In Vivo Imaging Uncovers the Migratory Behavior of Leukocytes within the Joints

Since leukocyte blood bone cells derived from the bone marrow, so they must enter tissue to cause inflammation. The control of this process represent a key point at which new therapeutics could be developed to attenuate inflammation. In general, leukocyte trafficking into inflamed tissue occurs in post-capillary venules and follow the adhesion cascade, which begins with the capture of free-flowing leukocyte by the vessel wall.

This is followed by rolling along the vessel wall in the direction pro. Arrest on the endothelium. So there is from adhesion and crawling in all directions on the vessel surface to locate a respective location for and transendothelial migration into specific location within the tissue.

Igniting inflammations. These distinct steps are regulated by combination of molecular signals including the chemoattractants. The chemoattractant activate leukocyte via ZP Cs and play critical roles in the migration cascade.

So to additional endpoint studies and in-vitro experiments have not provided insight in how chemoattractant and the chemoattractant receptor signaling to dynamically regulate leukocyte migratory behavior in-vivo. So recent advances in imaging technology have provided unprecedented views into immune cell migration in live animals, so greatly enhancing our understanding of the molecular regulation of immune cell trafficking in-vivo. So, recently, we have applied to investigate the molecular mechanism controlling neutrophil trafficking to sites of joint inflammations.

So our in-vivo joint imaging, so they build that new paradigm in type III hypersensitivity reactions within the joint. So, we're complement component C5a act as an initiator of inflammation while, so tissue-resident cells play more significant roles in inflammation in the current paradigm of type III hypersensitivity reactions. So, therefore, in-vivo imaging of the joint is a valuable technique for uncovering the migrated behavior of leukocyte and molecular dynamics with the inflamed joint.

So, we are sharing this protocol for in-vivo joint imaging to support research groups studying this process in inflamed joints. Prepare the dilution bovine type II collagen in 0.05 molar acetic acid by shaking overnight at four degree to create the emulsion mix collagen dilution with complete Freund adjuvant in a 1:2 ratio in a glass syringe. It could also be mixed by hand for approximately 20 minute.

This process generate heat, so cool the glass syringe containing emulsion on ice. A mice anesthetized before the immunizations. Shape the hair along the tail using clippers to prepare for immunization.

Slowly inject 100 microliter volume of the emulsion into the subcutaneous space near the base of the tail. Clinical scoring of arthritis and measurement of paw thickness are essential for assessing disease activity. Typically, arthritis clinical scoring and paw thickness measurements are taken every one to three days after the first immunizations.

Arthritis clinical score to this score is the sum of the scores for all four paws of each mouse. Scores are recorded as follows. So, zero is abnormal.

One is erythema and swelling of one digit. Two is erythema and the swelling of two digits or erythema and swelling of ankle joint. Three is erythema and swelling of more than three digits or swelling of two digits and ankle joint.

Four is erythema and severe swelling of the ankle, foot, and digit with deformity. So, paws thickness measurement is measured thickness of each paw using digital slide calipers. The average paw thickness measurement across all four paws is used for each mouse.

Mice are anesthetized and then so shape the mouse leg with clippers. Use hair removal cream to remove any remaining hair. Remove the cream with moist gauze.

Apply sterile corneal lubricant to both eyes to prevent corneal drying and reduce the risk of corneal oscillation. Under a surgical scope, make one centimeter incision along the skin using micro scissors. Cover the imaging earlier with 1.5%agarose with PBS.

The stage is set on the microscope. Adjust confocal microscope settings including laser power, gain, offset, and location. Once suitable location is identified, start image acquisition.

A representative result of CIA. Typically, the onset of arthritis is observed along day 28 with arthritis clinical score and paw thickness reaching a peak along day 35. The incidence of CIA on DBA 1-day mice is usually over 900 to 100%by day 35.

Here, the movie on the left shows the joint in control mice and the one on the right showed the joint in CIA mice. In these movies, the green cells represent Ly6G positive neutrophils. The orange cells indicate CD4 positive T-cells, and the white areas correspond to blood vessels.

On day 35, you can clearly see that more Ly6G positive neutrophils are present outside the blood vessels compared to CD4+T-cells. These neutrophils are highly motile, actively moving in multiple directions within the inflamed joint. Here, I'm showing the representative analysis of in-vivo imaging of the joints.

The graph on the left shows the number of newly sticking neutrophils, while the one on the right shows the number of newly transmigrated neutrophils during a 30 minutes recording. In the arthritic joints, the numbers of both newly sticking and transmigrated neutrophils are significantly higher than those observed in the control joints. In conclusion, we present our current protocol for in-vivo imaging of joints, which enables high-resolution, real-time visualization of leukocyte migratory behavior in living mice under physiological and pathological conditions.

This technique allows us to directly observe how immune cells interact within inflamed tissues, how they adhere to blood vessels walls transmigrate into the tissue, and orchestrate the inflammatory response. Through this approach, we can capture dynamic cellular behaviors that are otherwise invisible with conventional histological or in-vitro methods. By combining this imaging system with quantitative analysis and molecular markers, we are able to uncover key pathways that drive the initiation and progression of arthritis.

Importantly, the insights gained from these in-vivo observations not only deepen our understanding of immune cell dynamics, but also provide a scientific foundation or for identifying new therapeutic targets. As this technology continues to evolve, and as more researchers adapt and refine the methodology, we believe that in-vivo imaging will become an essential platform for studying complex immune-mediated diseases and a powerful breach connecting basic immunology with translational and clinical research. Ultimately, we hope that this approach will contribute to the development of innovative therapies for rheumatoid arthritis and other chronic inflammatory diseases in the near future.