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Therapeutic hypothermia (HT) is standard care for term infants with hypoxic–ischaemic (HI) encephalopathy. However, the efficacy of HT in preclinical models, such as the Vannucci model of unilateral HI in the newborn rat, is often greater than that reported from clinical trials. Here, we report a meta-analysis of data from every experiment in a single laboratory, including pilot data, examining the effect of HT in the Vannucci model. Across 21 experiments using 106 litters, median (95% CI) hemispheric area loss was 50.1% (46.0–51.9%; n = 305) in the normothermia group, and 41.3% (35.1–44.9%; n = 317) in the HT group, with a bimodal injury distribution. Median neuroprotection by HT was 17.6% (6.8–28.3%), including in severe injury, but was highly-variable across experiments. Neuroprotection was significant in females (p p = 0.07). Animals representing the median injury in each group within each litter (n = 277, 44.5%) were also analysed using formal neuropathology, which showed neuroprotection by HT throughout the brain, particularly in females. Our results suggest an inherent variability and sex-dependence of the neuroprotective response to HT, with the majority of studies in the Vannucci model vastly underpowered to detect true treatment effects due to the distribution of injury.

Background: Patients developing meningococcal septic shock reveal levels of Neisseria meningitidis (106-108/mL) and endotoxin (101-103 EU/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h. Objective: To investigate transcriptional profiles in heart, lungs, kidneys, liver, and spleen and immunostain key inflammatory cells and proteins in post mortem formalin-fixed, paraffin-embedded (FFPE) tissue samples from meningococcal septic shock patients. Patients and Methods: Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Differential expression of genes was detected using Affymetrix microarray analysis. Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. Inflammatory mediators were quantified in lysates from FF tissues. Results: The transcriptional profiles showed a complex pattern of protein-coding and non-coding RNAs with significant regulation of pathways associated with organismal death, cell death and survival, leukocyte migration, cellular movement, proliferation of cells, cell-to-cell signaling, immune cell trafficking, and inflammatory responses in an organ-specific clustering manner. The canonical pathways including acute phase response-, EIF2-, TREM1-, IL-6-, HMBG1-, PPAR signaling, and LXR/RXR activation were associated with acute heart, pulmonary, and renal failure. Fewer genes were regulated in the liver and particularly in the spleen. The main upstream regulators were TNF, IL-1β, IL-6, RICTOR, miR-6739-3p, and CD3. Increased numbers of inflammatory cells (CD68+, MPO+, CD3+, and CD20+) were found in lungs and heart. PAI-1 inhibiting fibrinolysis and MCP-1 attracting leukocyte were found significantly present in the septic tissue samples compared to the controls. Conclusions: FFPE tissue samples can be suitable for gene expression studies as well as immunostaining of specific cells or molecules. The most pronounced gene expression patterns were found in the organs with highest levels of Neisseria meningitidis DNA. Thousands of protein-coding and non-coding RNA transcripts were altered in lungs, heart and kidneys. We identified specific biomarker panels both protein-coding and non-coding RNA transcripts, which differed from organ to organ. Involvement of many genes and pathways add up and the combined effect induce organ failure.

Background Dysplasia and superficial esophageal cancer should initially be treated endoscopically. Little is known about post-procedural health-related quality of life (HRQL). The aim of this study was to present our results with endoscopic treatment and post-procedural HRQL. Materials and methods From June 2014 to December 2018, all patients treated with endoscopic mucosal resection (EMR) and/or radiofrequency ablation (RFA) for low-grade dysplasia (LGD), high-grade dysplasia (HGD), T1a and a minority of patients with T1b at Oslo University Hospital were prospectively included. In June 2019, all patients alive were scored according to the Ogilvie dysphagia score as well as the QLQ-C30 and QLQ-OG25 for assessment of HRQL. Results Eighty-six patients were treated out of whom 22 (26%) had LGD, 44 (51%) HGD, 13 (15%) T1a, and six patients (7%) T1b. Histology revealed adenocarcinoma in 18 (21%) and squamous cell carcinoma in one (1%), respectively. The mean follow-up was 22.9 months. Tumor regression or downstaging was archived in 78% of the patients with LGD, 66% of patients with HGD and in 89% of patients with T1a/b. Five patients (6%) had esophagectomy. There were few and no serious complications. The 90-days mortality was 1%. Fifty-two patients (88%) experienced no dysphagia (Ogilvie score 0). There was no difference in 11 out of the 15 variables in QLQ-C30 when compared to a non-cancerous reference population. Conclusions Endoscopic treatment is safe and efficient for treatment of dysplasia and superficial esophageal cancer. The two-years post-procedural level of HRQL and dysphagia was satisfactory.

Cannabidiol (CBD), a nonpsychoactive cannabinoid, has shown neuroprotective actions after neonatal hypoxia-ischemia (HI) in animals. We wanted to further explore the effects of CBD, alone and in conjunction with hypothermia, in a piglet model of global HI. Fifty-five anesthetized newborn piglets were randomized to either controls (n = 7) or HI (n = 48) by ventilation with 8% O2 until mean arterial blood pressure reached 20 mmHg and/or base excess reached −20 mmol/l. After resuscitation piglets were randomized to either: vehicle (VEH), CBD 1mg/kg, VEH+hypothermia (H) or CBD 1mg/kg+H (each n = 12). Piglets were euthanized 9.5 h after HI and plasma, urine, cerebrospinal fluid, and brain tissue were sampled for analysis. HI induced global damage with significantly increased neuropathology score, S100B in cerebrospinal fluid, hippocampal proton magnetic resonance spectroscopy biomarkers, plasma troponin-T, and urinary neutrophil gelatinase-associated lipocalin. CBD alone did not have any significant effects on these parameters while CBD+H reduced urinary neutrophil gelatinase-associated lipocalin compared with VEH+H (P < 0.05). Both hypothermic groups had significantly lower glutamate/N-acetylaspartate ratios (P < 0.01) and plasma troponin-T (P

BACKGROUND Surgery is considered necessary to achieve a cure for oesophageal cancer. Minimally invasive oesophageal resection is increasingly performed with the aim of reducing the number of complications compared with open surgery. The purpose of this study was to investigate postoperative complications, mortality and long-term survival following hybrid oesophageal resection by laparoscopy and thoracotomy.MATERIAL AND METHOD Patients with oesophageal cancer who underwent hybrid resection with curative intent at Oslo University Hospital Ulleval from 1 November 2007 to 1 June 2013 were included (n = 109). Complications were graded according to the Clavien-Dindo classification and survival figures were recorded.RESULTS Median age was 65 years, 79 % were men. Altogether 118 complications were recorded in 70 patients (64.2 %). Distribution of complications was 1.8 % for stage I, 29.4 % for stage II, 22.1 % for stage III and 11.0 % for stage IV. Anastomotic leakage occurred in 4.6 %. There was no postoperative mortality. The proportion of R0 resections with microscopic radicality was 91 % (n = 100). For the entire patient population, the estimated 5-year survival rate was 48 % (95 % CI 36 - 60 %), for R0 resection 51 % (38 - 63 %) and for R1-2 resection 0 %. Estimated median survival with R0-2, R0 and R1-2 resection was 55, 55 and 10 months (0 - 28 months), respectively. R status and stage had a significant bearing on survival.INTERPRETATION There was a low percentage of serious complications, no mortality and few anastomotic leakages after hybrid resection for oesophageal cancer. The 5-year survival rate was good.

In a consecutive hospital-based autopsy series, we examined the relationship between apolipoprotein E (apoE) and Alzheimer’s disease (AD) and investigated the clinicopathological relationship in AD. The study population included 99 patients (mean age 81 years) with AD-related neuropathological findings at death, of whom 83 were diagnosed with AD according to the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) classification, and a control group of patients without neurodegenerative disease (n = 1429). The patients were apoE genotyped and the density of β-amyloid senile plaques, neuritic plaques and neurofibrillary tangles was estimated in the cortex and hippocampus. The utility of immunohistochemical staining using an antibody directed against apoE4 in paraffin-embedded tissue was also evaluated. Among patients with “definite AD” according to CERAD, 65 % were e4 carriers, compared to 32 % among controls (p

BACKGROUND: Hyperthermia after hypoxia-ischaemia (HI) in newborn infants is associated with worse neurological outcomes. Loss of thermoregulation may also be associated with greater injury.METHODS: In the postnatal-day 7 (P7) rat, the effect of 5h of graded hyperthermia (38°C or 39°C) immediately after unilateral HI was compared to normothermia (NT, 37°C), and therapeutic hypothermia (TH, 32°C). Early (negative geotaxis) and late (staircase test) behavioural testing was performed, as well as neuropathology scoring in adulthood. Separately, P7 rats were exposed to HI, and individual nesting temperatures monitored before analysis of neuropathology at P14.RESULTS: Mortality increased as temperature was increased from 38°C (0%) to 39°C (50%) after HI. Hyperthermia also resulted in early behavioural deficits compared to NT animals. In adulthood, pathology scores in the thalamus, basal ganglia, cortex, and hippocampus increased as post-hypoxic temperature increased above NT. Significant global neuroprotection was seen in the TH group. However, no significant difference was seen between HI groups in the staircase test. One hour after HI, the core temperature of pups was inversely correlated with global pathology scores at P14.CONCLUSION: Early temperature is a significant determinant of injury after experimental HI. Spontaneous decreases in core temperature after HI may confound neuroprotection studies.Pediatric Research (2017); doi:10.1038/pr.2017.51.

Background. After perinatal asphyxia, the cerebellum presents more damage than previously suggested. Objectives. To explore if the antioxidant N-acetylcysteine amide (NACA) could reduce cerebellar injury after hypoxia-reoxygenation in a neonatal pig model. Methods. Twenty-four newborn pigs in two intervention groups were exposed to 8% oxygen and hypercapnia, until base excess fell to −20 mmol/l or the mean arterial blood pressure declined to n=12) or saline (vehicle-treated group, n=12). One sham-operated group (n=5) served as a control and was not subjected to hypoxia. Observation time after the end of hypoxia was 9.5 hours. Results. The intranuclear proteolytic activity in Purkinje cells of asphyxiated vehicle-treated pigs was significantly higher than that in sham controls (p=0.03). Treatment with NACA was associated with a trend to decreased intranuclear proteolytic activity (p=0.08), There were significantly less mutations in the mtDNA of the NACA group compared with the vehicle-treated group, 2.0 × 10−4 (±2.0 × 10−4) versus 4.8 × 10−5(±3.6 × 10−4, p<0.05). Conclusion. We found a trend to lower proteolytic activity in the core of Purkinje cells and significantly reduced mutation rate of mtDNA in the NACA group, which may indicate a positive effect of NACA after neonatal hypoxia. Measuring the proteolytic activity in the nucleus of Purkinje cells could be used to assess the effect of different neuroprotective substances after perinatal asphyxia.

Background: Pulmonary hypertension is a severe complication to chronic lung diseases, often with alveolar hypoxia. Inflammasomes are large multiprotein oligomers consisting of receptor molecules (e.g. NLR), an adaptor protein (ASC) and the effector enzyme caspase-1. Assembly of the inflammasome leads to activation of caspase-1 which cleaves interleukin (IL)-18 and IL-1β to their bioactive forms. Elevated levels of IL-18 and IL-1β are found in patients with pulmonary hypertension. Objective: To study the role of the effector caspase-1 in hypoxia-induced pulmonary hypertension. Methods: WT and caspase-1 −/- mice were exposed to 2 weeks of 10% oxygen. Right ventricular systolic pressure (RVSP) was measured with a microtipped transducer catheter. Muscularization of distal arteries was quantified by immunohistochemistry (IHC) methods on lung sections. PCR and Western blotting were performed for mRNA and protein detection. Results: Caspase-1 −/- mice exposed to hypoxia had significantly lower RVSP and RV weight/tibial ratio than WT hypoxic mice, indicating reduced pulmonary hypertension. Hypoxic caspase-1 −/- mice showed less degree of muscularization of distal pulmonary arteries compared to WT mice exposed to hypoxia. No differences in accumulation of collagen in adventitia were found. Expression of inflammatory mediators was measured, and a robust reduction in IL-6 was measured in mice depleted of caspase-1. IL-6 is the main activator of STAT3 phosphorylation at tyrosine 705 which was significantly reduced in caspase-1 deficiency. Conclusions: Caspase-1 regulates hypoxia-induced pulmonary hypertension through generation of IL-6 and phosphorylated STAT3 leading to muscularization of pulmonary arteries.

Background The pathophysiology and outcome of meningococcal septic shock is closely associated with the plasma level of N. meningitidis lipopolysaccharides (LPS, endotoxin) and the circulating level of meningococcal DNA. The aim of the present study was to quantify the number of N. meningitidis in different formalin-fixed, paraffin-embedded (FFPE) tissue samples and fresh frozen (FF) tissue samples from patients with systemic meningococcal disease (SMD), to explore the distribution of N. meningitidis in the body. Methods DNA in FFPE and FF tissue samples from heart, lungs, liver, kidneys, spleen and brain from patients with meningococcal shock and controls (lethal pneumococcal infection) stored at variable times, were isolated. The bacterial load of N. meningitidis DNA was analyzed using quantitative real-time PCR (qPCR) and primers for the capsule transport A (ctrA) gene (1 copy per N. meningitidis DNA). The human beta-hemoglobin (HBB) gene was quantified to evaluate effect of the storage times (2-28 years) and storage method in archived tissue. Results N. meningitidis DNA was detected in FFPE and FF tissue samples from heart, lung, liver, kidney, and spleen in all patients with severe shock. In FFPE brain, N. meningitidis DNA was only detected in the patient with the highest concentration of LPS in the blood at admission to hospital. The highest levels of N. meningitidis DNA were found in heart tissue (median value 3.6 × 107 copies N. meningitidis DNA/μg human DNA) and lung tissue (median value 3.1 × 107 copies N. meningitidis DNA/μg human DNA) in all five patients. N. meningitidis DNA was not detectable in any of the tissue samples from two patients with clinical meningitis and the controls (pneumococcal infection). The quantity of HBB declined over time in FFPE tissue stored at room temperature, suggesting degradation of DNA. Conclusions High levels of N. meningitidis DNA were detected in the different tissue samples from meningococcal shock patients, particularly in the heart and lungs suggesting seeding and major proliferation of meningococci in these organs during the development of shock, probably contributing to the multiple organ failure. The age of archived tissue samples appear to have an impact on the amount of quantifiable N. meningitidis DNA.

Objective: The aim of this study was to non-invasively explore new methods of ultrasound attenuation measurements in livers of patients with Non-Alcoholic-Fatty-Liver-Disease (NAFLD) and to measure the liver tissue elasticity. Material and method: Sixteen patients with NAFLD, twelve patients with liver fibrosis and fifteen healthy subjects were included. Echo Levels (ELs) in dB were measured at 2 and 7cm depths in the right liver to calculate the attenuation. ELs were measured in liver and right kidney tissue to calculate the Hepato-Renal Index (HRI). This index was calculated both as a difference, HRIdiff; (EL Liver –EL Kidney) and HRI-ratio; (EL Liver / EL Kidney) using built-in software of the ultrasound scanner. Liver tissue elasticity was measured using transient elastography (TE, Fibroscan®). NAFLD and liver fibrosis were confirmed by liver biopsy. Results: We found that HRI- diff was significantly higher in the NAFLD group compared with healthy subjects, 6.2 dB (0.8-11.4) vs.1. 9 dB (0.0-6.1), p=0.012. HRI- ratio was significantly lower between the same two groups, 0.9 dB (0.81.02) vs.1.01 dB (0.9-1.12), and p

Background: Cannabidiol (CBD) is considered a promising neuroprotectant after perinatal hypoxia-ischemia (HI). We have previously studied the effects of CBD 1 mg/kg in the early phase after global HI in piglets. In contrast to prior studies, we found no evidence of neuroprotection and hypothesized that higher doses might be required to demonstrate efficacy in this animal model. Objective: To assess the safety and potential neuroprotective effects of high-dose CBD. Methods: Anesthetized newborn piglets underwent global HI by ventilation with 8% O2 until the point of severe metabolic acidosis (base excess -20 mmol/L) and/or hypotension (mean arterial blood pressure ≤20 mm Hg). Piglets were randomized to intravenous treatment with vehicle (n = 9) or CBD (n = 13). The starting dose, CBD 50 mg/kg, was reduced if adverse effects occurred. The piglets were euthanized 9.5 h after HI and tissue was collected for analysis. Results: CBD 50 mg/kg (n = 4) induced significant hypotension in 2 out of 4 piglets, and 1 out of 4 piglets suffered a fatal cardiac arrest. CBD 25 mg/kg (n = 4) induced significant hypotension in 1 out of 4 piglets, while 10 mg/kg (n = 5) was well tolerated. A significant negative correlation between the plasma concentration of CBD and hypotension during drug infusion was observed (p < 0.005). Neuroprotective effects were evaluated in piglets that did not display significant hypotension (n = 9) and CBD did not alter the degree of neuronal damage as measured by a neuropathology score, levels of the astrocytic marker S100B in CSF, magnetic resonance spectroscopy markers (Lac/NAA and Glu/NAA ratios), or plasma troponin T. Conclusions: High-dose CBD can induce severe hypotension and did not offer neuroprotection in the early phase after global HI in piglets.

Immediate early gene nerve growth factor-induced clone B (NGFI-B), a nuclear receptor important for differentiation and apoptosis, is expressed in mice and rat cerebellum from an early stage of postnatal development. Following apoptotic stimuli NGFI-B translocates to mitochondria to initiate cell death processes. Controlled cell death is critical for correct cerebellar development. Immunohistochemical analysis of NGFI-B in sections of mice cerebella showed NGFI-B to be expressed in granule neurons in vivo at a time (P8-11) when apoptosis is known to occur. The importance of NGFI-B for apoptosis of cultured rat cerebellar granule neurons was investigated by inducing apoptosis with calcium ionophore A23187 (CaI, 0.1μM). Imaging studies of gfp-tagged NGFI-B confirmed that mitochondrial translocation of NGFI-B occurred following treatment with CaI and was reduced by addition of 9-cis-retinoic acid (1μM), a retinoid X receptor (RXR) agonist that prevents dimerization of RXR and NGFI-B that is known to occur before translocation. Consequently, 9-cis-retinoic acid partly reduced cell death. To address the causality of NGFI-B in apoptosis further, knock-down by siRNA was performed and it removed 85% of the NGFI-B protein. This resulted in a complete inhibition of apoptosis after CaI exposure. Together these findings suggest that NGFI-B plays a role in controlling correct cerebellar development.

BACKGROUND Surgery is considered necessary to achieve a cure for oesophageal cancer. Minimally invasive oesophageal resection is increasingly performed with the aim of reducing the number of complications compared with open surgery. The purpose of this study was to investigate postoperative complications, mortality and long-term survival following hybrid oesophageal resection by laparoscopy and thoracotomy.MATERIAL AND METHOD Patients with oesophageal cancer who underwent hybrid resection with curative intent at Oslo University Hospital Ullevål from 1 November 2007 to 1 June 2013 were included (n = 109). Complications were graded according to the Clavien-Dindo classification and survival figures were recorded.RESULTS Median age was 65 years, 79 % were men. Altogether 118 complications were recorded in 70 patients (64.2 %). Distribution of complications was 1.8 % for stage I, 29.4 % for stage II, 22.1 % for stage III and 11.0 % for stage IV. Anastomotic leakage occurred in 4.6 %. There was no postoperative mortality. The proportion of R0 resections with microscopic radicality was 91 % (n = 100). For the entire patient population, the estimated 5-year survival rate was 48 % (95 % CI 36 - 60 %), for R0 resection 51 % (38 - 63 %) and for R1-2 resection 0 %. Estimated median survival with R0-2, R0 and R1-2 resection was 55, 55 and 10 months (0 - 28 months), respectively. R status and stage had a significant bearing on survival.INTERPRETATION There was a low percentage of serious complications, no mortality and few anastomotic leakages after hybrid resection for oesophageal cancer. The 5-year survival rate was good.

Mice lacking glycosylated lysosomal membrane protein (Glmp gt/gt mice) have liver fibrosis as the predominant phenotype due to chronic liver injury. The Glmp gt/gt mice grow and reproduce at the same rate as their wild-type siblings. Life expectancy is around 18 months. Wild-type and Glmp gt/gt mice were studied between 1 week and 18 months of age. Livers were analyzed using histological, immunohistochemical, biochemical, and qPCR analyses. It was shown that Glmp gt/gt mice were not born with liver injury; however, it appeared shortly after birth as indicated by excess collagen expression, deposition of fibrous collagen in the periportal areas, and increased levels of hydroxyproline in Glmp gt/gt liver. Liver functional tests indicated a chronic, mild liver injury. Markers of inflammation, fibrosis, apoptosis, and modulation of extracellular matrix increased from an early age, peaking around 4 months of age and followed by attenuation of these signals. To compensate for loss of hepatocytes, the oval cell compartment was activated, with the highest activity of the oval cells detected at 3 months of age, suggesting insufficient hepatocyte proliferation in Glmp gt/gt mice around this age. Although constant proliferation of hepatocytes and oval cells maintained adequate hepatic function in Glmp gt/gt mice, it also resulted in a higher frequency of liver tumors in older animals. The Glmp gt/gt mouse is proposed as a model for slowly progressing liver fibrosis and possibly as a model for a yet undescribed human lysosomal disorder.

Numerous studies have addressed the association between the apolipoprotein E polymorphism and cardiovascular disease, but only a few reports are based on findings at autopsy. In the present retrospective study, we have used autopsy findings from a general hospital population to further investigate this issue.We collected information from 1522 consecutive autopsy reports (886 men, mean age 65.7 years; 636 women, mean age 69.7 years) conducted at Oslo University Hospital, Norway, in the period from 1996 to 2000. Cause of death and signs related to cardiovascular disease including the degree of atherosclerosis in the aorta and the coronary arteries, signs of myocardial infarction, heart weight, and signs of cerebrovascular disease were recorded. The patients were genotyped, and the apolipoprotein E allele frequencies (ɛ2, 8.0%; ɛ3, 72.6%; and ɛ4, 19.4%) were not statistically different from a group of healthy controls. Approximately 35% of the patients died from a cardiovascular disease. Genotypes differed significantly (P.05), with more ɛ4-carriers (34.3% vs. 29.6%) and fewer ɛ2-carriers (11.8% vs. 13.9%) among patients who died from cardiovascular disease compared to those who died from other causes. A similar distribution of genotypes was seen in patients recorded with myocardial infarction or cerebrovascular disease. There was an association between the presence of ɛ4 and atherosclerosis in the aorta and coronary arteries, but this did not reach statistical significance. Among patients with signs of coronary heart disease, standardized heart weights were significantly higher in ɛ2-carriers compared to ɛ4-carriers.The present autopsy study suggests that the risk of developing and dying from cardiovascular disease, including coronary heart disease and cerebrovascular disease, is influenced by the apolipoprotein E polymorphism.

Interleukin (IL)-18 is a pro-inflammatory cytokine suggested to be involved in the development of pulmonary emphysema and inflammation. Studies involving immunology and cancer have revealed that IL-18 can have synergistic effects with IL-12. We have studied the presence of IL-18 and IL-12 receptors (IL-18R/IL-12R) in the lungs and whether IL-18 and IL-12, alone or in combination, have the ability to initiate the induction of mediators related to the development of emphysema and inflammation. The expression of the IL-18R was abundant in lungs compared to other organs (heart, liver, and spleen), and the IL-12R was also expressed in lung tissue. Mice treated with i.p. injection of recombinant murine IL-18 or IL-12 expressed significantly higher pulmonary mRNA levels of the matrix degrading enzymes metalloproteinase (MMP) 12 and cathepsin S, in addition to interferon-γ, tumor necrosis factor-α, and CXC chemokine ligand 9 (CXCL9) (all P < .05) than controls (received PBS). Treatment with IL-18 and IL-12 in combination showed an even more pronounced induction of these mediators, as well as a significant increase in MMP-9, IL-6, IL-1β, and transforming growth factor-β (P < .05). Furthermore, cellular apoptosis in lung tissue was induced. Immunohistochemical analysis revealed T-cell infiltration in pulmonary vessels following co-stimulation. In summary, IL-18 and IL-12 exert a synergistic effect on the lungs by inducing MMPs, cathepsins S, and pro-inflammatory cytokines, which may promote pulmonary emphysema and inflammation. The synergy between IL-18 and IL-12 involves infiltration of T-cells in the lungs, possibly induced by the T-cell chemoattractant CXCL9.

Mild therapeutic hypothermia (HT) reduces brain injury in survivors after perinatal asphyxia. Recent guidelines suggest that resuscitation of term infants should be started with air, but supplemental oxygen is still in use. It is not known whether supplemental oxygen during resuscitation affects the protection offered by subsequent HT.Wilcoxon median (95% confidence interval) hippocampal injury scores (range 0.0-4.0; 0 to ≥90% injury) were 21% O(2) normothermia (NT): 2.00 (1.25-2.50), 21% O(2) HT: 1.00 (0.50-1.50), 100% O(2) NT: 2.50 (1.50-3.25), and 100% O(2) HT: 2.00 (1.25-2.50). Although HT significantly reduced hippocampal injury (B = -0.721, SEM = 0.297, P = 0.018), reoxygenation with 100% O(2) increased injury (B = +0.647, SEM = 0.297, P = 0.033). Regression constant B = 1.896, SEM = 0.257 and normally distributed residuals.We confirm an ~50% neuroprotective effect of therapeutic HT in the neonatal rat. Reoxygenation with 100% O(2) increased injury and worsened reflex performance. HT was neuroprotective whether applied after reoxygenation with air or 100% O(2). However, HT after 100% O(2) gave no net neuroprotection.In an established neonatal rat model, hypoxia-ischemia (HI) was followed by 30-min reoxygenation in either 21% O(2) or 100% O(2) before 5 h of NT (37 °C) or HT (32 °C). The effects of HT and 100% O(2) on histopathologic injury in the hippocampus, basal ganglia, and cortex, and on postural reflex performance 7 d after the insult, were estimated by linear regression.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a clinically defined neurological condition after lack of oxygen and often associated with cardiac dysfunction in term infants. Therapeutic hypothermia (HT) after birth is neuroprotective in infants with HIE. However, it is not known whether HT is also cardioprotective. Four newborn pigs were used in the pilot study and a further 18 newborn pigs [randomly assigned to 72 h normothermia (NT) or 24 h HT followed by 48 h NT] were subjected to global HIE insults. Serum cTnI was measured before and post the HIE insult. Blood pressure, inotropic support, blood gases, and heart rate (HR) were recorded throughout. Cardiac pathology was assessed from histological sections. Cooling reduced serum cTnI levels significantly in HT pigs by 6 h (NT, 1.36 ± 0.67; HT, 0.34 ± 0.23 ng/mL; p = 0.0009). After rewarming, from 24 to 30 h postinsult, HR and cTnI increased in the HT group; from HR[24 h] = 117 ± 22 to HR[30 h] = 218 ± 32 beats/min (p = 0.0002) and from cTnI[24 h] = 0.23 ± 0.12 to cTnI[30 h] = 0.65 ± 0.53 ng/mL, (p = 0.05). There were fewer ischemic lesions on cardiac examination (37%) in the HT group compared with the NT group (70%). HT (24 h) pigs did not have the postinsult cTnI increase seen in NT-treated pigs. There was a trend that HT improved cardiac pathology in this 3-d survival model.

Glucocorticoid (GC) treatment in premature infants may have detrimental effects on the immature brain. Here we show that GCs dexamethasone (Dex) and hydrocortisone (HC) reduce proliferation and induce differentiation of chicken embryo cerebellar neurons in vivo and in vitro. Granule neurons incorporating bromodeoxyuridine were reduced in the internal granular layer (IGL) after 24-h exposure to both substances on embryonic day 17, with Dex about 100-fold more potent than HC. The effects were blocked by GR antagonist RU 38486. Both GCs also increased the expression of neuronal differentiation markers microtubule-associated protein 2 (Map2) and neuronal nuclei protein (NeuN), measured by western blotting of whole cerebellar lysates and immunohistochemistry, respectively. Treatment of cerebellar granule neuron cultures with both GCs significantly reduced the percentage of proliferating-cell nuclear antigen (PCNA) positive neurons and increased NeuN positive neurons, with similar dose-response relationship as in vivo. The cytostatic agent cytosine arabinoside showed comparable effects both on proliferation and differentiation. In conclusion, the effects of Dex and HC on chicken cerebellar granule neuron proliferation are GR mediated and reflect their pharmacological potency. In addition, the effects on differentiation may be related to a cell cycle block per se, since cytosine arabinoside mimicked the effect of the GCs.

The chemokine receptor CCR7 regulates lymphocyte trafficking, and CCR7 deficiency induces infiltration of T and B cells adjacent to vessels in mouse lungs. Perivascular infiltration of T and B cells has also been found in human pulmonary arterial hypertension, and downregulation of the CCR7 receptor in circulating leukocytes of such patients has been observed. To investigate whether changes in the CCR7 system contribute to the pathogenesis of pulmonary hypertension, we utilized mice deficient of the CCR7 receptor. The cardiopulmonary and inflammatory responses of CCR7 depletion were evaluated in CCR7-deficient and wild-type mice. Measurements of cytokines upregulated in the animal model were also performed in patients with pulmonary hypertension and controls and in vascular smooth muscle cells. We found that mice lacking CCR7 had increased right ventricular systolic pressure, reduced pulmonary artery acceleration time, increased right ventricular/tibial length ratio, Rho kinase-mediated pulmonary vasoconstriction, and increased muscularization of distal arteries, indicating pulmonary hypertension. These mice also showed increased perivascular infiltration of leukocytes, consisting mainly of T and B cells, and increased mRNA levels of the inflammatory cytokines interleukin-12 and CX3CL1 within pulmonary tissue. Increased serum levels of interleukin-12 and CX3CL1 were also observed in patients with pulmonary hypertension, particularly in those with pulmonary hypertension associated with connective tissue disorder. In smooth muscle cells, interleukin-12 induced secretion of the angiogenic cytokine interleukin-8. We conclude that these results suggest a role for CCR7 in the development of pulmonary arterial hypertension, at least in some subgroups, possibly via pulmonary infiltration of lymphocytes and secretion of interleukin-12 and CX3CL1.

Serotonin syndrome is a potentially life-threatening reaction that occurs in patients using drugs that elevate the serotonin level in the body. Excess serotonergic activity in the CNS and peripheral serotonin receptors results in neuromuscular hyperactivity, mental changes and autonomic symptoms. Hyperthermia is a characteristic feature of the syndrome. We describe neuropathological findings from two cases of lethal serotonin syndrome, both patients presenting with hyperthermia and neuromuscular symptoms. One of the patients had been taking amitriptylin and mirtazapin and the other had used amitriptylin and citalopram. They died, respectively, 10 days and 2½ months after the onset of serotonin syndrome symptoms. Post-mortem examination of the brains showed subtotal loss of cerebellar Purkinje cells in both cases. In the case with shorter survival time, areas with partial loss of cerebellar granule cells were observed, whereas in the case with longer survival time general and extensive loss of granule cells was found. Cells in other areas of the brain known to be sensitive to hypoxic injury were not affected. Selective loss of Purkinje cells has previously been described in neuroleptic malignant syndrome and heatstroke, conditions that are characterized by hyperthermia. This suggests that hyperthermia may be a causative factor of brain damage in serotonin syndrome. This is the first report describing neuropathological findings in serotonin syndrome.

Background Therapeutic hypothermia has become the standard of care for newborns with hypoxic-ischemic encephalopathy in high and middle income countries. Docosahexaenoic acid (DHA) has neuroprotective properties of reducing excitotoxicity, neuroinflammation and apoptosis in rodent models. We aim to study whether post hypoxic administration of i.v. DHA will reduce H+MRS biomarkers and gene expression of inflammation and apoptosis both with and without hypothermia in a large animal model. Methods Fifty-five piglets were randomized to severe global hypoxia (N = 48) or not (Sham, N = 7). Hypoxic piglets were further randomized by factorial design: Vehicle (VEH), DHA, VEH + Hypothermia (HT), or DHA + HT. 5 mg/kg DHA was given intravenously 210 min after end of hypoxia. Two-way ANOVA analyses were performed with DHA and hypothermia as main effects. Results Cortical lactate/N-acetylaspartate (Lac/NAA) was significantly reduced in DHA + HT compared to HT. DHA had significant main effects on increasing N-acetylaspartate and glutathione in hippocampus. Therapeutic hypothermia significantly reduced the Lac/NAA ratio and protein expression of IL-1β and TNFα in hippocampus and reduced Troponin T in serum. Neuropathology showed significant differences between sham and hypoxia, but no differences between intervention groups. Conclusion DHA and therapeutic hypothermia significantly improve specific H+MRS biomarkers in this short-term follow up model of hypoxia-ischemia. Longer recovery periods are needed to evaluate whether DHA can offer translational neuroprotection.

Objectives Recent studies suggest that activin A, a member of the transforming growth factor (TGF) superfamily, is involved in the pathogenesis of liver disorders. We sought to explore its possible role in non-alcoholic fatty liver disease (NAFLD). Methods Serum levels of activin A and its natural inhibitor, follistatin, were measured in patients with NAFLD (n=70) and in control subjects (n=30). Gene expression was quantified in liver biopsies obtained from patients with NAFLD (n=13) and controls (n=6). Effects of activin A were examined in Huh7 (human hepatoma cell line) hepatocytes. Results Patients with NAFLD had significantly elevated serum levels of activin A and follistatin compared with healthy controls. In patients with non-alcoholic steatohepatitis (NASH, n=38), there were particularly high levels of activin A that were significantly related to the degree of hepatic fibrosis. Liver biopsies from NAFLD patients showed a markedly increased activin A-follistatin mRNA ratio, indicating increased hepatic activin A activity. In hepatocytes, activin A enhanced the expression of collagen and TGF-beta(1), promoted matrix metalloproteinase activity, induced mitochondrial beta-oxidation, downregulated fatty acid (FA) synthase activity, promoted decreased weight percentage of saturated FAs, and altered the composition of polyunsaturated FAs. Conclusions Our findings support the complex role of activin A in the pathogenesis of NAFLD, involving effects on fibrosis and lipid accumulation.

Experimental animal models indicate that complement contributes to tissue damage during brain ischaemia and stroke, but limited data are available for a role of the complement in human stroke. We, therefore, evaluated whether acute ischaemia leads to complement activation in human brain. Indirect immunohistochemical staining was performed on paraffin-embedded, formalin-fixed human brain from 10 patients and 10 controls. Complement components C1q, C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical pathway. C9, C-reactive protein and IgM were detected in necrotic zones. Marked CD59 and weak CD55 expression were found in normal brains, but these complement regulators were virtually absent in ischaemic lesions. Modest amounts of mannose-binding lectin (MBL), MBL-associated serine protease-2 and factor B were found in both ischaemic lesions and controls. These data suggest that increased deposition of complement components combined with decreased expression of complement regulators is a possible mechanism of tissue damage during ischaemia in human brain.

The antidiabetic agent metformin is regularly discussed as a promising treatment for non-alcoholic fatty liver disease (NAFLD), which is characterized by insulin resistance. However, the evidence for its beneficial effects is limited, and conflicting reports have been published. The purpose of this study was to conduct a randomized, double-blind, placebo-controlled trial to test whether metformin improves liver histology in patients with non-alcoholic fatty liver disease.Forty-eight patients with biopsy-proven NAFLD were randomized to treatment with metformin (n=24) or placebo (n=24) for 6 months. A second liver biopsy was obtained in all subjects who completed the trial (n=44). Data analyses are restricted to this group (per-protocol analyses). The primary outcome was changes in histologically assessed liver steatosis. Secondary outcomes were changes in NAFLD activity (NAS)-score, liver steatosis assessed by computed tomography (CT), liver transaminases, body-weight, metabolic variables and inflammatory markers.No significant differences between treatment with metformin or placebo were observed for changes in liver steatosis, assessed either histologically or by CT, NAS-score, liver transaminases or on markers of insulin resistance or inflammation. In contrast, beneficial effects of metformin were observed on changes in body-weight (p0.001), serum levels of cholesterol (p=0.004), LDL-cholesterol (p0.001), glucose (p=0.032) and on HbA1c (p=0.020).Treatment with metformin for 6 months was no better than placebo in terms of improvement in liver histology in patients with NAFLD. Nevertheless, the use of metformin could still be beneficial in this group as it is associated with a reduction in serum levels of lipids and glucose. (ClinicalTrials.gov number, NCT00303537).

Munkeby BH, de Lange C, Emblem KE, Bjørnerud A, Kro GAB, Andresen J, Winther-Larssen EH, Løberg EM, Hald JK. A piglet model for detection of hypoxic-ischemic brain injury with magnetic resonance imaging. Acta Radiol 2008;49:1049–1057. Background Early detection of hypoxic-ischemic (HI) injury in the asphyxic newborn is important because present prognostic factors are inadequate. Furthermore, therapeutic interventions may have additional benefit if initiated in time. Purpose To assess whether the use of a combined protocol including conventional magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI), diffusion tensor imaging (DTI), and proton MR spectroscopy (MRS) could detect pathological findings in a piglet model 7 hours after HI. Material and Methods Ten piglets were submitted to HI for 30 min followed by reoxygenation with 21% O2 for 7 hours. MRI at 1.5T was done prior to and 7 hours after the HI. Single-voxel proton MRS was performed, and apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were measured in the basal ganglia. MRS identified N-acetylaspartate (NAA), choline (Cho), creatine (Cr), and lactate (Lac). Histology and microtubule-associated protein 2 (MAP-2) staining was performed in the basal ganglia at the end of the experiment. Results Compared to baseline, ADC, NAA/Cho, and NAA/Cr were significantly reduced after 7 hours (P < 0.001, P = 00.01, and P = 00.05, respectively) and FA values were increased (P

High-potency glucocorticoids (GC) are used in the prophylaxis and treatment of neonatal bronchopulmonal dysplasia, but there is concern about side effects on the developing brain. Recently, the low-potency GC hydrocortisone (HC) has been suggested as an alternative to high-potency GC. We compared the neurotoxic effects of HC with the high-potency GC dexamethasone (DEX) in chicken cerebellum. A single dose of GC was injected into the egg at embryonic day 16 and the death of granule neurons in histologic sections of the cerebellar cortex was examined 24 h later. DEX and HC showed a similar dose-dependent induction of morphological apoptosis and caspase-3 activation in the internal granular layer. A doubling of the apoptosis rate compared to the basal rate was seen for the highest dose of DEX (5 mg/kg) and medium dose of HC (1 mg/kg). In cultures of embryonic chicken cerebellar granule cells, DEX and HC increased cell death and induced rapid caspase-3 activation in a similar dose-dependent manner. Transfection of granule cells with a luciferase reporter gene revealed that the dose needed for the activation of gene transcription (classical signalling pathway) with DEX was much lower than for HC. In conclusion, HC does not present itself as a safer drug than DEX in this model. In addition, it appears that DEX and HC induce apoptosis in immature granule neurons via a non-genomic (non-classical) mechanism.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract. The diagnosis of GIST is based on histology together with a panel of immunohistochemical markers; the most important is KIT (CD117). A total of 434 cases of GISTs were confirmed by histology and immunohistochemistry, and incorporated into tissue microarrays. Validation of histological features as well as the prognostic value of two immunohistochemical biomarkers (p16 and L1) was assessed. High mitotic rate, large tumor size, nuclear atypia, and small bowel primary site were all validated as negative prognostic factors in GISTs. Expression of p16 was significantly correlated with unfavorable prognosis, whereas L1 expression was not.

Xylose absorption testing has traditionally involved measurement of serum xylose and/or measurement of excreted xylose in urine. However, by enriching xylose with a 13C- or 14C-isotope, absorption of an oral xylose load will be reflected in the time-dependent pattern of 13CO2 or 14CO2 exhaled in breath. Our objectives were to evaluate the diagnostic properties of 13C-xylose and 14C-xylose breath tests in coeliac disease, and to develop a diagnostic breath test index.We reviewed data from 41 coeliac patients who underwent the 14C-xylose breath test before and after commencement of a gluten-free diet, and 60 coeliac patients who underwent the 13C-xylose breath test, 37 of whom repeated the test after starting a gluten-free diet. Coeliac patients were compared with healthy control subjects.Coeliac patients exhaled significantly less 13CO2 or 14CO2 than healthy controls during the first hour of the test and more isotope-labelled CO2 than control subjects after 3 h. Diagnostic accuracy was optimal with test duration of 210 min combining gas measurements at 30 min and 210 min in a simple fraction. This gas fraction index (30 min/210 min) distinguished between coeliac patients and healthy control subjects with 84-95% sensitivity and 87-94% specificity. After commencement of a gluten-free diet, the gas fraction index increased in most coeliac patients, but remained lower than that in healthy control subjects.13C-xylose- and 14C-xylose breath tests discriminate between coeliac patients and healthy control subjects with high sensitivity and specificity. The stable isotope 13C-xylose breath test has comparable diagnostic accuracy to the radioactive isotope 14C-xylose breath test and should be the preferred alternative to traditional xylose absorption tests.

Highlights • Hepatocellular carcinoma in ectopic liver tissue is extremely rare. • Multiple successful resections of recurrent ectopic hepatocellular carcinoma. • Postoperative follow-up every 6 month with AFP and abdominal CT imaging. • Early MRI of the liver to exclude intrahepatic disease., Introduction Hepatocellular carcinoma (HCC) in ectopic liver tissue is extremely rare. Presentation of case A 64-year-old woman presented initially with abdominal complaints. Computed tomography (CT) revealed a tumor in the diaphragm and laparoscopic resection of the tumor was performed. Histology showed HCC. During the next 4 years four more tumors, all of which showed HCC on histology and were located extrahepatically, was treated with laparoscopic resection. During this course the patient was followed with regular thoracoabdominal CT and measurement of serum alpha-fetoprotein (AFP). A negative magnetic resonance imaging (MRI) examination of the liver excluded a primary intrahepatic tumor. Discussion The literature available on ectopic HCC and the guidelines for management of HCC do not address the postoperative surveillance of patients undergoing curative treatment. A follow-up regime has been proposed by Hatzaras et al. (2014) to include cross-sectional imaging of the liver and measurement of serum AFP levels [1]. CT would be the preferred study of choice in a total radiologic investigation of the abdomen. While MRI is prone to artifacts due to movements, CT scans allows so rapid recordings that this no longer is an issue. An early investigation of the liver for intrahepatic HCC should nevertheless be performed early to exclude primary intrahepatic HCC. Conclusion We recommend that patients with ectopic HCC should be followed every 6 months with measurement of AFP and abdominal CT imaging. MRI of the liver should be performed early to exclude primary intrahepatic HCC.

Background: Inflammasomes are large multiprotein oligomers consisting of a receptor (such as NLRP3), the adaptor protein (ASC) and caspase-1. Assembly of the inflammasome activates caspase-1 which cleaves interleukin (IL)-18 and IL-1β to their bioactive forms. Elevated levels of IL-18 and IL-1β are found in patients with pulmonary hypertension, and increased levels of IL-18 have been found in alveolar hypoxia which leads to pulmonary hypertension. Objective: To study the role of ASC in chronic hypoxia-induced pulmonary artery remodelling. Methods: WT, NLRP3 -/- and ASC -/- mice were exposed to 3 months of hypoxia in a tightly sealed chamber containing 10% oxygen. Right ventricular systolic pressure (RVSP) was measured with a microtipped transducer catheter. To quantify muscularization and collagen content of arteries, lung sections were stained with von Willebrand factor, smooth muscle α-actin, acid fuchsin orange G-stain (AFOG) and Sirius Red. Results: ASC -/- mice exposed to hypoxia had significantly lower RVSP than WT hypoxic mice, indicating reduced pulmonary hypertension. RVSP of NLRP3 -/- exposed to hypoxia was not altered compared to WT hypoxia. Hypoxic ASC -/- mice showed less degree of muscularization and fibrosis around pulmonary arteries compared to WT mice exposed to hypoxia. Conclusions: Depletion of the inflammasome adaptor ASC diminish pulmonary artery remodelling in hypoxia-induced pulmonary hypertension.

Background: The NLRP3 inflammasome acts as an early mediator of inflammation by cleaving and releasing IL-1β and IL-18 from their proforms. Objective: The aim of this study was to describe NLRP3 activation and evaluate whether deficiency of NLRP3 protects against neonatal hypoxic ischemic brain damage. Methods: C57BL/6 and NLRP3-/- mice at P9 were subjected to unilateral common carotid ligation followed by hypoxia. RT-PCR was used on mRNA in five different subregions of the brain. Brain infarction was evaluated by histopathology and 2,3,5-triphenyltetrazolium chloride staining. Plasma levels of IL-18 were measured by ELISA. Double labeling immunohistochemistry was used to examine cell-specific NLRP3 expression. Results: NLRP3 was upregulated 24 h after hypoxia-ischemia (HI) in the hippocampus (2.6-fold), striatum (2.2-fold) and thalamus (2.3-fold). Brain infarction volumes were not statistically significantly different in NLRP3-/- mice compared to WT mice 24 h after HI, accompanied by no significant changes in plasma IL-18 levels. Three hours after HI, NLRP3 expression occurred in astrocytes located in the hippocampus and habenular nucleus of the thalamus. Microglia only showed scarce expression at this time point, but prominent NLRP3 expression 72 h after HI. Conclusion: Astrocytes are early mediators of NLRP3 activity. No early neuroprotective effect of NLRP3 deficiency in neonatal HI brain damage was shown.

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

The role of aberrant crypt foci (ACF) as preneoplastic lesions in colon carcinogenesis is not clear. In Min/+ mice and their wild-type littermates treated with azoxymethane (AOM), we previously identified a subgroup of flat ACF that seem more immediate precursors of tumors than the classical elevated ACF. In the present study, we identified a similar subgroup of flat ACF in AOM-treated A/J mice and compared them with nascent tumors and classical elevated ACF. At week 1 and 2 after birth, A/J mice were injected subcutaneously with AOM (10 mg/kg bw/injection). At weeks 7–14, we examined the luminal surface of unsectioned colon preparations stained with methylene blue in the inverse light microscope. The lesions were also examined by histopathology and immunohistochemistry. Surface examination revealed flat ACF, classical elevated ACF and nascent tumors. Since flat ACF were not observed as elevated structures, their bright blue appearance and compressed pit pattern of crypt openings seen with transillumination were used as criteria for their identification. Flat ACF and nascent tumors displayed a uniform picture of severe dysplasia, compressed pit pattern, overexpression of cytoplasmic/nuclear β-catenin and nuclear overexpression of cyclin D1. Apparently, flat ACF and tumors represented the same type of dysplastic lesions at different stages of crypt multiplication. In contrast, classical elevated ACF did not seem to be as clearly related to tumorigenesis. They infrequently (1/20) possessed severe dysplasia, overexpression of cytoplasmic/nuclear β-catenin, or nuclear overexpression of cyclin D1, and they did not have compressed crypt openings. Furthermore, flat ACF grew significantly faster than classical elevated ACF. In conclusion, our data indicate a development from flat ACF to adenoma characterized by aberrant activation of the Wnt signaling pathway and fast crypt multiplication. Classical elevated ACF do not seem to be as closely related to tumorigenesis. © 2005 Wiley-Liss, Inc.

Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1β, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC−/−, and NLRP3−/− mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC−/− mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P < 0.001), indicating a substantially reduced pulmonary hypertension in mice lacking ASC. Magnetic resonance imaging further supported these findings by demonstrating reduced right ventricular remodeling. RVSP of NLRP3−/− mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1β in WT and NLRP3−/− mice, this response was absent in ASC−/− mice. Moreover, ASC−/− mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension.

Objective Although serrated polyps may be precursors of colorectal cancer (CRC), prospective data on the long-term CRC risk in individuals with serrated polyps are lacking. Design In a population-based randomised trial, 12 955 individuals aged 50–64 years were screened with flexible sigmoidoscopy, while 78 220 individuals comprised the control arm. We used Cox models to estimate HRs with 95% CIs for CRC among individuals with ≥1 large serrated polyp (≥10 mm in diameter), compared with individuals with adenomas at screening, and to population controls, and multivariate logistic regression to assess polyp risk factors for CRC. Results A total of 103 individuals had large serrated polyps, of which 81 were included in the analyses. Non-advanced adenomas were found in 1488 individuals, advanced adenomas in 701. Median follow-up was 10.9 years. Compared with the control arm, the HR for CRC was 2.5 (95% CI 0.8 to 7.8) in individuals with large serrated polyps, 2.0 (95% CI 1.3 to 2.9) in individuals with advanced adenomas and 0.6 (95% CI 0.4 to 1.1) in individuals with non-advanced adenomas. A large serrated polyp was an independent risk factor for CRC, adjusted for histology, size and multiplicity of concomitant adenomas (OR 3.3; 95% CI 1.3 to 8.6). Twenty-three large serrated polyps found at screening were left in situ for a median of 11.0 years. None developed into a malignant tumour. Conclusions Individuals with large serrated polyps have an increased risk of CRC, comparable with individuals with advanced adenomas. However, this risk may not be related to malignant growth of the serrated polyp. Trial registration number The Norwegian Colorectal Cancer Screening trial is registered at clinicaltrials.gov (NCT00119912).

Rhombencephalitis due to Listeria monocytogenes is characterized by progressive cranial nerve palsies and subacute inflammation in the brain stem. In this paper, we report observations made on mice infected with L. monocytogenes. Unilateral inoculation of bacteria into facial muscle, or peripheral parts of a cranial nerve, induced clinical and histological signs of mainly ipsilateral rhombencephalitis. Similarly, unilateral inoculation of bacteria into lower leg muscle or peripheral parts of sciatic nerve was followed by lumbar myelitis. In these animals, intraaxonal bacteria were seen in the sciatic nerve and its corresponding nerve roots ipsilateral to the bacterial application site. Development of myelitis was prevented by transsection of the sciatic nerve proximally to the hindleg inoculation site. Altogether, our results support the hypothesis that Listeria rhombencephalitis is caused by intraaxonal bacterial spread from peripheral sites to the central nervous system.

Three to 12 h of mild hypothermia (HT) starting after hypoxia-ischemia is neuroprotective in piglets that are anesthetized during HT. Newborn infants suffering from neonatal encephalopathy often ventilate spontaneously and are not necessarily sedated. We aimed to test whether mild posthypoxic HT lasting 24 h was neuroprotective if the animals were not sedated. Thirty-nine piglets (median weight 1.6 kg, range 0.8-2.2 kg; median age 24 h, range 7-48 h) were anesthetized and ventilated and subjected to a 45-min hypoxic (FiO(2) approximately 6%) global insult (n = 36) or sham hypoxia (n = 3). On reoxygenation, 18 were maintained normothermic (NT, 39.0 degrees C) for 72 h, and 21 were cooled from 39 (NT) to 35 degrees C (HT) for the first 24 h before NT was resumed (18 experimental, three sham hypoxia). Cardiovascular parameters and intermittent EEG were documented throughout. The brain was perfusion fixed for neuropathology and five main areas examined using light microscopy. The insult severity (duration in minutes of EEG amplitude < 7 microV) was similar in the NT and HT groups, mean +/- SD (28 +/- 7.2 versus 27 +/- 8.6 min), as was the mean FiO(2) (5.9 +/- 0.7 versus 5.8 +/- 0.8%) during the insult. Six NT and seven HT piglets developed posthypoxic seizures that lasted 29 and 30% of the time, respectively. The distribution and degree of injury (0.0-4.0, normal-maximal damage) within the brain (hippocampus, cortex/white matter, cerebellum, basal ganglia, thalamus) were similar in the NT and HT groups (overall score, mean +/- SD, 2.3 +/- 1.5 versus 2.4 +/- 1.3) as was the EEG background amplitude at 3 h (13 +/- 3.5 versus 10 +/- 3.3 microV). The HT animals shivered and were more active. The sham control group (n = 3) shivered but had normal physiology and neuropathology. Plasma cortisol was significantly higher in the HT group during the HT period, 766 +/- 277 versus 244 +/- 144 microM at 24 h. Mild postinsult HT for 24 h was not neuroprotective in unsedated piglets and did not reduce the number of animals that developed posthypoxic seizures. Cortisol reached 3 times the NT value at the end of HT. We speculate that the stress of shivering and feeling cold interfered with the previously shown neuroprotective effect of HT. Research on the appropriateness of sedation during clinical HT is urgent.

Supplemental oxygen used during resuscitation can be detrimental to the newborn brain. The aim was to determine how different oxygen therapies affect gene transcription in a hypoxia-reoxygenation model.C57BL/6 mice (n = 56), postnatal day 7, were randomized either to 120 min of hypoxia 8% O2 followed by 30 min of reoxygenation with 21, 40, 60, or 100% O2, or to normoxia followed by 30 min of 21 or 100% O2. Affymetrix 750k expression array was applied with RT-PCR used for validation. Histopathology and immunohistochemistry 3 d after hypoxia-reoxygenation compared groups reoxygenated with 21 or 100% O2 with normoxic controls (n = 22).In total, ~81% of the gene expression changes were altered in response to reoxygenation with 60 or 100% O2 and constituted many inflammatory-responsive genes (i.e., C5ar2, Stat3, and Ccl12). Oxidative phosphorylation was downregulated after 60 or 100% O2. Iba1(+) cells were significantly increased in the striatum and hippocampal CA1 after both 21 and 100% O2.In the present model, hypoxia-reoxygenation induces microglial accumulation in subregions of the brain. The transcriptional changes dominating after applying hyperoxic reoxygenation regimes include upregulating genes related to inflammatory responses and suppressing the oxidative phosphorylation pathway.

Gender, body weight, and cardiovascular disease (CVD) are all variables known to influence human heart weight. The impact of cancer is less studied, and the influence of age is not unequivocal. We aimed to describe the relationship between body size and heart weight in a large autopsy cohort and to compare heart weight in patients with cancer, CVD, and other diseases.Registered information, including cause of death, evidence of cancer and/or CVD, heart weight, body weight, and height, was extracted from the autopsy reports of 1410 persons (805 men, mean age 66.5 years and 605 women, mean age 70.6 years). The study population was divided in four groups according to cause of death; cancer (n=349), CVD (n=470), mixed group who died from cancer and CVD and/or lung disease (n=263), and a reference group with patients who did not die from any of these conditions (n=328). In this last group, heart weight correlated only slightly better with body surface area than body weight, and nomograms based on body weight are presented. Compared to the reference group (mean heart weight: 426 g and 351 g in men and women, respectively), heart weight was significantly lower (men: P.05, women: P.001) in cancer patients (men: 392 g, women: 309 g) and higher (P.001) in patients who died from CVD (men: 550 g, women: 430 g). Similar results were obtained in linear regression models adjusted for body weight and age. Among CVD, heart valve disease had the greatest impact on heart weight, followed by old myocardial infarction, coronary atherosclerosis, and hypertension. Absolute heart weight decreased with age, but we demonstrated an increase relative to body weight.The weight of the human heart is influenced by various disease processes, in addition to body weight, gender, and age. While the most prevalent types of CVD are associated with increased heart weight, patients who die from cancer have lower average heart weight than other patient groups. The latter finding, however, is diminished when adjusting for body weight.The present study demonstrates that the weight of the human heart is influenced by various disease processes like cancer and CVD, in addition to body weight, gender and, possibly, age.

The aim of this study was to produce a neonatal piglet model which, avoiding vessel ligation, exposed the whole animal to hypoxia and produced dose-dependent clinical encephalopathy and neuropathologic damage similar to that seen after birth asphyxia. Twenty-three piglets were halothane-anesthetized. Hypoxia was induced in 19 piglets by reducing the fractional concentration of inspired oxygen (FiO2) to the maximum concentration at which the EEG amplitude was below 7 microV (low amplitude) for 17-55 min. There were transient increases in Fio2 to correct bradycardia and hypotension. Posthypoxia, the piglets were extubated when breathing was stable. Four were sham-treated controls. We aimed at 72-h survival; seven died prematurely due to posthypoxic complications. EEG and a videotaped itemized neurologic assessment were recorded regularly. We found that 95% of the animals showed neuropathologic damage. The duration of low amplitude EEG during the insult and the arterial pH at the end of the insult correlated with cortical/white matter damage; r = 0.75 and 0.81, respectively. Early postinsult EEG background amplitude (r = 0.86 at 3 h) and neurologic score (r = 0.79 at 8 h) correlated with neuropathology. Epileptic seizures in seven animals were always associated with severe neuropathologic damage. We conclude that EEG-controlled hypoxia and subsequent intensive care enabled the animals to survive with an encephalopathy which correlated with the cerebral hypoxic insult. The encephalopathy was clinically, electrophysiologically, and neuropathologically similar to that in the asphyxiated term infant. This model is suitable for examining mechanisms of damage and evaluation of potential protective therapies after birth asphyxia.

AIM: To determine whether moderate hypothermia, applied after a hypoxic-ischaemic insult in neonatal rats, reduces cerebral damage. METHOD: Unilateral hypoxic-ischaemic brain damage was induced in 7 day old rats by left carotid ligation, followed by 120 minutes of normothermic exposure to 8% O2, followed by random selection to three hours of hypothermia (rectal temperature, mean (SD), 32.5 (0.4) degrees C) or normothermia (38.3 (0.4) degrees C). One hundred and one animals were used for brain temperature or blood chemistry studies and 24 for survival studies (7 days) with neuropathology, including cell counting as outcome measures. RESULTS: Thirty sections from each brain were histologically examined with respect to distribution and pattern of damage and given a score from 0 to 4. Animals treated with hypothermia had significantly less damage than normothermic animals (score 0.5 (0.3) vs 1.8 (0.5)). CONCLUSIONS: Posthypoxic hypothermia reduces brain damage in awake, unrestrained 7 day old rats.

To evaluate the predictive value of transient elastography (TE), easy available biochemical scores and a combination of these to detect advanced liver fibrosis (i.e. fibrosis stage ≥F3) in patients with chronic liver disease of different etiologies.A valid TE was obtained in 418 patients with chronic liver disease of mixed etiologies during the period of 2007-2010. Reliable fibrosis staging and biochemical data for calculation of APRI, FIB4, and AST/ALT-ratio (AAR) were available in 187 cases of which 50 had clinical obvious cirrhosis. Logistic regression analyses were performed to investigate if biochemical scores were significant predictors of advanced fibrosis independent of TE.In the whole group, TE correlated significantly with APRI and FIB4 but not with AAR. In patients with TE ≥7 kPa, a new formula combining TE and FIB4 improved the accuracy for detecting advanced fibrosis. Area under the receiver operating characteristic curve (AUROC) and sensitivity for the combined formula was 0.94 and 0.92, respectively, as opposed to 0.90 and 0.87 for TE alone. After exclusion of cases with clinical obvious cirrhosis, only FIB4 was a significant predictor of advanced liver fibrosis independent of TE. A combined formula gave a marginally improved AUROC in this group.Our findings suggest that the combination of TE and FIB4 is useful in the prediction of advanced fibrosis. The effect of this combination was marginal when only asymptomatic patients were included. Larger studies are needed to see if this effect is statistically significant and to detect possible differences according to etiology.

ObjectiveFetuin A has been associated with insulin resistance and the metabolic syndrome. We therefore explored the role of fetuin A in nonalcoholic fatty liver disease (NAFLD).DesignCross-sectional and intervention studies.MethodsWe included 111 subjects with histologically proven NAFLD of whom 44 participated in a randomized, controlled trial with metformin. One hundred and thirty-one healthy subjects and 13 subjects undergoing hepatic surgery for metastatic cancer served as controls. Main outcome variables were circulating levels of fetuin A according to the presence of NAFLD, hepatic gene expression of fetuin A and key enzymes in glucose and lipid metabolism, and the effect of metformin on fetuin A levels in vivo and in vitro (HepG2 cells).ResultsFetuin A levels were significantly higher in NAFLD patients compared with controls (324±98 vs 225±75 mg/l, Pβ=174 (95% confidence interval: 110–234)) independent of body mass index, age, sex, fasting glucose, and triglycerides. Hepatic fetuin A mRNA levels correlated significantly with hepatic mRNA levels of key enzymes in lipid (sterol regulatory element-binding protein 1c, carnitine palmitoyltransferase 1) and glucose (phosphoenol pyruvate kinase 1, glucose-6-phosphatase) metabolism. Plasma fetuin A levels decreased significantly after metformin treatment compared with placebo (−40±47 vs 15±82 mg/l, P=0.008). Metformin induced a dose-dependent decrease in fetuin A secretion in vitro.ConclusionsFetuin A levels were elevated in NAFLD. Hepatic expression of fetuin A correlated with key enzymes in glucose and lipid metabolism. Metformin decreased fetuin A levels in vitro.

Objective: Cerebrovascular factors are crucially involved in the early injury after perinatal asphyxia. With magnetic resonance imaging (MRI) and ultrasonography (US), this study aimed to quantify microvascular perfusion changes due to hypoxia and resuscitation, by comparing contrast-enhanced ultrasound (CEUS) to dynamic susceptibility contrast-enhanced (DSC)-MRI and diffusion-weighted MRI. Material and Methods: Newborn piglets (n 12/17) were reoxygenated with 21% (n 6) or 100% O2 (n 6) after global hypoxia. Five piglets served as controls. CEUS and MRI were performed before, during, and up to 7 hours after hypoxia. Following are the perfusion parameters for CEUS: peak intensity (PI), area under the curve (AUC), time to peak (TTP), and upslope a and perfusion/diffusion parameters for MRI: relative cerebral blood volume, relative cerebral blood flow, mean transit time, and apparent diffusion coefficient were compared between different regions in the brain across time points and also compared with histology at the end. Results: In CEUS, compared with the control group, perfusion changed significantly over time, in the hyperoxic group in all regions for PI, AUC in all regions of interests. The changes presented mainly as decreased perfusion during and shortly after resuscitation: for PI in the basal ganglia (BG), cortex, and the whole brain with 50 to 60% (P 0.001); for AUC in the BG and cortex with 90% (P 0.02) and in the whole brain with 70% (P 0.004). In the injured brains (confirmed by histology), significant changes over time were seen in TTP and AUC with mainly increased perfusion during hypoxia: for TTP in the cortex, AUC in the BG and whole brain with 90 to 100% (P 0.04), and for TTP in the whole brain with 50% (P 0.02). DSC-MRI showed the same trends in perfusion with regard to relative cerebral blood volume as CEUS. In all pigs exposed to hypoxia, perfusion returned toward baseline values at 7 hours after hypoxia in both methods. Apparent diffusion coefficient decreased significantly after 7 hours in the injured brains in the BG from 114.6 1.2 10 5 mm 2 /s to 90.3 24 10 5 mm 2 /s (P 0.03). Conclusions: CEUS and DSC-MRI can detect an early temporal evolution of cerebral perfusion in perinatal hypoxia and resuscitation, reversible after 7 hours. Hyperoxic resuscitation caused early decreased cerebral perfusion, not present in the normoxic group. The combined use of CEUS and DSC-MRI can provide important diagnostic information and give new insights into perinatal vascular hypoxia mechanisms.

We recently developed a (13)C-sorbitol breath test ((13)C-SBT) as an alternative to the H(2)-sorbitol breath test (H(2)-SBT) for coeliac disease. In this study we compared the diagnostic properties of the H(2)-SBT and the (13)C-SBT in follow-up of coeliac disease.Twenty-seven coeliac patients on a gluten-free diet (GFD) performed the breath tests. All had been tested before treatment in the initial study of the (13)C-SBT, in which 39 untreated coeliac patients, 40 patient controls, and 26 healthy volunteers participated. Five gram sorbitol and 100 mg (13)C-sorbitol were dissolved in 250 ml tap water and given orally. H(2), CH(4) and (13)CO(2) were measured in end-expiratory breath samples every 30 min for 4 h. Increased H(2) concentration ≥20 ppm from basal values was used as cut-off for the H(2)-SBT. Sixty minutes values were used as diagnostic index in the (13)C-SBT.(13)CO(2) levels at 60 min increased in 20/26 treated coeliac patients (77%) after GFD, but were significantly lower than in control groups. Out of 20 patients who had a positive H(2)-SBT before GFD, 12 had a negative H(2)-SBT after GFD. Peak H(2) concentrations were not correlated with (13)C-SBT results.The study confirms the sensitivity of a one-hour (13)C-SBT for small intestinal malabsorption. The (13)C-SBT has superior diagnostic properties compared with the H(2)-SBT in follow-up of coeliac disease.

Investigation of uncertain celiac disease (CD) in patients already on a gluten-free diet (GFD) is difficult. We evaluated HLA-DQ2-gliadin tetramers for detection of gluten-specific T cells in peripheral blood and histological changes in the duodenum after a short gluten challenge as a diagnostic tool.HLA-DQ2+ individuals on a GFD for at least 4 weeks were investigated; 35 with uncertain diagnosis, 13 CD patients, and 2 disease controls. All participants had a challenge with four slices of gluten-containing white bread, daily for 3 days (d1-d3). An esophagogastroduodenoscopy with biopsy sampling was done on d0 and d4. Biopsies were scored according to revised Marsh criteria. Peripheral blood CD4+ T cells were isolated, stained with HLA-DQ2-gliadin peptide tetramers, and analyzed by flow cytometry on d0 and d6.After challenge, a positive tetramer test was seen in 11/13 CD patients. Four of these subjects also showed typical histological changes on challenge. Of the 35 patients with uncertain diagnosis, 3 were diagnosed with CD. Two of these three patients had both positive tetramer staining and histological changes in biopsies after challenge.Tetramer staining for gluten-specific T cells is a sensitive method in detecting an immune response in CD patients after a short gluten challenge. The prevalence of CD in the group with self-prescribed GFD was about 10%.

Mild hypothermia can attenuate the development of brain damage after asphyxia. Supplemental oxygen during resuscitation increases generation of reactive oxygen species, compared to room air. It is unknown if supplemental oxygen affects hypothermic neuroprotection. We studied the early effects of hyperoxic reoxygenation and subsequent hypothermia on tissue oxygenation, microcirculation, inflammation and brain damage after global hypoxia. Anesthetized newborn pigs were randomized to control (n=6), or severe global hypoxia (n=46). Three pigs died during hypoxia or reoxygenation. After 20-min reoxygenation with room air (n=22) or 100% oxygen (n=21), pigs were randomized to normothermia (deep rectal temperature 39 degrees C, n=22) or total body cooling (35 degrees C, n=21) for 6.5 h before the experiment was terminated. We demonstrated a differential effect of post-hypoxic hypothermia between animals reoxygenated with 100% oxygen and with room air, with reduced damage only in hypothermic animals reoxygenated with 100% oxygen (P=0.001). Hyperoxic reoxygenation resulted in a significant overshoot in striatal oxygen tension, without affecting microcirculation. Inflammatory response after the insult did not differ between groups. The results indicate an early protective effect of hypothermia which may vary with oxygen level used during reoxygenation.