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 nätverk tillväxt och remodellering är kritiska aspekter av sårläkning, inflammation, diabetesretinopati, tumörtillväxt och andra sjukdomstillstånd 1, 2. Nätverk tillväxten vanligen tillskrivs angiogenes, definierad som tillväxt av nya fartyg från redan existerande kärl. Den angiogena processen är också direkt kopplat till arteriogenes, definierad som den kapillära förvärv av en perivaskulär cell beläggning och fartyg utvidgningen. Naturligtvis är angiogenes komplex och involverar flera spelare på cellulär och molekylär nivå 3. Att förstå hur en mikrovaskulär nätverket växer kräver att identifiera de rumsliga och tidsmässiga dynamik längs hierarkin i ett nätverk under tidsförloppet av angiogenes. Denna information är avgörande för utvecklingen av terapier som syftar till att manipulera kärltillväxt.
Den exteriorisering som beskrivs i denna artikel är en enkel, reproducerbar modell för stimulerabiträda vid angiogenes i råtta tarmkäxet. Det var anpassad från sårläkande modeller på råtta tarmkäxet 4-7, och är ett alternativ till att stimulera angiogenes i tarmkäxet via ip injektioner av proangiogena medel 8, 9. Den exteriorisering modellen är attraktivt eftersom det kräver minimal kirurgiskt ingrepp och ger dramatiska, reproducerbara ökning av kapillär groddar, vaskulär området och vaskulär densitet under en relativt kort tid kurs i en vävnad som gör det möjligt att två-dimensionell visualisering av hela mikrovaskulära nät ner till enskild cellnivå. Det stimulerade tillväxten speglar naturliga angiogena svar i en fysiologisk miljö utan inblandning av utländska angiogena molekyler. Med immunohistokemiska märkningsmetoder är denna modell har visat sig vara ytterst användbara för att identifiera nya cellulära händelser som är involverade i angiogenes. Utredarna kan lätt korrelera angiogena statistik under tiden loppet av ombyggnad med tiden specific dynamik, såsom cellulära fenotypiska förändringar eller cellulära interaktioner 4, 5, 7, 10, 11.
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.
The authors 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).
Name | Company | Catalogue Number | Additional Comments |
Drape | Cardinal Health | 4012 | 12″x12″ Bio-Shield Regular Sterilization Wraps |
Noyes Micro Scissor | Roboz Surgical Instrument | 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 Instrument | RS-5135 | Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4″ Length |
Graefe Forcep | Roboz Surgical Instrument | RS-5130 | Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4″ Length |
4-0 suture | ETHICON | 699G | (1.5 metric) ETHILON Nylon Suture Black Monofilament |
5-0 suture | ETHICON | 8556 | (1.0 metric) PROLENE Polyprolene Suture Blue Monofilament |
7-0 suture | ETHICON | 1647G | (0.5 metric) ETHILON Nylon Suture Black Monofilament |
Castroviejo Micro Needle Holder | Fine Science Tools | 12060-02 | Tip Width:0.6mm Clamping Length:5mm Length:9cm Straight tip |
Castroviejo Needle Holder | Fine Science Tools | 12565-14 | Tip Shape:Straight Tip Width:1.5mm Clamping Length:10mm Scissors:No Lock:Yes Length:14cm Serrated:Yes |
Scalpel Handle | Roboz Surgical Instrument | 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.
Name | Company | Catalogue Number | Additional Comments |
Beuthanasia | Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply) | MWI #: 011168 | Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg 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 #: 009307 | Anased 100 mg/ml |
Saline | Hospira Inc. | 94-217-JT | |
PBS | SIGMA | 011M8207 | |
Saponin | Sigma | BCBB4080 | |
PECAM (CD31) | BD Pharmingen | 553371 | |
Streptavidin-CY3 | Jackson ImmunoResearch | 016-160-084 | |
BSA | Jackson ImmunoResearch | 096555 | |
VECTASTAIN Elite ABC | Vector Laboratories | PK-6100 | |
Vector Nova Red | Vector Laboratories | SK-4800 | |
VectaMount | Vector Laboratories | H-500 |
Table of Specific Reagents