The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.
Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.
Coverage of exposed tendon, bone and implant material in reconstructive surgery relies on the use of flaps. A flap is a block of tissue that is transferred on its vascular pedicle that guarantees arterial inflow and venous outflow. Despite broad expertise and the availability of a variety of flaps to be transferred, ischemia-induced complications ranging from wound breakdown to total tissue loss are still encountered. Whereas conservative treatment and healing by secondary intention can be expected after minor tissue necrosis, significant flap necrosis usually requires surgical revision, including debridement, wound conditioning and secondary reconstruction. This increases morbidity, prolongs hospital stay and consequently leads to increased health care costs.
Flaps with an undefined pattern of vasculature or randomly perfused areas in the distal zone most remote from the arterial inflow are particularly prone to ischemic damage. Accordingly, numerous experimental and clinical studies have evaluated the development of necrosis in both, axial pattern flaps (defined blood supply) and random pattern flaps (undefined blood supply)1-3. The main findings are commonly based on macroscopic evaluation of the size of the necrotic area. In order to assess the causes and mechanisms of tissue necrosis more in detail, several studies focused on the analysis of microcirculation. Different techniques have been used to measure tissue perfusion, including the analysis of tissue oxygen tension using polarographic electrodes4-5, as well as the measurement of blood flow using laser Doppler flowmetry6-7, dye diffusion8, and microspheres9-10. These techniques, however, only allow for measuring indirect parameters of tissue perfusion and do not enable any morphological analysis of the microhemodynamic processes within an individual area of interest of a flap.
Sandison is known to be the first who has used a transparent chamber for prolonged in vivo studies, which he performed in rabbits11. In 1943 — approximately 20 years later — Algire was the first to adapt such a transparent chamber to be applicable in mice in order to study the behavior of micro-implants of tumor cells12. Due to the fact that mice are so-called loose skin animals and after some technical refinements over the following years, Lehr and co-workers were able to adapt such a dorsal skinfold chamber developing a smaller and lighter titanium chamber. This chamber enabled evaluation using intravital fluorescence microscopy, a technique that allows direct and repetitive visualization of a number of morphologic and microcirculatory features and their changes over time under different physiological and pathophysiological conditions, such as ischemia-reperfusion injury13.
In the investigation of perfusion of skin, muscle and bone flaps under normal and pathological conditions two trends occurred: First, the “acute” flap models that do not use the dorsal skinfold chamber such as the pedicled ear flap in the mouse14, the laterally based island skin flap in the hamster15 and the pedicled composite flap in the rat16. Second, the “chronic” flap model where the combination of a flap with a dorsal skinfold chamber permits repetitive microcirculatory analyses over several days with intravital fluorescence microscopy. It consists of a randomly perfused musculocutaneous flap that is integrated in the skinfold chamber of the mouse 17. Its width-to-length ratio was chosen that a situation of acute persistent ischemia consistently results in ~50% flap tissue necrosis 10 to 14 days after flap elevation. This reproducible extent of tissue necrosis allows further evaluation of both, protective (i.e., development of less necrosis) and detrimental factors (i.e., development of more necrosis) on flap pathophysiology. During the last years, several experimental publications demonstrating the effect of different pre-, peri- and post-conditioning procedures, including the administration of tissue-protective substances18-24 and the local application of physiologic stressors such as heat25 and shockwaves26, have emerged.
The quantitative analyses of necrosis, microvascular morphology and microcirculatory parameters can further be correlated to immunohistochemical analyses and protein assays. Different proteins and molecules including vascular endothelial growth factor (VEGF), nitric oxide synthases (NOS), nuclear factor kappa B (NF-κB) and heat shock proteins (HSP-32: heme-oxygenase 1 (HO-1) and HSP-70) have been shown to play a role in tissue protection. Based on this chamber flap model, two modifications have been developed in order to analyze neovascularization and microcirculation during skin graft healing27 and angiogenic developments in a pedicled flap with axial pattern perfusion28. We present a reproducible and reliable model that includes an ischemically challenged musculocutaneous flap in the mouse skinfold chamber. This model allows visualization and quantification of the microcirculation and hemodynamics by intravital epi-fluorescence microscopy.
NOTE: Prior to implementation of the presented model, the corresponding animal protection laws must be consulted and permission must be obtained from the local authorities. In this work, all experiments were performed in conformity with the guiding principles for research involving animals and the German legislation on protection of animals. The experiments were approved by the local animal care committee.
1. Animal Preparation and Surgical Elevation of the Flap
2. Intravital Epifluorescence Microscopy
3. Analysis of Recorded Data
NOTE: With the use of a computer-assisted image analysis system quantify all recorded parameters off-line as follows29.
4. Postoperative Care
5. Euthanasia and Explantation of the Skinfold Chamber
Necrosis
The main endpoint of this model — tissue necrosis following flap elevation (i.e., induction of acute persistent ischemia) — is repeatedly measured and illustrated macroscopically as shown in Figure 3 over a period of 10 days. Final demarcation of flap necrosis usually occurs between day 5 and 7 after surgery and is characterized by a red fringe, i.e., zone of vasodilation and microvascular remodeling, developing between the proximal vital and the distal necrotic zone of the flap (Fig. 3D-F). Untreated control mice usually develop a necrotic area of about 50% after 10 days that can be divided into three distinct areas: the well perfused proximal, the intercalated and critically perfused central area and the necrotic distal area (Fig. 3F). Previous studies of our group, as mentioned in the introduction, have shown protective effects after different preconditioning procedures, which are characterized by a shift of the critically perfused area from the central zone towards the distal zone of the flap.
Vessel parameters
Microvascular diameters and red blood cell (RBC)-velocity of arterioles and venules of various diameters as well as of neighboring capillaries are measured and the resulting volumetric blood flow is calculated off-line. This allows correlating both morphological and functional changes of distinct microvessels from day 1 to day 10 (Fig. 4A-B). In addition, the functional density of the RBC-perfused capillaries, the parameter for nutritive perfusion of the tissue, is analyzed. The capillaries are usually oriented in-parallel and present with diameters between 3-5 µm under physiologic conditions (Fig. 4C). Blood flow, functional capillary density and eventually oxygen tension of the tissue gradually decrease from proximal to distal to reach a threshold “of viability” (Fig. 4C-E), where the capillaries are not perfused anymore (Fig. 4E).
Microvascular remodeling and angiogenesis
Remodeling with dilation and increased tortuosity of the microvessels can be observed in all experimental groups undergoing flap preparation (i.e., induction of acute persistent ischemia: Fig. 4F). However, in this model, the ischemic stimulus alone is not enough to induce angiogenesis as for example seen in a variety of preconditioning experiments with the hormone erythropoietin (Fig. 4G-H). The new functional microvascular networks that usually develop from microvascular buds and sprouts emanating perpendicularly from the pre-existing in-parallel arranged capillaries are first visible between day 3 and 5.
Inflammatory response (leukocyte-endothelial interaction and apoptosis)
Acute persistent ischemia usually induces a considerable inflammatory response in untreated animals that is represented by adhering leukocytes to the microvascular endothelium. This inflammatory response is characterized by rolling (i.e., intermittent adhesion of leukocytes to the vascular endothelium) and sticking (i.e., firm adhesion of leukocytes to the vascular endothelium) leukocytes (Fig. 5A-B). In addition, an ischemia induced increase of apoptotic cells can be seen in all control animals with the characteristic signs of nuclear condensation, fragmentation and margination (Fig. 5C-D). Both, leukocyte-endothelial interaction and apoptosis are signs for the ischemia-induced inflammatory response and gradually increase with progressive microvascular dysfunction and decreased oxygen tension of the tissue (i.e., along the “ischemic” axis of the flap proximal to distal (Fig. 5A-D)).
Figure 1. Illustration of the titanium chamber frame and all of its work pieces. (A) Disassembled frame consisting of two frame parts, three screws, five nuts, one piece of foam, a cover glass and a snap ring. (B) Required tools to assemble the frame including a hex nut driver, a snap ring plier and a wire cutter. A screwdriver is not required but recommended if chambers will be used several times. (C) Assembled single chamber parts. Left: Back side of the frame with two screws and attached nuts on the lower two holes. The back side is used to suture the frame onto the skin and bears the flap. Right: Assembled front side of the frame with attached foam to guarantee tightness. Note that all three screw-holes are kept free of foam. Make sure that none of the foam will be pressed into the observation window, which bears the cover glass. (D) Schematic assembled chamber frame without dorsal skinfold.
Figure 2. Illustration of the operative flap procedure and its implementation in the titanium dorsal skinfold chamber of the mouse. (A) Elevated trans-illuminated doubled dorsal skinfold of the mouse to visualize vascular architecture for outlining the position of the dorsal skinfold chamber. (B) The backside of the chamber's titanium frame has been positioned and aligned with the vessels. The incision of two holes for screws to attach the front side of the frame has been made. (C and D) Outlining of the flap on the dorsal skin laterally: The width-to-length ratio is 15 mm to 11 mm while centralizing the two perpendicularly arising vessels. A 2 mm distance to the contralateral side (unmarked area between thin flap outline and thick border) will be used to grasp the skin with forceps to elevate the flap. For the observation of the backside skin through the chamber window, additional tissue has to be removed (hatched area). (E) Elevated laterally based skin flap, demonstrating the randomly arranged vascular architecture originating from the base of the flap. The hatched area has been cut out, but is still attached to the flap and is removed in the next step (hanging skin below the forceps). (F) Mounting the back side of the chamber frame. The skin surface of the flap is on the backside and the “raw” surface under the glass window side. The chamber`s screws are stuck through the incised holes on both sides of the dorsal skinfold. (G) The surrounding skin, anteriorly and posteriorly, of the flap is fixed in the holes of the upper chamber rim. (H) The skin flap is stretched out and also sutured to the back side of the frame. To avoid dehydration of the flap, 0.9% sodium chloride solution is dripped onto the flap repeatedly. (I) The flap and the surrounding skin is completely fixed in the holes of the upper chamber rim. The flap is sutured back laterally into the adjacent dorsal skin to guarantee tightness of the chamber. (J) Mounting the foam-bearing counterpart of the frame, surrounding the observation window. (K) Completely mounted chamber: A cover glass is attached to the observation windows and sealed with a snap ring. A third screw is attached to the top of the frame for additional tightness. The window in the chamber allows repeated analyses of the microvasculature of the flap by intravital microscopy. For easier access during microscopy the three screws are shortened. (L) Awake and moving mouse with mounted dorsal skinfold chamber.
Figure 3. Presentation of the morphological development and demarcation of the flap necrosis at days 0 (immediately after flap preparation, A), 1 (B), 3 (C), 5 (D), 7 (E) and 10 (F). Final necrotic demarcation takes place between days 5 and 7 (D and E). Controls show a distinct zone of demarcation within the central flap area, including a red fringe and a white falx (double arrowhead and asterisk in E), reflecting a hyperemic response and microvascular remodeling as well as a non-perfused but potentially viable area delineating tissue necrosis developing distally (E). In panel F, the flap tissue is divided in three distinct areas by 2 horizontal lines: the well perfused proximal zone (at the base of the image), the critically perfused central zone (including the red fringe an the white “falx lunatica” corresponding to the penumbra in ischemic brain tissue after stroke injury) and the necrotic distal zone (circumferentially marked by red border). Magnification 16X.
Figure 4. Intravital epi-fluorescence microscopy displaying images of arteriovenular (AV) bundles (A, B), capillary fields (C, D, E) and morphological changes such as remodeling (F) and angiogenesis (G, H). Intravital microscopy showing the same AV-bundle in a control animal (A, B) at day 1 (A) and 10 (B). Note the absence of dilatory response in controls over the whole observation period in both arteriolar (a) and venular (v) diameter. Images C, D and E demonstrate parallelly arranged capillaries in the well perfused proximal zone (C), critically perfused central transition zone (D) and the non-perfused necrotic distal zone (E) of the ischemic flap tissue. In the critically perfused central zone control mice only show vascular remodeling (F), characterized by in parallel arranged capillaries with dilation and increased tortuosity. In contrast, mice receiving erythropoietin before flap elevation as a preconditioning regimen show newly formed and perpendicularly arising capillaries, which are clearly distinguishable from normal pre-existing capillaries (G, H). This angiogenic response has not been observed in controls. Contrast enhancement with fluorescent dextran 150,000. Magnification 80X.
Figure 5. Intravital epi-fluorescence microscopy displaying AV-bundles in the well perfused proximal flap zone (A) and the critically perfused ischemic central transition zone of the tissue (B). Note the increased presence of adhering leukocytes (arrows) in postcapillary venules (v) and arterioles (a) within the ischemic flap zone (B) when compared the healthy proximal zone (A). Contrast enhancement with Rhodamine. Magnification 80X. Intravital epi-fluorescence microscopy displaying nuclear condensation indicating apoptotic cell death within the proximal (C) and critically perfused central flap area (D) of controls. An increased number of apoptotic cells (white arrows) is observed within the more ischemic central transition zone (D) when compared to the proximal zone (C). Contrast enhancement with Bisbenzimide. Magnification 250X.
In order to decrease ischemic complications and thereby improve the clinical outcome, more detailed knowledge of pathophysiologic processes in critically perfused flap tissue is required. The development of new animal models that mimic acute persistent ischemia is therefore mandatory. Accordingly, we were able to develop an easily reproducible and reliable model allowing for repetitive morphological, dynamic and functional real-time evaluation of various parameters of muscle and skin vasculature that can be correlated with immunohistochemical and molecular analysis of the sampled flap tissue.
The surgical procedure does not require any specific surgical skills, though practice and some manual dexterity is necessary. A learning curve of about 25-30 operated animals is usually necessary to master the mounting of the dorsal skinfold chamber and flap preparation properly. In experienced hands, time for surgery averages 35 minutes. Crucial steps of flap preparation include the correct placement and outline of the flap in the center of the chamber’s window with accurate positioning of the main vessels to be transected (using trans-illumination) and the meticulous removal of the gelatin-like layer above the panniculus carnosus using image-magnification to improve the image quality.
Since flap dimensions are given in millimeters, we recommend using a sliding caliper for correct flap marking. If there are any difficulties while positioning the horizontally arranged dominant vessels within the chamber’s window, one should rather choose a more distal than a more proximal position of the vessels. The correct positioning of these vessels that transverse the chamber’s window, guarantees transection of these while elevating the flap from distal to proximal and results in a randomly perfused flap. In other words, failure to transect these major vessels will result in an axial perfusion pattern without necrosis of the tissue under the window. The final crucial step during surgical preparation concerns the removal of the gelatin-like areolar tissue layer. Experience has shown that excessive removal damages the subjacent panniculus carnosus and so the delicate horizontally arranged muscular capillaries. Contrariwise, conservative removal results in edema-formation and eventually limited visibility of the regions of interest during microscopy. If all these steps are mastered with care, the flap very constantly develops approximately 50% necrosis 10 days after flap elevation. This allows studying various therapeutic strategies that may improve or worsen survival of the flap tissue. Finally, the method of intravital fluorescence microscopy is a well-established procedure to evaluate microvascular tissue perfusion and can be learned very quickly.
The major drawback of this model is the limited observation time of the tissue within the chamber’s window of approximately 10 to 14 days following flap preparation. This is due to the progressive loosening of the skin proximal to the dorsal skinfold chamber that finally results in sideways tilting of the chamber. Rarely, the sandwich-like, fixed skin pulls out of the chamber’s frame and renders microscopy impossible. Since necrosis is most often fully demarcated between day 5 and 7 after surgery, this does not really compromise data acquisition before tilting of the chamber or pulling out of the skin within the chamber.
Advantages of the model include easy reproducibility and the potential use of genetically modified animals. The relatively small size of the window that can be assessed might however compromise the significance in translating the results into human conditions. In addition, there are anatomical differences between loose skinned animals and humans. While the animals and surgical tools have a reasonable cost-effectiveness, the hard- and software necessary to perform microscopy (epi-illumination microscope, high-resolution camera, fluorescent dyes and special software for off-line data analysis) represents a bigger investment.
Conclusion:
We present a time and cost-effective animal model in mice that allows visualization and quantification of microcirculatory parameters at high resolution. This approach represents an ideal method to analyze critically perfused musculocutaneous flap tissue and the underlying cellular mechanisms. Morphological changes of regions of interest can be repeatedly investigated and correlated with functional changes on a microvascular and cellular level. After intravital epi-fluorescence microscopy, the tissue can be further processed using histological and molecular approaches.
The authors have nothing to disclose.
We thank Katharina Haberland for image editing. Funding: The senior author received a KKF Grant from the Technische Universität München to set up a new research laboratory.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
C57Bl/6 mice 6-8w 20-22g | Charles River | ||
depilation cream | Veet | any depilation cream | |
titanium chamber | Irola | 160001 | Halteblech M |
slotted cheese head screw | Screws and More | 842210 | DIN84 M2x10 |
hexagon full nut | Screws and More | 93422 | DIN934 M2 |
snap ring | Schaefer-Peters | 472212 | DIN472 J12x1,0 |
cover glass | Volab | custom-made cover glass 11,8mm in diameter | |
fixing foam | tesamoll | 05559-100 | tesamoll Standard I-Profile |
ketamine hydrochloride | Parke Davis | Ketavet® | |
dihydroxylidinothiazine hydrochloride | Bayer | Rompun® | |
Buprenorphin | Essex Pharma | Temgesic® | |
Saline 0,9% | |||
desinfection alcohol | |||
Vicryl 5-0 | Ethicon | V 490 H | |
Ethilon 5-0 | Ethicon | EH 7823 H | |
1ml syringes | |||
surgical skin marker with flexible ruler | Purple surgical | PS3151 | any surgical skin marker and flexible ruler |
pointed scissors | |||
Micro-Scissors | |||
normal scissors | |||
2 clamps | |||
fine anatomic forceps | |||
micro-forceps | |||
hex nuter driver | wiha | 1018 | |
screwdriver | wiha | 685 | |
snap ring plier | Knipex | 4411J1 | 12-25mm |
wire cutter | Knipex | 70 02 160 | Wire cutter is used to cut screws short; 160mm |
trans-illumination light | IKEA | 501.632.02 | LED light Jansjö; any light |
magnification glasses | |||
intravital microscope | Zeiss | 490035-0001-000 | Scope.A1.Axiotech |
LED system | Zeiss | 423052-9501-000 | Colibri.2 |
LED module 365nm | Zeiss | 423052-9011-000 | |
LED module 470nm | Zeiss | 423052-9052-000 | |
LED module 540-580nm | Zeiss | 423052-9121-000 | |
Filter set 62 62 HE BFP + GFP + HcRed | Zeiss | 489062-9901-000 | range 1: 350-390nm excitation wavelength split 395 / 402-448nm; range 2: 460-488nm, split 495nm / 500-557nm; range 3: 567-602nm, split 610nm / 615-infinite |
Filter set 20 Rhodamine | Zeiss | 485020-0000-000 | 540-552nm, split 560, emission 575-640nm |
2,5x objective NA=0,06 | Zeiss | 421020-9900-000 | A-Plan 2,5x/0.06 |
5x objective NA=0,16 | Zeiss | 420330-9901-000 | EC Plan-Neofluar 5x/0.16 M27 |
10x objetive NA=0,30 | Zeiss | 420340-9901-000 | EC Plan-Neofluar 10x/0.30 M27 |
20x objective NA=0.50 | Zeiss | 420350-9900-000 | EC Plan-Neofluar 20x/0.50 M27 |
50x objective NA=0,55 | Zeiss | 422472-9960-000 | LD Epiplan-Neofluar 50x/0.55 DIC 27 |
ZEN imaging software | Zeiss | ZenPro 2012 | |
CapImage | Dr. Zeintl | ||
Fluorescein isothiocyanate-dextran | Sigma-Aldrich | 45946 | |
bisBenzimide H 33342 trihydrochloride | Sigma-Aldrich | B2261 | harmful if swallowed; causes severe skin burns and eye damage, may cause repiratory irritat |
Rhodamine 6G chloride | Invitrogen | R634 | harmful if swallowed; may cause genetic defects; may cause cancer; may damage fertility or the unborn child |
Pentobarbital | Merial | Narcoren® |