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

Summary

In vivo imaging of the joints is a valuable technique for uncovering the migratory behavior of leukocytes and molecular dynamics within the inflamed joints. Here, we present a detailed protocol for in vivo imaging of the joints using a murine model of type II collagen-induced arthritis (CIA).

Abstract

Leukocytes are blood-borne cells derived from the bone marrow that must migrate into tissues to mediate inflammation. Controlling this migration, therefore, represents a critical point for the development of new therapeutic strategies to attenuate inflammation. Traditional endpoint studies have not allowed us to fully dissect the migratory behavior of leukocytes in vivo. Recent advances in imaging technology, however, have provided unprecedented insights into immune cell migration in live animals, greatly enhancing our understanding of the molecular regulation of immune cell trafficking.

Here, we present a detailed protocol for in vivo imaging of the joints using a murine type II collagen-induced arthritis (CIA) model. Mice are immunized with type II collagen emulsified in complete Freund's adjuvant, and arthritis progression is monitored by clinical scoring and paw thickness measurements. At the peak of arthritis, leukocytes are visualized in vivo using confocal microscopy in transgenic mice expressing fluorescent proteins or in wild-type mice after intravenous injection of fluorescently labeled antibodies and high-molecular-weight Dextran for vessel staining. Careful surgical exposure of the joint, application of agarose, and proper positioning of the leg allow stable long-term imaging, typically lasting several hours. This approach enables real-time analysis of leukocyte arrest, crawling, and transendothelial migration within inflamed joints.

This protocol will be valuable for research groups investigating leukocyte trafficking in arthritis and other inflammatory diseases. It may also serve as a platform for preclinical evaluation of therapies targeting immune cell migration.

Introduction

Since leukocytes are blood-borne cells derived from the bone marrow, they must enter tissue to cause inflammation¹. The control of this process represents a key point at which new therapeutics could be developed to attenuate inflammation². In general, leukocyte trafficking into inflamed tissue occurs in postcapillary venules and follows the adhesion cascade, beginning with the capture of free-flowing leukocytes by the vessel wall. Furthermore, the process is followed by leukocytes rolling along the vessel wall in the direction of flow, leukocyte arrest on the endothelium, release from adhesion, and crawling in all directions on the vessel surface to locate a receptive site for transendothelial migration (TEM) into a specific location within the tissue^1,3.

These distinct steps are regulated by a combination of molecular signals, including the selectins (for rolling), integrins (for arrest, crawling), and chemoattractants (CAs) (for arrest, possibly crawling, and TEM)³. CAs activate leukocytes via seven transmembrane spanning G protein-coupled chemoattractant receptors (CARs), and play critical roles in the adhesion cascade by upregulating integrin affinity (leading to arrest) and inducing TEM. However, the exact mechanisms of TEM are not fully understood. Traditional endpoint studies and in vitro experiments have not provided insight into how CA-CAR signaling dynamically regulates leukocyte migratory behavior in vivo.

Recent advances in imaging technology have provided unprecedented views into immune cell migration in live animals, greatly enhancing our understanding of the molecular regulation of immune cell trafficking in vivo^4,5,6. For instance, this technology has elucidated the molecular control of neutrophil trafficking to sites of tissue inflammation, such as thermal liver injury⁷ and laser skin injury⁸. Therefore, we have applied to investigate the molecular mechanisms controlling neutrophil trafficking to sites of joint inflammation⁹. The in vivo joint imaging described here revealed that complement component C5a receptor 1 (C5aR1) is crucial for initiating neutrophil adhesion to joint endothelium, igniting inflammation within the joints. C5aR1 facilitated integrin-dependent neutrophil arrest and spreading, which was followed by TEM mediated by leukotriene B4 receptor, and crawling driven by CC chemokine receptor 1 (CCR1). CXC chemokine receptor 2 (CXCR2) played a role in the later stages of neutrophil TEM in the inflamed joints¹⁰. In addition, in vivo joint imaging revealed a new paradigm in type III hypersensitivity reactions within the joint, where C5a acts as an initiator of inflammation, while tissue-resident cells play more significant roles in inflammation in the current paradigm of type III hypersensitivity reactions^10,11.

In addition, we applied in vivo imaging of the joint to study the molecular dynamics of C5a within the joint. C5a loaded onto the joint tissue in wild-type mice was rapidly transported into the vascular lumen, while in C5aR2-deficient mice, C5a remained entirely within the synovial tissue¹². These data demonstrate that C5aR2, expressed on the joint endothelium, transports C5a into the vessel lumen, a process necessary for C5aR1-dependent neutrophil adhesion in the joints¹³.

In vivo imaging of the joints is applicable to all models of inflammatory arthritis and allows for long-term observation (usually a couple of hours), although visualization of deep tissue (>1 mm) might be limited depending on the microscope used. Taken together, this technique provides a powerful tool to investigate the migratory behavior of leukocytes and molecular dynamics within the inflamed joints. We are sharing this protocol for in vivo joint imaging to support research groups studying this process in inflamed joints.

Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee of St. Marianna University School of Medicine (Approval No. TG240926-2 and TG250801-2) and conducted in accordance with institutional and national guidelines.

1. Preparation of mouse model of arthritis

NOTE: Animal models of arthritis have provided valuable tools for understanding the pathogenesis of the disease and for developing new therapies¹⁴. While several mouse models of arthritis have unique advantages and limitations, type II collagen-induced arthritis (CIA) has been widely used in studies on the pathogenesis of rheumatoid arthritis (RA)¹⁴. In this section, we describe the protocol of CIA models of arthritis (Supplies and instruments are shown in Figure 1).

1. Prepare a dilution of bovine type II collagen in 0.05 M acetic acid (final concentration 4 mg/mL) by shaking overnight at 4 °C.
2. To create the emulsion, mix the collagen dilution (4 mg/mL in 0.05 M acetic acid) with complete Freund's adjuvant (CFA) in a 1:1 ratio in a glass syringe. This yields a final collagen concentration of 2 mg/mL in the emulsion. Vigorously mix by hand for approximately 20 min. This process generates heat, so cool the glass syringe containing the emulsion on ice. Use a beaker to check the consistency of the emulsion during preparation.
   NOTE. Store any remaining collagen dilution at -80 °C. This can be used for the second immunization.
3. Transfer the emulsion to a 1 mL glass syringe equipped with a 26 x 1/2 G needle, and keep it on ice until immunization.
4. Anesthetize 8-week-old DBA1/J Mice (male or female) with intraperitoneal injections of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and vetorphale (5 mg/kg).
   NOTE. Transgenic mice often have a C57BL6 background. Chicken collagen is typically used for CIA in C57BL6 mice; however, the incidence and severity of CIA in C57BL6 mice are lower than in DBA/1J mice. If using transgenic mice on a C57BL6 background, consider conducting a pilot study to assess CIA incidence and severity.
5. Shave the hair around the tail using clippers to prepare for immunization.
6. Slowly inject 100 µL volume of the emulsion (containing 200 µg of collagen) into the subcutaneous space along the caudal dorsum (near the base of the tail) (Figure 2).
7. Repeat steps 1.2 to 1.6 at day 21 for the second immunization (Figure 2).

2. Evaluation of arthritis clinical score and measurement of paw thickness

NOTE: 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 1-3 days after the first immunization.

1. Arthritis clinical score: Record the sum of the scores for all four paws of each mouse. Use the following scoring system:
   0 = Normal
   1 = Erythema and swelling of one digit
   2 = Erythema and swelling of two digits or erythema and swelling of the ankle joint
   3 = Erythema and swelling of more than three digits or swelling of two digits and ankle joints
   4 = Erythema and severe swelling of the ankle, foot, and digits with deformity.
   NOTE. A swollen ankle joint is defined as a paw thickness greater than 4 mm.
2. Paw thickness measurement: Measure the thickness of each paw using digital slide calipers. Use the average paw thickness across all four paws of each mouse.

3. In vivo imaging of Joints

NOTE: In vivo imaging of joints allows us to observe leukocyte migration into the joints and analyze their behavior within the tissue in live mice. To visualize cells, transgenic mice genetically engineered to express fluorescent proteins in specific cells, as well as mice injected intravenously with anti-cell marker antibodies, are used. The supplies used are shown in Figure 3.

1. Anesthetize arthritogenic mice intraperitoneally with medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and vetorphale (5 mg/kg).
2. Shave the mouse leg with clippers and use hair removal cream (<60 s) to remove any remaining hair. Avoid prolonged application to prevent skin irritation.
3. Remove the cream with moist gauze (Figure 4A). Ensure all hair is removed to avoid undesirable autofluorescence in the imaging area. Repeat if required.
4. Apply sterile corneal lubricant is applied to both eyes during anesthesia to prevent corneal desiccation or ulceration.
5. Under a surgical scope, make a 1 cm incision along the skin using microscissors (Figure 4B).
   NOTE. This step is critical. Surgery can trigger leukocyte (especially neutrophil) recruitment. Avoid any damage that might cause artifacts. Due to angiogenesis in arthritic joints, these joints are prone to bleeding. Multiple practice sessions may be needed for successful surgery.
6. Position the arthritic joints on the plastic plate (Figure 4C).
   NOTE. Tissue adhesive can be used to help fix the mouse's leg in place. This study utilizes clay to secure a glass slide to a plastic plate.
7. Cover the imaging area with approximately 1 mL of 1.5% agarose with phosphate buffered saline (PBS) (Figure 4D).
   NOTE. Ensure the agarose has cooled to prevent tissue injury, which may promote leukocyte recruitment to the imaging area.
8. Set the stage on the microscope.
   NOTE. Use a heating pad to maintain the mouse's body temperature during prolonged imaging sessions.
9. Adjust confocal microscope settings (laser power, gain, offset, location, etc). Here, perform imaging on a confocal microscope with a 25 water-immersion objective lens, NA 1.1. Set the laser excitation at 488 nm, 561 nm, and 640 nm, and collect the emission at 488.0 nm, 561.6 nm, and 638.5 nm.
   1. Check the scan rate of the microscope. Use a pixel resolution of 512 x 512, with 5 optical sections, a Z stack of 4 µm, and a cycle time of 15 s to allow analysis of individual motile cells over sufficient time to obtain meaningful data.
10. Once a suitable location is identified, start image acquisition.
    NOTE. If using anti-cell marker antibodies to label leukocytes, inject the antibodies intravenously 3 min before imaging begins. For blood vessel visualization, inject Qdots, CD31 antibody, or Dextran simultaneously. For example, inject approximately 2 µg of antibodies per mouse of Ly6G for neutrophils and CD4 for T cells, along with approximately 80 µg of 680-250k Dextran per mouse for blood vessel staining. Antibodies and Dextran are usually dissolved in 100 µL of sterile PBS. Be aware that some antibodies may deplete leukocytes, so check that the selected antibodies do not reduce leukocyte counts.
11. Process the ND2 file images acquired with the confocal microscope using the General Analysis 3 (GA3) module of NIS-Elements image analysis software (version 6.20.00). Perform the extraction of vascular regions and background subtraction. Following the processing of all time frames, export the images as a video.
    NOTE. As part of the analysis, count the number of sticking and extravasated cells.
12. After completion of imaging, either allow the animal to recover as a survival procedure or humanely euthanize it, depending on the experimental design. If survival is intended, gently close the surgical site and provide appropriate postoperative care in accordance with institutional animal care guidelines.
    NOTE: In this study, all animals were humanely euthanized immediately after imaging in compliance with institutional and national guidelines.

4. Statistical analysis

1. Present the data as mean ± standard error of the mean (SEM). Perform statistical analysis using Student's t-test for two-group comparisons or one-way ANOVA for multiple groups, followed by Tukey's post hoc test. Consider a p-value < 0.05 as statistically significant.
   NOTE: All analysis was performed using GraphPad Prism version 10.

Results

Here, 8-week-old male DBA/1J mice are immunized with an emulsion containing bovine type II collagen and CFA in a 1:1 ratio on day 0 and day 21. Arthritis clinical scoring and paw thickness measurements are performed every 1-3 days between day 0 and 42. Typically, the onset of arthritis is observed around day 28, with the arthritis clinical score and paw thickness reaching a peak around day 35 (Figure 5A-C). The incidence of CIA on DBA/1J mice is usually over...

Discussion

In vivo imaging of joints, unlike traditional endpoint studies, offers unprecedented insights into dynamic biological processes in live mice. For instance, this technique enables us to understand how four different chemoattractant receptors - C5aR1, BLT1, CCR1, and CXCR2 - collaborate to initiate neutrophil recruitment into inflamed joints in arthritis^10,11. Additionally, in vivo imaging has revealed a novel functional role for C5aR2 in transpo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
29 G x 1/2"	Terumo	SS-10M2913
0.1 M acetic acid	Fujifilm	016-18835
1 mL plastic syringe	TOP	19531
1 mm syringe for grease	Terumo	SS-01T
18 G x 1 1/2"	Terumo	NN-1838R
26 G x 1/2"	Terumo	NN-2613S
680-250 kDa dextran	Biotium	80135
Agarose	invitrogen	16520-100
Bovine type II collagen	Collagen Research Center	K42
Clay	debika	93186
Complete freund’s adjuvant (CFA)	Chondrex	7001
Confocal microscope	Nikon	A1
Cover glass	Matsunami Glass IND.,Ltd	C024401
Digital slide caliper	Mitsutoyo	500-180-30
Falcon tube (15 mL)	Corning	NA
Falcon tube (50 mL)	Corning	NA
FITC-conjugated anti-mouse Ly6G antibody	Biolegend	127606
Forceps	Roboz	RS-5070
Forceps	NAPOX	MA-48-2
Glass syringe and connector	Tsubasa Industry Co., Ltd	S7P
Grease	Toray Dow Corning Silicone	NA
Hair clipper	Wahl	WA5109
Hair clipper	Wahl	9966
Hair removal crème	Kracie	N/A
Medetomidine	meiji	N/A
Micro-scissors	Roboz	RS-5610
Micro-scissors	Roboz	RS-5630
Midazolam	Sandoz	N/A
Moist gauze	Nippon Paper Crecia Co., Ltd	S-200
NIS-Elements image analysis 6.20.00	Nikon	NA
PBS in Falcon tube (50 mL)	Shimadzu Diagnostics Corporation	5913
PE conjugated anti-mouse CD4 antibody	Biolegend	100408
Plastic plate	Corning	351029
Prism 10.3.1	GraphPad	NA
Slide glass	Muto Pure Chemicals Co.,Ltd	5115
Surgical scope	Olympus	VMZ
Tissue bond	3M	1469SB
Vetorphale	meiji	N/A