Ranna A. Rozenfeld

Clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: 2007 update from the American College of Critical Care Medicine

Background:The Institute of Medicine calls for the use of clinical guidelines and practice parameters to promote “best practices” and to improve patient outcomes. Objective:2007 update of the 2002 American College of Critical Care Medicine Clinical Guidelines for Hemodynamic Support of Neonates and Children with Septic Shock. Participants:Society of Critical Care Medicine members with special interest in neonatal and pediatric septic shock were identified from general solicitation at the Society of Critical Care Medicine Educational and Scientific Symposia (2001–2006). Methods:The Pubmed/MEDLINE literature database (1966–2006) was searched using the keywords and phrases: sepsis, septicemia, septic shock, endotoxemia, persistent pulmonary hypertension, nitric oxide, extracorporeal membrane oxygenation (ECMO), and American College of Critical Care Medicine guidelines. Best practice centers that reported best outcomes were identified and their practices examined as models of care. Using a modified Delphi method, 30 experts graded new literature. Over 30 additional experts then reviewed the updated recommendations. The document was subsequently modified until there was greater than 90% expert consensus. Results:The 2002 guidelines were widely disseminated, translated into Spanish and Portuguese, and incorporated into Society of Critical Care Medicine and AHA sanctioned recommendations. Centers that implemented the 2002 guidelines reported best practice outcomes (hospital mortality 1%–3% in previously healthy, and 7%–10% in chronically ill children). Early use of 2002 guidelines was associated with improved outcome in the community hospital emergency department (number needed to treat = 3.3) and tertiary pediatric intensive care setting (number needed to treat = 3.6); every hour that went by without guideline adherence was associated with a 1.4-fold increased mortality risk. The updated 2007 guidelines continue to recognize an increased likelihood that children with septic shock, compared with adults, require 1) proportionally larger quantities of fluid, 2) inotrope and vasodilator therapies, 3) hydrocortisone for absolute adrenal insufficiency, and 4) ECMO for refractory shock. The major new recommendation in the 2007 update is earlier use of inotrope support through peripheral access until central access is attained. Conclusion:The 2007 update continues to emphasize early use of age-specific therapies to attain time-sensitive goals, specifically recommending 1) first hour fluid resuscitation and inotrope therapy directed to goals of threshold heart rates, normal blood pressure, and capillary refill ≤2 secs, and 2) subsequent intensive care unit hemodynamic support directed to goals of central venous oxygen saturation >70% and cardiac index 3.3–6.0 L/min/m2.

Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B

We previously reported that neuronal nitric oxide synthase (nNOS) is the predominant NOS in the intestine. Inducible NOS (iNOS), an enzyme involved in the inflammatory response, is regulated by cytokines via the transcriptional factor NF‐κB. We examined a new mechanism of intestinal iNOS regulation with respect to the role of nNOS and its effect on NF‐κB. Young Sprague‐Dawley rats were treated for 4 days with 1) saline, 2) 7‐nitroindazole (7‐NI, specific nNOS inhibitor), 3) 7‐NI + pyrrolidine dithiocarbam‐ate (PDTC, NF‐κB inhibitor), or 4) PDTC. Intestinal iNOS mRNA, NF‐κB activity, and the tissue content of the regulatory IκBα were examined. We found that 7‐NI‐treated animals had higher intestinal NF‐κB (p50‐p65) activity, lower IκBα content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS activity compared with controls. All of these changes were abolished when PDTC was given together with 7‐NI. PDTC alone had no effect. 7‐NI induces a delayed increase in intestinal myeloperoxidase activity (after elevation in NF‐κB and iNOS), which could be abrogated by PDTC. We conclude that in normal rat small intestine, nNOS suppresses the gene expression of iNOS through NF‐κB down‐regulation and that nNOS suppression leads to IκBα degradation, NF‐κB activation, and iNOS expression.—Qu, X.‐w., Wang, H., De Plaen, I. G., Rozenfeld, R. A., Hsueh, W. Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B. FASEB J. 15, 439‐446 (2001)

American College of Critical Care Medicine Clinical Practice Parameters for Hemodynamic Support of Pediatric and Neonatal Septic Shock

Objectives: The American College of Critical Care Medicine provided 2002 and 2007 guidelines for hemodynamic support of newborn and pediatric septic shock. Provide the 2014 update of the 2007 American College of Critical Care Medicine “Clinical Guidelines for Hemodynamic Support of Neonates and Children with Septic Shock.” Design: Society of Critical Care Medicine members were identified from general solicitation at Society of Critical Care Medicine Educational and Scientific Symposia (2006–2014). The PubMed/Medline/Embase literature (2006–14) was searched by the Society of Critical Care Medicine librarian using the keywords: sepsis, septicemia, septic shock, endotoxemia, persistent pulmonary hypertension, nitric oxide, extracorporeal membrane oxygenation, and American College of Critical Care Medicine guidelines in the newborn and pediatric age groups. Measurements and Main Results: The 2002 and 2007 guidelines were widely disseminated, translated into Spanish and Portuguese, and incorporated into Society of Critical Care Medicine and American Heart Association/Pediatric Advanced Life Support sanctioned recommendations. The review of new literature highlights two tertiary pediatric centers that implemented quality improvement initiatives to improve early septic shock recognition and first-hour compliance to these guidelines. Improved compliance reduced hospital mortality from 4% to 2%. Analysis of Global Sepsis Initiative data in resource rich developed and developing nations further showed improved hospital mortality with compliance to first-hour and stabilization guideline recommendations. Conclusions: The major new recommendation in the 2014 update is consideration of institution—specific use of 1) a “recognition bundle” containing a trigger tool for rapid identification of patients with septic shock, 2) a “resuscitation and stabilization bundle” to help adherence to best practice principles, and 3) a “performance bundle” to identify and overcome perceived barriers to the pursuit of best practice principles.

Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor

Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca(2+)-dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT-PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine.

The role of xanthine oxidase in platelet activating factor induced intestinal injury in the rat

Background Xanthine oxidase (XO) is an important source of reactive oxygen species in the small intestine. Aims To examine the interaction of platelet activating factor (PAF), XO, and neutrophils in mediating intestinal injury in rats. Methods Two doses of PAF were used to induce either reversible hypotension, or irreversible shock with intestinal necrosis. The activities of XO, and its precursor xanthine dehydrogenase (XD), in both the whole intestinal tissue and epithelial cells, were measured. XO was localised by histochemical staining. Results PAF dose dependently induced an increase in XO activity, predominantly in the ileal epithelium, without altering the total activity of XD+XO. Most of the XD to XO conversion was via proteolysis. PAF induced XO activation and intestinal injury were prevented by prior neutrophil depletion. PAF induced XO activation is probably not due to reperfusion, as XO activation preceded the recovery of mesenteric flow. Allopurinol pretreatment substantially inhibited intestinal neutrophil sequestration induced by high dose (but not low dose) PAF. Conclusions PAF rapidly activates intestinal XO through proteolytic XD-XO conversion, predominantly in the ileal epithelium. This effect is mediated by neutrophils. XO activation promotes PAF induced polymorphonuclear leucocyte sequestration in the intestine.

Endotoxin, but not platelet-activating factor, activates nuclear factor-κB and increases IκBα and IκBβ turnover in enterocytes

Bacterial endotoxin (lipopolysaccharide; LPS) and platelet‐activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)‐κB in the intestine, which up‐regulates many pro‐inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF‐κB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF‐κB activation in IEC‐6 (a non‐transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF‐κB mostly as p50–p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF‐κB‐dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF‐κB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IκBα and IκBβ. We found that TNF causes rapid degradation of IκBα and IκBβ. In contrast, LPS did not change the levels of IκBα and IκBβ up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IκBα and IκBβ, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IκBα and IκBβ and therefore activates NF‐κΒ via at least two mechanisms: initially, through an IκB‐independent mechanism, and later, via an increased turnover of the inhibitor IκB. NF‐κΒ activation precedes the gene expression of iNOS (assayed by reverse transcription–polymerase chain reaction), suggesting that LPS up‐regulates iNOS via this transcription factor.

Roles of nitric oxide synthases in platelet-activating factor-induced intestinal necrosis in rats.

OBJECTIVE To examine the role of constitutive and inducible nitric oxide synthases (cNOS and iNOS) in platelet-activating factor (PAF)-induced shock and intestinal injury. DESIGN Prospective, randomized, controlled experimental study. SETTING Hospital research laboratory. SUBJECTS Young adult male Sprague-Dawley rats were anesthetized and studied. INTERVENTIONS Rats were injected with PAF, either alone or after the following pretreatments: a) selective iNOS inhibitors aminoguanidine or S-methylisothiourea; b) 3-morpholinosydnonimine, a NO donor; c) S-methylisothiourea + 3-morpholinosydnonimine; and d) antineutrophil antibody (to deplete neutrophils). MEASUREMENTS AND MAIN RESULTS Blood pressure, hematocrit, white blood cell counts, intestinal injury, and intestinal cNOS and iNOS activities were assessed. We found that: a) cNOS is the predominant NOS in the intestine and its activity is inversely correlated to the level of tissue injury; b) there is a time-dependent increase in cNOS activity in sham-operated animals, which was abolished by PAF; c) Western blotting and immunohistochemistry showed iNOS present in the normal intestine, localizing mainly in crypt cells; d) iNOS inhibitors attenuated PAF-induced injury in animals with high cNOS activity, but had no protective effect in animals with low cNOS activity; e) 3-morpholinosydnonimine, alone or together with S-methylisothiourea, alleviated PAF-induced injury; and f) neutrophil depletion blocked the suppressive effect of PAF on cNOS and prevented injury. CONCLUSIONS We conclude that cNOS and iNOS play different roles in PAF-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of iNOS inhibitors.

Lysozyme-Modified Probiotic Components Protect Rats against Polymicrobial Sepsis: Role of Macrophages and Cathelicidin-Related Innate Immunity

Severe sepsis is associated with dysfunction of the macrophage/monocyte, an important cellular effector of the innate immune system. Previous investigations suggested that probiotic components effectively enhance effector cell functions of the immune system in vivo. In this study, we produced bacteria-free, lysozyme-modified probiotic components (LzMPC) by treating the probiotic bacteria, Lactobacillus sp., with lysozyme. We showed that oral delivery of LzMPC effectively protected rats against lethality from polymicrobial sepsis induced by cecal ligation and puncture. We found that orally administrated LzMPC was engulfed by cells such as macrophages in the liver after crossing the intestinal barrier. Moreover, LzMPC-induced protection was associated with an increase in bacterial clearance in the liver. In vitro, LzMPC up-regulated the expression of cathelicidin-related antimicrobial peptide (CRAMP) in macrophages and enhanced bactericidal activity of these cells. Furthermore, we demonstrated that surgical stress or cecal ligation and puncture caused a decrease in CRAMP expression in the liver, whereas enteral administration of LzMPC restored CRAMP gene expression in these animals. Using a neutralizing Ab, we showed that protection against sepsis by LzMPC treatment required endogenous CRAMP. In addition, macrophages from LzMPC-treated rats had an enhanced capacity of cytokine production in response to LPS or LzMPC stimulation. Together, our data suggest that the protective effect of LzMPC in sepsis is related to an enhanced cathelicidin-related innate immunity in macrophages. Therefore, LzMPC, a novel probiotic product, is a potent immunomodulator for macrophages and may be beneficial for the treatment of sepsis.

Interaction of platelet-activating factor, spleen and atrial natriuretic peptide in plasma volume regulation during endotoxaemia in rats

1 We studied endotoxin (lipopolysaccharide, LPS)‐induced platelet‐activating factor (PAF) production in various visceral organs, and the effect of PAF antagonists or splenectomy on LPS‐induced changes. 2 PAF production in response to LPS was highest in the spleen, followed by ileum, heart, lung and kidneys. None was found in the liver. The splenic response was rapid, reaching 10 times the basal level at 30 min. The increased PAF content in each organ was unrelated to the enzyme activity of either macrophages or neutrophils. 3 LPS‐induced hypotension and haemoconcentration were largely prevented by PAF antagonists and splenectomy. 4 Plasma volume fell, and plasma atrial natriuretic peptide (ANP) rose, after LPS administration. Splenectomy or pretreatment with PAF antagonists almost completely prevented these LPS‐induced changes at 30 min, but only partially reversed them at 90 min. 5 These results suggest that during endotoxaemia: (a) the spleen is the site of the highest endogenous PAF production; (b) the initial release of ANP is dependent on the production of endogenous PAF, and a PAF‐ANP interaction mediates the early plasma volume reduction; (c) plasma volume reduction as well as ANP release depend on the spleen; (d) PAF mediated the hypotensive response and its action in the spleen; and (e) sequestered neutrophils are probably not the main source of PAF in the spleen.

Tetrahydrobiopterin prevents platelet-activating factor-induced intestinal hypoperfusion and necrosis: Role of neuronal nitric oxide synthase.

Objective:We reported previously that neuronal nitric oxide synthase (nNOS) is the predominant NOS in rat small intestine and is down-regulated by platelet-activating factor (PAF). The severity of the bowel injury induced by PAF is inversely related to its suppressing effect on nNOS. Here, we investigated whether intestinal perfusion is regulated by nNOS and whether tetrahydrobiopterin, a co-factor and stabilizer of nNOS, reverses PAF-induced intestinal hypoperfusion and injury. Setting:Animal laboratory. Design:We first examined nNOS regulation of splanchnic blood flow by measuring the perfusion of the heart, lung, ileum, and kidney in rats after a nNOS inhibitor. We then examined the protective effect of tetrahydrobiopterin on PAF-induced bowel injury, mesenteric hypoperfusion, and systemic inflammation. Subjects:Adult male Sprague-Dawley rats. Intervention:In part 1 of the experiment, rats were given 7-nitroindazole (a specific nNOS inhibitor, 50 mg·kg−1·day−1). In part 2 of the experiment, rats were treated with tetrahydrobiopterin (20 mg/kg) 5 mins before and 30 mins after PAF challenge (2.2 &mgr;g/kg, intravenously) Measurements:Perfusion of the heart, lung, ileum, and kidney was measured at 1 and 4 days after 7-nitroindazole, using fluorescent microspheres. Intestinal injury and inflammation (myeloperoxidase content), blood perfusion, calcium dependent-NOS activity, and systemic inflammation (hypotension and hematocrit increase) were assessed 1 hr after PAF with and without tetrahydrobiopterin treatment. Results:In part 1 of the experiment, 7-nitroindazole induced a long-lasting reduction of blood perfusion and inducible NOS expression selectively in the ileum but not in nonsplanchnic organs such as heart, lungs, and kidneys. In part 2, tetrahydrobiopterin protected against PAF-induced intestinal necrosis, hypoperfusion, neutrophil influx, and NOS suppression. It also reversed hypotension and hemoconcentration. Sepiapterin (2 mg/kg, stable tetrahydrobiopterin precursor) also attenuated PAF-induced intestinal injury. Conclusions:We conclude that nNOS selectively regulates intestinal perfusion. Tetrahydrobiopterin prevents PAF-induced intestinal injury, probably by stabilizing nNOS and maintaining intestinal perfusion.