Measure Of Neutrophil Infiltration Research Articles

Effects of Repeated LPS Infusion on Hematologic, Metabolic, and Intestinal Histology Parameters

Objectives were to characterize effects of repeated lipopolysaccharide infusion on circulating metabolites, white blood cells, and intestinal histology in pigs. Ten crossbred female pigs (168 ± 2 kg) were reproductively synchronized and jugular catheterized. The experimental period, during the follicular phase of the estrous cycle, lasted 5 d and pigs were assigned to either control (CON; 3 mL i.v. saline; n=4) or lipopolysaccharide (LPS; 0.1 μg/kg BW LPS i.v.; Escherichia coli 055:B5 Sigma, St. Louis, Missouri; n=6) with treatments administered 4x/d at 0000, 0600, 1200, and 1800h. Each animal was limit‐fed 2.7 kg of feed once daily at 0600 h. Blood samples for metabolites and a complete blood count were obtained daily at 1730h. Rectal temperatures were measured 2x/d at 0530 and 1730h and condensed into daily averages. Animals were sacrificed on d 6 beginning at 0600h and segments of jejunum, ileum, and colon were collected and analyzed for morphology and were stained for myeloperoxidase (a measure of neutrophil infiltration). Circulating platelets were decreased 31% (P<0.01) and total white blood cell count tended to be decreased 10% (P=0.07) in LPS pigs relative to CON pigs. Lipopolysaccharide binding protein was increased 37% in LPS pigs relative to CON pigs (P=0.05) and was increased 60% on d 1 of the experiment relative to CON pigs (P = 0.02). There was a treatment by day interaction for neutrophils, lymphocytes, monocytes, and eosinophils where on d 1 only, neutrophils were increased 58% in LPS pigs relative to CON pigs (P<0.01) and lymphocytes, monocytes, and eosinophils were decreased by 61, 93, and 81%, respectively in LPS pigs relative to CON pigs (P<0.01 for all). Similar treatment by day differences in blood metabolites were observed where on d 1, insulin, blood urea nitrogen, β‐hydroxybutyric acid, and ionized calcium increased 36, 14, 13, and 7%, respectively, in LPS pigs relative to CON pigs (P=0.03, P=0.07, P=0.02, and P<0.01, respectively). There was no treatment by day interaction for blood glucose; however, it tended to be increased 6% in LPS pigs on d 1 relative to CON pigs (84 vs 79 mg/dL; P=0.08). Rectal temperature did not differ between the two treatments throughout the entire study (38.5 vs 38.6°C; P=0.71). In the jejunum, a tendency for reduced goblet cell area was observed (2.2 vs 5.6%; P=0.06) as well as an increase in myeloperoxidase stained area (1.03 vs 0.87%; P<0.01) in LPS pigs relative to CON pigs. No other differences in histological measurements were observed. In conclusion, LPS only altered metabolism on d 1 of the 5 d injection period. Platelets were the only measurement consistently affected throughout the 5 d. Jejunum was the only intestinal segment affected by repeated LPS exposure as determined by a tendency for reduced goblet cell area and an increase in myeloperoxidase stained area.

Short-term effect of ascorbate on bacterial content, plasminogen activator inhibitor-1, and myeloperoxidase in septic mice

Sepsis, a potential risk associated with surgery, leads to a systemic inflammatory response including the plugging of capillary beds. This plugging may precipitate organ failure and subsequent death. We have shown that capillary plugging can be reversed rapidly within 1 h by intravenous injection of ascorbate in mouse skeletal muscle. It is unknown whether, in parallel with this effect, ascorbate negatively affects the protective responses to sepsis involving the fibrinolytic and immune systems. We hypothesized that treatment with ascorbate for 1 h does not alter bacterial content, plasminogen activator inhibitor 1 (PAI-1), and neutrophil infiltration in lung, kidney, spleen, and liver (organs with high immune response) of septic mice. Sepsis was induced by feces injection into the peritoneum. Mice were injected intravenously with ascorbate at 6 h (10 mg/kg), and samples of peritoneal fluid, arterial blood, and organs collected at 7 h were subjected to analyses of bacterial content, PAI-1 messenger RNA and enzymatic activity, and myeloperoxidase (MPO) (a measure of neutrophil infiltration). Sepsis increased bacterial content in all fluids and organs and increased PAI-1 messenger RNA and enzymatic activity in the lung and liver. Sepsis increased the myeloperoxidase level in the lung and liver, and lowered it in the spleen. Except for decreasing the bacterial content in blood, these responses to sepsis were not altered by ascorbate. The rapid effect of ascorbate against capillary plugging in the septic mouse skeletal muscle is not accompanied by alterations in PAI-1 or myeloperoxidase responses in the organs with high immune response.

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.

Abstract 4202: Sex disparities in the cutaneous response to acute ultraviolet light exposure

Abstract Epidemiological evidence has identified a 3-fold increase in squamous cell skin cancer incidence in men compared to women. The historical explanation for this sex-bias is increased occupational exposure to ultraviolet (UV) light. Our studies demonstrated, however, that when chronically exposed to equivalent doses of UV light, male mice developed more skin tumors that were larger and more aggressive than those arising in female mice. Additionally, the skin of male mice acutely exposed to UV light had higher levels of oxidative DNA damage and decreased total antioxidant capacity compared to females (Thomas-Ahner, JM Cancer Research 2007). The hypothesis for the current study is that the topical application of anti-inflammatory and/or antioxidant compounds may differentially affect the acute UVB induced inflammatory response in the sexes. To test this hypothesis, female and male Skh-1 mice were exposed to 2240J/m2 UVB light followed immediately by topical application of either an anti-inflammatory compound (Solaraze), an antioxidant (Vitamin E), or a combination of the 2 compounds. Animals were sacrificed at 24 and 48 hrs following a single exposure and topical treatment. Skin was analyzed for myeloperoxidase (MPO) activity as a measure of neutrophil infiltration, catalase activity, and immunohistochemically for the presence of p53 protein in the epidermis. In response to UVB exposure, vehicle treated female mice had significantly increased MPO activity compared to male mice. Topical treatments with Solaraze, Vitamin E or the combination significantly reduced MPO levels in female mice but had no effects on the already low levels of UV-induced MPO in male skin. Catalase is a key enzyme used for detoxifying hydrogen peroxide in the skin. Baseline levels of catalase were lower in male skin compared to female skin and UVB exposure significantly reduced catalase in both sexes. Topical treatment with Solaraze significantly increased male catalase activity to levels similar to female unirradiated skin while vitamin E partially restored catalase activity in female mice but had no effect in male skin. The single exposure to UVB induced higher numbers of p53 positive epidermal cells in male skin, however at this time point topical treatment had no effect on UVB-induced p53 levels in either sex. Our data suggests that UVB differentially effects inflammation in the sexes and that topical application of anti-inflammatory or anti-oxidant compounds have differential effects on markers of an acute inflammatory response in the skin. Future studies will evaluate the effects of additional compounds and delivery vehicles following both acute and chronic UVB exposure. This work was supported by NIH NCI CA133629. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4202.

Role of nitric oxide in lung injury associated with experimental acute pancreatitis.

This study evaluated the effect of varying the synthesis of nitric oxide with sodium nitroprusside or N-nitro-L-arginine methyl ester (L-NAME) in a pancreatitis-lung injury model. Rats (n = 45) were randomized to control or caerulein-induced pancreatitis groups, treated with saline, sodium nitroprusside (0.4 micrograms/kg) or L-NAME (10 mg/kg). Myeloperoxidase activity was used as a measure of neutrophil infiltration. Wet to dry (W:D) lung weight and bronchoalveolar lavage (BAL) protein concentrations were used to assess vascular leakage. Pancreatitis was shown to induce pulmonary neutrophil influx: mean(s.e.m.) myeloperoxidase activity 6.79(0.5) units/g in caerulein-treated animals versus 2.08(0.5) units/g in controls (P < 0.001). Animals with pancreatitis showed increased microvascular leakage compared with controls (mean(s.e.m.) W:D lung weight 7.01(0.5) versus 2.85(0.2), P < 0.001; BAL protein concentration 2539(222) versus 347(32) micrograms/ml, P < 0.001). Compared with the saline-treated pancreatitis group, these changes were reduced by sodium nitroprusside (mean(s.e.m.) myeloperoxidase activity to 2.5(0.4) units/g, P < 0.001; W:D lung weight to 3.8(0.37), P < 0.001; BAL protein concentration 1389(182) micrograms/ml, P < 0.05). L-NAME exacerbated the pancreatitis-induced pulmonary oedema (W:D lung weight increased to 11.96(0.6), P < 0.001), protein leakage (BAL protein concentration rose to 3707(309) micrograms/ml, P < 0.05) and neutrophil infiltration (myeloperoxidase activity increased to 9.01(0.3) units/g, P < 0.05). These data suggest that, in vivo, nitric oxide inhibits pancreatitis-induced lung injury, possibly in part by inhibiting pulmonary neutrophil influx.

Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice.

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.

Activated protein C attenuates skeletal muscle ischaemia reperfusion injury.

<h2>Abstract</h2> <b>Aims:</b> Pharmacological modulation of skeletal muscle reperfusion injury after an ischaemic insult may improve limb salvage rates and prevent the associated systemic sequelae. Activated Protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties that has been extensively studied in the setting of sepsis. The purpose of our study was to evaluate the effects of APC on skeletal muscle ischaemia reperfusion injury. <b>Methods:</b> Adult male Sprague Dawley rats (n = 24) were randomised into three groups: control group, I/R group treated with normal saline and I/R group treated with APC. Bilateral hind-limb ischaemia was induced by rubber band application proximal to the level of the greater trochanters for two hours. Treatment groups received either normal saline or APC prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Skeletal muscle injury was assessed based on myeloperoxidase content as a measure of neutrophil infiltration and wet to dry ratio as a measure of oedema formation. Histological analysis was also performed on the muscle. <b>Results:</b> APC significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle (Table). TABLE—ABSTRACT 28Skel muscle wet:dry ratioSkel muscle MPO (U/g)Max twitch contraction (N/g)Tetanic contraction at 50 HZ (N/g)Control group3.17 ± 0.100.74 ± 0.032.45 ± 0.264.21 ± 0.26I/R saline3.40 ± 0.140.95 ± 0.131.52 ± 0.202.88 ± 0.53APC3.21 ± 0.110.82 ± 0.071.86 ± 0.183.52 ± 0.36P valueP < 0.03P < 0.02P < 0.01P < 0.01 These findings were supported by our histological findings. Statistical significance was determined using variance analysis. <b>Conclusion:</b> Activated Protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury.

Lung retrieval from non–heart beating cadavers with the use of a rat lung transplant model

Lungs retrieved from cadavers after death and circulatory arrest may alleviate the critical shortage of lungs for transplant. We report a rat lung transplantation model that allows serial measurement of arterial blood gases after left single lung transplantation from non-heart beating donors. Twelve Sprague-Dawley rats underwent left lung transplantation with a vascular cuff technique. Donor rats were anesthetized with intraperitoneal injection of pentobarbital, heparinized, intubated via tracheotomy, and then killed with pentobarbital. Lungs were retrieved immediately or after 2 hours of oxygen ventilation after death (tidal volume 1 mL/100 g, rate 40/min FIO2 = 1.0, positive end-expiratory pressure 5 cm H2O). Recipient rats were anesthetized, intubated, and ventilated. The carotid artery and jugular vein were cannulated for arterial blood gases and infusion of Ringer's lactate (4 mL/h). Anesthesia was maintained with halothane 0.2%, and recipient arterial blood gases were measured at 4 and 6 hours after lung transplantation after snaring the right pulmonary artery for 5 minutes. Animals were put to death 6 hours after lung transplantation, and portions of transplanted lungs were frozen in liquid nitrogen and assayed for wet/dry ratio, myeloperoxidase as a measure of neutrophil infiltration, and conjugated dienes as a measure of free radical-mediated lipid peroxidation. Arterial PO2 and wet/dry ratio were not significantly different in recipients of non-heart beating donor lungs retrieved immediately after death or after 2 hours of oxygen ventilation. Significant neutrophil infiltration was observed in recipients of non-heart beating donor lungs retrieved 2 hours after death from oxygen-ventilated donors. Strategies to ameliorate reperfusion injury may allow for successful lung transplantation from non-heart beating donors.

Effect of dietary vitamin E on spontaneous or nitric oxide donor-induced mutations in a mouse tumor model.

Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence in humans, but no firm conclusions about a protective effect can be drawn from these studies. Recently, we reported a statistically significant correlation in the Mutatect mouse tumor model between the number of neutrophils and the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus. We have now used this model to investigate vitamin E's effect on the hprt mutation rate. Mutatect cells were grown in mice as subcutaneous tumors for 2-3 weeks, the tumor cells were recovered, and 6-thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration. Vitamin E (2 IU/kg body weight) was provided in the diet for 3-4 weeks. In some experiments, glyceryl trinitrate (100 mg/kg body weight) was also administered as a source of nitric oxide. All statistical tests were two-sided. Mouse tumors from the Mutatect MN-11 cell line exhibited a 3.2-fold higher median mutation frequency than the same cells in culture (P:<. 0001); vitamin E reduced this frequency by 24.9% (P: =.01). Mutatect TM-28-derived tumors (which secrete interleukin 8) were heavily infiltrated with neutrophils and had a correspondingly high mutation frequency; in two separate experiments, vitamin E reduced the median mutation frequency by 68.9% (P: =.0019) and 84.1% (P: =.011) and myeloperoxidase levels by 75.3% (P: =.0002) and 75.5% (P: =.026), respectively. Glyceryl trinitrate increased the mutation frequency in MN-11 tumors, and vitamin E reduced the median frequency by 61.4% (P: =.058). Dietary vitamin E afforded strong protection against both spontaneously arising and nitric oxide-induced mutations. Two separate protective mechanisms by vitamin E may be operating: scavenging of a nitric oxide-related genotoxic species and altering the infiltration of neutrophils into tumors.

Release of Tumor Necrosis Factor after Pulmonary Artery Occlusion and Reperfusion

Cytokines may function as mediators of reperfusion tissue injury in lungs. Because the lung contains resident macrophages that can serve as potential sources of cytokines, we examined the possibility that pulmonary artery occlusion by reperfusion is a factor in mediating the release of cytokines. After left lung ischemia induced by a 24-h period of left pulmonary artery occlusion, we observed a transient increase in TNF-alpha concentration in lung effluent in rabbits during the period reperfusion. The peak TNF-alpha levels ranged from 55 to 335 pg/ml, and a mean peak time was at 45 to 60 min after the initiation of reperfusion. The TNF-alpha concentrations then decreased towards baseline. TNF-alpha was detected in control plasma or in plasma from sham-operated animals. Less than 10 pg/ml of endotoxin was detected in any samples. Lung tissue myeloperoxidase content, a measure of neutrophil infiltration, increased progressively during the 2-h reperfusion period. The time course of generation of TNF-alpha preceded the maximal rise in lung tissue myeloperoxidase activity. The data show that lung ischemia/reperfusion results in transient generation of TNF-alpha, which is known to mediate neutrophil sequestration. Neutrophil sequestration and resulting lung injury after reperfusion may be dependent on generation of TNF-alpha at the onset of reperfusion.