Number Of Infiltrating Neutrophils Research Articles

Topical combined Phyllanthus emblica Linn. and simvastatin improves wound healing in diabetic mice by enhancing angiogenesis and reducing neutrophil infiltration.

The present study aimed to investigate the effects of combined Phyllanthus emblica Linn. (PE) and simvastatin (SIM) on diabetic wounds in male BALB/C mice. Bilateral full thickness wound excisions were performed in the control and diabetic groups (45 mg/kg streptozotocin, intraperitoneally injected daily for 5 days). The diabetic mice received daily treatment with four different types of cream: Vehicle [diabetes mellitus (DM) + Vehicle group], 100% PE (DM + PE group), 5% SIM (DM + SIM group) and combined 100% PE + 5% SIM (DM + Combination group) for 4, 7 and 14 days. The tissue malondialdehyde (MDA) and IL-6 protein levels, the number of infiltrated neutrophils, and the percentages of wound closure (%WC), capillary vascularity (%CV) and re-epithelialization (%RE) were subsequently measured. The results indicated that in the DM + Combination group, %CV and %WC were significantly increased when compared with the DM + Vehicle group on days 7 and 14. The tissue MDA content on day 14, and the number of infiltrated neutrophils on days 4 and 7 were significantly reduced in the DM + Combination group compared with those in the DM + Vehicle group. Furthermore, a strong positive correlation was revealed between %CV and %WC in the five groups on day 7 (r=0.736; P=0.0003). These findings indicated that topical application of combined PE and SIM could enhance wound healing by upregulating angiogenesis and reducing neutrophil infiltration in mice with diabetic wounds.

Abstract 3180: Targeting LRG1 to normalize tumor vasculature and enhance therapeutic efficacy

Abstract In recent years improving tumor vascular function, to render the tumour microenvironment less permissive and to improve delivery of therapeutics, has gained traction due to a growing body of supportive evidence. Identifying suitable targets that are tractable, ubiquitous and safe, however, has proven to be more challenging. Almost a decade ago we reported that a secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), was induced in ocular neovascular complications and contributed to the formation of dysfunctional neovessels1. More recently, we have reported that LRG1 is expressed in experimental and human tumors, and that its inhibition with a function-blocking antibody improves outcome in multiple primary2 and metastatic3 models of cancer. Crucially, we found that LRG1 blockade normalizes tumor vessels and enhances the efficacy of cisplatin, adoptive T cell and checkpoint inhibitor therapy, and that a humanized version of our blocking antibody named Magacizumab effectively inhibits tumour growth both alone and as an antibody-drug-conjugate4.Using animal models and in vitro assays we present here further data in support of LRG1 as a promising target for the treatment of solid cancers. We have further investigated the effects of LRG1 blockade on immune cell infiltration using flow cytometric and immunohistochemical analysis. In subcutaneous B16 melanoma-bearing mice treated with a PD1 checkpoint inhibitor, antibody blockade of LRG1 significantly enhanced the infiltration of CD3+ and CD8+ T cells, with the latter exhibiting a more activated phenotype as evidenced by higher GrzB, reduced PD1hi expression and increased proliferation. We also observed a reduction in the Tregs:Th ratio and a higher number of MHCII+ cells. These outcomes were also observed in LLC tumors alongside a reduction in the number of infiltrated neutrophils. In preliminary studies, where we investigated LRG1 blockade in the Rag1 mouse, we observed no effect suggesting that the downstream mode of action is mediated by impacting the immune system. Finally, to test the development of our humanized anti-LRG1 antibody Magacizumab, that only recognizes human LRG1, we demonstrated its efficacy in B16F0 tumors grown in a human LRG1 knock-in mouse. These studies provide compelling evidence that LRG1 is a novel, legitimate and potentially efficacious target for the treatment of various human solid cancers.

EMMPRIN Promotes the Expression of MMP-9 and Exacerbates Neurological Dysfunction in a Mouse Model of Intracerebral Hemorrhage.

Extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to be a vital inflammatory mediator in several neurological and neurodegenerative diseases. However, the role of EMMPRIN in intracerebral hemorrhage (ICH) remains unexplored. In this study, we aimed to exploit a highly selective monoclonal anti-EMMPRIN antibody to functionally inhibit EMMPRIN activity and thus that of MMPs as the downstream effector. To induce ICH pathology, adult C57BL/6 male mice were injected with collagenase type VII or saline as control into the right basal ganglia and were euthanized at different time points. The anti-EMMPRIN monoclonal antibody was intravenously injected once daily for 3days to block the expression of EMMPRIN initiating at 4h post-ICH. Western blot and immunofluorescence analysis results revealed that EMMPRIN expression was significantly increased surrounding the hematoma at 3 and 7d time points after ICH when compared to the saline treated control group. EMMPRIN expression was co-localized with GFAP (astrocytes) and Iba1 (microglia) at 3d time point post-ICH, but not in the control group mice. The co-localization of EMMPRIN with CD31 in endothelial cells occurred in both groups and was higher in the ICH brain. However, EMMPRIN expression was not detected in neurons from either group. The inhibition of EMMPRIN reduced the expression of MMP-9, the number of infiltrated neutrophils, the degree of brain injury and promoted neurological recovery after ICH. In conclusion, EMMPRIN could mediate the upregulation of MMP-9 and exacerbate neurological dysfunction in a mouse model of experimental ICH. Furthermore, blocking EMMPRIN reduced brain injury and subsequently promoted neurological recovery in ICH mice brains. These outcomes highlight that inhibition of EMMPRIN can be a potential therapeutic intervention strategy to regulate MMP-9's pathological roles during ICH.

Pharmacological evidence for the involvement of ryanodine receptors in halothane-induced liver injury in mice

Drug-induced liver injury (DILI) is a major safety concern in drug development. Halothane (HAL), an inhaled anesthetic, induces severe and idiosyncratic liver injury. Ryanodine receptors (RyR) are major intracellular calcium release channels found on the plasma membrane of the endoplasmic reticulum (ER). It has been reported that disordered hepatic calcium homeostasis is a feature of HAL-induced liver injury (HILI) in guinea pigs. However, there are no reports on whether RyR could mediate the pathogenesis of HILI. The aim of the present study was to investigate the effect of RyR on HILI. Ryanodine (RYA, RyR agonist, 50 μg/kg, i.p.) was administered to BALB/c female mice 1 h before HAL administration (15 mmol/kg, i.p.), which significantly elevated plasma transaminase levels and induced severe hepatic inflammation and necrosis. In contrast, dantrolene sodium (DAN, RyR antagonist) treatment significantly suppressed HILI in a dose- and time-dependent manner and alleviated liver damage. The number of infiltrated neutrophils in the liver were higher in the group treated with HAL + RYA than in the group treated with HAL alone, while DAN treatment decreased neutrophil infiltration in HILI. The hepatic mRNA levels of proinflammatory cytokines; chemokines; and factors related to danger signals, neutrophils, oxidative and ER stress, pro-apoptosis, and RyR were significantly increased with RYA pretreatment, whereas these levels were decreased with DAN treatment. These results suggest that RYA exacerbates HILI, and DAN exerts a protective effect against HILI. Hence, our study provides a novel insight regarding the effect of RyR in the mechanism underlying HILI.

Tanshinone IIA alleviates brain damage in a mouse model of neuromyelitis optica spectrum disorder by inducing neutrophil apoptosis

BackgroundNeuromyelitis optica spectrum disorder (NMOSD), an autoimmune astrocytopathic disease associated with the anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions primarily located on the optic nerves and spinal cord. Tanshinone IIA (TSA), an active natural compound extracted from Salvia miltiorrhiza Bunge, has profound immunosuppressive effects on neutrophils.ObjectiveThe present study aimed to evaluate the effect of TSA on NMOSD mice and explore the underlying mechanisms. Mice were initially administered TSA (pre-TSA group, n = 20) or vehicle (vehicle group, n = 20) every 8 h for 3 days, and then NMOSD model was induced by intracerebral injection of NMOSD-immunoglobulin G (NMO-IgG) and human complement (hC). In addition, post-TSA mice (n = 10) were administered equal dose of TSA at 8 h and 16 h after model induction. At 24 h after intracerebral injection, histological analysis was performed to assess the inhibitory effects of TSA on astrocyte damage, demyelination, and neuroinflammation in NMOSD mice, and western blotting was conducted to clarify the effect of TSA on the NF-κB and MAPK signaling pathways. Furthermore, flow cytometry and western blotting were conducted to verify the proapoptotic effects of TSA on neutrophils in vitro.ResultsThere was a profound reduction in astrocyte damage and demyelination in the pre-TSA group and post-TSA group. However, prophylactic administration of TSA induced a better effect than therapeutic treatment. The number of infiltrated neutrophils was also decreased in the lesions of NMOSD mice that were pretreated with TSA. We confirmed that prophylactic administration of TSA significantly promoted neutrophil apoptosis in NMOSD lesions in vivo, and this proapoptotic effect was mediated by modulating the caspase pathway in the presence of inflammatory stimuli in vitro. In addition, TSA restricted activation of the NF-κB signaling pathway in vivo.ConclusionOur data provide evidence that TSA can act as a prophylactic agent that reduces NMO-IgG-induced damage in the mouse brain by enhancing the resolution of inflammation by inducing neutrophil apoptosis, and TSA may serve as a promising therapeutic agent for neutrophil-associated inflammatory disorders, such as NMOSD.

Tranexamic Acid Protects Ovary and Testis Functions and Ameliorates Osteoporosis in Mice

Introduction: In a rapidly aging society, the number of people suffering from osteoporosis keeps increasing. However, effective prevention strategies for osteoporosis are not yet currently available. Objective: In this study, we examined the ameliorative effects of tranexamic acid on osteoporosis in 24-month-old mice. Methods: During the study period, mice were orally administered tranexamic acid 3 times per week. Results: Bone mineral density, which is a parameter of osteoporosis, was improved following tranexamic acid administration. In addition, female mice evidenced a stronger phenotypic improvement than male mice. In female mice treated with tranexamic acid, ovary abnormalities were reduced. Furthermore, the levels of transforming growth factor-β, hyaluronic acid, CD44, reactive oxygen species, and apoptosis, as well as the number of infiltrated neutrophils and macrophages in the ovary were lower than those in the control or solvent-administered mice. In addition, 17β-estradiol levels in blood increased when compared with the control or solvent-treated mice. In addition, administration of tranexamic acid to 24-month-old male mice decreased the level of apoptosis in the testis. However, the levels of 17β-estradiol and testosterone in blood increased compared with the control or solvent-administered mice. Conclusions: The use of tranexamic acid had an ameliorative effect on osteoporosis, possibly by protecting ovaries and testes.

CD44‐Targeted Monoclonal Antibody is Anti‐Inflammatory in an In‐Vivo Model of Monosodium Urate Crystal Induced Peritonitis

CD44‐Targeted Monoclonal Antibody is Anti‐Inflammatory in an <i>In‐Vivo</i> Model of Monosodium Urate Crystal Induced Peritonitis

PM2.5 Exposure in the Respiratory System Induces Distinct Inflammatory Signaling in the Lung and the Liver of Mice.

Fine particulate matter 2.5 (PM2.5) is a harmful air pollutant currently threatening public health. Although many studies have been performed on the general negative effects of PM2.5 in mice and humans, the migration patterns of various immune cells in response to PM2.5 exposure remain unclear. In this study, we aimed to investigate the immune cell migratory response in the lung and the liver of intratracheally PM2.5-inoculated mice. To investigate the migration trajectory of immune cells in the lung and the liver tissues of mice, we employed microscopic tools including two-photon intravital imaging, histological analysis, and transmission electron microscopy. Our data from two-photon intravital imaging showed that there was no significant difference in the number of infiltrated neutrophils in the lung and the liver of PM2.5-treated mice, compared to the nontreated condition. However, from the histological analysis and the transmission electron microscopy after vascular perfusion to remove intravascular leukocytes, we observed that some leukocytes were frequently observed in the lung and the liver of PM2.5-treated mice. Interestingly, quantification of leukocyte population using flow cytometry showed significant increase of neutrophils and macrophages in the lung, but not much in the liver, 24 h post-PM2.5 treatment. These data imply that two-photon intravital imaging of the lung and the liver actually visualized neutrophils, which were adherent to the luminal side of the vasculature. We then conducted mRNA microarray analysis to further observe how PM2.5 affects gene expression patterns in the lung and the liver. PM2.5 treatment changed the mRNA expression associated with the IL-17 signaling pathway in the lung and changed the mRNA expression associated with metabolic pathways in the liver. In summary, these results suggest that the immune response in the lung is distinctly regulated from that in the liver under acute PM2.5-induced inflammation and that these organs consequently are regulated via distinct signaling pathways.

Muscle regeneration is disrupted by cancer cachexia without loss of muscle stem cell potential.

Cancer cachexia is a severe, debilitating condition characterized by progressive body wasting associated with remarkable loss of skeletal muscle weight. It has been reported that cancer cachexia disturbs the regenerative ability of skeletal muscle, but the cellular mechanisms are still unknown. Here, we investigated the skeletal muscle regenerative process in mouse colon-26 (C26) tumor cell-bearing mice as a C26 cancer cachexia model. Although the proliferation and differentiation abilities of muscle stem cells derived from the C26 tumor cell-bearing mice were sustained in vitro, the proliferation and differentiation were severely impaired in the cachexic mice. The numbers of both macrophages and mesenchymal progenitors, which are critical players in muscle regeneration, were reduced in the cancer cachexic mice, indicating that the skeletal muscle regeneration process was disrupted by cancer cachexia. Furthermore, the number of infiltrated neutrophils was also reduced in cancer cachexia mice 24 hours after muscle injury, and the expression of critical chemokines for muscle regeneration was reduced in cancer cachexia model mice compared to control mice. Collectively, although the ability to regeneration of MuSCs was retained, cancer cachexia disturbed skeletal muscle regenerative ability by inhibiting the orchestrated muscle regeneration processes.

Caspase recruitment domain-containing protein 9 (CARD9) knockout reduces regional ischemia/reperfusion injury through an attenuated inflammatory response.

Ischemic heart disease remains a leading cause of morbidity and mortality in the United States. Interventional reperfusion induces further damage to the ischemic myocardium through neutrophil infiltration and acute inflammation. As caspase recruitment domain-containing protein 9 (CARD9) plays a critical role in innate immune response and inflammation, we hypothesized that CARD9 knockout would provide protection against ischemia and reperfusion (I/R) injury through attenuation of acute inflammatory responses. C57BL/6 wild-type (WT) and CARD9-/- mice were subjected to 45 min left anterior descending (LAD) coronary artery occlusion followed by 24-h reperfusion. Area at risk (AAR) and infarct size were measured by Evans blue and triphenyltetrazolium chloride (TTC) staining. Frozen heart sections were stained with anti-mouse GR-1 antibody to detect infiltrated neutrophils. Concentrations of cytokines/chemokines TNF-α, IL-6, CXCL-1 and MCP-1 were determined in heart tissue homogenate and serum by ELISA assay. Western immunoblotting analyses were performed to measure the phosphorylation of p38 MAPK. Our results indicate that following I/R, infarct size was significantly smaller in CARD9-/- mice compared to WT. The number of infiltrated neutrophils was significantly lower in CARD9-/- mice compared to WT. Levels of TNF-α, IL-6, CXCL-1 and MCP-1 were significantly reduced in heart tissue and serum from CARD9-/- mice compared to WT. CARD9-/- mice also exhibited significantly lower levels of phosphorylated p38 MAPK. Taken together, our results suggest that CARD9 knockout protects the heart from ischemia/reperfusion (I/R) injury, possibly through reduction of neutrophil infiltration and attenuation of CARD9-associated acute inflammatory signaling.

Naringenin ameliorates LPS-induced acute lung injury through its anti-oxidative and anti-inflammatory activity and by inhibition of the PI3K/AKT pathway.

The aim of the present study was to explore the effect of naringenin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a mouse model, as well as the underlying mechanism. The animals were randomly assigned to four groups: PBS-treated healthy control (Control), LPS-induced ALI (LPS), vehicle-treated ALI (LPS + Vehicle), and naringenin-treated ALI (LPS + Nar) group. Naringenin (100 mg/kg) was administered orally for 4 consecutive days, starting 3 days prior to induction of ALI. The survival rates of mice, lung wet/dry weight ratios, lung injury score, protein levels of bronchoalveolar lavage fluid (BALF), lactate dehydrogenase (LDH) activity in the BALF, lung myeloperoxidase (MPO) activity, the number of infiltrated neutrophils and reactive oxygen species (ROS) levels (H2O2 and malondialdehyde) were assessed. In addition, the serum and BALF levels of inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and macrophage inflammatory protein 2] were determined, along with the total and the phosphorylated protein levels of phosphatidylinositol 3-hydroxy kinase (PI3K) and AKT in lung tissues. The results showed that naringenin pre-treatment significantly increased the survival rate, improved histopathologic changes, alleviated pulmonary edema and lung vascular leak, downregulated the levels of ROS and reduced neutrophil infiltration as well as the levels of inflammatory cytokines in the serum and BALF. Moreover, naringenin pre-treatment reduced the total and the phosphorylated protein levels of PI3K and AKT. The present study suggested that naringenin pre-treatment ameliorated LPS-induced ALI through its anti-oxidative and anti-inflammatory activity and by inhibition of the PI3K/AKT pathway in mice.

An iminosugar-based heparanase inhibitor heparastatin (SF4) suppresses infiltration of neutrophils and monocytes into inflamed dorsal air pouches

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.

Abstract TMP52: Inactivated Microglia and Inhibition Neutrophil Infiltration Reduce Brain Injury Following Intracerebral Hemorrhage in Mice

Introduction: Microglia become activated and monocytes become macrophages following intracerebral hemorrhage (ICH). They are often called “microglia/macrophage” since it is difficult to differentiate between them. The neutrophils also infiltrate into the injured brain after ICH. Those cells are capable of releasing numerous detrimental mediators. Hypothesis: The activation of microglia/macrophage and the infiltrated neutrophils promote brain injury after ICH and that inhibition both of them attenuates brain injury following ICH. Methods: 10-μl of autologous blood obtained from tail was introduced into the right striatum of adult male mice to produce ICH injury. We used transgenic CD11b thymidine kinase (CD11b-TK) mice where proliferating microglia/macrophage were removed by ganciclovir treatment. Functional grade purified anti-mouse Ly6G/Gr-1 antibodies were used to reduce the neutrophil infiltration. Groups of CD11b-TK mice were treated with ganciclovir and/or anti-mouse Ly6G/Gr-1 antibodies. These mice were killed at 3 days after ICH injury and brain sections were stained for various parameters. Results: ICH injury resulted in activation of microglia/macrophages through 1-7 days of ICH insult. The area of brain damage peaked at 2-3 days; the number of dying neurons peaked at 1-3 days, the number of infiltrated neutrophils peaked at 1-3 days, while the activation of microglia/macrophages peaked at days 3-4. These findings led us to choose day 3 for further studies. The activation of microglia/macrophage was significantly reduced in CD11b-TK transgenic mice with ganciclovir treatment after ICH. The numbers of infiltrated neutrophils were significantly reduced with anti-mouse Ly6G/Gr-1 antibodies treatment after ICH. Correspondingly, the area of brain damage and the extent of neuronal death were minimal in CD11b-TK mice with ganciclovir and anti-LyG6/Gr-1 antibodies treatment after ICH. Conclusions: Both activated microglia/macrophages and the infiltrated neutrophils after ICH promote brain injury. Thus inhibition of their activities reduces ICH brain injury. These results shed light on the advent of new medications for ICH patients, including microglia deactivators and the antibodies to alleviate neutrophil infiltration.

TPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis.

We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9−/−) BMC (group 2), MMP-9−/− receiving MMP-9−/− BMC (group 3), and MMP-9−/− receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI.

Fetal exposure to maternal inflammation does not affect postnatal development of genetically-driven ileitis and colitis.

Background: Chronic inflammatory disorders have been increasing in incidence over the past decades following geographical patterns of industrialization. Fetal exposure to maternal inflammation may alter organ functions and the offspring's disease risk. We studied the development of genetically-driven ileitis and colitis in response to maternal inflammation using mouse models. Methods: Disease susceptible (Tnf ΔARE/+ and IL10−/−) and disease-free (Tnf +/+ and IL10−/+) offspring were raised in inflamed and non-inflamed dams. Ileal, caecal and colonic pathology was evaluated in the offspring at 8 or 12 weeks of age. Ly6G-positive cells in inflamed sections from the distal ileum and distal colon were analysed by immunofluorescence microscopy. Gene expression of pro-inflammatory cytokines was measured in whole tissue specimens by quantitative PCR. Microarray analyses were performed on laser microdissected intestinal epithelium. Caecal bacterial communities were assessed by Illumina sequencing of 16S rRNA amplicons. Results: Disease severity, the number of infiltrated neutrophils as well as Tnf and Il12p40 mRNA expression were independent of maternal inflammation in the offspring of mouse models for ileitis (Tnf ΔARE/+) and colitis (IL10−/−). Although TNF-driven maternal inflammation regulated 2,174 (wild type) and 3,345 (Tnf ΔARE/+) genes in the fetal epithelium, prenatal gene expression patterns were completely overwritten after birth. In addition, co-housing experiments revealed no change in phylogenetic diversity of the offspring's caecal microbiota in response to maternal inflammation. This is independent of the offspring's genotype before and after the onset of tissue pathology. Conclusions: Disease risk and activity in mouse models of chronic ileitis and colitis was independent of the fetal exposure to maternal inflammation. Likewise, maternal inflammation did not alter the diversity and composition of offspring's caecal microbiota, clearly demonstrating that changes of the gene expression program in the fetal gut epithelium were not relevant for the development of chronic inflammatory disorders in the gut.

Myeloperoxidase Exacerbates Secondary Injury by Generating Highly Reactive Oxygen Species and Mediating Neutrophil Recruitment in Experimental Spinal Cord Injury

An animal study using myeloperoxidase-knockout (MPO-KO) mice to examine the in vivo role of myeloperoxidase (MPO) in spinal cord injury (SCI). To clarify the influence of MPO on inflammatory cell infiltration, tissue damage, and functional recovery after SCI. MPO is considered to be important in spreading tissue damage after SCI because it generates strong neurotoxic oxidant hypochlorous acid (HOCl). However, the direct involvement of MPO in the pathophysiology of SCI remains to be elucidated. To compare the inflammatory reaction, tissue damage, and neurological recovery after SCI, a moderate contusion injury was created at the ninth thoracic level in MPO-KO mice and wild-type mice. A HOCl-specific probe solution was injected into the lesion epicenter to assess the spatiotemporal production of MPO-derived HOCl. Inflammatory reactions were quantified by flow cytometry and quantitative real-time polymerase chain reaction, and tissue damage was evaluated by an immunohistochemical analysis. The motor function recovery was assessed by the open-field locomotor score. Prominent production of HOCl was observed during the hyperacute phase of SCI at the lesion site in the wild-type mice; however, little expression was observed in the MPO-KO mice. In this phase, the number of infiltrated neutrophils was significantly reduced in the MPO-KO mice compared with the wild-type mice. In addition, significant differences were observed in the expression levels of proinflammatory cytokines and apoptosis-related genes between 2 groups. In the histological sections, fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells and more spared myelin were observed at the lesion site in MPO-KO mice. Consistent with these results, better functional recovery was observed in the MPO-KO mice than in the wild-type mice after SCI. These results clearly indicated that MPO exacerbated secondary injury and impaired the functional recovery not only by generating strong oxidant HOCl, but also by enhancing neutrophil infiltration after SCI.

Thromboxane receptor signalling in renal ischemia reperfusion injury

F2-isoprostanes are formed by oxidative modification of arachidonic acid and are the gold standard for detection of oxidative stress in vivo. F2-isoprostanes are biologically active compounds that signal through the thromboxane A2 (TP) receptor; infusion of F2-isoprostanes reduces glomerular filtration in the kidney by constricting afferent arterioles. This study investigated whether endogenous F2-isoprostanes contribute to the pathogenesis of ischemic acute kidney injury, which is associated with oxidative stress and reduced glomerular filtration. TP receptor knockout mice—that lack F2-isoprostanes and thromboxane A2 signalling—and wild-type control mice underwent 30 min of renal ischemia and 24 h of reperfusion. Kidney dysfunction, histological injury and the number of infiltrated neutrophils were similar between the two mouse strains, whereas TP receptor knockout mice had significantly more apoptotic cells and tissue lipid peroxidation than their wild-type counterparts. F2-isoprostanes and thromboxane B2 were readily detectable in urine collections after surgery. The findings indicate that F2-isoprostanes and thromboxane A2 signalling do not contribute critically to the pathogenesis of ischemic acute kidney injury and more generally provide evidence against a prominent role for F2-isoprostanes signalling in exacerbating acute disease states associated with oxidative stress.

PREVENTION OF HEMORRHAGIC SHOCK-INDUCED INTESTINAL TISSUE INJURY BY GLUTAMINE VIA HEME OXYGENASE-1 INDUCTION

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.

Hemin Treatment Abrogates Monocrotaline-Induced Pulmonary Hypertension

Treatment of rats with monocrotaline (MCT), a pyrrolizidine alkaloid plant toxin, is known to cause pulmonary hypertension (PH), and it has been used as a useful experimental model of PH. Recent findings suggested that pulmonary inflammation may play a significant role in the pathogenesis of MCT-induced PH. We also demonstrated that, following MCT administration to rats, there was a significant and sustained increase in the pulmonary expression of heme oxygenase-1 (HO-1), which is known to be induced by various oxidative stresses, including inflammation and free heme, and is thought to be essential in the protection against oxidative tissue injuries. In this study, we administered hemin (ferriprotoporphyrin chloride, 30 micromol/kg b.w., subcutaneously), a potent inducer of HO-1, every 3 days to rats following subcutaneous administration of MCT (60 mg/kg) and examined its effect on MCT-induced PH and pulmonary inflammation. MCT administration caused pulmonary arterial wall thickening with marked elevation of right ventricular pressure, in association with prominent pulmonary inflammation as revealed by the increase in gene expression of tumor necrosis factor-alpha and the number of infiltrated neutrophils in the lung. In contrast, hemin treatment of MCT-administered animals, which led to a further increase in pulmonary HO-1 mRNA expression, significantly ameliorated MCT-induced PH as well as tissue inflammation. These findings suggest that hemin treatment ameliorates MCT-induced PH possibly mediated through induction of pulmonary HO-1 which leads to the attenuation of pulmonary inflammation.

Epidermal Development and Wound Healing in Matrix Metalloproteinase 13-Deficient Mice

Degradation of the extracellular matrix, which is an indispensable step in tissue remodelling processes such as embryonic development and wound healing of the skin, has been attributed to collagenolytic activity of members of the matrix metalloproteinase family (MMPs). Here, we employed mmp13 knockout mice to elucidate the function of MMP13 in embryonic skin development, skin homeostasis, and cutaneous wound healing. Overall epidermal architecture and dermal composition of non-injured skin were indistinguishable from wild-type mice. Despite robust expression of MMP13 in the early phase of wound healing, wild-type and mmp13 knockout animals did not differ in their efficiency of re-epithelialization, inflammatory response, granulation tissue formation, angiogenesis, and restoration of basement membrane. Yet, among other MMPs also expressed during wound healing, MMP8 was found to be enhanced in wounds of MMP13-deficient mice. In summary, skin homeostasis and also tissue remodelling processes like embryonic skin development and cutaneous wound healing are independent of MMP13 either owing to MMP13 dispensability or owing to functional substitution by other collagenolytic proteinases such as MMP8.