This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.
Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).
Ischemia-reperfusion injury (IRI) occurs when there is a transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is thought to be one of the contributing factors to the pathophysiology of pressure ulcers, where prolonged bed rest predisposes long-term hospital patients to injury. In these patients, both the skin and the underlying muscles are constantly exposed to weight pressure exerted over areas of bony prominence, resulting in localized injuries that, if left untreated, may become necrotic1.
The damages involved in an IRI are twofold. During ischemia, the occlusion of blood vessels leads to a drastic drop of oxygen delivery to the tissues. This results in a decrease of ATP and pH, which inactivates ATPases involved in cellular metabolism. In turn, cellular calcium levels spike, and stressed or damaged cells undergo apoptosis or necrosis2. The release of intracellular contents or damage associated molecular patterns (DAMP), like HMGB1, contributes to the inflammatory response3. The second insult occurs during reperfusion. Although oxygen and pH levels are restored during reperfusion, this results in the generation of reactive oxygen species (ROS), which leads to the oxidation of intracellular lipids, DNA, and proteins. Consequently, pro-inflammatory mediators are activated, which sets off a secondary inflammatory response that involves the recruitment of immune cells to the inflammatory site2. While the cascade of biochemical events leading up to the inflammatory response has been well described, the spatial and temporal regulation of the immune cell activities are not well understood.
Here, we describe a robust IR model on mouse ear skin using simple magnet clamping. Coupled with multiphoton intravital imaging (MP-IVM), we established a model to study the in vivo inflammatory responses that occur after reperfusion takes place. The rationale behind the development and use of this technique is to try to understand how both interstitial and infiltrating cells respond to IR in real time.
Existing models of IR using the clamping technique on the skin flank are highly invasive, as they require the surgical implantation of steel plates in the skin flank, making them less-than-ideal for immunological studies4. A similar non-invasive clamping technique has been described in the mouse skin flank5,6. However, because of the incorporation of the intravital imaging component in this method, we instead chose the ear skin as the targeted IR site, as it circumvents movements due to breathing and offers stability during imaging7,8. Moreover, leukocyte subsets that span the interstitium are identical between the ear skin and the skin flank, although the numbers and proportions may vary slightly9. Thus, the ear skin represents an ideal imaging site.
In addition, most data retrieved from these IRI models are limited to macroscopic evaluations (grading of ulcers) and microscopic analyses of endpoint inflammatory indicators10. Using this model, real-time visualization of the cellular response of neutrophils after reperfusion in the skin of a fluorescent reporter mouse is enabled. A previously published intravital ear imaging model is utilized8 with additional modifications (Figures 1, 2).
All experiments dealing with live animals were conducted in accordance to all relevant animal use and care guidelines and regulations.
1. Choice of Fluorescent Reporter Mice
2. Mouse Anesthesia
3. Depilation
4. Induction of Ischemia and Reperfusion Injury
5. Injection of Blood Vessel Labeling Agents
6. Placement of the Ear on the Imaging Platform
7. Multiphoton Microscope Setup and Imaging Parameters
Note: This protocol uses a single beam, multiphoton microscope with a tunable (680 – 1,080 nm) Ti:Sa laser (3.3 W at 800 nm; pulse length of 140 fs; 80 MHz repetition rate) with a 20X water objective (NA = 1.0) for intravital imaging studies.
8. Terminating the Experiment
9. Image Analysis
Note: Data generated from the imaging experiment can be visualized by different software packages.
This protocol uses a custom-built ear skin imaging platform, as shown in Figure 1. Several features of this platform are specifically designed to facilitate imaging while maintaining physiological settings. Placing the ear on the heated brass platform not only maintains the ear at a physiological temperature of 35 °C, but it also isolates the ear from inevitable movements due to breathing. The addition of a metal clip on the brass platform creates a gap to prevent the coverslip holder from exerting weight on the ear, thereby maintaining uninterrupted blood flow. The stage platform is also designed to accommodate a heating pad that will maintain the mouse at 37 °C throughout the imaging procedure.
The coverslip holder (Figure 2) is designed to allow the coverslip to be attached onto the metal holder with the help of vacuum grease. The coverslip holder is connected to a stand that allows for flexible adjustment in the vertical and horizontal axes with the help of the adaptor. This way, the user can first accurately adjust the position of the coverslip over the ear and subsequently fix that position by tightening the screws on the adaptor.
In this protocol, we have described the use of magnets to simulate ischemia and reperfusion. Figure 3b displays representative views of the ears before and immediately after ischemia. Under physiological conditions, major blood vessels can be visualized macroscopically. The pinching effect of the magnets temporarily stems the blood flow, which can be observed by a transient blanching effect (black arrow) when the magnets are removed.
In this particular demonstration, imaging is focused on the edge of the ischemic zone (Figure 3c). This interface between the ischemic (clamped) and non-ischemic (clamp-free) zone is usually marked by massive Evans Blue leakage into the interstitium (white solid arrow), while the ischemic zone at early time points is devoid of any Evans Blue signal (white arrowhead) (Figure 3d). This creates important landmarks for intravital imaging studies and helps to maintain consistency between independent experiments.
The data shows that, in response to an IR insult, infiltrating neutrophils exit into the interstitium from intact blood vessels lining the edge of the ischemic zone and migrate towards the injury site (Figure 3d and Movie 1). We expect a delayed response of neutrophils migrating into the interstitium as compared to other inflammation models due to a lack of viable perfusing blood vessels at earlier time points post-reperfusion. To fully characterize these cellular activities, cell tracking analysis may be employed to understand their function in vivo. Speed, mean displacement, and chemotactic index are some of the potential read-outs that may be obtained from the cell tracking analysis.
Figure 1. Schematic Diagram of a Custom-made Ear Skin Stage for Intravital Multiphoton Imaging. (a) Top view of the ear imaging stage with its dimensions. (b) Frontal view and dimensions. Note that the brass plate is thicker at the sides, in order to provide stability to the platform, and thinner in the middle, to minimize contact area with the skin, ensuring uninterrupted blood flow when the ear is folded over the edge of the slit and rested on the platform. (c) Side view of the curved holder that pins down the feedback probe. The heating pad can be fixed onto the platform using masking tape (not shown). Modified from Li et al., Nat Protoc, 20128. Please click here to view a larger version of this figure.
Figure 2. Design of the Coverslip Holder; Side and Top View. The coverslip holder holds the coverslip in a fixed position and thus reduces z-drift. Please click here to view a larger version of this figure.
Figure 3. The IR Model. (A) Schematic diagram showing the positioning of magnets. (B) Photos showing a pre-ischemic (left) and a post-ischemic ear (right; right ear) after magnet clamping. (C) Schematic of the ear showing the region imaged with respect to where the magnet had been placed. (D) Maximum intensity z-projected snapshots from a time-lapse sequence showing neutrophil infiltration post-reperfusion in a LysM-eGFP mouse. Elapsed time shown in hh:mm format. Scale Bar: 100 µm. Please click here to view a larger version of this figure.
Movie 1. Neutrophil Recruitment in IR Injury. A time-lapse sequence of maximum projection showing neutrophil (green) infiltration in response to reperfusion in LysM-eGFP albino mouse ear skin after 1.5 h of ischemia. At earlier time points, the lack of Evans blue signal (red) indicates temporary vessel occlusion after ischemia. Collagen (blue, second harmonic generation). Please click here to view this video. (Right-click to download.)
Significance
IR is one of the leading causes of skin pressure ulcers. The early stages (I and II) of pressure ulcers describe the condition of the human skin (as compared to the underlying subcutaneous tissues and muscles). However, an understanding of the immunological etiology is still lacking. Here, we present a simple and robust IR model on mouse ear skin in order to address this gap. We simulate ischemia by clamping the mouse ear between two magnets and subsequently study the downstream immune responses after magnet removal (reperfusion). By using magnets to generate a constant pressure on each ear for a fixed period of time, inducing ischemia, we are able to ensure reproducibility and consistency between experiments.
The strength of this non-invasive IR model also lies in its ability to demonstrate, in real time, in vivo immune responses by utilizing a well-established model of intravital imaging of the ear skin, which offers more stability during imaging as compared to the skin flank procedure. Some of the current skin flank models involve surgical procedures that may alter the immune landscape of the skin4. In addition, imaging the skin flank itself presents a technical challenge, as respiratory movements may contribute to motion-related artifacts during imaging5,6. Our current model circumvents these problems.
In addition to studying the physiological responses of immune cells to IR, this model may also be applicable to pathophysiological settings like diabetes mellitus, where diabetes may worsen IR injury through increased oxidative stress. Understanding the immune responses will contribute to an understanding of wound healing.
Critical Steps
Once the mouse is under anesthesia, thermoregulation is impaired and the core body temperature drops drastically. To prevent hypothermia, external heating is essential to maintain the core body temperature at 37 °C. The ear, located distally to the body core, is physiologically cooler by 1 – 2 °C and must be kept at 35 °C. This consistency in heating will also ensure reproducible read-outs of cell dynamics. At the same time, the heating system must be checked to ensure that the mouse is not over-heating throughout the entire length of anesthesia.
The use of albino mice (BALB/c and C57BL/6-C2J) for all skin imaging studies is highly recommended to avoid pigment-induced speckling injuries. If albino mice are not available, decreasing the laser power during image acquisition may alleviate the problem for pigmented mice. However, further optimization may be needed, as a reduced depth of tissue penetration may result in a poorer acquisition outcome.
The ear skin is very delicate; hence, care must be taken to avoid any circumstances that may cause inflammation. Examples of improper techniques include, but are not limited to, the following: 1) leaving the depilatory cream on for an excessive amount of time over what is recommended; 2) applying excessive friction on the ear when bringing the ear through the slit of the ear imaging platform or when detaching the ear from the masking tape; and 3) overheating the ear, either due to a faulty heating system or a misplaced temperature feedback probe that may be recording ambient temperature instead of the mouse core temperature.
Modifications and Troubleshooting
Most of the troubleshooting pertaining to the ear preparation procedure has been previously listed8. If the ear is imaged at a later time point post-reperfusion, the ear may swell beyond the 0.5-mm gap created by the metal clip to accommodate the thickness of the ear (Figure 1). In this scenario, the placement of the coverslip over the ear may hinder blood flow, which is evident when the macroscopically visible blood vessels disappear from view. This must be avoided by creating a larger gap between the coverslip holder and the brass platform. This can be done in two ways, either by manually adjusting the height of the coverslip holder (Figure 2) or by using a thicker metal clip.
Limitations of the Technique
In this protocol, we used Evans blue to determine the location of the ischemic zone, since leakage of the Evans blue in the interstitium marks the boundary of the ischemic zone at early time points post-ischemia. Evans Blue is commonly used as a blood vessel labeling agent. However, under inflammatory conditions, vascular integrity is severely compromised. This results in the leakage of Evans Blue into the interstitium. In this model, we observe a drastic loss of contrast between blood vessels and interstitium soon after reperfusion. As such, in studies where leukocyte-endothelial interactions are involved, we recommend experimenting with other blood vessel labeling agents of different sizes (e.g., dextrans, etc.).
Future Applications
Besides utilizing the LysM-eGFP mouse to demonstrate how neutrophils respond to skin IR, this model can also be used for studying other leukocyte responses, both in infiltrating (monocytes) and in resident interstitial (macrophages) leukocytes. This model can also be extended to other studies examining blood and lymphatic integrity, as well as leukocyte-endothelial interactions after an IR injury. Coupled with the ability to visualize collagen as SHG signals through MP-IVM, it would also be interesting to monitor collagen integrity after IR or during wound healing. In summary, we have set up a non-invasive, robust model of IR for intravital imaging in order to study immune responses in the mouse ear skin.
The authors have nothing to disclose.
We thank Thomas Graf for providing us with the LysM-eGFP mice.
Mice strains | |||
Lysozyme-GFP C57BL/6 | Thomas Graf, Center for Genomic Regulation | ||
C57BL/6-C2J | Jackson Laboratories | 000058 | To be crossed with Lysozyme-GFP to generate albino Lysozyme-GFP for skin imaging |
Name | Company | Catalog Number | Comments |
Reagents | |||
PBS | |||
Viaflex 0.9% (wt/vol) saline | Baxter Healthcare | F8B1323 | |
Ketamine (100 mg ml−1 ketamine hydrochloride | Parnell | Ketamine is a controlled drug and all relevant local regulations should be followed | |
Ilium Xylazil-20 (20 mg ml−1 xylazine hydrochloride) | Troy Laboratories | Xylazil-20 is a controlled drug and all relevant local regulations should be followed. | |
Evans blue (10 mg ml−1 in PBS or saline) | Sigma-Aldrich | 46160 | |
Ultrapurified water | |||
Name | Company | Catalog Number | Comments |
Equipment | |||
Insulin syringe with needle | BD | 328838 | |
Transfer pipettes | Biologix Research Company | 30-0135 | |
3M paper masking tape | 3M | 2214 | |
Deckglaser microscope cover glass (22 mm × 32 mm) | Paul Marienfeld | 101112 | |
Curved splinter forceps | Aesculap, B. Braun Melsungen | BD312R | |
Veet hair removal cream | Reckitt Benckiser | ||
Medical cotton-tipped applicators | Puritan Medical Products Company | 806-WC | |
C-fold towels | Kimberly-Clark | 20311 | |
Kimwipes delicate task wipes | Kimtech Science | 34155 | |
Gold-plated, N42-grade neodymium magnets, 12mm in diameter and 2mm thick | first4magnets | F656S | |
Plastic guide, 10cm by 1.5cm (polyvinyl chloride material) | fold in half lengthwise, bind with masking tape and slot magnet in | ||
High vacuum grease | Dow Corning | ||
Name | Company | Catalog Number | Comments |
Microscope | |||
TriM Scope II single-beam two-photon microscope | LaVision BioTec | ||
Tunable (680–1,080 nm) Coherent Chameleon Ultra II One Box Ti:sapphire laser (≥3.3 W at 800 nm; pulse length of 140 fs, 80 MHz repetition rate) | Coherent | ||
Water-dipping objectives (20×, NA = 1.0) | Olympus | XLUMPLFLN20xW | |
Name | Company | Catalog Number | Comments |
Miscroscope filter and mirror sets (for imaging GFP, SHG, Evans Blue) | |||
495 long-pass | Chroma | T495LPXR | |
560 lomg-pass | Chroma | T560LPXR | |
475/42 band-pass | Semrock | FF01-475/42-25 | |
525/50 band-pass | Chroma | ET525/50m | |
655/40 band-pass | Chroma | NC028647 | |
Name | Company | Catalog Number | Comments |
Skin-imaging stage platform (refer to diagram for assembly) | |||
A metal base plate (126 mm × 126 mm × 1 mm) | |||
A brass platform for the ear (79 mm × 19 mm; 1 mm thickness at side, 0.5 mm thickness in the middle; Fig. 1) with slit (1.7 mm × 1 mm; 1.5 mm away from long edge) | |||
Two plastic blocks (10 mm in height)—for heat insulation | |||
Curved holder, for positioning the control thermistor on the ear platform | |||
Interface cable CC-28 with DIN connector and thermistors, one for the temperature control and the other for the temperature monitor | (Warner Instruments (Harvard Apparatus) | 640106 | connect the interface cable to both resistive heater blocks set at 35°C |
Resistive heater blocks RH-2 | (Warner Instruments (Harvard Apparatus) | 640274 | Resistive heater blocks can heat the brass ear platform up to over 100 °C within minutes. Ensure that the control thermistor has been properly secured in the holder in order to avoid overheating. |
Temperature controller TC-344B for the ear platform | (Warner Instruments (Harvard Apparatus) | 640101 | |
Temperature controller TR-200 for mouse heating pad | Fine Science Tools | 21052-00 | Unit is no longer for sale. Ask manufacturer for alternatives |
Power supply for TR-200 | Fine Science Tools | 21051-00 | Unit is no longer for sale. Ask manufacturer for alternatives |
Heating pad | Fine Science Tools | 21060-00 | Unit is no longer for sale. Ask manufacturer for alternatives. |
Animal rectal probe | Fine Science Tools | 21060-01 | Unit is no longer for sale. Ask manufacturer for alternatives. After connecting the rectal probe and heating pad to the temperature controller TR-200, set the temperature to 37 °C |
Name | Company | Catalog Number | Comments |
Coverslip holder | |||
2 plastic rods, 1 cm in diameter, 10 cm in length | |||
1 plastic adaptor with holes drilled to accommodate rods (refer to diagram) | |||
3 plastic tightening screws for keeping plastic rods in place | |||
1 metal plate, 6 cm x 2.5 cm, with a 2 cm square cut at 1 end, 2 mm edge away from short edge | |||
1 pair of nut and bolt for attaching metal plate to plastic rod | |||
1 acrylic base (4 cm x 5 cm x 1.5 cm) with magnet to hold coverslip holder on skin-imaging stage platform. 1 rod is permanently fixed onto base. | |||
Name | Company | Catalog Number | Comments |
Imaging analysis software | |||
Imaris v8.1.2 | Bitplane |