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Low-level expression and limited role for the inducible isoform of nitric oxide synthase in the vascular hyporeactivity and mortality associated with cecal ligation and puncture in the rat.

Expression of the inducible isoform of nitric oxide synthase (iNOS) contributes to the hypotension and vascular hyporeactivity in various models of shock induced by bacterial lipopolysaccharide (LPS). However, the role of iNOS in response to shock caused by live bacteria is more controversial. In the present study, we investigated the role of iNOS in a rat model of cecal ligation and puncture (CLP). CLP resulted in increased plasma nitrite/nitrate levels (up to 59 microM at 24 h) and increased pulmonary iNOS activity (up to 71 fmoles/mg/min at 12 h) and caused a significant vascular hyporeactivity at 18 h. The degree of NO production and iNOS induction was approximately 30% of that observed several hours after administration of LPS in the same species, and the degree of vascular hyporeactivity was less than that observed after LPS injection. Selective inhibition of iNOS with mercaptoethylguanidine (MEG) reduced plasma nitrite/nitrate levels, but did not prevent the development of vascular hyporeactivity, and did not improve survival in this model of CLP. Thus, CLP-induced sepsis causes low-level induction of iNOS, but factors other than iNOS are the crucial determinants of the vascular failure and mortality in this model.

Animals↗

Cecal ligation and double puncture impairs heat shock protein 70 (HSP-70) expression in the lungs of rats.

Induction of the heat shock response may improve outcome from pathophysiological disturbances. This improvement is associated with and believed to result from expression of heat shock protein (HSP)-70. Therefore, we examined the temporal expression of HSP-70 in an animal model of acute respiratory distress syndrome (ARDS) secondary to fecal peritonitis. Specifically, we hypothesize that sepsis in rats impairs pulmonary HSP-70 expression. ARDS was induced in adolescent rats via cecal ligation and double puncture (2CLP). Sham-operated animals served as controls. Lung tissue was collected 0, 3, 6, 16, 24, and 48 h after 2CLP and sham operation. Northern blot hybridization analysis was performed to detect steady-state HSP-70 messenger ribonucleic (mRNA) levels. HSP-70 protein levels were determined via immunoblotting and immunohistochemistry. Mortality after 2CLP was 50% at 24 h and 75% at 48 h. Northern blot hybridization analysis revealed no significant change in steady-state HSP-70 mRNA levels in lung at any time after 2CLP. HSP-70 steady-state mRNA levels increased after sham operation and was higher than values in 2CLP at 6, 16, and 24 h. HSP-70 protein levels did not change over time in either group. Thus, the expression of HSP-70 does not change after 2CLP. Although lack of an increase in protein levels may be adaptive after sham operation, it is not appropriate after 2CLP. Therefore, failed HSP-70 expression represents a form of pulmonary epithelial dysfunction that may contribute to lung injury in sepsis.

Animals↗

Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture.

Sepsis remains a serious clinical problem despite intense efforts to improve survival. Experimental animal models of sepsis have responded dramatically to immunotherapy blocking the activity of cytokines. Despite these preclinical successes, human clinical trials have not demonstrated any improvement in survival. We directly compared the mortality, morbidity, and immunopathology in two models of sepsis, one due to lipopolysaccharide (LPS) and the other to cecal ligation and puncture (CLP). BALB/c mice were injected intraperitoneally with 250 microg of LPS or subjected to CLP with an 18-gauge needle. Both models yielded similar mortality (> 85%) and morbidity. Additionally, neutropenia and lymphopenia developed in both groups. Plasma and peritoneal levels of cytokines (TNF, IL-1, IL-6, and the chemokines KC and MIP-2) were measured at 1.5, 4, and 8 h after challenge. LPS induced substantially higher levels of cytokines in both compartments with peak levels between 1.5 and 4 h that began to decline at 8 h. In contrast, cytokine levels in the CLP model were continuing to increase at the 8 h-time point and often exceeded the LPS-induced values at this time. Our data demonstrate that the LPS and CLP models have similar mortality but significant differences in the kinetics and magnitude of cytokine production. Immunotherapy for sepsis based on cytokine production after LPS challenge is misdirected because the LPS model does not accurately reproduce the cytokine profile of sepsis.

Animals↗

Effect of sodium arsenite on iNOS expression and vascular hyporeactivity associated with cecal ligation and puncture in the rat.

Induction of the heat shock response protects animals from either endotoxemia or peritonitis. In endotoxemia, heat shock protein (HSP) induction is associated with reversal of vascular hyporeactivity and inhibition of iNOS expression. Recent studies suggest differences in the inflammatory mechanisms during endotoxemia and peritonitis animal models and their response to therapeutic interventions. We therefore studied the effect of the HSP inducer sodium arsenite (SA) on vascular reactivity and iNOS expression in rats undergoing cecal ligation and puncture (CLP). CLP resulted in suppression of the pressor effect of norepinephrine (NE) in vivo (measured by changes in blood pressure in response to NE boluses) and ex vivo (changes in contraction force in isolated mesenteric arteries in response to NE concentrations), and in the expression of iNOS protein. Pretreatment of the rats with SA resulted in reversal of CLP-induced vascular hyporeactivity in vivo and ex vivo, and inhibition of iNOS expression after 22 h. SA pretreatment improved 7-day survival after CLP from 18.2% to 70% (P < 0.005). Glucocorticoid receptor inhibition did not affect the effect of HSP induction on iNOS expression. The similarity of the effect of HSP on vascular reactivity and iNOS expression in two distinct sepsis models suggests that this effect may be clinically important and that a causative relationship between HSP induction, iNOS inhibition, and reversal of vascular reactivity is likely.

Animals↗

Antioxidant amplifies antibiotic protection in the cecal ligation and puncture model of microbial sepsis through interleukin-10 production.

Preadministration of antioxidants such as pyrrolidine dithiocarbamate (PDTC) and phenyl N-tert-butyl nitrone (PBN) protects animals from lethality in sepsis models. However, the requirement of preadministration greatly diminishes the clinical significance of these studies. Although the synthetic antioxidant PBN has been shown to effectively protect rodents from lethality in endotoxemia (lipopolysaccharide [LPS] model), preliminary screening indicates that pre- or postadministration of PBN does not protect in the rat cecal ligation and puncture (CLP) model. We show in this report that in a rat CLP model, the administration of PBN (150 mg/kg, 30 min after CLP) followed by the antibiotic imipenem (IMP; 10 mg/kg, 1 h after CLP) significantly increased survival compared with other single treatment groups. Previously, we have shown that PBN's protection in a rat LPS model is mediated by the overproduction of the anti-inflammatory cytokine interleukin (IL)-10. We show in this study that the increase in survival found in the PBN + IMP-treated group was abrogated by immunoneutralization with anti-IL-10 antibody, indicating that endogenous IL-10 is an effective protective factor. Plasma LPS levels were shown to be elevated after imipenem treatment, and the increased LPS level could have assisted to overproduce endogenous IL-10, as in the case of the PBN-treated LPS model. Statistical analysis indicated that the increase of IL-10 in PBN + IMP-treated group at early time period has significant association to the improvement of survival.

Animals↗

Recommendations for the use of routine bone marrow aspiration and lumbar punctures in the follow-up of patients with retinoblastoma.

PURPOSE: Patients with metastatic retinoblastoma have a poor outcome. Hope that early detection of extraocular spread will improve survival has led to routine monitoring with bone marrow and cerebrospinal fluid (CSF) examinations. In light of cost and patient morbidity, the clinical utility of this practice is questioned. PATIENTS AND METHODS: We have performed 254 serial bone marrow aspirations and 164 lumbar punctures in 60 children with retinoblastoma. RESULTS: Two patients with extensive intraocular disease at diagnosis developed positive bone marrow aspirations, although no patient died of distant metastasis. Three patients developed positive CSF examinations. All had neurologic symptoms at the time of CSF positivity. CONCLUSIONS: We recommend performing staging bone marrow and CSF evaluations only in patients with clinical, histologic, or radiologic evidence of local or systemic extension (Pratt stage III-IV), or in patients presenting with one Reese-Ellsworth group V eye and retrolaminar or extrascleral extension of their tumor. We recommend limiting follow-up bone marrow and CSF evaluations to patients who develop objective signs and symptoms of metastatic or regionally recurrent disease.

Biopsy, Needle↗

Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.

OBJECTIVE: To evaluate protein C and other factors associated with the septic response as predictors of mortality in a clinically relevant animal model of sepsis. DESIGN: Laboratory investigation. SETTING: Eli Lilly and Company discovery research laboratory. SUBJECTS: Forty female Sprague Dawley rats weighing 245-265 g. INTERVENTIONS: Polyethylene catheters were surgically implanted into the femoral vein and sepsis was induced by cecal ligation and puncture (CLP). A solution of 5% dextrose in 0.9 % saline was continuously infused via femoral catheters immediately following surgery. Blood sampling was done before surgery and at 6 and 20 hrs after surgery. Rats were then monitored for survival out to 4 days. MEASUREMENTS AND MAIN RESULTS: Blood collections were used to measure blood glucose, bacteremia, plasma protein C, D-dimer, hormones, chemokines, cytokines, and myoglobin (as a marker of organ damage). Mortality was categorized into three groups: early death (before 30 hrs post-CLP), late death (after 30 hrs post-CLP), and survivors (96 hrs post-CLP). Compared with survivors, early death rats had statistically significant differences in 30 variables indicative of severe inflammation, coagulopathy, and muscle damage including less bacterial clearance, hypoglycemia, lower plasma protein C, higher plasma D dimer, higher plasma cytokine/ chemokines, and higher plasma myoglobin concentrations. Twenty variables had a moderate to strong correlation with time of death. Receiver operator characteristic curves generated from a simple logistic regression model indicated that KC and macrophage inflammatory protein-2, rodent homologues of the human growth related oncogene CXC chemokine family, and protein C were the best predictors of mortality in this model. CONCLUSIONS: The data from this study indicate that an early decrease in protein C concentration predicts poor outcome in a rat sepsis model. The data further indicate that increases in the CXC chemokines macrophage inflammatory protein-2 and KC precede poor outcome.

Animals↗

Sophorolipids block lethal effects of septic shock in rats in a cecal ligation and puncture model of experimental sepsis.

OBJECTIVE: Sophorolipids, a family of natural and easily chemoenzymatically modified microbial glycolipids, are promising modulators of the immune response. The potential of the therapeutic effect of sophorolipids was investigated in vivo in a rat model of sepsis and in vitro by analysis of nitric oxide and cytokine production. DESIGN: Prospective, randomized animal study. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats, 200-240 g. INTERVENTIONS: Intra-abdominal sepsis was induced in vivo in 166 rats via cecal ligation and puncture (CLP); 60 rats were used to characterize the model. The remaining rats were treated with sophorolipids or vehicle (dimethylsulfoxide [DMSO]/physiologic saline) by intravenous (iv) tail vein or intraperitoneal (IP) injection immediately post-CLP (25/group). Survival rates were compared at 36 hrs after surgery. In vitro, macrophages were cultured in lipopolysaccharide (LPS) +/- sophorolipid and assayed for nitric oxide (NO) production and gene expression profiles of inflammatory cytokines. In addition, splenic lymphocytes isolated from CLP rats +/- sophorolipid treatment (three per group) were analyzed for cytokine production by RNase protection assay. MEASUREMENTS AND MAIN RESULTS: CLP with 16-gauge needles optimized sepsis induction and resultant mortality. Sophorolipid treatment improved rat survival by 34% (iv) and 14% (IP) in comparison with vehicle controls (p < .05 for iv treatment). Sophorolipids decreased LPS-induced macrophage NO production by 28% (p < .05). mRNA expression of interleukin (IL)-1beta was downregulated by 42.5 +/- 4.7% (p < .05) and transforming growth factor (TGF)-beta1 was upregulated by 11.7 +/- 1.5% (p < .05) in splenocytes obtained 6 hrs postsophorolipid treatment. LPS-treated macrophages cultured 36 hrs with sophorolipids showed increases in mRNA expression of IL-1alpha (51.7%), IL-1beta (31.3%), and IL-6 (66.8%) (p < .05). CONCLUSIONS: Administration of sophorolipids after induction of intra-abdominal sepsis significantly decreases mortality in this model. This may be mediated in part by decreased macrophage NO production and modulation of inflammatory responses.

Animals↗

Cecal ligation and puncture in rats interrupts the circadian rhythms of corticosterone and adrenocortical responsiveness to adrenocorticotrophic hormone.

OBJECTIVE: To determine the altered patterns of pituitary-adrenal activity and impaired adrenocortical sensitivity to adrenocorticotrophic hormone (ACTH) in the cecal ligation and puncture (CLP) model of sepsis. DESIGN: Prospective, controlled experiment. SETTING: Basic science laboratory. SUBJECTS: Sprague-Dawley male rats 300-450 g. INTERVENTIONS: Indwelling arterial catheters and CLP with either an 18-(CLP18) or a 21-gauge needle or sham surgery. MEASUREMENTS AND MAIN RESULTS: Plasma ACTH and corticosterone recovered most rapidly after sham surgery and least rapidly after CLP18. From postoperative day 4 am through day 7, a robust diurnal rhythm of corticosterone (p < .001) with a modest rhythm of ACTH (p < .01) occurred only in sham rats, and the slope of the regression between plasma corticosterone and ACTH increased from am to pm after sham surgery (p < .05) but not after CLP. Corticosterone in response to intravascular ACTH (3, 10, and 250 ng/kg) 2 hrs after dexamethasone (0.25 mg/kg) only showed an am to pm difference after sham surgery. The pm sham responses to all doses of ACTH were greater (p < .01) than the respective am sham responses that were not different from the respective am or pm responses after CLP. Corticosterone after 10 ng/kg ACTH in the pm decreased as plasma macrophage migration inhibitory factor and IL-6 increased after CLP (r = -.691 and r = -.813, respectively; p < .02 in each case). CONCLUSIONS: The adrenocortical sensitivity to ACTH in the pm after CLP is suppressed progressively with the intensity of inflammation. This suppression appears to be a major factor in the interruption of circadian patterns of hormonal secretion in sepsis.

Adrenal Cortex↗

Canine fossa puncture and clearance of the maxillary sinus for the severely diseased maxillary sinus.

OBJECTIVES: To assess the impact of canine fossa puncture (CFP) with clearance of the maxillary sinus during endoscopic sinus surgery on the subjective and objective outcomes as defined by quality of life questionnaires, endoscopic findings, endoscopic aspirate for culture and sensitivity, and magnetic resonance imaging (MRI) evaluation. STUDY DESIGN: Retrospective, cross-sectional study. METHODS: Twenty-five patients who had undergone canine fossa were compared to 12 patients who had undergone routine endoscopic middle meatal clearance of the maxillary sinus. At an average postoperative time point of 19.9 months, all patients were administered the chronic sinusitis survey (CSS) and a visual analogue scale (VAS). At this visit, patients underwent nasendoscopy, sampling of the ostial region for bacterial and fungal cultures, and MRI of the sinuses. RESULTS: There were 42 sides for analysis from 25 patients on the study arm; the control arm comprised 12 patients with completely opacified maxillary sinuses and 17 sides for analysis. The CSS symptom subscore was better in the CFP group (Mann-Whitney test, P = .02). The overall VAS score was less in the CFP group (Mann-Whitney test, P = .01), indicating better symptom control. No opacification was evident in 61.9% of patients in the CFP group compared with 11.8% in the control arm (chi, P < .001). CONCLUSION: CFP is a useful technique to address the severely diseased maxillary sinus and is associated with significantly better symptom control compared with the transnasal middle meatal antrostomy clearance of the maxillary sinus.

Adult↗

Pain, position, and stylet styles: infant lumbar puncture practices of pediatric emergency attending physicians.

OBJECTIVES: Lumbar punctures (LPs) are common emergency department (ED) procedures. Few pediatric studies exist to define training, guide practice, or indicate preferred methods for infants. While pain control is recommended, no recent studies indicate prevalence of analgesic use since the advent of topical anesthetics. We surveyed academic pediatric ED physicians to assess training and technique preferences and to highlight pain control usage. METHODS: A total of 398 physicians were randomly selected from the 621 e-mail accessible members of the AAP Section on Pediatric Emergency Medicine. Questions concerning physician training, analgesia, and technique were either sent by regular mail or via e-mail link to a Web-based survey. RESULTS: Of 359 deliverable surveys, there were 188 physician responses (52.4%) with differential response rates by survey format [58 e-mail (36%) and 130 regular mail responses (66%); P < 0.05]. Almost a third will advance the needle without the stylet in place. Two-thirds of physicians do not routinely use analgesia for neonatal LPs. Those using pain control were trained more recently (median 12 years vs. 15 years postresidency; P = 0.04). Analgesia use was the most common practice changed since residency. CONCLUSIONS: Analgesia is underused for infant LPs. Advancing the needle without a stylet is not uncommon. Response rate to regular mail surveys was much higher.

Adult↗

Iron overload before cecal ligation and puncture increases mortality.

Iron metabolism is dysregulated in critically ill patients. A mouse model of dysregulated iron metabolism was used to examine the consequence of iron loading upon sepsis. Mice deleted in the hfe gene (hfe-/-) abnormally accumulate iron in tissue; defects in the human hfe gene are clinically expressed as hemochromatosis. Hfe-/- mice and wild-type counterparts were randomized to receive either high- or low-iron diets for 2 weeks. After iron loading, mice were subjected to cecal ligation and puncture (CLP), a clinically relevant animal model of intra-abdominal sepsis. A preliminary (but underpowered) study suggested that iron-loaded hfe-/- mice had increased mortality as compared with hfe-/- mice fed a low-iron diet. There was no difference between wild-type and hfe-/- mice fed a low-iron diet or between wild-type mice fed hihg- and low-iron diets. A subsequent, appropriately powered study showed that iron-loaded hfe-/- mice had significantly higher mortality from intra-abdominal sepsis than hfe-/- mice fed a low-iron diet. Iron loading was confirmed through chemical assay of iron concentration in hepatic tissue. Animals with dysregulated iron handling, loaded with iron and subjected to CLP, had double the mortality of animals with normal iron levels. Critical care patients often have altered iron metabolism. In clinical practice, critically ill patients may receive iron through direct administration and the transfusion of blood products. Iron therapy may adversely affect the clinical outcome from sepsis.

Administration, Oral↗

Hemodynamic and cardiac contractile function during sepsis caused by cecal ligation and puncture in mice.

Sepsis is among the leading causes of death in the critically ill, yet the pathophysiology of sepsis is incompletely understood. Genetically engineered mice offer a unique opportunity to explore the cellular and molecular pathogenesis of sepsis. However, the hemodynamic responses of mice during sepsis are not completely understood because of the difficulty in performing cardiovascular measurements in mice. We used a 1.4-F pressure and conductance catheter to measure hemodynamics in wild-type C57BL/6J mice during sepsis caused by cecal ligation and puncture. Septic mice exhibited significant hypothermia compared with the sham group. In addition, there was a progressive decrease in mean arterial blood pressure and systemic vascular resistance in septic mice as well as an increase in stroke volume and cardiac output. Sepsis also caused a significant time-dependent impairment of left ventricular function as indicated by decreased dp/dtmax and dp/dtmin. The slope of end systolic pressure volume relationship also decreased over time, as did the time varying maximum elastance and preload-recruitable stroke work of the left ventricle. In conclusion, septic mice exhibit hemodynamic alterations during sepsis that are similar to those observed in humans. The miniaturized conductance catheter allows for effective measurements of hemodynamic function in septic mice and provides measurements that cannot be obtained using other cardiovascular monitoring techniques.

Animals↗

Cecal ligation and puncture.

The model of cecal ligation and puncture (CLP) in rodents has been used extensively to investigate the clinical settings of sepsis and septic shock. This model produces a hyperdynamic, hypermetabolic state that can lead to a hypodynamic, hypometabolic stage, and eventual death. Blood cultures are positive for enteric organisms very early after CLP. The model has been widely used over the past 26 years and is highly versatile in adapting to a range of severity and testing objectives. It is inexpensive to prepare and technically straightforward. Aspects of sepsis research investigated using CLP include energetics, metabolism, resuscitation, antibiotic therapy, microbial factors, cardiovascular responses, immune function, mediator release, and cytokine expression patterns. The challenge of the small circulating blood volume in rodents can be overcome by using micromethods that enable analysis of small volumes, or alternatively, by using a large number of animals to obtain serial samples.

Animals↗

Chemically modified tetracycline (COL-3) improves survival if given 12 but not 24 hours after cecal ligation and puncture.

Sepsis can result in excessive and maladaptive inflammation that is responsible for more than 215,00 deaths per year in the United State alone. Current strategies for reducing the morbidity and mortality associated with sepsis rely on treatment of the syndrome rather than prophylaxis. We have been investigating a modified tetracycline, COL-3, which can be given prophylactically to patients at high risk for developing sepsis. Our group has shown that COL-3 is very effect at preventing the sequelae of sepsis if given before or immediately after injury in both rat and porcine sepsis models. In this study, we wanted to determine the "treatment window" for COL-3 after injury at which it remains protective. Sepsis was induced by cecal ligation and puncture (CLP). Rats were anesthetized and placed into five groups: CLP (n = 20) = CLP without COL-3, sham (n = 5) = surgery without CLP or COL-3, COL3@6h (n = 10) = COL-3 given by gavage 6 h after CLP, COL3@12h (n = 10) = COL-3 given by gavage 12 h after CLP, and COL3@24h (n = 20) = COL-3 given by gavage 24 h after CLP. COL-3 that was given at 6 and 12 h after CLP significantly improved survival as compared with the CLP and the CLP@24h groups. Improved survival was associated with a significant improvement in lung pathology assessed morphologically. These data suggest that COL-3 can be given up to 12 h after trauma and remain effective.

Animals↗

The nature and incidence of complications of peripheral arterial puncture.

Follow-up of the sites of 839 peripheral arterial punctures has confirmed the technique as safe and free from major sequelae. Minor complications, mainly bruising and tenderness, occurred at 39% of sites; this incidence is much less than that previously reported following short-term arterial cannulation. At only 2% of sites was there any significant pain or tenderness. There was a small incidence of pulse diminution particularly at the radial site but no case of arterial occlusion. The use of a smaller needle (23 s.w.g.) significantly reduced the overall incidence of complications.

Arteries↗

Observations on deliberate dural puncture with a Tuohy needle: pressure measurements.

Pressure recordings were made during passage of a Tuohy needle from the interspinous ligament to the subarachnoid space for lumbar drain insertion. Epidural space pressures were always positive. Negative pressures were seen only at the moment of entry into the subarachnoid space. These were artefactual and were caused by tenting of the dura by the blunt Tuohy needle. Use of a closed measurement system such as this facilitates the development of large transdural pressure gradients because of the inability of the epidural space pressure to equilibrate with atmospheric pressure. This may contribute to ease of dural puncture.

Cerebrospinal Fluid Pressure↗

Delayed cutaneous fluid leak from the puncture hole after removal of an epidural catheter.

A case of a delayed but persistent cutaneous fluid leak from a puncture hole following removal of an epidural catheter is presented. The fluid was subsequently found not to be cerebrospinal but interstitial oedema fluid. This case demonstrates the importance of performing simple investigations on any such fluid before ordering more complex tests.

Anesthesia, Epidural↗