol>
3. Mesentery Tissue Culture for Time-Lapse Studies
<ol>
	<li>Transfer autoclaved culture supplies (see section 1.1) and tissues to a sterile laminar flow hood.</li>
	<li>Use tweezers to transfer each tissue atop a polycarbonate filter membrane. Grab tissues by the fat pad to avoid damaging the vasculature.</li>
	<li>Quickly spread the tissue using the fat pad, being careful not to touch the window. Invert the insert with the tissue into the bottom of a 6-well plate and cover with 3 mL of media (Figure 1). Typical media used for this procedure includes Minimum Essential Media (MEM) with 1% Penicillin Streptomycin (PenStrep) and 10% Fetal Bovine Serum (FBS). Media can be supplemented with other serums and/or growth factors to stimulate angiogenesis.</li>
	<li>Repeat steps 3.2 - 3.3 for each tissue and culture in standard incubator conditions (5% CO2, 37 &#176;C) for up to 5 days.</li>
</ol>
4. Time-Lapse Imaging of Mesentery Tissue
<ol>
	<li>On the day of imaging, supplement the media in each well with conjugated BSI-Lectin and incubate under standard culture conditions for 30 min. Wash tissues twice with lectin-free media. BSI-Lectin stain will remain visible on the mesentery tissue for up to 3 days in culture.</li>
	<li>Transfer the plate to a microscope stage. Identify blood and lymphatic vessels based on their morphology and network structure.</li>
	<li>Locate a desired network region on each tissue and take images. Take note of the imaging location to ensure the same region will be captured for subsequent images. If using a motorized stage, document the coordinates.</li>
	<li>Return tissues to the incubator and continue to culture until desired end point. Repeat steps 4.1 - 4.3 as needed depending on desired experimental time points.</li>
</ol>
5. Tissue Immunolabeling
<ol>
	<li>BSI-Lectin Labeling
	<ol>
		<li>Incubate tissues for 30 min at 37 &#176;C with 1:40 FITC-conjugated lectin in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media. For rinses, add media and then immediately replace.</li>
	</ol>
	</li>
	<li>Live/Dead Labeling
	<ol>
		<li>Incubate tissues for 10 min at 37 &#176;C with 1:500 2 mM ethidium homodimer-1 and 1:500 1 mM calcein AM in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media.</li>
	</ol>
	</li>
	<li>BSI-Lectin/NG2 Labeling
	<ol>
		<li>Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.</li>
		<li>Fix tissues in cold methanol for 30 min at -20 &#176;C. Wash tissues with PBS (3 x 10 min).</li>
		<li>For primary antibody labeling incubate tissues for 1 h at room temperature with 1:100 rabbit polyclonal NG2 antibody and 5% normal goat serum (NGS). Wash tissues with PBS (3 x 10 min).</li>
		<li>For secondary antibody labeling incubate tissues for 1 h at room temperature with 1:100 goat anti-rabbit CY2-conjugated antibody (GAR-CY2) and 5% NGS. Wash tissues with PBS (3 x 10 min).</li>
		<li>Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS. For rinses, add PBS and then immediately replace.</li>
		<li>To mount the slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.</li>
	</ol>
	</li>
	<li>LYVE-1/PECAM Labeling
	<ol>
		<li>Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.</li>
		<li>Fix tissues in cold methanol for 30 min at -20 &#176;C. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For primary antibody labeling incubate tissues for 1 h at room temperature with 1:200 mouse monoclonal biotinylated CD31 antibody and 1:100 rabbit polyclonal LYVE-1 antibody in PBS + 0.1% saponin + 2% bovine serum albumin (BSA) + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1:500 CY3-conjugated streptavidin antibody and 1:100 GAR-CY2 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>To mount slides, cover tissues with 50:50 PBS and glycerol solution and place a coverslip on top. Seal the slide edges using nail polish.</li>
	</ol>
	</li>
	<li>BrdU/BSI-Lectin Labeling
	<ol>
		<li>Add 1 mg/mL BrdU to media and replace tissue media with BrdU solution. Incubate for 2 h at 37 &#176;C.</li>
		<li>Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.</li>
		<li>Fix tissues in cold methanol for 30 min at -20 &#176;C. Wash tissues with PBS (3 x 10 min).</li>
		<li>Denature tissue DNA in 2 M HCl for 1 h at 37 &#176;C. Wash tissues in PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For primary antibody labeling, incubate tissues for 1 h at room temperature with 1:100 monoclonal mouse anti-BrdU in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1:100 goat anti-mouse Cy3-conjugated antibody (GAM-Cy3) in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS.</li>
		<li>To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.</li>
	</ol>
	</li>
	<li>BSI-Lectin/CD11b labeling
	<ol>
		<li>Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.</li>
		<li>Fix tissues in cold methanol for 30 min at -20 &#176;C. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For primary antibody labeling incubate tissues for 1 h at room temperature with 1:100 mouse anti-rat CD11b in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>For secondary antibody labeling incubate tissues for 1 h at room temperature with 1:100 GAM-Cy3 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).</li>
		<li>Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS.</li>
		<li>To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol documents a method for using the rat mesentery culture model as an ex vivo tool for time-lapse imaging of microvascular network growth. Previous work in our laboratory has established the use of our model for 1) angiogenesis8, 2) lymphangiogenesis8, 3) pericyte-endothelial cell interactions8, and 4) anti-angiogenic drug testing9. The ability for imaging cultured rat mesentery tissues at multiple time points offers a quantitative assay for evaluating tissue-specific growth responses and the tracking of cell-cell interactions during various angiogenic stimuli. The increased proliferation of endothelial cells during angiogenesis and the presence of pericytes are consistent with our previous work8 and validate the dynamic interactions between multiple cell types during angiogenesis in cultured rat mesenteric tissues.
Compared to commonly used tissue culture models and in vitro cell culture systems, the rat mesentery culture model is unique because growth occurs within an intact, real microvascular network. Consider in contrast the aortic ring assay, which was established to study angiogenic sprouting from aortic segments in a collagen gel10. While sprouting in the aortic ring involves multiple cell types, capillary sprouts grow out of the excised segments of the aorta, which is very different from the in vivo scenario. The brain slice model is another ex vivo model, but it is void of lymphatic vessels. Moreover, the brain slice model has not been shown to be capable of time-lapse imaging before and after angiogenic stimulation11. Another ex vivo model that has been recently introduced is the retina culture model. The advantage of the retina model is that angiogenesis occurs from intact microvascular networks within the tissue12,13. For these models, GFP-transgenic mice strains can be used to be able to observe capillary sprouting over time, but unfortunately, the mouse mesentery is avascular14, eliminating the GFP-transgenic mice mesentery substitution for rat mesentery, as utilized in our model. Furthermore, we show that a simple lectin labeling of rat mesentery tissues in culture is sufficient to determine network growth at different time points and in comparison to the other ex vivo models, our model allows for simultaneous observation of both blood and lymphatic endothelial cells.
BSI-lectin was used in this paper to visualize microvascular networks and detect angiogenic responses. Lectin is a protein structure that binds to glycoproteins on endothelial cells and was selected for this protocol due to its short incubation time compared to endothelial antibody markers. Lectin is less expensive than antibodies and it does not require fixing; it can also be easily mixed in the culture media and replaced with fresh media after the incubation period ends. While future studies are needed to elucidate the potential effects of the lectin labeling technique on the angiogenic process, our representative results (Figure 4) demonstrate that robust angiogenesis can be induced and previous work9 demonstrates that angiogenesis in lectin labeled networks can be inhibited via targeting Vascular Endothelial Growth Factor (VEGF). Antibody markers can potentially be used as an alternative labeling approach when there is a need for more specific markers, or when there is a need to investigate other cell types that are present in the microvascular networks such as smooth muscle cells, pericytes, and nerves. Another potential method for visualizing cells would be gene transfection.
The advantage of using the time-lapse rat mesentery model has been highlighted in the representative results for this protocol. The comparison of images before and after treatment reduces issues of variability that influence non-paired statistical analysis. The explant specific responses varied from 20% to 233% increase in vessel density and from 40% to 3,500% increase in sprout density. The specific causes for this variation remain unknown, but measuring growth in the same tissue over time present the ability to confirm tissue specific responses.
Comparative analysis of images at different time points during microvascular growth also allows for tracking endothelial cells. For example, our lab has identified vascular islands as endothelial cell segments in the vicinity of microvascular networks that are disconnected from nearby networks15,16. To confirm that these islands connect to the nearby network in response to angiogenic stimuli, the rat mesentery culture model was used. As shown in Figure 5, vascular islands were tracked after tissue stimulation with basic Fibroblast Growth Factor (bFGF) or VEGF plus Platelet-Derived Growth Factor (PDGF). We have also shown similar results post serum stimulation (data not shown here). After the stimulation of angiogenesis, the originally disconnected vascular islands can be found connected to nearby networks.
Other potential applications of the rat mesentery culture model could leverage the ability to investigate the relationships between lymphatic and blood vessels and their respective endothelial cells and the tracking of interstitial cell fate. Time-lapse images of the same microvascular networks before and after stimulation with 10% serum in this model provided examples of potential lymphatic-to-blood vessel integration (Figure 6). Before stimulation, lymphatic and blood vessels were distinguished based on vessel morphology. After stimulation, lymphatic versus blood vessel identity became less clear. The potential for lymphatic/blood endothelial cell interactions is supported by the observation of PECAM+/LYVE-1- blood endothelial cells connecting with PECAM+/LYVE-1+ lymphatic endothelial cells (data not shown here). These observations support the use of the rat mesentery culture model for investigating lymphatic/blood endothelial cell plasticity. Figure 6A also highlights the lectin labeling of apparent interstitial cells. While this labeling is inconsistent and heterogeneous from tissue to tissue, it does emphasize the presence of endogenous tissue resident cells. CD11b labeling of cultured tissues (Figure 7) suggests that these lectin-positive interstitial cells could be a sub-set of macrophages. Given the emerging interest in macrophage involvement in angiogenesis20, an additional strength of the model could be its use to track macrophage dynamics over time.
Much like other ex vivo models, a current limitation of studying angiogenesis in the rat mesentery culture model is the lack of blood flow. Shear stress caused by blood flow has been shown to play a role in endothelial cell morphology and proliferation as well as angiogenesis17,18,19. For the representative results presented in Figure 4, the absence of shear stress alone may have been sufficient to induce an angiogenic response in the cultured networks. However, we know that based on our initial publication characterizing the rat mesentery culture model8, that media supplementation causes increased angiogenesis versus media alone. Future studies incorporating flow within the cultured microvascular networks are undoubtedly needed to more closely mimic the in vivo scenario. Potential approaches for incorporating flow might include cannulation of network feeding arterioles or even cannulation of further upstream arteries within the fat border of mesenteric windows. However, despite the lack of flow, the viability of multiple cell types, the maintenance of blood and lymphatic microvascular networks, and cell proliferation during angiogenesis supports the rat mesentery culture model&#39;s relative increased level of complexity compared to cell based in vitro models.
In conclusion, this protocol describes a simple, reproducible ex vivo method for imaging angiogenic responses in intact microvascular networks. Such a method offers an alternative to cell based in vitro models for evaluating angiogenic cell dynamics at specific locations within a network environment. The method also offers a novel tool for investigating angiogenesis, lymphangiogenesis and blood/lymphatic mis-patterning simultaneously.

Microvascular network growth and remodeling are common denominators for tissue function, wound healing, and multiple pathologies and a key process&#160;is angiogenesis, defined as the growth of new blood vessels from existing ones1,2. For tissue engineering new vessels or designing angiogenic based therapies, understanding the importance of the cellular dynamics involved in angiogenesis is critical. However, this process is complex. It can vary at specific locations within a microvascular network and involves multiple cell types (i.e. endothelial cells, smooth muscle cells, pericytes, macrophages, stem cells) and multiple systems (lymphatic networks and neural networks). Although in vitro models have contributed tremendously to examining the relationship between different cells involved in angiogenesis3, their physiological relevance can be undermined due to their limited complexity and the fact that they do not closely reflect an in vivo scenario. To overcome these limitations, three-dimensional culture systems3, ex vivo tissue models4, microfluidic systems5,6, and computational models7 have been developed and introduced in recent years. However, there is still a need for a model with time-lapse capability to investigate angiogenesis in intact microvascular networks ex vivo. The establishment of new time-lapse models for angiogenesis studies with that level of complexity will provide an invaluable tool to understand the underlying mechanisms regulating angiogenesis and to improve therapies.
A potential model that enables the ex vivo investigation of angiogenesis across an intact microvascular network is the rat mesentery culture model8. In recent work, we have demonstrated that blood and lymphatic microvascular networks remain viable after culture. More importantly, the rat mesentery culture model can be used to investigate functional pericyte-endothelial cell interactions, blood and lymphatic endothelial cell connections, and time-lapse imaging. The objective of this paper is to provide our protocol for the time-lapse imaging method. Our representative results document the multiple cell types that remain viable after the stimulation of angiogenesis with serum and offer examples of using this method for quantifying tissue specific angiogenic responses as well as endothelial cell tracking studies.

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

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

After 3 days in culture, tissues were labeled with a live/dead viability/cytotoxicity kit to demonstrate the viability of the microvasculature in the rat mesentery culture model (Figure 2A). The majority of cells present in the mesentery remained viable in the culture where endothelial cells were identified based on their location in microvascular segments. Endothelial cell proliferation was also confirmed by lectin/BrdU labeling (Figure 2D). Smooth muscle cell and pericyte presence along vessels was confirmed with NG2 labeling (Figure 2B). Labeling for LYVE1 and PECAM identified branching lymphatic and blood microvascular networks and confirmed the maintained lymphatic versus blood endothelial cell phenotype (Figure 2C).
The time-lapse feature of this model was utilized by labeling the microvascular networks with BSI-lectin at different time points and imaging the same region within the network over time; this capability is particularly valuable for investigating tissue specific angiogenic responses. The supplementation of media with 10% serum caused a robust angiogenic response after 3 days of stimulation. Additionally, new vessel segments and capillary sprouts were identified by day 5 of stimulation (Figure 3). The time-lapse imaging method allowed for the quantitative comparison of network regions before and after stimulation (Figure 4). For this representative study, which corroborates our previous results9, the number of vessels per vascular area and the number of capillary sprouts per vascular area were quantified from one 4X image per tissue. Blood vessel segments were defined as lectin-positive blood endothelial cell segments present between two branch points and capillary sprouts were defined as blind ended segments originating from a host vessel. Time-lapse comparison of network regions also enabled tracking of endothelial cell segments (Figure 5) and identification of blood/lymphatic vessel mis-patterning (Figure 6). Labeling of cultured tissues for lectin and CD11b additionally confirmed the presence of interstitial resident macrophages (Figure 7) in remodeling networks.
<img alt="Figure 1" src="/files/ftp_upload/55183/55183fig1.jpg" /> 
Figure 1. Mesenteric windows were located by pulling out the small intestine through a surgical stage. The surgical stage was designed and made by 3-D printing. The elliptical hole in the center is approximately 2 in by 1 in (A). The mesenteric windows were then spread out on top of a membrane insert, and the insert was inverted and put into a well (B). Scale bar = 2 cm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55183/55183fig2.jpg" /> 
Figure 2. Blood vessels remain viable in the rat mesentery culture model. Live/dead assay performed after culture showed a high ratio of live cells (green) to dead cells (red) specifically along the blood vessels (A). Mesentery tissues were labeled with lectin and anti-NG2, to identify pericytes (red) alongside vessels (green) and to confirm that different types of cells are present in the post-culture tissues (B). Tissues were also labeled against PECAM/LYVE-1 to identify blood (red) vessels from lymphatic (green) vessels (C). To investigate if microvascular cells undergo proliferation in culture, mesentery tissues were labeled with lectin/anti-BrdU. On capillary segments labeled with lectin (green), multiple cells were confirmed to be proliferative (red) (D). Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55183/55183fig3.jpg" /> 
Figure 3. Time-lapse imaging of the rat mesentery enables observing microvascular remodeling over the course of the culture. A robust angiogenic response was observed after 3 (B) and 5 days (C) of culture with 10% serum stimulation. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55183/55183fig4.jpg" /> 
Figure 4. Microvascular networks in the rat mesentery culture model were imaged before and after angiogenesis. Comparison of the same network labeled with lectin on day 0 and day 3 (A, B) post-stimulation with 10 % serum identifies new vessels. Lectin also labels a population of unidentified interstitial cells. Quantification of vessel density (C, D) and the number of capillary sprouts per vascular area (E, F) confirmed an increase in both metrics for each tissue. C, E) Before (day 0) and after (day 3) comparisons per tissue. D, F) Comparison between day 0 and day 3 averages using a paired Student&#39;s t-test confirmed a significant difference in both the average number of vessel segments (p &#60; 0.0001) and the average number of sprouts (p &#60; 0.00001) per vascular area. White bars represent day 0, and black bars represent day 3. Values are averages &#177; SEM. For this representative analysis, 13 tissues were harvested from 2 rats. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55183/55183fig5.jpg" /> 
Figure 5. The rat mesentery culture model can be used for investigating vascular island fate and incorporation into nearby networks. Using time-lapse imaging, vascular islands,&#160;defined as disconnected endothelial segments, were identified on day 0 and their connection to the nearby network was confirmed by day 3 post-angiogenic stimulation. Mesentery tissues were stimulated with bFGF (A, B) and VEGF/PDFG-BB (C, D). Hollow arrows show disconnected segments on day 0 and solid arrows represent island connection to the network. Arrowheads indicate the location of connections between a vascular island and the nearby network. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55183/55183fig6.jpg" /> 
Figure 6. Time-lapse images demonstrate the ability to observe lymphatic and blood vessel patterning. Lymphatic (l) vessels can be distinguished from arterioles (a) and venules (v) based on labeling morphology on day 0 (A). On day 5 post-stimulation with 10% serum, lymphatic morphology is lost and vessels appear to have integrated with the nearby angiogenic blood vessels (B). Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55183/55183fig7.jpg" /> 
Figure 7. Macrophages remain present in cultured rat mesenteric tissues. Lectin/CD11b co-labeling of tissues cultured for 3 days with 10% serum suggest that lectin positive interstitial cells are a subset of macrophages. (A) A representative image of BSI-lectin labeling. (B) Cd11b labeling in the same field of view. (C) The merged image. The arrows identify examples of co-labeling. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55183/55183fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

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

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

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

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

an-ex-vivo-method-for-time-lapse-imaging-cultured-rat-mesenteric

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.8K Views.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

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

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Immunology and Infection

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

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

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

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

Biology

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

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

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

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

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

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

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

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

Medicine

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

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

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

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

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

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

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.

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 ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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

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