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

Summary

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.

Abstract

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.

Introduction

Microvascular network growth and remodeling are common denominators for tissue function, wound healing, and multiple pathologies and a key process is angiogenesis, defined as the growth of new blood vessels from existing ones^1,2. 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, ste....

Protocol

All animal experiments and procedures were approved by the Tulane University's Institutional Animal Care and Use Committee (IACUC).

1. Surgical Procedure Setup

1. 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.
2. Ster....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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