Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

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...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+model+for+anti-angiogenic+drug+testing+on+intact+microvascular+networks.">An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.</a> PLoS One. 10 (3), e0119227 (2015).</li><li>Nicosia, R. F., Ottinetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+microvessels+in+serum-free+matrix+culture+of+rat+aorta.+A+quantitative+assay+of+angiogenesis+in+vitro.">Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.</a> Lab Invest. 63 (1), 115-122 (1990).</li><li>Hutter-Schmid, B., Kniewallner, K. M., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+as+a+model+to+study+angiogenesis+of+brain+vessels.">Organotypic brain slice cultures as a model to study angiogenesis of brain vessels.</a> Front Cell Dev Biol. 3, 52 (2015).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat Protoc. 5 (10), 1659-1665 (2010).</li><li>Unoki, N., Murakami, T., Ogino, K., Nukada, M., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+retinal+angiogenesis+reveals+decreased+development+and+progression+of+neovascular+sprouting+by+anecortave+desacetate.">Time-lapse imaging of retinal angiogenesis reveals decreased development and progression of neovascular sprouting by anecortave desacetate.</a> Invest Ophthalmol Vis Sci. 51 (5), 2347-2355 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Kelly-Goss, M. R., Sweat, R. S., Azimi, M. S., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+islands+during+microvascular+regression+and+regrowth+in+adult+networks.">Vascular islands during microvascular regression and regrowth in adult networks.</a> Front Physiol. 4, 108 (2013).</li><li>Kelly-Goss, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+along+vascular+islands+during+microvascular+network+growth.">Cell proliferation along vascular islands during microvascular network growth.</a> BMC Physiol. 12, 7 (2012).</li><li>Skalak, T. C., Price, R. 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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 poi...

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 border="0" cellpadding="0" cellspacing="0" width="679">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:165px;">Drape</td>
			<td style="width:183px;">Cardinal Health</td>
			<td style="width:132px;">4012</td>
			<td style="width:199px;">12&#8221;x12&#8221; Bio-Shield Regular Sterilization Wraps</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Scalpel Handle</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
			<td style="width:199px;">Scalpel Handle, #3; Solid; 4&#34; Length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Surgical Blade</td>
			<td>Cincinnati Surgical</td>
			<td>0110</td>
			<td>Stainless Steel; Size 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Dish (60mm)</td>
			<td>Thermo Scientific</td>
			<td>130181</td>
			<td>10/Sleeve</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:165px;">Graefe Forcep (curved tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Graefe Forcep (straight tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:165px;">Noyes Micro Scissor</td>
			<td style="width:183px;">Roboz Surgical Instrument</td>
			<td style="width:132px;">RS-5677</td>
			<td style="width:199px;">Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Gauze Pads</td>
			<td>FisherBrand</td>
			<td>13-761-52</td>
			<td style="width:199px;">Non-Sterile Cotton Gauze Sponges; 4&#34;x4&#34; 12-Ply</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Cotton-Tippled Applicators</td>
			<td>FisherBrand</td>
			<td>23-400-124</td>
			<td style="width:199px;">6&#34; Length; Wooden Shaft; Single Use Only</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">6-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-49</td>
			<td style="width:199px;">Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Syring 5ml</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Sterile Bowl</td>
			<td>Medical Action Industries Inc.</td>
			<td>01232</td>
			<td style="width:199px;">32 oz. Peel Pouch; Blue; Sterile Single Use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:165px;">6-Well Plate Inserts (CellCrown Inserts)</td>
			<td>SIGMA</td>
			<td>Z681792-3EA</td>
			<td>6-Well Plate Inserts; Non-Sterile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Polycarbonate Filter Membrane</td>
			<td>SIGMA</td>
			<td>TMTP04700</td>
			<td style="width:199px;">Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&#160;&#181;m, 47&#160;mm, White Plain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Beuthanasia</td>
			<td style="width:183px;">Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td style="width:199px;">Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Ketamine</td>
			<td style="width:183px;">Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Xylazine</td>
			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Anased 100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td>2F7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Invitrogen</td>
			<td>11095080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Saponin&#160;</td>
			<td>SIGMA</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>S25372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Povidone-Iodine</td>
			<td>Operand</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hydrochloric Acid</td>
			<td>SIGMA</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">FITC-conjugated Lectin</td>
			<td>SIGMA</td>
			<td>L9381-2MG</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td>
			<td>SIGMA</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
			<td>SIGMA</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</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 muscl...

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 ...

The overall goal of this experiment is to demonstrate the use of the rat mesenteric culture model for the time-lapse investigation of microvascular network re-modeling ex vivo to evaluate parasite endothelial cell interactions, blood lymphatic endothelial cell connections, or anti-angiogenic drug effects. This method can help answer key questions in the field of microvascular remodeling, including how do microvasculars know where to grow, and what is the fate of endothelial cell segments. The main advantage of this technique is that real, intact microvascular networks can be imaged before and after growth ex vivo.Demonstrating the procedure will be Sadegh Azimi, a graduate student in our laboratory who developed the method. Begin by placing an ethanol-sterilized plexiglass platform on top of a sterile, absorbent under-pad and an anesthetized adult male rat on top of the platform. Disinfect the shaved abdominal region of the rat with two 70%isopropyl alcohol wipes, followed by povidone-iodine.Starting one inch below the sternum, use a scalpel to make a three-quarter to one and one-quarter inch incision in the gut through the skin, connective tissue, and muscle. Then, place a drape with a precut hole over the animal and place a surgical stage over the drape, taking care to align the openings in both materials with the incision. Using sterile cotton-tipped applicators, pull the ileum through the surgical stage opening along with six to eight mesenteric windows.Use a sterile syringe to drip 37 degrees Celsius sterile saline onto the exposed tissues as needed. When all of the windows have been externalized, use tweezers to grasp a fat pad and use scissors to harvest the windows, leaving a two millimeter fat border around each mesenteric tissue. Take care that the tweezers do not touch the mesenteric windows by only touching the surrounding fat borders.Wash each window one time in 37 degree Celsius PBS and one time in 37 degree Celsius medium as it is collected. Returning the ileum to the abdominal cavity after all of the windows have been isolated. In a sterile laminar flow hood, use tweezers to transfer each mesenteric tissue by the fat border onto the polycarbonate filter membrane of a cell culture plate insert.Use the fat pad to quickly spread the tissue samples, taking care not to touch the windows. Then, invert the insert and place into one well of a six well plate so the tissue lays flat on the bottom of the well. Cover the tissue with three millimeters of medium, and transfer the remaining windows to the plate in the same manner.Then, culture the mesentery windows under standard tissue culture incubator conditions for up to five days. On the day of imaging, supplement the cultures with conjugated BSI lectin and return the plate to the incubator. After 30 minutes, wash the samples two times with lectin-free medium and transfer the plate to a fluorescent microscope stage.Identify the blood and lymphatic vessels based on their morphology and network structures. Then, locate a network region of interest on each tissue to obtain images, taking note of the location to ensure that the same region is captured in subsequent images. When all of the images have been obtained, return the samples to the incubator until the next imaging session or the appropriate experimental endpoint.After three days in culture, the majority of the cells present in the mesentery are viable, with the presence of the endothelial cells confirmed by their location in the microvascular segments. Endothelial cell proliferation can also be confirmed by lectin/BrdU labeling, while the presence of smooth muscle cells and parasites along the vessels can be confirmed with NG2 labeling. Branching lymphatic and blood microvascular network labeling further confirms the maintenance of the lymphatic cell phenotype in the cultured tissue.Supplementation of the mesentery window tissue culture medium with 10%serum for three days induces a robust angiogenic response with new vessel segments and capillary sprouts identified after five days of stimulation. The time lapse imaging allows quantification of the number of vessels per vascular area and the number of capillary sprouts per vascular area from one four x image per tissue. Time lapse comparisons of network regions also enable the tracking of endothelial cell segments and the identification of blood and lymphatic vessel mis-patterning.Further, lectin and Cd11b labeling additionally confirms the presence of interstitial resident macrophages in the remodeling networks. Once mastered, this technique should be able to be completed in under four hours. After watching this video, you should have a good understanding of how to harvest, culture, and time-lapse image rat mesenteric tissues.

Research

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