Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease. We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section. IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Dystrophin deficiency promotes leukocyte recruitment in mdx mice.

BACKGROUND: A growing body of evidence defines inflammation as a hallmark feature of disease pathogenesis of Duchenne muscular dystrophy. To tailor potential immune modulatory interventions, a better understanding of immune dysregulation in Duchenne muscular dystrophy is needed. We now asked whether dystrophin deficiency affects the cascade of leukocyte recruitment. METHODS: We performed intravital microscopy on the cremaster muscle of wild-type and dystrophin-deficient mdx mice. Recruitment was triggered by preparation alone (traumatic inflammation) or in combination with scrotal TNFα injections. Neutrophilic infiltration of the cremaster muscle was assessed on tissue sections. Integrin expression on circulating neutrophils and serum levels of pro-inflammatory cytokines were measured by flow cytometry. RESULTS: Mdx mice show increased rolling and adhesion at baseline (traumatic inflammation) and a more profound response upon TNFα injection compared with wild-type animals. In both models, neutrophilic infiltration of the cremaster muscle is increased. Upregulation of the integrins LFA-1 and Mac-1 on circulating leukocytes and pro-inflammatory cytokines IL-6 and CCL2 in the serum points toward systemically altered immune regulation in mdx mice. CONCLUSION: We are the first to show exaggerated activation of the leukocyte recruitment cascade in a dystrophin-deficient organism in vivo.

LPS-induced maternal inflammation promotes fetal leukocyte recruitment and prenatal organ infiltration in mice.

BACKGROUND: A pro-inflammatory intrauterine milieu accounts for increased perinatal morbidity and mortality. We asked how maternal inflammation as seen in endotoxemia affects fetal leukocyte recruitment in vivo during late gestation. METHODS: Inflammation was induced in pregnant LysEGFP-mice by intraperitoneal LPS injection between gestational day 14 and 18 (E14-E18). After 20 h, intravital fluorescence microscopy was performed on fetal yolk sac venules to examine leukocyte rolling (number of rolling cells/min) and adhesion (>30 s). Infiltration of neutrophils into chorion/amnion, lung, and kidney were quantified by immunofluorescence microscopy. RESULTS: At high doses (2 × 1 mg/kg), LPS triggered preterm birth (PTB) and intrauterine fetal death (IUFD), with early gestations at high risk of IUFD and late gestations prone to PTB. Lower LPS dosing (2 × 0.25 mg/kg) did not induce labor, but promoted maternal and fetal cytokine production, as well as neutrophilic infiltration of fetal membranes, as seen in chorioamnionitis (CAM). Baseline fetal leukocyte recruitment increased throughout gestation, and maternal inflammation further augmented adhesion at E16-E18. Enhanced leukocyte recruitment ultimately translated into prominent infiltration of fetal lung and kidney. CONCLUSION: LPS-induced maternal endotoxemia promotes IUFD, PTB, and fetal leukocyte recruitment depending on gestational age. Our proposed model may serve as a platform to test novel perinatal immune modulators.

RAGE controls leukocyte adhesion in preterm and term infants.

BACKGROUND: Insufficient leukocyte recruitment may be one reason for the high incidence of life-threatening infections in preterm infants. Since the receptor of advanced glycation end products (RAGE) is a known leukocyte adhesion molecule and highly expressed during early development, we asked whether RAGE plays a role for leukocyte recruitment in preterm and term infants. METHODS: Leukocyte adhesion was analyzed in dynamic flow chamber experiments using isolated leukocytes of cord blood from extremely premature (<30 weeks of gestation), moderately premature (30-35 weeks of gestation) and mature neonates (>35 weeks of gestation) and compared to the results of adults. For fluorescent microscopy leukocytes were labeled with rhodamine 6G. In the respective age groups we also measured the plasma concentration of soluble RAGE (sRAGE) by ELISA and Mac-1 and LFA-1 expression on neutrophils by flow cytometry. RESULTS: The adhesive functions of fetal leukocytes significantly increase with gestational age. In all age groups, leukocyte adhesion was crucially dependent on RAGE. In particular, RAGE was equally effective to mediate leukocyte adhesion when compared to ICAM-1. The plasma levels of sRAGE were high in extremely premature infants and decreased with increasing gestational age. In contrast, expression of ß2-Integrins Mac-1 and LFA-1 which are known ligands for RAGE and ICAM-1 did not change during fetal development. CONCLUSION: We conclude that RAGE is a crucial leukocyte adhesion molecule in both preterm and term infants.

RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner.

BACKGROUND: The receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the ß2-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated ß2-integrin-dependent leukocyte adhesion in RAGE-/- and Icam1-/- mice in different cremaster muscle models of inflammation using intravital microscopy. RESULTS: We demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo. CONCLUSION: Our results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.