Name: In Vivo Imaging Uncovers the Migratory Behavior of Leukocytes within the Joints (Video) | JoVE Journal
Uploaded: 2025-12-09T14:30:00.000+00:00
Duration: 10 min 10 s
Description: Learn in vivo imaging of murine joints using a type II collagen-induced arthritis model to visualize leukocyte migration and molecular dynamics. This protocol enables real-time observation of immune cell behavior in inflamed tissues.

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5 minutes

The work of Y.M. is supported by the Japanese Society for the Promotion of Science (JSPS) KAKENHI [grant number JP22K08531, JP25K02574]; Japan Agency for Medical Research and Development (AMED) [Grant Number 25bm1423033]; The Mochida Memorial Foundation for Medical and Pharmaceutical Research; The JCR Grant for Promoting Research for FRONTIER and The Mitsubishi Foundation.

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).
<ol>
	<li>Prepare a dilution of bovine type II collagen in 0.05 M acetic acid (final concentration 4 mg/mL) by shaking overnight at 4 &#176;C.</li>
	<li>To create the emulsion, mix the collagen dilution (4 mg/mL in 0.05 M acetic acid) with complete Freund&#39;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 &#176;C. This can be used for the second immunization.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Shave the hair around the tail using clippers to prepare for immunization.</li>
	<li>Slowly inject 100 &#181;L volume of the emulsion (containing 200 &#181;g of collagen) into the subcutaneous space along the caudal dorsum (near the base of the tail) (Figure 2).</li>
	<li>Repeat steps 1.2 to 1.6 at day 21 for the second immunization (Figure 2).</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Anesthetize arthritogenic mice intraperitoneally with medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and vetorphale (5 mg/kg).</li>
	<li>Shave the mouse leg with clippers and use hair removal cream (&#60;60 s) to remove any remaining hair. Avoid prolonged application to prevent skin irritation.</li>
	<li>Remove the cream with moist gauze (Figure 4A). Ensure all hair is removed to avoid undesirable autofluorescence in the imaging area. Repeat if required.</li>
	<li>Apply sterile corneal lubricant is applied to both eyes during anesthesia to prevent corneal desiccation or ulceration.</li>
	<li>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.</li>
	<li>Position the arthritic joints on the plastic plate (Figure 4C). 
	NOTE. Tissue adhesive can be used to help fix the mouse&#39;s leg in place. This study utilizes clay to secure a glass slide to a plastic plate.</li>
	<li>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.</li>
	<li>Set the stage on the microscope. 
	NOTE. Use a heating pad to maintain the mouse&#39;s body temperature during prolonged imaging sessions.</li>
	<li>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.
	<ol>
		<li>Check the scan rate of the microscope. Use a pixel resolution of 512 x 512, with 5 optical sections, a Z stack of 4 &#181;m, and a cycle time of 15 s to allow analysis of individual motile cells over sufficient time to obtain meaningful data.</li>
	</ol>
	</li>
	<li>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 &#181;g of antibodies per mouse of Ly6G for neutrophils and CD4 for T cells, along with approximately 80 &#181;g of 680-250k Dextran per mouse for blood vessel staining. Antibodies and Dextran are usually dissolved in 100 &#181;L of sterile PBS. Be aware that some antibodies may deplete leukocytes, so check that the selected antibodies do not reduce leukocyte counts.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Present the data as mean &#177; standard error of the mean (SEM). Perform statistical analysis using Student&#39;s t-test for two-group comparisons or one-way ANOVA for multiple groups, followed by Tukey&#39;s post hoc test. Consider a p-value &#60; 0.05 as statistically significant. 
	NOTE: All analysis was performed using GraphPad Prism version 10.</li>
</ol>

<ol><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+migration+into+inflamed+tissues%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Leukocyte migration into inflamed tissues.</a> Immunity. 41 (5), 694-707 (2014).</li>
<li>Miyabe, Y., Lian, J., Miyabe, C., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines+in+rheumatic+diseases%3A+pathogenic+role+and+therapeutic+implications%5BTitle%5D)+AND+%22Nat+Rev+Rheumatol%22%5BJournal%5D)">Chemokines in rheumatic diseases: pathogenic role and therapeutic implications.</a> Nat Rev Rheumatol. 15 (12), 731-746 (2019).</li>
<li>Griffith, J. W., Sokol, C. L., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines+and+chemokine+receptors%3A+positioning+cells+for+host+defense+and+immunity%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">Chemokines and chemokine receptors: positioning cells for host defense and immunity.</a> Annu Rev Immunol. 32, 659-702 (2014).</li>
<li>Murooka, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-infected+T+cells+are+migratory+vehicles+for+viral+dissemination%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">HIV-infected T cells are migratory vehicles for viral dissemination.</a> Nature. 490 (7419), 283-287 (2012).</li>
<li>Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427 (6970), 154-159 (2004).</li>
<li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy%3A+visualizing+immunity+in+context%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21 (3), 315-329 (2004).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+function+and+fate+of+neutrophils+in+sterile+injury+and+repair%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Visualizing the function and fate of neutrophils in sterile injury and repair.</a> Science. 358 (6359), 111-116 (2017).</li>
<li>Lammermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+swarms+require+LTB4+and+integrins+at+sites+of+cell+death+in+vivo%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo.</a> Nature. 498 (7454), 371-375 (2013).</li>
<li>Miyabe, Y., Kim, N. D., Miyabe, C., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+chemokine+control+of+neutrophil+migration+in+vivo+in+a+murine+model+of+inflammatory+arthritis%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Studying chemokine control of neutrophil migration in vivo in a murine model of inflammatory arthritis.</a> Methods Enzymol. 570, 207-231 (2016).</li>
<li>Miyabe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+C5a+receptor+is+the+key+initiator+of+neutrophil+adhesion+igniting+immune+complex-induced+arthritis%5BTitle%5D)+AND+%22Sci+Immunol%22%5BJournal%5D)">Complement C5a receptor is the key initiator of neutrophil adhesion igniting immune complex-induced arthritis.</a> Sci Immunol. 2 (7), eaaj2195(2017).</li>
<li>Sadik, C. D., Miyabe, Y., Sezin, T., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+critical+role+of+C5a+as+an+initiator+of+neutrophil-mediated+autoimmune+inflammation+of+the+joint+and+skin%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin.</a> Semin Immunol. 37, 21-29 (2018).</li>
<li>Miyabe, Y., Miyabe, C., Mani, V., Mempel, T. R., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atypical+complement+receptor+C5aR2+transports+C5a+to+initiate+neutrophil+adhesion+and+inflammation%5BTitle%5D)+AND+%22Sci+Immunol%22%5BJournal%5D)">Atypical complement receptor C5aR2 transports C5a to initiate neutrophil adhesion and inflammation.</a> Sci Immunol. 4 (35), eaav5951(2019).</li>
<li>Kohl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Igniting+the+flame+in+arthritis%3A+C5aR2+controls+endothelial+transcytosis+of+C5a%5BTitle%5D)+AND+%22Sci+Immunol%22%5BJournal%5D)">Igniting the flame in arthritis: C5aR2 controls endothelial transcytosis of C5a.</a> Sci Immunol. 4 (35), eaax0352(2019).</li>
<li>Miyabe, Y., Miyabe, C., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LTB4+and+BLT1+in+inflammatory+arthritis%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">LTB4 and BLT1 in inflammatory arthritis.</a> Semin Immunol. 33, 52-57 (2017).</li>
<li>Miyabe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Necessity+of+lysophosphatidic+acid+receptor+1+for+development+of+arthritis%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Necessity of lysophosphatidic acid receptor 1 for development of arthritis.</a> Arthritis Rheum. 65 (8), 2037-2047 (2013).</li>
</ol>

Y.M. received grant/research support from Chugai Pharmaceutical Co., Ltd.

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 arthritis10,11. Additionally, in vivo imaging has revealed a novel functional role for C5aR2 in transpo...

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 tissue1,3.
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 vivo4,5,6. 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 reactions10,11.
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 (&#62;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.

<table><tbody><tr><td>29 G x 1/2&quot;</td><td>Terumo</td><td>SS-10M2913</td><td></td></tr><tr><td>0.1 M acetic acid</td><td>Fujifilm</td><td>016-18835</td><td></td></tr><tr><td>1 mL plastic syringe</td><td>TOP</td><td>19531</td><td></td></tr><tr><td>1 mm syringe for grease</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>18 G x 1 1/2&quot;</td><td>Terumo</td><td>NN-1838R</td><td></td></tr><tr><td>26 G x 1/2&quot;</td><td>Terumo</td><td>NN-2613S</td><td></td></tr><tr><td>680-250 kDa dextran</td><td>Biotium</td><td>80135</td><td></td></tr><tr><td>Agarose</td><td>invitrogen</td><td>16520-100</td><td></td></tr><tr><td>Bovine type II collagen</td><td>Collagen Research Center</td><td>K42</td><td></td></tr><tr><td>Clay</td><td>debika</td><td>93186</td><td></td></tr><tr><td>Complete freund&amp;rsquo;s adjuvant (CFA)</td><td>Chondrex</td><td>7001</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>A1</td><td></td></tr><tr><td>Cover glass</td><td>Matsunami Glass IND.,Ltd</td><td>C024401</td><td></td></tr><tr><td>Digital slide caliper</td><td>Mitsutoyo</td><td>500-180-30</td><td></td></tr><tr><td>Falcon tube (15 mL)</td><td>Corning</td><td>NA</td><td></td></tr><tr><td>Falcon tube (50 mL)</td><td>Corning</td><td>NA</td><td></td></tr><tr><td>FITC-conjugated anti-mouse Ly6G antibody</td><td>Biolegend</td><td>127606</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-5070</td><td></td></tr><tr><td>Forceps</td><td>NAPOX</td><td>MA-48-2</td><td></td></tr><tr><td>Glass syringe and connector</td><td>Tsubasa Industry Co., Ltd&amp;nbsp;</td><td>S7P</td><td></td></tr><tr><td>Grease</td><td>Toray Dow Corning Silicone</td><td>NA</td><td></td></tr><tr><td>Hair clipper</td><td>Wahl</td><td>WA5109</td><td></td></tr><tr><td>Hair clipper</td><td>Wahl</td><td>9966</td><td></td></tr><tr><td>Hair removal cr&amp;egrave;me</td><td>Kracie</td><td>N/A</td><td></td></tr><tr><td>Medetomidine</td><td>meiji</td><td>N/A</td><td></td></tr><tr><td>Micro-scissors</td><td>Roboz</td><td>RS-5610</td><td></td></tr><tr><td>Micro-scissors</td><td>Roboz</td><td>RS-5630</td><td></td></tr><tr><td>Midazolam</td><td>Sandoz</td><td>N/A</td><td></td></tr><tr><td>Moist gauze</td><td>Nippon Paper Crecia Co., Ltd</td><td>S-200</td><td></td></tr><tr><td>NIS-Elements image analysis 6.20.00</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>PBS in Falcon tube (50 mL)</td><td>Shimadzu Diagnostics Corporation</td><td>5913</td><td></td></tr><tr><td>PE conjugated anti-mouse CD4 antibody</td><td>Biolegend</td><td>100408</td><td></td></tr><tr><td>Plastic plate</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>Prism 10.3.1</td><td>GraphPad</td><td>NA</td><td></td></tr><tr><td>Slide glass</td><td>Muto Pure Chemicals Co.,Ltd</td><td>5115</td><td></td></tr><tr><td>Surgical scope</td><td>Olympus</td><td>VMZ</td><td></td></tr><tr><td>Tissue bond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Vetorphale&amp;nbsp;</td><td>meiji</td><td>N/A</td><td></td></tr></tbody></table>

in vivo imaging uncovers the migratory behavior of leukocytes within the joints

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

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.

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...

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).

Leukocytes are blood-borne cells derived from the bone marrow that must migrate into tissues to mediate inflammation. Controlling this migration, ...

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.

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Research

JoVE Journal

Immunology and Infection

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad.  We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Tracking Mouse Bone Marrow Monocytes In Vivo

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

Learn in vivo imaging of murine joints using a type II collagen-induced arthritis model to visualize leukocyte migration and molecular dynamics. This protocol enables real-time observation of immune cell behavior in inflamed tissues.

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

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