Functions and regulation of lipocalin-2 in gut-origin sepsis: a narrative review.

Lipocalin-2 (Lcn2), an innate immune protein, has come to be recognized for its roles in iron homeostasis, infection, and inflammation. In this narrative review, we provide a comprehensive description based on currently available evidence of the clinical implications of Lcn2 and its therapeutic potency in gut-origin sepsis. Lcn2 appears to mitigate gut barrier injury via maintaining homeostasis of the microbiota and exerting antioxidant strategy, as well as by deactivating macrophages and inducing immune cell apoptosis to terminate systemic hyper-inflammation. We propose that development of a therapeutic strategy targeting lipocalin-2 could be highly promising in the management of gut-origin sepsis.

Deep anesthesia worsens outcome of rats with inflammatory responses.

OBJECTIVE: We tested the hypothesis that deep anesthesia with sevoflurane, known as a potent immunomodulator, for 4 h would worsen the 24-h outcomes of rats through modulation of the inflammatory responses. METHODS: Forty-nine male Wistar rats, administered low dose of lipopolysaccharide (0.5 mg/kg) intravenously to elicit moderate inflammatory responses mimicked mild surgical stress, underwent one minimum alveolar concentration (MAC) or 2 MAC sevoflurane anesthesia for 4 h. The 24-h survival rate, arterial blood gases, plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-α concentrations, and rate of T lymphocyte apoptosis in spleen were evaluated. We further examined the effects of hypotension and TNF-α discharge on the survival rate. RESULTS: The survival rate in 2 MAC group was significantly lower accompanied with decreased base excess and increased level of cytokines (IL-6, TNF-α) compared to 1 MAC group. The apoptosis rate did not differ between the two groups. Neither norepinephrine infusion to restore hypotension nor administration of anti-TNF-α antibody improved the outcome in the 2 MAC group. CONCLUSIONS: Deep anesthesia with sevoflurane even for a short-term period augments the release of inflammatory cytokines evoked by inflammatory insults like surgical stress, impairs the acid-base balance, and subsequently deteriorates the outcomes.

Neutrophil gelatinase-associated lipocalin regulates gut microbiota of mice.

BACKGROUND AND AIM: Because neutrophil gelatinase-associated lipocalin (NGAL) is known to provide significant bacteriostatic effects during infectious conditions, we tested the hypothesis that this protein is up-regulated and secreted into the intraluminal cavity of the gut under critically ill conditions and is thus responsible for the regulation of bacterial overgrowth. METHODS: With our institutional approval, male C57BL/6J mouse (6-7 weeks) were enrolled and applied for lipopolysaccharide or peritonitis model compared with naïve control. We assessed NGAL protein concentrations in intestinal lumen and up-regulation of NGAL expression in intestinal tissues in in vivo as well as ex vivo settings. Simultaneously, we examined the effects of NGAL protein administration on the growth of Escherichia coli (E. coli) in in vivo and in vitro experimental settings. The localization of NGAL in intestinal tissues and lumen was also assessed by immunohistological approach using NGAL antibody. RESULTS: Both lipopolysaccharide and peritonitis insults evoked the marked up-regulation of NGAL mRNA and protein levels in gut tissues such as crypt cells. In addition, the administration of NGAL protein significantly inhibited the outgrowth of enteric E. coli under both in vitro and in vivo conditions, accompanied by histological evidence. CONCLUSION: Neutrophil gelatinase-associated lipocalin protein accompanied by apparent bacteriostatic action accumulated in the intestinal wall and streamed into the mucosal layer during critically ill state, thereby possibly shaping microbiota homeostasis in the gut.

The effects of epidural anesthesia on growth of Escherichia coli at pseudosurgical site: the roles of the lipocalin-2 pathway.

BACKGROUND: Neutrophil-derived lipocalin-2 exerts bacteriostatic effects through retardation of iron uptake by the Gram-negative organisms like Escherichia coli. We tested the hypothesis that the expression of lipocalin-2, a bacteriostatic protein, was upregulated by induction of surgical site infection (SSI) with E coli in healthy and diseased rats and that epidural anesthesia modulated its expression. METHODS: Male Wistar rats were randomized into a healthy or disease group, the latter of which was administered lipopolysaccharide. Both groups were further divided into 3 subgroups, the control, saline, and lidocaine groups: group healthy control (n = 10), healthy saline (n = 10), and healthy lidocaine (n = 10) versus group disease control (n = 15), disease saline (n = 18), and disease lidocaine (n = 19), respectively. While saline was epidurally administered to the control and saline groups, lidocaine was administered to the lidocaine groups. Except for the control groups, E coli was injected to the pseudosurgical site to mimic SSI after abdominal surgery. Plasma concentrations of inflammatory cytokine and lipocalin-2 were measured. At 72 hours, the surgical site tissues were obtained to evaluate mRNA expression of lipocalin-2 and E coli DNA expression. RESULTS: All disease subgroups showed markedly increased plasma inflammatory cytokines versus the healthy subgroups. Among the disease subgroups, plasma concentrations of lipocalin-2 and tissue mRNA expression of lipocalin-2 were significantly increased in group disease lidocaine versus the others. Concurrently, E coli DNA expression in the tissue specimens was also significantly lower in group disease lidocaine as compared with group disease saline. CONCLUSIONS: Epidural anesthesia was associated with an increase in the expression lipocalin-2 and a decrease in the expression of E coli DNA at pseudosurgical sites in sick but not healthy rats. These observations suggest a potential mechanism by which epidural anesthesia could reduce the risk of SSI.

Inhaled nitric oxide improves outcomes after successful cardiopulmonary resuscitation in mice.

BACKGROUND: Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia/reperfusion injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR). METHODS AND RESULTS: Adult male mice were subjected to potassium-induced CA for 7.5 minutes whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 ppm NO for 23 hours. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4 of 13), depressed neurological and left ventricular function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24 hours after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23 hours starting 1 hour after CPR attenuated neurological and left ventricular dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11 of 13; P=0.003 versus mice breathing air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum nitrate/nitrite levels. Deficiency of the α1 subunit of soluble guanylate cyclase, a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR. CONCLUSIONS: These results suggest that NO inhalation after CA and successful CPR improves outcome via soluble guanylate cyclase-dependent mechanisms.

Delayed paraplegia after spinal cord ischemic injury requires caspase-3 activation in mice.

BACKGROUND AND PURPOSE: Delayed paraplegia remains a devastating complication after ischemic spinal cord injury associated with aortic surgery and trauma. Although apoptosis has been implicated in the pathogenesis of delayed neurodegeneration, mechanisms responsible for the delayed paraplegia remain incompletely understood. The aim of this study was to elucidate the role of apoptosis in delayed motor neuron degeneration after spinal cord ischemia. METHODS: Mice were subjected to spinal cord ischemia induced by occlusion of the aortic arch and left subclavian artery for 5 or 9 minutes. Motor function in the hind limb was evaluated up to 72 hours after spinal cord ischemia. Histological studies were performed to detect caspase-3 activation, glial activation, and motor neuron survival in the serial spinal cord sections. To investigate the impact of caspase-3 activation on spinal cord ischemia, outcome of the spinal cord ischemia was examined in mice deficient for caspase-3. RESULTS: In wild-type mice, 9 minutes of spinal cord ischemia caused immediate paraplegia, whereas 5 minutes of ischemia caused delayed paraplegia. Delayed paraplegia after 5 minutes of spinal cord ischemia was associated with histological evidence of caspase-3 activation, reactive astrogliosis, microglial activation, and motor neuron loss starting at approximately 24 to 48 hours after spinal cord ischemia. Caspase-3 deficiency prevented delayed paraplegia and motor neuron loss after 5 minutes of spinal cord ischemia, but not immediate paraplegia after 9 minutes of ischemia. CONCLUSIONS: The present results suggest that caspase-3 activation is required for delayed paraplegia and motor neuron degeneration after spinal cord ischemia.

Hydrogen sulfide improves survival after cardiac arrest and cardiopulmonary resuscitation via a nitric oxide synthase 3-dependent mechanism in mice.

BACKGROUND: Sudden cardiac arrest (CA) is one of the leading causes of death worldwide. We sought to evaluate the impact of hydrogen sulfide (H(2)S) on the outcome after CA and cardiopulmonary resuscitation (CPR) in mouse. METHODS AND RESULTS: Mice were subjected to 8 minutes of normothermic CA and resuscitated with chest compression and mechanical ventilation. Seven minutes after the onset of CA (1 minute before CPR), mice received sodium sulfide (Na(2)S) (0.55 mg/kg IV) or vehicle 1 minute before CPR. There was no difference in the rate of return of spontaneous circulation, CPR time to return of spontaneous circulation, and left ventricular function at return of spontaneous circulation between groups. Administration of Na(2)S 1 minute before CPR markedly improved survival rate at 24 hours after CPR (15/15) compared with vehicle (10/26; P=0.0001 versus Na(2)S). Administration of Na(2)S prevented CA/CPR-induced oxidative stress and ameliorated left ventricular and neurological dysfunction 24 hours after CPR. Delayed administration of Na(2)S at 10 minutes after CPR did not improve outcomes after CA/CPR. Cardioprotective effects of Na(2)S were confirmed in isolated-perfused mouse hearts subjected to global ischemia and reperfusion. Cardiomyocyte-specific overexpression of cystathionine gamma-lyase (an enzyme that produces H(2)S) markedly improved outcomes of CA/CPR. Na(2)S increased phosphorylation of nitric oxide synthase 3 in left ventricle and brain cortex, increased serum nitrite/nitrate levels, and attenuated CA-induced mitochondrial injury and cell death. Nitric oxide synthase 3 deficiency abrogated the protective effects of Na(2)S on the outcome of CA/CPR. CONCLUSIONS: These results suggest that administration of Na(2)S at the time of CPR improves outcome after CA possibly via a nitric oxide synthase 3-dependent signaling pathway.

Protective effects of nitric oxide synthase 3 and soluble guanylate cyclase on the outcome of cardiac arrest and cardiopulmonary resuscitation in mice.

OBJECTIVES: Despite advances in resuscitation methods, survival after out-of-hospital cardiac arrest remains low, at least in part, due to postcardiac arrest circulatory and neurologic failure. To elucidate the role of nitric oxide (NO) in the recovery from cardiac arrest and cardiopulmonary resuscitation (CPR), we studied the impact of NO synthase (NOS3)/cGMP signaling on cardiac and neurologic outcomes after cardiac arrest and CPR. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Mice. INTERVENTIONS: Female wild-type (WT) mice, NOS3-deficient mice (NOS3-/-), NOS3-/- mice with cardiomyocyte-specific overexpression of NOS3 (NOS3-/-CSTg), and mice deficient for soluble guanylate cyclase alpha1 (sGCalpha1-/-) were subjected to potassium-induced cardiac arrest (9 min) followed by CPR. Cardiac and neurologic function and survival were assessed up to 24 hrs post-CPR. MEASUREMENTS AND MAIN RESULTS: Cardiac arrest and CPR markedly depressed myocardial function in NOS3-/- and sGCalpha1-/- but not in WT and NOS3-/-CSTg. Neurologic function score and 24 hrs survival rate was lower in NOS3-/- and sGCalpha1-/- compared with WT and NOS3-/-CSTg. Detrimental effects of deficiency of NOS3 or sGCalpha1 were associated with enhanced inflammation of heart and liver and increased cell death in heart, liver, and brain that were largely prevented by cardiomyocyte-restricted NOS3 overexpression. CONCLUSIONS: These results demonstrate an important salutary impact of NOS3/sGC signaling on the outcome of cardiac arrest. Myocardial NOS3 prevented postcardiac arrest myocardial dysfunction, attenuated end-organ damage, and improved neurologic outcome and survival. Our observations suggest that enhancement of cardiac NOS3 and/or sGC activity may improve outcome after cardiac arrest and CPR.

One-lung ventilation in a patient with Fontan circulation undergoing diaphragmatic plication surgery: a case report.

BACKGROUND: Surgeries requiring one-lung ventilation (OLV) in patients with Fontan circulation pose great challenges. However, little information is available regarding the safety of OLV in Fontan patients when hemidiaphragmatic paralysis is present. CASE PRESENTATION: A 41-year-old woman who underwent repeated Fontan procedures was re-admitted to our hospital because of worsening shortness of breath. As left hemidiaphragmatic paralysis was considered to be contributing to her symptom, an open thoracic left diaphragmatic plication surgery was scheduled. A preoperative pulmonary artery angiogram revealed a remarkably reduced blood flow to the left lung. The surgeon requested OLV during the surgery. Despite our concern regarding the impact of OLV on the Fontan circulation, OLV did not result in major hemodynamic changes. CONCLUSION: OLV can be safely implemented in patients with hemidiaphragmatic paralysis with preserved Fontan circulation. Preoperative pulmonary artery angiography may provide useful information for estimating the impact of OLV on the Fontan circulation.

Beta-Blocker Therapy Preserves Normal Splenic T-Lymphocyte Numbers Reduced in Proportion to Sepsis Severity in a Sepsis Model.

Lymphocyte cell death contributes to sepsis-induced immunosuppression, leading to poor prognosis. This study examined whether sepsis severity and beta-blocker therapy could affect the degree of T-lymphocyte cell death in a mouse model of sepsis. In the first control study, 20 animals were allocated to 4 groups: control group with sham operation (group C, n = 5) and 3 groups with cecum ligation and puncture (CLP) performed at 3 different sites: proximal, middle, and distal cecum (groups CLP-P, CLP-M, and CLP-D, respectively; n = 5 in each group). Their spleens were resected under general anesthesia 24 hours after CLP, and the total number of normal splenic T lymphocytes per mouse and the percentage of apoptotic T lymphocytes were evaluated using flow cytometry. In the second experimental study, the effect of the beta-blocker esmolol was examined in CLP-P (group CLP-PE vs. CLP-P; n = 5 in each group). The total normal splenic T-lymphocyte numbers per mouse significantly decreased in proportion to CLP severity (group C, 18.6 × 106 (15 × 106-23.6 × 106); CLP-D, 9.2 × 106 (8.8 × 106-9.8 × 106); CLP-M, 6.7 × 106 (6.3 × 106-7.0 × 106); and CLP-P, 5.3 × 106 (5.1 × 106-6.8 × 106)). Beta-blocker therapy restored T-lymphocyte numbers (group CLP-PE vs. CLP-P; 6.94 ± 1.52 × 106 vs. 4.18 ± 1.71 × 106; p=0.027) without affecting apoptosis percentage. Beta-blocker therapy might improve sepsis-induced immunosuppression via normal splenic T-lymphocyte preservation.

Cesarean hysterectomy in a hybrid operating room for placenta percreta: a report of three cases.

BACKGROUND: Placenta percreta is the most severe abnormality in invasive placenta and often treated with cesarean hysterectomy. Endovascular embolization for placental abnormality is known to reduce bleeding from the placental bed and from the abnormal neovasculature surrounding the uterus. We describe three cases of placenta percreta treated with uninterrupted cesarean hysterectomy and embolization performed using a hybrid operating room (HOR). CASE DESCRIPTION: Cases were two placenta previa percretas and an impending uterine rupture with placenta percreta, treated with elective cesarean hysterectomy in HOR. Planned conversion of spinal to general anesthesia was performed after the fetal delivery. Immediate embolic devascularization of abnormal neovasculature was directly observed and facilitated adhesiolysis. Surgical blood losses were 1850 g, 2500 g, and 1180 g, respectively. CONCLUSION: Cesarean hysterectomy combined with endovascular embolization in the HOR for placenta percreta is an advantageous option to enhance patient safety by multidisciplinary approach without patient transfer.

Targeting Oxygen-Sensing Prolyl Hydroxylase for Metformin-Associated Lactic Acidosis Treatment.

Metformin is one of the most widely used therapeutics for type 2 diabetes mellitus and also has anticancer and antiaging properties. However, it is known to induce metformin-associated lactic acidosis (MALA), a severe medical condition with poor prognosis, especially in individuals with renal dysfunction. Inhibition of prolyl hydroxylase (PHD) is known to activate the transcription factor hypoxia-inducible factor (HIF) that increases lactate efflux as a result of enhanced glycolysis, but it also enhances gluconeogenesis from lactate in the liver that contributes to reducing circulating lactate levels. Here, we investigated the outcome of pharmaceutical inhibition of PHD in mice with MALA induced through the administration of metformin per os and an intraperitoneal injection of lactic acid. We found that the PHD inhibitors significantly increased the expression levels of genes involved in gluconeogenesis in the liver and the kidney and significantly improved the survival of mice with MALA. Furthermore, the PHD inhibitor also improved the rate of survival of MALA induced in mice with chronic kidney disease (CKD). Thus, PHD represents a new therapeutic target for MALA, which is a critical complication of metformin therapy.

Persistent hyperglycemia modulates gut immune function and microbiota in rats.

BACKGROUND: Since hyperglycemia-induced cellular dysfunction could be associated with alterations of the immune system, we tested the hypothesis that hyperglycemia augments the aberrant immune responses such as inflammation and differentiation of CD4(+) T lymphocytes in the mesenteric lymph nodes (MLNs), and induces alterations of microbiota both under physiological and pathological conditions. METHODS: Male Wistar rats were randomly allocated into 4 groups: Control and Endotoxemia (lipopolysaccharide, LPS 1 mg/kg) with or without hyperglycemia. The hyperglycemia groups were administered glucose solution (10-40 %), while the normoglycemia groups were administered saline. Alterations of the mRNA expressions of inflammatory cytokines and CD4(+) T lymphocyte transcriptional factor expressions in the MLNs, and those of the intestinal microbiota were analyzed at 24 hr. RESULTS: Hyperglycemia was kept approximately 250-350 mg/dL during the 24 hr study period. At the end of 24 hr, hyperglycemia augmented the mRNA expressions of interleukin (IL)-1ß and IL-6 in the MLNs, while both the helper T (Th) 2 and regulatory-T (Treg) transcriptional factors were simultaneously up-regulated under non-endotoxemic condition. LPS injection significantly modulated the obligate anaerobe bacterial populations of the Bacteroidetes class, and altered the population sizes of the Clostridium perfringens and the Bacteroides fragilis subgroup. Hyperglycemia did not enhance these alterations of the microbiota evoked by LPS, although it did modify the bacterial populations of the L. reuteri subgroup and staphylococci in healthy condition without endotoxemia. CONCLUSIONS: The present study indicates that both gut immune function and microbiota are significantly modulated by persistent hyperglycemia.

Sodium sulfide prevents water diffusion abnormality in the brain and improves long term outcome after cardiac arrest in mice.

AIM OF THE STUDY: Sudden cardiac arrest (CA) is one of the leading causes of death worldwide. Previously we demonstrated that administration of sodium sulfide (Na(2)S), a hydrogen sulfide (H(2)S) donor, markedly improved the neurological outcome and survival rate at 24 h after CA and cardiopulmonary resuscitation (CPR) in mice. In this study, we sought to elucidate the mechanism responsible for the neuroprotective effects of Na(2)S and its impact on the long-term survival after CA/CPR in mice. METHODS: Adult male mice were subjected to potassium-induced CA for 7.5 min at 37°C whereupon CPR was performed with chest compression and mechanical ventilation. Mice received Na(2)S (0.55 mgkg(-1) i.v.) or vehicle 1 min before CPR. RESULTS: Mice that were subjected to CA/CPR and received vehicle exhibited a poor 10-day survival rate (4/12) and depressed neurological function. Cardiac arrest and CPR induced abnormal water diffusion in the vulnerable regions of the brain, as demonstrated by hyperintense diffusion-weighted imaging (DWI) 24 h after CA/CPR. Extent of hyperintense DWI was associated with matrix metalloproteinase 9 (MMP-9) activation, worse neurological outcomes, and poor survival rate at 10 days after CA/CPR. Administration of Na(2)S prevented the development of abnormal water diffusion and MMP-9 activation and markedly improved neurological function and long-term survival (9/12, P<0.05 vs. Vehicle) after CA/CPR. CONCLUSION: These results suggest that administration of Na(2)S 1 min before CPR improves neurological function and survival rate at 10 days after CA/CPR by preventing water diffusion abnormality in the brain potentially via inhibiting MMP-9 activation early after resuscitation.