Antineutrophil Serum Research Articles

The role of neutrophils in pain: systematic review and meta-analysis of animal studies.

The peripheral inflammatory response is an attractive therapeutic target for pain treatment. Neutrophils are the first circulating inflammatory cells recruited to sites of injury, but their contribution to pain outcomes is unclear. We performed a systematic review and meta-analysis of original preclinical studies, which evaluated the effect of preemptive neutrophil depletion on pain outcomes (PROSPERO registration number: CRD42022364004). Literature search (PubMed, January 19, 2023) identified 49 articles, which were meta-analyzed using a random-effects model. The risk of bias was evaluated using SYRCLE's tool. The pooled effect considering all studies showed that neutrophil depletion induced a consistent pain reduction. Inflammatory, joint, neuropathic, and visceral pain showed significant pain alleviation by neutrophil depletion with medium-large effect sizes. However, muscle and postoperative pain were not significantly alleviated by neutrophil depletion. Further analysis showed a differential contribution of neutrophils to pain outcomes. Neutrophils had a higher impact on mechanical hyperalgesia, followed by nociceptive behaviors and mechanical allodynia, with a smaller contribution to thermal hyperalgesia. Interspecies (mice or rats) differences were not appreciated. Analyses regarding intervention unveiled a lower pain reduction for some commonly used methods for neutrophil depletion, such as injection of antineutrophil serum or an anti-Gr-1 antibody, than for other agents such as administration of an anti-Ly6G antibody, fucoidan, vinblastine, CXCR1/2 inhibitors, and etanercept. In conclusion, the contribution of neutrophils to pain depends on pain etiology (experimental model), pain outcome, and the neutrophil depletion strategy. Further research is needed to improve our understanding on the mechanisms of these differences.

Early neutrophil infiltration is critical for inflammation-sensitized hypoxic-ischemic brain injury in newborns

Neutrophils are the most abundant leukocytes and usually the first immune cell-type recruited to a site of infection or tissue damage. In asphyxiated neonates, elevated peripheral neutrophil counts are associated with poorer neurological outcomes. Induced neutropenia provides brain protection in animal models of neonatal hypoxic-ischemic (HI) injury, but the anti-neutrophil serum used in past studies heavily cross-reacts with monocytes, thus complicating the interpretation of results. Here we examined neutrophil influx and extravasation, and used a specific anti-Ly6G antibody for induced neutropenia against lipopolysaccharide (LPS)-pretreated HI injury in murine neonates, a model for inflammation-sensitized hypoxic-ischemic encephalopathy (HIE). As early as 6 h after the LPS/HI insult, the mRNAs for neutrophil-recruiting and mitogenic chemokines ascended in the ipsilateral hemisphere, coinciding with immuno-detection of neutrophils. However, neutrophils mainly resided within blood vessels, exhibiting signs for neutrophil extracellular traps (NETs), before 48 h post-LPS/HI. Prophylactic anti-Ly6G treatment blocked the brain infiltration of neutrophils, but not monocytes or lymphocytes, and markedly decreased LPS/HI-induced pro-inflammatory cytokines, matrix metalloproteinase 9 (MMP-9), and brain tissue loss. In contrast, anti-Ly6G treatment at 4 h post-LPS/HI failed to prevent the influx of neutrophils and brain damage. Together, these results suggest important pathological functions for early-arriving neutrophils in inflammation-sensitized HIE.

NONPRODUCTIVE COUGH AS PREAMBLE OF RESPIRATORY FAILURE IN A PATIENT WITH HYPEREOSINOPHILIC SYNDROME

SESSION TITLE: Tuesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM INTRODUCTION: Hypereosinophilic syndrome (HES) is a rare disorder characterized by peripheral blood eosinophilia of 〖1.5 x 10〗ˆ3/μL or higher and evidence of end organ manifestations attributable to the eosinophilia and not otherwise explained in the clinical setting. HES has an unknown prevalence, most often affects people age 20 through 50. Few patients with prolonged eosinophilia develop organ dysfunction that characterizes HES. CASE PRESENTATION: This is the case of a healthy 52-year-old man without history of significant systemic illness who presents with non-productive cough, daily febrile episodes and left upper extremity pain for a period 2 months. On physical examination, lungs are clear upon chest auscultation and left sided axillary pain is elicited upon arm abduction. Initial blood work revealed WBC 〖40.62 x 10〗ˆ3/μL; eosinophil count 〖23.56 x 10〗ˆ3/μL, elevated sedimentation rate 112 mm/hr. Initial diagnosing testing for HES was performed. Complete blood count was remarkable for increasing trend in leukocytes and eosinophils. Routine chemistries, including liver function tests were within normal limits. Quantitative serum immunoglobulin levels, including IgE were normal. Serum troponin, echocardiogram, electrocardiogram, Pulmonary function tests revealed no abnormalities. Bone marrow biopsy including cytogenetics had no myeloproliferative abnormalities. Flexible bronchoscopy with bronchoalveolar lavage revealed eosinophils count less than 3%, no evidence of bacteria, viral or fungal infection. Serology examination for diagnosis of parasitic infection, HIV testing, antineutrophil antibodies ANCA, Serum tryptase and B12 levels, PET scan, lymph node biopsy, EBV viral load were completely negative. The patient’s eosinophilia continued to rise, peaking at 56.00 x 103/μL, despite high-dose prednisone therapy (200 mg by mouth daily), he went in to respiratory failure, cardiac arrest and developed multi organ dysfunction. Hydroxyurea (1 g daily) was added. He improved after the addition of hydroxyurea, and a decrease trending of his eosinophil count was seen. Hydroxyurea was discontinued, and prednisone was tapered. DISCUSSION: HES is a rare heterogeneous disease, any organ system is vulnerable to get affected. The symptoms are common in many other medical problems, making an initial diagnosis more difficult. Sometimes no underlying cause can be found for an observed eosinophilia, even after an extensive diagnostic evaluation. In the absence of an identifiable reactive cause for the eosinophilia, a diagnosis of idiopathic HES can be established if there is evidence of organ involvement and dysfunction. CONCLUSIONS: Our case illustrates the importance of considering diagnosis in patients with significant eosinophilia. Have a high index of suspicion in the background of persistent peripheral eosinophilia; after exclusion of secondary causes. Reference #1: https://doi.org/10.1182/asheducation-2018.1.326 ASH Education Book November 30, 2018 vol. 2018 no. 1 326-331 Reference #2: Klion AD. How I treat hypereosinophilic syndromes. Blood. 2015;126(9):1069-1077. Reference #3: Khoury P, Abiodun AO, Holland-Thomas N, Fay MP, Klion AD. Hypereosinophilic syndrome subtype predicts responsiveness to glucocorticoids. J Allergy Clin Immunol Pract. 2018;6(1):190-195. DISCLOSURES: No relevant relationships by Hector Cintron-Colon, source=Web Response No relevant relationships by Ricardo Fernandez, source=Web Response No relevant relationships by Vanessa Fonseca Ferrer, source=Web Response No relevant relationships by WIlliam Marrero, source=Web Response No relevant relationships by Luis Piñeiro, source=Web Response

Serologic and fecal markers to predict response to induction therapy in dogs with idiopathic inflammatory bowel disease.

BackgroundLittle information is available of markers that assess the disease course in dogs with idiopathic inflammatory bowel disease (IBD).ObjectivesEvaluate relationship between disease severity and serum and fecal biomarkers in dogs with idiopathic IBD before and after treatment.AnimalsSixteen dogs with idioptahic IBD and 13 healthy dogs.MethodsProspective case control study. Canine IBD activity index (CIBDAI) clinical score, serum concentrations of C‐reactive protein (CRP), perinuclear antineutrophil cytoplasmic antibodies (pANCA), and serum and fecal canine calprotectin (cCP) were measured before and after 21 days of treatment.ResultsSerum CRP (median 3.5 mg/L; range: 0.1‐52.4 mg/L), fecal cCP (median 92.3 μg/g; range: 0.03‐637.5 μg/g), and CIBDAI scores significantly increased in dogs with IBD before treatment compared with serum CRP (median 0.2 mg/L; range: 0.1‐11.8 mg/L; P < .001), fecal cCP (median 0.67 μg/g; range: 0.03‐27.9 μg/g; P < .001) and CIBDAI (P < .001) after treatment. No significant associations between CIBDAI scores and before or after treatment serum biomarkers. There was a significant association between fecal cCP and CIBDAI scores before treatment (rho = 0.60, P = .01). CRP and fecal cCP significantly decreased after treatment (median 3.5 mg/L v. 0.2 mg/L; P < .001 and 92.3 μg/g v. 0.67 μg/g; P = .001, respectively).Conclusions and Clinical ImportanceOur data indicate that measurement of fecal cCP concentration is a useful biomarker for noninvasive evaluation of intestinal inflammation. Dogs with severe signs of GI disease more often have abnormal markers than dogs having less severe disease.

Mechanisms Underlying Combined Effects of Hypertension (HTN) and Hypercholesterolemia (HCh) on Microvascular Function

HTN and HCh are known to initiate inflammatory and thrombogenic responses in the microvasculature. However, little is known about whether, and how, combinations of risk factors (e.g., HTN+HCh) alter microvascular function. In this study we examined how the combined actions of HTN and HCh on the microvasculature differ from the responses to either risk factor alone. Intravital video microscopy was used to monitor the adhesion and emigration of leukocytes and the oxidation of dihydrorhodamine (a measure of oxidative stress) in cremaster muscle post‐capillary venules of wild type (WT) mice that were either infused with angiotensin II (AngII) for 2 wks (HTN), placed on a high cholesterol diet (HCh), or both (HTN+HCh). Either HTN or HCh alone promoted the recruitment of leukocytes and enhanced the production of reactive oxygen species (ROS) in venules. However, the combination of HTN and HCh produced changes in leukocyte recruitment and ROS production that were greatly attenuated compared to HTN alone. The changes in leukocyte adhesion and ROS production were not observed in HTN+HCh mice rendered neutropenic with anti‐neutrophil serum. The inhibitory effect of HCh on the AngII mediated responses was not mimicked by administration of an apoA‐I mimetic (4F). These findings indicate that HCh counteracts the proinflammatory effects of AngII via a mechanism that involves diminished ROS production. (Supported by HL26441).

Am80, a retinoic acid receptor agonist, ameliorates murine vasculitis through the suppression of neutrophil migration and activation

Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS). Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody-positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay. Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide. Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.

Role of flavin-containing-monooxygenase-dependent neutrophil activation in thioacetamide-induced hepatic inflammation in rats

Thioacetamide is widely used in industry and is known to be one of the most potent hepatotoxicants in experimental animals. We investigated the involvement of flavin-containing monooxygenase (FMO)-dependent hepatic-neutrophil activation and the release of proinflammatory mediators in thioacetamide-induced hepatic injury in rats. Thioacetamide (100mg/kg, intraperitoneally) increased, within 12h, hepatic serum aspartate transferase and alanine transferase levels, tumor necrosis factor-α production, interleukin-1β and nitrite levels, and myeloperoxidase activity. Rabbit anti-neutrophil serum markedly inhibited all thioacetamide-altered parameters. In addition, FMO-competitive inhibitor methimazole reduced thioacetamide-induced myeloperoxidase activity, hepatic tumor necrosis factor-α, interleukin-1β, nitrite, inducible nitric oxide synthase, and hepatic damage in thioacetamide-treated rats. Thus, we conclude that FMO-dependent hepatic neutrophil activation initiates the release of proinflammatory mediators in thioacetamide-treated rats.

Hydrogen sulfide preconditioning or neutrophil depletion attenuates ischemia-reperfusion-induced mitochondrial dysfunction in rat small intestine

The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.

Roles of Inflammation and the Activated Protein C Pathway in the Brain Edema Associated With Cerebral Venous Sinus Thrombosis

Increased blood-brain barrier (BBB) permeability, brain edema, and hemorrhage are important consequences of cerebral venous sinus thrombosis (CVST). The objective of this study was to define the role of the protein C pathway in the BBB permeability and edema elicited by experimental CVST. The role of neutrophil recruitment was also evaluated. Edema, BBB permeability, leukocyte-endothelial cell adhesion (LECA) and inflammatory cytokine levels were monitored in a murine model of CVST. The role of activated protein C (APC) was assessed in wild type mice (WT) receiving APC neutralizing antibody and in endothelial protein C receptor overexpressing mice (EPCR-tg). Neutrophil involvement was evaluated using an anti-CD18 antibody (Ab) and antineutrophil serum. Brain edema and increases in BBB permeability and LECA were noted 48 hours after CVST. APC immunoblockade exacerbated these responses, while EPCR-tg exhibited blunted responses, as did WT treated with either antineutrophil serum or the CD18 Ab. The protein C pathway protects the brain against the deleterious microvascular responses to CVST, a response that appears to be linked to the recruitment of inflammatory cells.

Hepatocyte Transplantation-Induced Liver Inflammation Is Driven by Cytokines-Chemokines Associated With Neutrophils and Kupffer Cells

Hepatocyte transplantation-induced liver inflammation impairs cell engraftment. We defined whether proinflammatory cytokines and chemokines played roles in regulation of hepatocyte engraftment in the liver. We performed studies over up to 3 weeks in rat hepatocyte transplantation systems. Expression of 84 cytokine-chemokine genes was studied by quantitative real-time polymerase chain reactions. Expression of selected up-regulated genes was verified by immunohistochemistry. Hepatic recruitment of neutrophils was demonstrated by myeloperoxidase activity assays, and Kupffer cell activation was established by carbon phagocytosis assays. The role of neutrophils and Kupffer cells in regulating expression of cytokine-chemokine genes as well as cell engraftment was determined by cell depletion studies. Within 6 hours after syngeneic cell transplantation, expression of 25 cytokine-chemokine genes increased by 2- to 123-fold, P < .05. These genes were largely associated with activated neutrophils and macrophages, including chemokine ligands, CXCL1, CXCL2, CCL3, CCL4; chemokine receptors, CXCR1 or CXCR2, CCR1, CCR2; and regulatory cytokines tumor necrosis factor alpha and interleukin-6. Inflammatory cells in the liver immunostained for CCR1, CCR2, CXCR1, and CXCR2, which indicated that up-regulated messenger RNA was appropriately translated. When neutrophils and Kupffer cells were depleted with neutrophil antiserum and gadolinium chloride, respectively, before transplanting cells, cell transplantation-induced cytokine-chemokine responses were attenuated. Virtually all abnormalities subsided in animals treated with neutrophil antiserum plus gadolinium chloride. Moreover, depletion of neutrophils or Kupffer cells improved engraftment of transplanted cells. Cell transplantation-induced liver inflammation involves proinflammatory cytokine-chemokine systems capable of modulation by neutrophils and Kupffer cells. This offers new directions for optimizing cell therapy strategies.

Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis.

Studies have implicated tumour necrosis factor-alpha (TNF-alpha) in type-II collagen (CII)-induced arthritis (CIA), a well established animal model of human rheumatoid arthritis. Precisely how TNF is involved in CIA is not yet clear. In this study the effects of TNF on CIA were examined, independent of its potential effects on the immune response, by performing peri-articular injection of TNF in combination with passive immunization of rats. A sub-arthritic dose (5 mg) of affinity-purified anti-CII IgG, which alone was insufficient to induce spontaneous clinical arthritis, was used throughout the study. Obvious clinical arthritis that persisted for several days was rapidly induced by injections of 100 ng TNF into hindpaws of rats that were passively immunized shortly before the TNF injection. Injections of TNF in non-immunized control rats did not induce clinical arthritis, nor did buffer-only injections in passively immunized controls. The clinical arthritic response was a local phenomenon, limited only to the TNF-injected hindpaws. No swelling was observed in the opposite, buffer-injected hindpaws, indicating the effects of TNF were not systemic. Depletion of peripheral blood phagocytes with anti-rat neutrophil antiserum before passive immunization completely abolished the ability of TNF to induce clinical arthritis, identifying phagocytic cells as the essential target cells in evoking this arthritic response. A role for complement activation was also demonstrated in this model through the use of a soluble recombinant version of CD35, the cell surface complement receptor type-1 (sCR1, BRL55730), which significantly reduced TNF-induced arthritis in phagocyte-replete rats.

Mechanisms of platelet and leukocyte recruitment in experimental colitis

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.

Acute Pancreatitis: Bench to the Bedside

Acute Pancreatitis: Bench to the Bedside

The Post-Surgical Inflammatory Response Provokes Enhanced Tumour Recurrence: A Crucial Role for Neutrophils

Background/Aims: Peritoneal trauma activates a cascade of peritoneal defence mechanisms responsible for postoperative intra-abdominal tumour recurrence. After peritoneal trauma, inflammatory cells and soluble factors are present in the abdominal cavity and can be captured in lavage fluids. The present study evaluated which component enhances intra-abdominal tumour recurrence. Furthermore, we evaluated which inflammatory cells are present and studied the influence of anti-neutrophil serum (ANS) on peritoneal tumour recurrence. Methods: In a peritoneal trauma model in rats, postoperative lavage fluids were collected and separated into cellular and supernatant components. Both components were injected in naïve rats together with CC531s colon carcinoma cells. In a second experiment, rats were treated with one or three doses of ANS. Results: Intraperitoneal injection of naïve recipients with inflammatory cells or supernatant resulted in significant tumour recurrence. Severe peritoneal trauma provoked significant intra-abdominal neutrophil influx which could be prevented by ANS. Treatment with one dose did not affect blood cell counts and significantly reduced tumour recurrence. Treatment with three doses of ANS decreased blood lymphocytes, monocytes, and neutrophils and induced tumour load. Conclusions: Neutrophils play a crucial role in postoperative adhesion and growth of spilled tumour cells after surgical peritoneal trauma. Prevention of peritoneal neutrophil influx reduces local tumour recurrence.

Molecular characterization of post-thrombotic syndrome

The post-thrombotic syndrome represents a poorly understood and significant vascular health problem. This review focuses on our current understanding of the pathogenesis of post-thrombotic syndrome. We emphasize the cellular and molecular mechanisms that are responsible for the critical components of post-thrombotic syndrome. These include the initiation of deep venous thrombosis, the pathogenesis of elevated venous pressure, and the factors responsible for nonhealing of venous stasis ulcers.

Neutrophil ablation with anti-serum does not protect against phase 2 ventricular arrhythmias in anaesthetised rats with myocardial infarction

Arrhythmias, including ventricular fibrillation (VF), occur in two phases after coronary obstruction, the first during the reversible stage of acute myocardial ischaemia (phase 1) and the second during evolution of the infarct (phase 2). We tested the hypothesis that phase 2 arrhythmias are mediated by actions of neutrophils accumulating within the infarct. Male rats (n=10 per group) were randomized to receive 2 ml/kg i.p. of either rabbit anti-rat neutrophil anti-serum or normal rabbit serum. After 17 h, single stage left coronary artery ligation was performed under pentobarbitone anaesthesia, and ischaemia was maintained for 240 min. Anti-serum pretreatment caused almost total neutropenia, reducing neutrophils in circulating blood from 2096+/-274x10(3) to 8+/-8x10(3) per ml (p<0.05). It also blocked neutrophil accumulation in the infarct, reducing cardiac myeloperoxidase activity from 74.7+/-27.4 to 9+/-3 mU per mg protein (p<0.05). Despite this, there was no significant difference between control and anti-serum-treated rats in the incidence of phase 2 VF (30% in each group) tachycardia (VT; 60% vs 80%) or number of ventricular premature beats (VPBs). Neutrophil accumulation within the evolving myocardial infarct does not mediate phase 2 VF.

Depletion of neutrophils reduces neuronal degeneration and inflammatory responses induced by quinolinic acid in vivo

The use of anti-neutrophil serum (anti-PMN) to induce neutropenia has been assessed for neuroprotection, modulation of microgliosis and astrogliosis, effects on oxidative stress, and intactness of blood-brain barrier (BBB) following injection of the excitotoxin quinolinic acid (QUIN) into rat striatum. At 1 day following QUIN injection, considerable striatal neurodegeneration was measured (Fluoro-Jade B marker). At this time, marked microgliosis (OX-42 marker) and astrogliosis (GFAP marker) were evident in QUIN-injected striatum. Treatment of QUIN-injected animals with anti-PMN protected neurons (48% reduction of striatal neuron loss) and inhibited microgliosis (61% reduction) and astrogliosis (43% reduction) compared with QUIN injection alone. Anti-PMN treatment was effective in decreasing levels of superoxide anions (by 42%) compared with QUIN alone; in addition, expressions of the neutrophil enzyme myeloperoxidase and the neutrophil oxidant 3-chlorotyrosine were markedly reduced (by 79 and 72%, respectively) with neutrophil depletion. QUIN-induced leakiness in BBB was indicated by elevated striatal levels of the blood protein fibrinogen, a result confirmed using Evans blue dye; anti-PMN was effective in reducing BBB permeability. Measurements from QUIN-injected animals directly confirmed anti-PMN efficacy in diminishing numbers of circulating neutrophils. Longer term neuroprotection and reduced microgliosis were also observed at 7 days post-injection of anti-PMN; at this time, anti-PMN-treated rats also demonstrated an improved apomorphine-induced rotational performance. We conclude that anti-PMN treatment could serve as a novel strategy to prevent leakiness to BBB, reduce gliosis, and protect striatal neurons in excitotoxin-injected brain.

The Role of Neutrophils and Oxygen Free Radicals in Post-Operative Adhesions

Postoperative intra-abdominal adhesion formation remains a major surgical problem. Surgery induces an inflammatory reaction, which is responsible for adhesion formation. Neutrophils and their oxygen-free radicals are key mediators in the early post-operative inflammatory response. The present study evaluates the effect of either blocking the influx of neutrophils or its products by scavenging oxygen-free radicals on adhesion formation. Reproducible rat models were used to induce post-surgical intra-abdominal adhesions. In the first experiment anti-neutrophil serum (ANS) was used to prevent neutrophils from entering the peritoneal cavity after surgery. In a second experiment superoxide dismutase (SOD), catalase, and mannitol were tested, to scavenge the superoxide, hydrogen peroxide, and hydroxyl radicals, respectively. In positive control groups 69 to 76% of the area of interest contained adhesions. In all experimental groups, except for mannitol, a significant reduction in post-surgical adhesion formation could be achieved. ANS reduced adhesion formation by 38% (P < 0.001) and SOD/catalase by 42% (P < 0.01). Mannitol could not reduce adhesion formation. Intra-abdominal influx of neutrophils after surgical peritoneal trauma plays an important role in post-operative adhesion formation. Preventing the intra-abdominal influx of neutrophils in the early post-operative inflammatory reaction can reduce adhesion formation, but an even more selective approach, by scavenging its products, proved as efficient.

Neutrophil infiltration is implicated in the sustained thermal hyperalgesic response evoked by allergen provocation in actively sensitized rats

It has been proposed that allergen provocation induces hyperalgesia but the involvement of immunoglobulin E and leukocytes remains poorly understood. Here, we have compared the profile of allergen-evoked thermal hyperalgesic response in both passively and actively sensitized rats, and investigated the role of leukocytes in allergen-evoked nociception. Wistar rats were passively sensitized with an intraplantar injection of immunoglobulin E anti-dinitrophenylated bovine serum albumin monoclonal antibody (0.5 μg/paw), and challenged with dinitrophenylated bovine serum albumin (0.5 μg/paw) 24 h later. Alternatively, the animals were actively sensitized with a mixture of Al(OH) 3 and ovalbumin and challenged intraplantarly with ovalbumin (12 μg/paw) 14 days later. We found that the thermal hyperalgesic responses set in very rapidly and with comparable intensity in both passively and actively sensitized rats. However, while in the former group the response was shorter, peaking within 1 h and reducing thereafter, a marked plateau was observed from 1 to 6 h post-challenge in the latter group. Actively sensitized rats also had higher neutrophil influx in the plantar tissue, as attested by both myeloperoxidase activity and histological analysis. Treatment of actively sensitized rats with either fucoidin (10 mg/kg, i.v) or anti-rat neutrophil antiserum (i.p.) reduced neutrophil accumulation and the late hyperalgesic response noted from 3 to 6 h post-challenge. Thus, we conclude that though immunoglobulin E-mediated mechanisms can cause thermal hyperalgesia, components of the cellular immune reaction are crucial in order to amplify and sustain the immediate hyperalgesic response triggered by allergen, in a process dependent on neutrophil recruitment.

Antecedent ethanol ingestion (EPC) prevents postischemic arteriolar endothelium‐dependent dilatory (EDD) dysfunction: Role of leukocyte emigration

Our aims were to determine whether EPC 24 hrs prior to ischemia (25 min) and reperfusion (60 min) (I/R) would prevent leukocyte/endothelial cell interactions (LEI) in postcapillary venules and impaired EDD responses in upstream arterioles and if so, whether I/R-induced LEI and EDD are causally linked. The small bowel was subjected to I/R, with leukocyte rolling, adhesion, and emigration monitored at min 20–30 and 50–60 of reperfusion by intravital microscopic techniques. Mesenteric arteriolar responses to graded increases in acetylcholine concentrations were then assessed. I/R markedly increased LEI, events that were exclusively confined to postcapillary venules, and reduced arteriolar vasodilator responses to acetylcholine. EPC or depletion of circulating granulocytes with anti-neutrophil serum (ANS) prior to I/R prevented postischemic LEI and EDD. Treatment with phalloidin completely prevented I/R-induced leukocyte emigration, without influencing postischemic leukocyte rolling and adhesion, and preserved EDD responses. However, treatment with carbenoxolone, a gap junction inhibitor, was without effect. The latter results suggest that postischemic EDD dysfunction occurs by a mechanism that requires leukocyte emigration but may not involve an ascending propagated signal via gap junctions between cells in the microvascular network. Since EPC also prevented I/R-induced LEI and EDD dysfunction, it appears likely that antecedent ethanol ingestion preserves EDD responses via inhibition of leukocyte emigration. NIH (HL 54797 and DK 43785).