Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic.

Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Imaging peripheral microcapillary networks in humans using nailfold capillaroscopy (NFC) has highlighted the importance of microcirculation as a target organ system in a wide range of systemic illnesses¹. Capillaroscopy involves the use of a microscope to magnify and visualize vessels in the nailfold in vivo. As such, it is a technique widely used in the clinic to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic^2,3, cardiac⁴, ocular (e.g., glaucoma)^5,6, and endocrine diseases (e.g., hypertension and diabetes mellitus^7,8). Morphological changes in the nailfold capillaries, including hemorrhages, increased vessel tortuosity, and avascular regions, are readily detected using NFC. These morphological abnormalities represent pathological processes such as excessive or deficient microvascular remodeling^9,10. NFC is a useful diagnostic tool for detecting these pathologies. Additionally, this technique is useful in the assessment of drug efficacy¹¹.

However, translating clinical NFC findings to animal models of disease is challenging for many reasons. Visualizing microvasculature in animals is typically invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as microcomputed tomography^12,13, coherence tomography angiography¹⁴, and photoacoustic imaging techniques¹⁵. Since peripheral microvascular pathology is evident in a broad range of systemic and central nervous system diseases, including myocardial infarction¹⁶, hypertension¹⁷, age-related neurodegenerations of the central nervous system such as Alzheimer's disease¹⁸, and optic neuropathies such as glaucoma¹⁹, a non-invasive, cost-effective in vivo visualization technique is highly beneficial.

Capillaroscopy has been used to evaluate the nailfold microvasculature in animal models, including guinea pigs²⁰ and mice²¹, thus demonstrating its capability as a non-invasive imaging tool. Here, we apply capillaroscopy to a different part of the nail, the nailbed. Harnessing the transparency of the mouse nail, nailbed capillaroscopy introduces a novel location for the visualization of peripheral microvasculature. In comparison to NFC, which is particularly useful for monitoring blood cell motion^21,22, the nailbed capillaroscopy protocol described here provides a larger area for better observation of microvascular morphology and structure. We provide a protocol that allows researchers to easily and inexpensively assess the morphology of mouse nailbed microvasculature, which is a novel location for non-invasive peripheral vascular imaging. This protocol provides representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains (SV129/S6 and C57/B6J). We show that nailbed capillaroscopy is an inexpensive, non-invasive microvascular imaging modality. Further studies using this exploratory method will be essential to apply nailbed capillaroscopy to a wide range of mouse models of disease where peripheral microvascular abnormalities are evident in pathology.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Vanderbilt University Medical Center and Massachusetts General Hospital.

1. Preparation of mouse nails for imaging

NOTE: For optimal vessel clarity and skin recovery, allow at least 24 h before imaging.

1. To enable unobstructed imaging of mouse nailbeds, remove fur from the rodent paws at least 24 h before capillaroscopy imaging (Figure 1A). To remove fur from mouse paws, follow steps 1.1.1-1.1.6.
   1. Sedate the animal using inhaled isoflurane anesthesia (2% isoflurane in 5% carbon dioxide/95% oxygen). Confirm that the mouse is adequately anesthetized by pinching the foot pad on both hind feet. If properly anesthetized, there should be no jerking motion (pedal withdrawal reflex.) If there is a reflex movement, allow the mouse more time under isoflurane to become fully anesthetized. Re-test for the pedal withdrawal reflex and continue.
   2. After the mouse is properly anesthetized, apply a lubricant eye gel or sterile non-medicated ophthalmic ointment to both eyes to prevent drying of the cornea while under anesthesia.
   3. Maintaining the animal under anesthesia using a nose cone, apply a generous amount of hair removal cream to the entire paw using an applicator. Take care to cover the entire paw and nailbed areas, as shown in Figure 1B.
   4. Leave hair removal cream on the paw for 2 min at room temperature (RT).
   5. Carefully clean away hair removal cream by gently rubbing it with a clean tissue.
   6. Wash the paw in lukewarm, sterile water.
      NOTE: Paws should be free of fur and nails unobstructed for imaging, as shown in Figure 1C.
   7. Return the mouse to its cage to ensure a safe recovery from anesthesia. Do not leave the mouse unattended until it has regained consciousness. Once the mouse has regained consciousness, return it to the original cage with the company of other animals.

2. In vivo nailbed capillaroscopy imaging

1. Following a minimum of 24 h recovery time post-fur removal, set up the capillaroscopy equipment in a temperature-controlled room (maintained between 21.5-22.5 °C), as shown in Figure 2A.The complete setup for nailbed imaging includes 1) isoflurane anesthesia equipment, 2) an anesthesia nose cone, 3) an adjustable animal stage, 4) a capillaroscopy microscope, and 5) a laptop with video software for imaging.
2. Sedate the animal using inhaled isoflurane anesthesia (2% isoflurane in 5% carbon dioxide/95% oxygen).
   1. Confirm that the mouse is adequately anesthetized by pinching the foot pad on both hind feet. If properly anesthetized, there should be no jerking motion (pedal withdrawal reflex.) If there is a reflex movement, return to step 1.1.1 and allow the mouse more time to be fully anesthetized. Then, re-test for the pedal withdrawal reflex and continue.
   2. After the mouse is properly anesthetized, apply lubricant eye gel or sterile non-medicated ophthalmic ointment to both eyes to prevent drying of the cornea during anesthesia.
3. Maintaining the animal under sedation, position the hind paw volar side up on top of the lab tape platform below the objective, as shown in Figure 2B, zoom.
4. Spread the toes gently to separate nails under the microscope objective using an applicator. Ensure nailbeds are separated from one another for optimal vessel imaging.
   NOTE: Figure 2C shows the complete imaging setup showing vessel picture on laptop video software.
5. To reduce glare and improve focus, generously apply immersion oil (corn oil) to the paw, ensuring complete nail coverage. Add white tape or similar under the mouse paw to enhance contrast and improve visualization of the vessel bed (Figure 3; arrow 3).
6. Focus on the nail on the second digit of the hind paw; in mice, this is the largest nail and easiest to image. To focus the mouse nail, use the x and y stage adjustors (Figure 3; arrow 4) and magnification wheel (up to 280x on this instrument; Figure 3; arrow 1).
7. Turn the objective to reduce the positioning of glare to bring the nail vessel network into view (Figure 3; arrow 2).
   NOTE: If vessels become difficult to see or imaging time becomes extended, generously reapply the immersion oil. Font paws of mice are smaller than the hind paws; thus, it is recommended that imaging is carried out on hind paws.
8. Connect the capillaroscope to a laptop computer via a universal serial bus (USB) connection.
9. Open the Debut video software application on the laptop.
   NOTE: Verify that the device is properly connected to the laptop in settings.
10. On the laptop screen, visualize what is being magnified by the capillaroscope and focus on the nailbed by adjusting the x and y stage adjustors and the magnification wheel to obtain a clear image.
11. Once the microscope is focused and a clear image of the vessel network in the nailbed is seen, record a video by hitting the red record button in Debut or a similar video software program (Figure 2C).
12. Save each video to the appropriate project folder and label each video accordingly.

3. Saving nailbed images

1. Open the nailbed video in the software and manually choose a frame in the video where vessels are in clear focus.
2. Using the Screenshot tool on the computer, take a screenshot of the debut video screen showing clear vasculature in the nail. Save image.
3. Open the screenshot image in ImageJ software by clicking File and Open; select the file from its destination folder.
4. If required, adjust the brightness and contrast by selecting Image > Adjust > Brightness/Contrast. This tool can help alter the contrast of the images to better visualize the vessel morphology.
5. Once the image has been adjusted, click Set in the Brightness/Contrast tool.
6. Save the image as a TIFF file by clicking on File and then Save as.

Using the capillaroscopy method described here, nailbed vascular morphology can be easily imaged, as shown in Figure 4A. Typical nailbed vasculature in a mouse exhibits three consistent features, as highlighted in Figure 4B: each nailbed has 1) an afferent vessel, 2) an efferent vessel, and 3) a network of capillaries connecting both the afferent and efferent vessels. To demonstrate the consistency of nailbed morphology, we show in Figure 4C a representative nailbed capillary network in a wild-type mouse on an SV129/S6 background and in Figure 4D a wild-type mouse on a C57/B6J background.

Figure 1: Nail preparation for capillaroscopy imaging. (A) Example mouse paw with arrows illustrating fur obscuring nails. (B) Application of hair removal cream- apply generously to the mouse hind paw, taking care to cover the entire paw. (C) Example mouse paw after hair removal; note - nails are no longer obstructed by fur and are ready for imaging in 24 hours. Please click here to view a larger version of this figure.

Figure 2: Capillaroscopy setup. (A) Photograph of capillaroscopy equipment showing complete setup for nailbed imaging: (1) isoflurane anesthesia equipment, (2) anesthesia nose cone, (3) adjustable animal stage, (4) capillaroscopy microscope, and (5) laptop with video software for imaging. (B) Example mouse placement for procedure; the mouse is kept under sedation by the nose cone, and the hind foot is placed volar side-up for imaging under objective (see zoom). (C) Complete imaging setup showing vessel picture on laptop video software. Please click here to view a larger version of this figure.

Figure 3: Capillaroscope adjustment and suggestions. Photograph of capillaroscopy equipment to illustrate adjustable options to improve image quality. (1) Use the knob to adjust the z position to bring the objective towards the mouse nail, (2) twist the objective to adjust the amount of glare from the light on nails, (3) apply white or colored laboratory tape to maximize contrast for better vessel network viewing, (4) utilize capillaroscopy microscope x and y stage adjustment for animal positioning. Please click here to view a larger version of this figure.

Figure 4: Typical nailbed vasculature morphology in mice. (A) Example of mouse nailbed image acquired by capillaroscopy and (B) typical nailbed morphology consists of three elements: 1) an afferent vessel, 2) an efferent vessel, and 3) a capillary network that exists between elements 1 and 2. Nailbed morphology remains consistent between wild-type mouse strains, including mice on a (C) SV129/S6 and (D) C57/B6J background. Please click here to view a larger version of this figure.

In summary, we provide a protocol allowing researchers to easily and inexpensively assess the morphology of mouse nailbed microvasculature, a novel location for non-invasive peripheral vascular imaging. Like NFC methods used in guinea pigs²⁰ and mice²¹, the major strength of the protocol described here is that it allows for quick and non-invasive evaluation of peripheral microvasculature in mouse models of disease. This could be particularly useful for studies that involve peripheral vascular abnormalities, such as rheumatic diseases^2,3 and endocrine diseases including hypertension and diabetes mellitus^7,8.

Harnessing the transparency of the mouse nail, nailbed capillaroscopy introduces a novel location for the visualization of peripheral microvasculature. In comparison to NFC, which is particularly useful for monitoring blood cell motion^21,22, the nailbed capillaroscopy protocol described here provides a larger area for better observation of microvascular morphology and structure.

To ensure optimal imaging, we recommend paying attention to the steps that prepare the nail for imaging. Effective hair removal is imperative to successful nailbed imaging; hair obscuring the nail will lead to imaging failure. We also recommend that hair removal is completed at least 24 h before imaging; this is to allow any irritation from the hair removal procedure to resolve. To limit variability that may occur in using this method, we recommend that the second digit of the hind paw be used for imaging due to its larger size. This also maintains consistency between animals. Furthermore, the generous application of immersion oil helps to improve the resolution of the small nailbed capillary networks. Finally, applying appropriately colored laboratory tape beneath the nail before imaging also improves contrast for visualization of vessels.

Despite the efficacy of the technique in resolving gross nailbed vessel morphology, there are some limitations to the technique as outlined here. Due to the nature of the imaging interface and the size of the vessels, resolution of blood flow or blood cell velocity is not possible using this protocol. Further work optimizing vessel image capture may enable automated image analysis approaches in the future, which would be valuable in further characterizing peripheral microvascular features. Secondly, the protocol described requires anesthesia of mice to capture nailbed images. Anesthesia changes microvessel morphology over long periods of time (after 65 min)²³. Although the time under anesthesia is considerably shorter using this method, researchers should be mindful of the time taken to complete the procedure from mouse to mouse to ensure consistency and reduce variability in the data collected.

Peripheral vessel imaging is often invasive and expensive^12,13; however, the protocol described here helps to overcome these limitations to allow visualization of peripheral vessel morphology quickly, cheaply, and easily. In conclusion, the exploratory method described in this protocol could be implemented in future studies that aim to characterize morphological abnormalities in the mouse nailbed in models of peripheral vascular disease.

Unrelated to this work, Dr. Pasquale was a paid consultant to Twenty Twenty. Unrelated to this work, Clara Cousins is a paid consultant to Cartography Biosciences. The other authors have nothing to disclose.

This work was funded by unrestricted departmental funds awarded to Lauren K. Wareham. Dr. Pasquale is supported by The Glaucoma Foundation (NYC) and by an unrestricted challenge grant from Research to Prevent Blindness (NYC).