Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

One of our lab's interests is vascular dysfunction in neurodegenerative diseases. We aim to detect and monitor peripheral microvascular pathology via capillaroscopy to gain insight into its potential role in central nervous system diseases. Detecting microvascular dysfunction in animals is often invasive, carried out ex vivo, or expensive. With this protocol, researchers can easily and inexpensively assess the nailbed microvasculature and translate clinical nailfold capillaroscopy findings to animal models. We introduce a novel location for visualizing mirroring peripheral microvasculature, the nailbed. In comparison to nail fold capillaroscopy, nailbed capillaroscopy provides a larger area for better observation of microvascular morphology and structure.

[Instructor] To begin position the anesthetized mouse on the operating table for the procedure. Then apply lubricant eye gel or sterile non-medicated ophthalmic ointment to both eyes to prevent the cornea from drying. Then, maintaining the animal under anesthesia using a nose cone, apply a generous amount of hair-removal cream to the entire paw using an applicator. Ensure complete coverage of the paw and nailbed areas. After leaving the cream for two minutes, carefully clean away the hair-removal cream with a clean tissue, and wash the paw in lukewarm sterile water. 24 hours post fur removal, set up the capillaroscopy equipment in a temperature-controlled room maintained between 21.5 and 22.5 degrees Celsius. Connect the capillaroscope to a laptop computer via USB connection. Open the Debut video software application on the laptop. After the mouse is properly anesthetized, apply lubricant eye gel or sterile non-medicated ophthalmic ointment to both eyes. Place white laboratory tape or a similar background material under the paw to enhance contrast and improve vessel visualization. Now position the hind paw volar-side up on the lab tape platform, below the objective. Gently spread the toes to separate the nails under the microscope objective using an applicator. Ensure the nailbeds are clearly separated for optimal vessel imaging. Visualize what is being magnified by the capillaroscope and focus on the nailbed by adjusting the X-and Y-stage adjusters and the magnification wheel to obtain a clear image. To reduce glare and improve focus, generously Apply immersion oil to the paw, ensuring complete nail coverage. Focus on the nail of the second digit of the hind paw, as this is the largest nail, and easiest to image in mice. Adjust the X- and Y-stage adjusters and magnification wheel to bring the vessel network into focus. Adjust the objective to minimize glare and bring the nail vessel network into view. To record a video, hit the red Record button in Debut. Save each video to the appropriate project folder, and label each video accordingly. Next, open the ImageJ software, and drag the file into the software interface to load the image. To adjust the brightness and contrast, select image, followed by Adjust and Brightness/Contrast. This tool helps alter the contrast of the images to better visualize vessel morphology. Once the image has been adjusted, click Set in the Brightness/Contrast tool. Finally, go to File, Save As, and select Tiff to save the image. Nailbed vascular morphology was successfully imaged using the capillaroscopy method. Typical mouse nailbed vasculature consisted of an afferent vessel, an efferent vessel, and a network of capillaries connecting both. The nailbed capillary network was consistent across different mouse strains, as demonstrated in wild-type mice on an SV129/S6 background and a C57/B6J background.