1Department of Biomedical Engineering, Tulane University, 2Department of Biomedical Engineering, University of Virginia, 3Center for Stem Cell Research and Regenerative Medicine, Tulane University
Yang, M., Stapor, P. C., Peirce, S. M., Betancourt, A. M., Murfee, W. L. Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis. J. Vis. Exp. (63), e3954, doi:10.3791/3954 (2012).
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.
1. Surgical Procedure Set-Up Notes
2. Rat Mesentery Exteriorization Model
3. Tissue Harvesting and Fixation
4. Tissue Immunolabeling
5. Representative Results
Representative images of rat mesentery tissues immunohistochemically labeled for PECAM are displayed in Figure 3. PECAM labeling identifies all vessel types along the hierarchy of remodeling microvascular networks and can be used to quantify angiogenic metrics at specific time points post stimulation. PECAM labeling also allows for the determination of arterioles versus venules. Feeding arterioles typically exhibit smaller diameters and elongated endothelial cell morphology compared to paired venules (Figure 4). Capillaries and capillary sprouts can be identified based on their vessel diameter and relative position within a network. Typical characteristics of remodeling networks include increased capillary sprouting, vessel density, vascularized area and venular tortuosity. Quantification of various angiogenic metrics identifies the time course of network growth (Figure 5). Capillary sprouting from pre-existing vessels, peaks between Day 3 and Day 5 and returns to the unstimulated level by Day 10. This transient increase in sprouting is followed by increases in vascular density and vascularized area. As evidence for the remodeling of larger vessels in this model, the number of arteriole and venule segments also increases over the time course.
In our laboratory, this model has been used to identify cellular phenotypic changes at specific time point during this remodeling process10, 11. For example, class III β-tubulin identifies pericytes along angiogenic vessels (Figure 6). In unstimulated tissues, class III β-tubulin expression is nerve specific. In contrast, during the peak of capillary sprouting, class III β-tubulin is expressed by perivascular cells. This type of result highlights the use of this simple and robust angiogenic model to identify novel cell types involved in the angiogenic process.
Figure 1. Images of the plastic stage pre- and post-modification. The pre-modified stage is a 100 mm Petri dish. Modifications include an elliptical hole cut in the center and the subsequent addition of modeling clay or silicon glue to the hole's edge for the creation of a raised, smooth surface. This surface provides an inner boundary that facilitates superfusion of the exteriorized mesenteric windows. Scale bar is 1 cm.
Figure 2. Image of exteriorized mesenteric region. Mesenteric windows are defined as the thin translucent membranes between the artery/vein pairs feeding the small intestine. During the exteriorization duration, the mesenteric region is laid out and immersed in saline inside a modified Petri dish. Inert yellow modeling clay provides a smooth surface for the mesentery to be pulled through the pre-cut hole. Scale bar is 1 cm.
Figure 3. Representative images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. PECAM labeling identified the hierarchy of microvascular networks including, arterioles (A), venules (V) and capillaries (C). Post stimulation, microvascular networks displayed an increase in capillary sprouting (arrow heads) and vessel density. Scale bar is 100 μm.
Figure 4. Representative images of arteriole/venule pairs within adult rat mesenteric microvascular networks. In both images, arterioles (A) can be differentiated from venules (V) based on a smaller relative diameter and elongated endothelial cell morphology. Scale bars are 20 μm.
Figure 5. Representative quantification of angiogenic metrics over the time course of microvascular growth post mesentery exteriorization. A) Vascular area per tissue area. B) Number of capillary sprouts per vascular area. C) Total vascular length per vascular area. *represents significant difference compared to unstimulated group. Statistical comparisons were made using a One-Way ANOVA followed by Dunn's test. (p < 0.05). UN represents unstimulated.
Figure 6. Representative fluorescent images of mesenteric microvascular networks from unstimulated tissues and tissues at 3 and 10 days post exteriorization of the mesentery. Immunofluorescent PECAM labeling (red) identifies endothelial cells, and class III β-tubulin labeling (green) identifies nerves (arrow heads) and perivascular cells (arrows). Perivascular cells transiently upregulate class III β-tubulin during capillary sprouting. In unstimulated microvascular networks, class III β-tubulin is nerve specific and does not identify perivascular cells. 3 days post stimulation, class III β-tubulin positively labels perivascular cells along microvessels. By day 10, class III β-tubulin expression pattern begins to return to the unstimulated scenario. Scale bar is 50 μm.
Figure 7. Images supporting the feasibility of tracking pre-labeled locally applied cells during microvascular network growth stimulated by the mesentery exteriorization. Cells were superfused over mesenteric windows during the 20-minute exteriorization period. 1 day post-surgery, DiI labeled cells (red) were observed in the dame focal plane with PECAM positive microvessels (green). A, B) Examples of DiI labeled bone marrow cells exhibiting rounded and elongated morphologies. In some cases (arrows) cells were elongated along microvessels. C) Example of a cluster of DiI labeled mesenchymal stem cells near the tip of a capillary sprout (arrow). Scale bars are 50 μm (A), and 20 μm (B, C).
The exteriorization model was reported in 2006 and is adapted from previous mechanical injury rat mesentery models of angiogenesis4-7 and produces similar results to well established i.p. injection models that take advantage of the rat mesentery9. The 20 minute exteriorization time was experimentally determined to produce a robust angiogenic response. While this time period could be varied, it does allow for local application of angiogenic inhibitors4 for mechanistic studies and direct application of exogenous cells for cell lineage studies. Feasibility of cell incorporation into remodeling mesenteric tissue is supported by preliminary studies in our laboratory using pre-labeled bone marrow cells and mesenchymal stem cells (Figure 7), and by the success of investigating the fate of human adipose-derived stromal cells injection i.p.14. In our laboratory, we have used this model to identify pericyte phenotypic changes over the time course of the angiogenic response10 and to assess the angiogenic potential during pathological conditions, such as hypertension12. The angiogenic response and cell phenotype changes associated with this model can also be observed in other rat mesenteric angiogenic models including chronic hypoxia exposure10, 11.
A limitation of the exteriorization model is that the exact triggering mechanisms of angiogenesis are unknown. Exteriorization of the mesentery has been linked to mast cell degranulation and increased histamine levels6, yet further investigation is required to gain more insight. The angiogenic stimulus is undoubtedly multi-factorial, producing a robust remodeling response across the hierarchy of a microvascular network. While the unknown mechanisms remain a major critique of this model, its reproducibility and simplicity make it attractive for identifying cellular dynamics involved in the inherently complex capillary sprouting process. The reproducibility of the model is supported by comparable angiogenic metrics over the time course of microvascular network growth across multiple rat strains (male Wistar and female Sprague-Dawley) in previously published studies from our laboratory10, 12. Since, the majority of adult rat mesenteric tissues are vascularized, the model also allows for multiple tissues to be examined per animal. Unfortunately, this model is not obviously applicable to genetic mouse models as mouse mesenteric windows have less native vascularization and, in our experience, commonly lack observable branching networks. Future applications include the investigation of vessel functionality during angiogenesis using intra-vital microscopy at specific time points and the investigation of related cellular dynamics involved in lymphangiogenesis and neurogenesis. Though the extent of native vascularization per mesenteric window seems to be roughly proportional with age, we have observed branching microvascular networks in male Wistar rats as young as 4-5 weeks old. These observations suggest that the exteriorization model could also be applied to compare the angiogenic differences across ages.
We have nothing to disclose.
This work was supported by the Board of Regents of the State of Louisiana LEQSF(2009-12)-RD-A-19 (PI: W.L. Murfee) and the Tulane Hypertension and Renal Center of Excellence funded by NIH grant P20RR017659-08 (PI: L. Gabriel Navar).
|Drape||Cardinal Health||4012||12"x12" Bio-Shield Regular Sterilization Wraps|
|Noyes Micro Scissor||Roboz Surgical Instruments Co.||RS-5677||Noyes Micro Dissecting Spring Scissors;
Straight, Sharp-Blunt Points; 13mm Cutting Edge;0.25mm Tip Width, 4 1/2" Overall Length
|Graefe Forcep||Roboz Surgical Instruments Co.||RS-5135||Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4" Length|
|Graefe Forcep||Roboz Surgical Instruments Co.||RS-5130||Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4" Length|
|4-0 suture||Ethicon Inc.||699G||(1.5 metric) ETHILON Nylon Suture Black Monofilament|
|5-0 suture||Ethicon Inc.||8556||(1.0 metric) PROLENE Polyprolene Suture Blue Monofilament|
|7-0 suture||Ethicon Inc.||1647G||(0.5 metric) ETHILON Nylon Suture Black Monofilament|
|Castroviejo Micro Needle Holder||Fine Science Tools||12060-02||Tip Width:0.6mm
|Castroviejo Needle Holder||Fine Science Tools||12565-14||Tip Shape:Straight
|Scalpel Handle||Roboz Surgical Instruments Co.||RS-9843||Scalpel Handle, #3; Solid; 4" Length|
|Sterile Surgical Blade||Cincinnati Surgical||0110||Stainless Steel; Size 10|
|Petri Dish||Fisher Scientific||08-757-13||Beveled Ridge, Slippable|
|Table of Specific Surgical Materials and Tools.|
|Beuthanasia||Merck & Co.||MWI #: 011168||Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium|
|Ketamine||Fort Dodge Animal Health||MWI #: 000680||Kateset 100 mg/ml|
|Xylazine||Lloyd, Inc.||MWI #: 009307||Anased 100 mg/ml (Ordered from MWI Veterinary Supply)|
|PECAM (CD31)||BD Biosciences||553371|
|VECTASTAIN Elite ABC||Vector Laboratories||PK-6100|
|Vector Nova Red||Vector Laboratories||SK-4800|
|Table of Specific Reagents|