Peer Teaching in Undergraduate Medical Education: What are the Learning Outputs for the Student-Teachers? A Systematic Review.

Introduction: To achieve quality in medical education, peer teaching, understood as students taking on roles as educators for peers, is frequently used as a teaching intervention. While the benefits of peer teaching for learners and faculty are described in detail in the literature, less attention is given to the learning outputs for the student-teachers. This systematic review focuses on the learning outputs for medical undergraduates acting as student-teachers in the last decade (2012-2022). Aim: Our aim is to describe what learning outputs student-teachers have from peer teaching, and map what research methods are used to assess the outputs. We defined learning outputs in a broad sense, including all types of learning experiences, intended and non-intended, associated with being a peer teacher. Methods: A literature search was conducted in four electronic databases. Title, abstract and full text were screened by 8 independent reviewers and selection was based on predefined eligibility criteria. We excluded papers not describing structured peer teaching interventions with student-teachers in a formalized role. From the included articles we extracted information about the learning outputs of being a student-teacher as medical undergraduate. Results: From 668 potential titles, 100 were obtained as full-texts, and 45 selected after close examination, group deliberation, updated search and quality assessment using MERSQI score (average score 10/18). Most articles reported learning outputs using mixed methods (67%). Student-teachers reported an increase in subject-specific learning (62%), pedagogical knowledge and skills (49%), personal outputs (31%) and generic skills (38%). Most articles reported outputs using self-reported data (91%). Conclusion: Although there are few studies that systematically investigate student-teachers learning outputs, evidence suggests that peer teaching offers learning outputs for the student-teachers and helps them become better physicians. Further research is needed to enhance learning outputs for student-teachers and systematically investigate student-teachers' learning outputs and its impact on student-teachers.

Exercise reduces intramuscular stress and counteracts muscle weakness in mice with breast cancer.

BACKGROUND: Patients with breast cancer exhibit muscle weakness, which is associated with increased mortality risk and reduced quality of life. Muscle weakness is experienced even in the absence of loss of muscle mass in breast cancer patients, indicating intrinsic muscle dysfunction. Physical activity is correlated with reduced cancer mortality and disease recurrence. However, the molecular processes underlying breast cancer-induced muscle weakness and the beneficial effect of exercise are largely unknown. METHODS: Eight-week-old breast cancer (MMTV-PyMT, PyMT) and control (WT) mice had access to active or inactive in-cage voluntary running wheels for 4 weeks. Mice were also subjected to a treadmill test. Muscle force was measured ex vivo. Tumour markers were determined with immunohistochemistry. Mitochondrial biogenesis and function were assessed with transcriptional analyses of PGC-1α, the electron transport chain (ETC) and antioxidants superoxide dismutase (Sod) and catalase (Cat), combined with activity measurements of SOD, citrate synthase (CS) and ß-hydroxyacyl-CoA-dehydrogenase (ßHAD). Serum and intramuscular stress levels were evaluated by enzymatic assays, immunoblotting, and transcriptional analyses of, for example, tumour necrosis factor-α (TNF-α) and p38 mitogen-activated protein kinase (MAPK) signalling. RESULTS: PyMT mice endured shorter time and distance during the treadmill test (~30%, P < 0.05) and ex vivo force measurements revealed ~25% weaker slow-twitch soleus muscle (P < 0.001). This was independent of cancer-induced alteration of muscle size or fibre type. Inflammatory stressors in serum and muscle, including TNF-α and p38 MAPK, were higher in PyMT than in WT mice (P < 0.05). Cancer-induced decreases in ETC (P < 0.05, P < 0.01) and antioxidant gene expression were observed (P < 0.05). The exercise intervention counteracted the cancer-induced muscle weakness and was accompanied by a less aggressive, differentiated tumour phenotype, determined by increased CK8 and reduced CK14 expression (P < 0.05). In PyMT mice, the exercise intervention led to higher CS activity (P = 0.23), enhanced ß-HAD and SOD activities (P < 0.05), and reduced levels of intramuscular stressors together with a normalization of the expression signature of TNFα-targets and ETC genes (P < 0.05, P < 0.01). At the same time, the exercise-induced PGC-1α expression, and CS and ß-HAD activity was blunted in muscle from the PyMT mice as compared with WT mice, indicative that breast cancer interfere with transcriptional programming of mitochondria and that the molecular adaptation to exercise differs between healthy mice and those afflicted by disease. CONCLUSIONS: Four-week voluntary wheel running counteracted muscle weakness in PyMT mice which was accompanied by reduced intrinsic stress and improved mitochondrial and antioxidant profiles and activities that aligned with muscles of healthy mice.

Neutrophils in chronic inflammatory diseases.

Chronic inflammation is a component of many disease conditions that affect a large group of individuals worldwide. Chronic inflammation is characterized by persistent, low-grade inflammation and is increased in the aging population. Neutrophils are normally the first responders to acute inflammation and contribute to the resolution of inflammation. However, in chronic inflammation, the role of neutrophils is less well understood and has been described as either beneficial or detrimental, causing tissue damage and enhancing the immune response. Emerging evidence suggests that neutrophils are important players in several chronic diseases, such as atherosclerosis, diabetes mellitus, nonalcoholic fatty liver disease and autoimmune disorders. This review will highlight the interaction of neutrophils with other cells in the context of chronic inflammation, the contribution of neutrophils to selected chronic inflammatory diseases, and possible future therapeutic strategies.

Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force.

The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD+ levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ectopically expressed NDUFA4L2 caused a ~20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD+ , which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.

Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Acute lung injury (ALI) and respiratory distress can develop as a consequence of sepsis with pathogens such as group A Streptococcus (GAS). In the pathogenesis of sepsis-associated ALI, endothelial barrier disruption brought on by phagocyte activation is considered a causative factor. Here, we find that sevuparin, a heparinoid with low anticoagulant activity, prevents neutrophil-induced lung plasma leakage in a murine model of systemic inflammation evoked by heat-killed GAS (hkGAS). Furthermore, using human neutrophils and endothelial cell monolayers, we demonstrate that sevuparin inhibits hkGAS-induced endothelial barrier disruption by neutralizing the activity of neutrophil-derived proteins. By mass spectrometry of neutrophil secretion, we identify proteins, including serprocidins, S100 proteins, and histone H4, that interact with sevuparin and that are responsible for the disruptive effect on endothelial integrity. Collectively, our results demonstrate the critical role of neutrophil-derived proteins in vascular hyperpermeability caused by GAS and suggest sevuparin as a potential therapeutic in acute neutrophilic inflammation.-Rasmuson, J., Kenne, E., Wahlgren, M., Soehnlein, O., Lindbom, L. Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.

Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Neutrophil recruitment and plasma exudation are key elements in the immune response to injury or infection. Activated neutrophils stimulate opening of the endothelial barrier; however, the underlying mechanisms have remained largely unknown. In this study, we identified a pivotal role of the proinflammatory kallikrein-kinin system and consequent formation of bradykinin in neutrophil-evoked vascular leak. In mouse and hamster models of acute inflammation, inhibitors of bradykinin generation, and signaling markedly reduced plasma exudation in response to chemoattractant activation of neutrophils. The neutrophil-driven leak was likewise suppressed in mice deficient in either the bradykinin B2 receptor or factor XII (initiator of the kallikrein-kinin system). In human endothelial cell monolayers, material secreted from activated neutrophils induced cytoskeletal rearrangement, leading to paracellular gap formation in a bradykinin-dependent manner. As a mechanistic basis, we found that a neutrophil-derived heparin-binding protein (HBP/azurocidin) displaced the bradykinin precursor high-molecular-weight kininogen from endothelial cells, thereby enabling proteolytic processing of kininogen into bradykinin by neutrophil and plasma proteases. These data provide novel insight into the signaling pathway by which neutrophils open up the endothelial barrier and identify the kallikrein-kinin system as a target for therapeutic interventions in acute inflammatory reactions.-Kenne, E., Rasmuson, J., Renné, T., Vieira, M. L., Müller-Esterl, W., Herwald, H., Lindbom, L. Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation

Neutrophil recruitment and plasma exudation are key elements in the immune response to injury or infection. Activated neutrophils stimulate opening of the endothelial barrier; however, the underlying mechanisms have remained largely unknown. In this study, we identified a pivotal role of the proinflammatory kallikrein-kinin system and consequent formation of bradykinin in neutrophil-evoked vascular leak. In mouse and hamster models of acute inflammation, inhibitors of bradykinin generation, and signaling markedly reduced plasma exudation in response to chemoattractant activation of neutrophils. The neutrophil-driven leak was likewise suppressed in mice deficient in either the bradykinin B-2 receptor or factor XII (initiator of the kallikrein-kinin system). In human endothelial cell monolayers, material secreted from activated neutrophils induced cytoskeletal rearrangement, leading to paracellular gap formation in a bradykinin-dependent manner. As a mechanistic basis, we found that a neutrophil-derived heparin-binding protein (HBP/azurocidin) displaced the bradykinin precursor high-molecular-weight kininogen from endothelial cells, thereby enabling proteolytic processing of kininogen into bradykinin by neutrophil and plasma proteases. These data provide novel insight into the signaling pathway by which neutrophils open up the endothelial barrier and identify the kallikrein-kinin system as a target for therapeutic interventions in acute inflammatory reactions.Kenne, E., Rasmuson, J., Renne, T., Vieira, M. L., Muller-Esterl, W., Herwald, H., Lindbom, L. Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation

Neutrophil recruitment and plasma exudation are key elements in the immune response to injury or infection. Activated neutrophils stimulate opening of the endothelial barrier; however, the underlying mechanisms have remained largely unknown. In this study, we identified a pivotal role of the proinflammatory kallikrein-kinin system and consequent formation of bradykinin in neutrophil-evoked vascular leak. In mouse and hamster models of acute inflammation, inhibitors of bradykinin generation, and signaling markedly reduced plasma exudation in response to chemoattractant activation of neutrophils. The neutrophil-driven leak was likewise suppressed in mice deficient in either the bradykinin B-2 receptor or factor XII (initiator of the kallikrein-kinin system). In human endothelial cell monolayers, material secreted from activated neutrophils induced cytoskeletal rearrangement, leading to paracellular gap formation in a bradykinin-dependent manner. As a mechanistic basis, we found that a neutrophil-derived heparin-binding protein (HBP/azurocidin) displaced the bradykinin precursor high-molecular-weight kininogen from endothelial cells, thereby enabling proteolytic processing of kininogen into bradykinin by neutrophil and plasma proteases. These data provide novel insight into the signaling pathway by which neutrophils open up the endothelial barrier and identify the kallikrein-kinin system as a target for therapeutic interventions in acute inflammatory reactions.Kenne, E., Rasmuson, J., Renne, T., Vieira, M. L., Muller-Esterl, W., Herwald, H., Lindbom, L. Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Muscle Weakness in Rheumatoid Arthritis: The Role of Ca2+ and Free Radical Signaling.

In addition to the primary symptoms arising from inflammatory processes in the joints, muscle weakness is commonly reported by patients with rheumatoid arthritis (RA). Muscle weakness not only reduces the quality of life for the affected patients, but also dramatically increases the burden on society since patients' work ability decreases. A 25-70% reduction in muscular strength has been observed in pateints with RA when compared with age-matched healthy controls. The reduction in muscle strength is often larger than what could be explained by the reduction in muscle size in patients with RA, which indicates that intracellular (intrinsic) muscle dysfunction plays an important role in the underlying mechanism of muscle weakness associated with RA. In this review, we highlight the present understanding of RA-associated muscle weakness with special focus on how enhanced Ca2+ release from the ryanodine receptor and free radicals (reactive oxygen/nitrogen species) contributes to muscle weakness, and recent developments of novel therapeutic interventions.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation.

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection.

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

Assessing large-vessel endothelial permeability using near-infrared fluorescence imaging--brief report.

OBJECTIVE: Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation. APPROACH AND RESULTS: To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E-deficient mice by Western diet for 4 weeks. Evans blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evans blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction-absorbance methodology for Evans blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α-stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evans blue dye uptake in (apolipoprotein E-deficient) mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry. CONCLUSIONS: The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions.

BACKGROUND: Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell-derived mediators into the circulation. OBJECTIVES AND METHODS: We report here that a plasma protease cascade, the factor XII-driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. RESULTS: Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation. CONCLUSIONS: In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

Renal effects of treatment with a TLR4 inhibitor in conscious septic sheep.

INTRODUCTION: Acute kidney injury (AKI) is a common and feared complication of sepsis. The pathogenesis of sepsis-induced AKI is largely unknown, and therapeutic interventions are mainly supportive. In the present study, we tested the hypothesis that pharmacological inhibition of Toll-like receptor 4 (TLR4) would improve renal function and reduce renal damage in experimental sepsis, even after AKI had already developed. METHODS: Sheep were surgically instrumented and subjected to a 36-hour intravenous infusion of live Escherichia coli. After 12 hours, they were randomized to treatment with a selective TLR4 inhibitor (TAK-242) or vehicle. RESULTS: The E. coli caused normotensive sepsis characterized by fever, increased cardiac index, hyperlactemia, oliguria, and decreased creatinine clearance. TAK-242 significantly improved creatinine clearance and urine output. The increase in N-acetyl-beta-D-glucosaminidas, a marker of tubular damage, was attenuated. Furthermore, TAK-242 reduced the renal neutrophil accumulation and glomerular endothelial swelling caused by sepsis. These effects were independent of changes in renal artery blood flow and renal microvascular perfusion in both cortex and medulla. TAK-242 had no effect per se on the measured parameters. CONCLUSIONS: These results show that treatment with a TLR4 inhibitor is able to reverse a manifest impairment in renal function caused by sepsis. In addition, the results provide evidence that the mechanism underlying the effect of TAK-242 on renal function does not involve improved macro-circulation or micro-circulation, enhanced renal oxygen delivery, or attenuation of tubular necrosis. TLR4-mediated inflammation resulting in glomerular endothelial swelling may be an important part of the pathogenesis underlying Gram-negative septic acute kidney injury.

Factor XII: a drug target for safe interference with thrombosis and inflammation.

Data from experimental animal models revealed an essential role for factor XII (FXII) in thrombotic occlusive diseases. In contrast to other blood coagulation factors, deficiency in the protease is not associated with abnormal bleeding from injury sites (hemostasis) in patients or in animals. Cumulatively, these findings suggest that FXII could be targeted as a new method of anticoagulation that is devoid of bleeding risks. An FXIIa-neutralizing antibody, 3F7, has been developed that inhibited thrombosis in an extracorporeal membrane oxygenation (ECMO) system as efficiently as heparin. However, in sharp contrast to heparin, 3F7 treatment was not associated with an increase in therapy-associated hemorrhage. In this review, we summarize current knowledge of FXII physiology and pharmacology.

Vitamin D3 and phenylbutyrate promote development of a human dendritic cell subset displaying enhanced antimicrobial properties.

A promising strategy in the fight against multidrug-resistant pathogens is the induction of endogenous AMPs, with compounds such as VitD3 and PBA. These compounds display an array of immunomodulatory effects that remain to be investigated in further detail to establish their role in the clearance of infection and possible modulation of AMP expression. Here, we have investigated the effects of VitD3 and PBA on human monocyte-DC differentiation and found that VitD3 and PBA promote the development of a stretched CD14⁺/CD1a⁻ DC subset. This subset produced enhanced levels of ROS and human cathelicidin; furthermore, it displayed enhanced killing capacity of Staphylococcus aureus compared with control DCs. When experiments were performed in WT and cathelicidin-deficient mice, we established that a ROS-producing, stretched DC subset was also induced in mouse-derived cells, independent of cathelicidin expression. However, in contrast to the human DCs, enhanced cathelicidin expression and enhanced antimicrobial activities were not found in the murine VitD3/PBA DC subset. In conclusion, the results of this study show that VitD3 and PBA induce a human DC subset that is effective against infection. These results promote further research into the use of these compounds as an antimicrobial treatment strategy.

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice.

BACKGROUND: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. METHODS: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. RESULTS: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. CONCLUSION: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice.

Immune cell recruitment to inflammatory loci is impaired in mice deficient in basement membrane protein laminin alpha4.

For leukocytes to penetrate the vessel wall, they need to interact sequentially with the endothelial lining and the perivascular BM. The matrix protein laminin-411 is a major constituent of the vascular BM. The laminin alpha4 chain is a component of laminin-411 and has structural and signaling functions. Here, we addressed the role of BM laminin alpha4 in leukocyte recruitment to inflammatory loci. We used several recruitment models in Lam4(-/-) and WT mice to determine whether lack of laminin-411 in the perivascular BM influences extravasation of inflammatory cells. Recruitment of all major leukocyte subsets (neutrophils, monocytes, and lymphocytes) was reduced in Lam4(-/-) mice compared with WT. With the use of intravital microscopy, we concluded that this decrease was a result of impaired diapedesis through the vessel wall, as neither leukocyte adhesion to the endothelial lining nor migration in extravascular tissue was hampered in Lam4(-/-) mice. Collectively, our data suggest a reduced ability of immune cells to penetrate the vessel wall in mice deficient in laminin alpha4.

Distinct infiltration of neutrophils in lesion shoulders in ApoE-/- mice.

Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T-lymphocytes in lesions. However, recent data suggest that neutrophils also may be of importance in atherogenesis. Here, we use apolipoprotein E (ApoE)-deficient mice with fluorescent neutrophils and monocytes (ApoE(-/-)/Lys(EGFP/EGFP) mice) to specifically study neutrophil presence and recruitment in atherosclerotic lesions. We show by flow cytometry and confocal microscopy that neutrophils make up for 1.8% of CD45(+) leukocytes in the aortic wall of ApoE(-/-)/Lys(EGFP/EGFP) mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. However, neutrophils accumulate at sites of monocyte high density, preferentially in shoulder regions of lesions, and may even outnumber monocyte/macrophages in these areas. Furthermore, intravital microscopy established that a majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque. These data demonstrate neutrophilic granulocytes as a major cellular component of atherosclerotic lesions in ApoE(-/-) mice and call for further study on the roles of these cells in atherogenesis.