A protocol for a lung neovascularization model in rodents

Abstract

By providing insight into the cellular events of vascular injury and repair, experimental model systems seek to promote timely therapeutic strategies for human disease. The goal of many current studies of neovascularization is to identify cells critical to the process and their role in vascular channel assembly. We propose here a protocol to analyze, in an in vivo rodent model, vessel and capillary remodeling (reorganization and growth) in the injured lung. Sequential analyses of stages in the assembly of vascular structures, and of relevant cell types, provide further opportunities to study the molecular and cellular determinants of lung neovascularization.

Figure 1

Equipment setup. (a) Overview of Plexiglas chamber in which rodents breathe a high oxygen tension. Animals are housed in standard cages in the main chamber. An air-lock with a connecting door to the main chamber and an exterior door facilitate transfer of cages and food and water during the course of the exposure schedule. Gloved portholes assist in the transfer of these materials in and out of the main chamber. Note the location of the flowmeter, the oxygen analyzer and the valves that direct the flow of gas from adjacent liquid oxygen/nitrogen tanks. (b) Illustration of gas supply valves connecting the flowmeter and oxygen analyzer to the main chamber and airlock. A tube carries mixed gas from the flowmeter to the main supply valve (1) where it is directed either to the main chamber or to a calibration outlet for sampling the oxygen tension (by a glove test). Gas drawn from the main chamber, or from the airlock, is directed via a sensor supply valve (2) through a container of Drierite dessicant (not shown) to the analyzer. Gas sampled by the analyzer is returned to the main chamber, or air-lock, via a sensor return valve (3).

Figure 2

Drawing illustrating the inflation apparatus to distend lungs initially with a 3% paraformaldehyde/0.1% gluteraldehyde solution (shown in blue). Fixative is delivered to the tracheal catheter at 23 cm H₂Opressure (determined by gravity) and to the vascular catheter at 100 cm H₂O (determined by positive pressure from a peristaltic pump). Note: use a standard pressure monometer to predetermine the height of the mercury column needed for the pump to generate this pressure. The catheters connect first to a luer adapter, or a luer-adapted needle, and then, via a 4-way stopcock, to the tubing delivering fixative from each reservoir. Hemostat clamps (indicated by black bars) regulate the flow of fixative through the delivery tubes from each reservoir. A gate clip (heavy black bar) positioned on the tubing from the peristaltic pump is used to obtain/regulate pressure in the vascular reservoir.

Figure 3

Image and cartoon of fixative-distended rodent (rat) lung. (a) Note the sharply distended peripheral lung margins denoting well-inflated lungs. (b) Optimal tissue blocks are collected from peripheral zones (red) of the right and left lung. These zones provide the maximum number of small lung vessels (<100 μm in diameter) for analysis, while avoiding the central large airway and vascular structures that enter at the hilum and subsequently branch throughout the lung.

Figure 4

Illustration of vessel wall thickening by brightfield microscopy and by TEM in normal rat lung and in the rat lung after breathing high oxygen. (a-d) Brightfield microscopy; (e-h) TEM. Representative examples of wall thickening by perivascular cells in alveolar wall vessels present in 1-μm-thick Epon/araldite sections stained with toluidine blue: (a)D0, 45-μm-diameter vessel; (b)D28,31-μm-diameter vessel (note the development of a single elastic lamina, see arrows); (c and d)D28, 21-μm-diameter vessel (note lumen encroachment by cells). Original magnification: ×630. (Reproduced with permission from ref. 4.) Representative images of wall thickening by perivascular cells in alveolar wall vessels obtained by TEM: (e)23-μm-diameter vessel, (f-h) 17-, 15-, 18-μm-diameter vessels, respectively. Typically, in the early stage of breathing high oxygen, vessel wall thickness is close to normal and consists mainly of endothelial cells (e, D4). Atthis time, cell injury by high oxygen causes edema to widen the interstitium surrounding the vessel (*). Later (f-h, D28), wall thickness increases as endothelial cells are surrounded by interstitial fibroblasts aligning as perivascular cells (indicated by red dots). Alv=alveolus, EC=endothelial cell, IFB=interstitial fibroblast. Original magnification: (e) ×1,416, (f) ×1,662, (g) ×1,416,(h) ×1,662.

Figure 5

Illustration of alveolar-capillary membrane structure in the normal rat lung and in the rat lung after breathing high oxygen. The alveolar capillary membrane of normal lung is characterized by its complex, open (lace-like) structure (a, arrows). After 4 weeks in high oxygen, regions of the membrane now lack patent capillary segments (b, arrows). These are demonstrated by TEM to consist of avascular zone and residual capillary structures. After 4 weeks in high oxygen, 1 week of weaning and then 4 weeks of breathing air, patent capillary segments are restored— resulting in an alveolar capillary membrane structurally close to normal (c, arrows). Alv=alveolus. Original magnification: ×400.