Long-term cognitive and psychiatric outcomes of acute respiratory distress syndrome managed with Extracorporeal Membrane Oxygenation.

BACKGROUND: Cognitive dysfunction is often reported in patients who have experienced acute respiratory distress syndrome (ARDS). Extra Corporeal Membrane Oxygenation (ECMO) therapy is increasingly used to manage ARDS patients in ICU, transforming survival rates. However, few studies have examined cognitive outcomes. METHODS: We examined self-reported cognitive complaints, psychiatric outcomes and neuropsychological test performance in survivors of severe hypoxaemia managed with VV-ECMO, at 18-24 month follow-up, compared with a group of healthy controls. RESULTS: Over 70% of ECMO-treated patients (N = 46) complained of difficulty in at least one aspect of cognition on self-report measures (study 1). However, a much lower frequency of cognitive impairment was found on formal neuropsychological testing (study 2). Mean neuropsychological test scores of the ECMO group (N = 24) did not significantly differ from healthy controls (N = 23) after controlling for depression. Less than 30% of ECMO-treated patients showed impairments in anterograde memory, and deficits on general IQ or executive function were seen in <17% of patients. However, we observed high levels of self-reported anxiety and depression in the ECMO-treated patients. CONCLUSIONS: Cognitive outcomes in ECMO-treated patients were generally good, with preserved neuropsychological function in the majority of patients, despite severe hypoxaemia and high rates of self-reported difficulties. However, we saw high levels of mental health symptoms in these patients, highlighting a need for psychological support.

Use of microarray analysis to study gene expression in the avian epiphyseal growth plate.

Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate.

Ovocleidin (OC 116) is present in avian skeletal tissues.

Ovocleidin (OC-116), a protein identified in eggshell matrix, was found to be expressed in avian growth plate chondrocytes. Because OC-116 has been reported to be a member of a family of related phosphoprotein genes clustered on avian chromosome 4, we expanded our search to other skeletal tissues. Using Western blotting, we found OC-116 in the matrix of chick cortical bone and laying hen medullary bone as well as in hypertrophic chondrocyte lysates. Furthermore, other members of this family (bone sialoprotein, dental matrix protein-1, and osteopontin) were also present in the eggshell matrix. Reverse transcription-PCR was used to confirm the presence of the OC-116 gene in bone tissues as well as the expression of bone sialoprotein and dental matrix protein-1 in uterine tissue. These results, in combination with those of other laboratories, show that this family of phosphoproteins is found in a wide variety of avian mineralized tissues.

Immunolocalization of vascularization factors in normal, tibial dyschondroplasia and rachitic cartilage.

Tibial dyschondroplasia is a skeletal abnormality characterized by an avascular plug of cartilage in the metaphysis of the long bones of rapidly growing birds. An avascular, thickened growth plate also occurs in vitamin D/calcium-deficient rickets. This research was conducted to evaluate tibial dyschondroplasia and rachitic tissue for factors shown to be important in normal growth plate vascularization. Immunohistochemical analysis demonstrated the presence of vascular endothelial growth factor, its receptor Flk-1, and matrix metalloproteinases MMP-9 and MMP-13 to be present in all tissues examined. In most cases, immunostaining was intracellular, except near blood vessels where the matrix was also stained. The results suggest that, for the four proteins examined, disruption of their expression is not a key factor in the development of these avascular cartilage lesions in avians. The relationship of these observations to the pathology of tibial dyschondroplasia is discussed.

Tibial dyschondroplasia 40 years later.

Tibial dyschondroplasia is a disease of rapid growth rate that occurs in many avian species. It is characterized by an avascular lesion in which the life span of the growth plate chondrocyte is essentially doubled. A characteristic pattern of gene expression and gene product localization has emerged that mimics the pattern observed with endoplasmic reticulum (ER) stress in growth plate chondrocytes. This activates a cell-survival mechanism called autophagy. The initial phases of this mechanism appear to originate in the avascular transition zone of the growth plate. Because specific genes and gene products are associated with autophagy and ER stress, it should now be possible to identify the mechanisms involved in the development of this cartilage abnormality. The potential biochemical pathways responsible for initiating ER stress are discussed.

Strontium administration in young chickens improves bone volume and architecture but does not enhance bone structural and material strength.

Genetic selection for rapid body growth in broiler chickens has resulted in adverse effects on the skeletal system exemplified by a higher rate of cortical fractures in leg bones. Strontium (Sr) has been reported to have beneficial effects on bone formation and strength. We supplemented the diet of 300-day-old chicks with increasing dosages of Sr (0%, 0.12%, or 0.24%) to study the capacity of the element to improve bone quality and mechanical integrity. Treatment with Sr increased cortical bone volume and reduced bone porosity as measured by micro-computed tomography. The higher level of Sr significantly reduced bone Ca content (34.7%) relative to controls (37.2%), suggesting that Sr replaced some of the Ca in bone. Material properties determined by the three-point bending test showed that bone in the Sr-treated groups withstood greater deformation prior to fracture. Load to failure and ultimate stress were similar across groups. Our results indicate that Sr treatment in rapidly growing chickens induced positive effects on bone volume but did not improve the breaking strength of long bones.

Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate.

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens.

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

The effect of changes in arterial PCO2 on neuroendocrine function in man.

There is evidence that changes in arterial P(CO(2)) (P(a,CO(2))), as well as P(O(2)), influence neuroendocrine function. The hyponatraemia and fluid retention (cor pumonale) seen in chronic obstructive pulmonary disease (COPD) and type II respiratory failure is associated with increased vasopressin release. This study examines the specific effects of altered P(a,CO(2)) on hormone release from the posterior and anterior pituitary. The study was performed in 20 ventilated ICU patients in the late recovery phase of their illness. None had primary respiratory disease. Control blood samples were taken and the alveolar ventilation was then adjusted to allow the P(a,CO(2)) increase or decrease for a period of 3 h, during which time further blood samples were taken for the determination, by radioimmmunoassy of vasopressin, oxytocin, growth hormone and cortisol. Urine output and electrolyte concentrations were also measured. Circulating concentrations of growth hormone and oxytocin increased with increasing P(a,CO(2)). Vasopressin release showed a similar pattern up to a P(a,CO(2)) of approximately 6.0 kPa, above which vasopressin concentrations were inversely related to P(a,CO(2)). There was no significant effect on cortisol concentrations. No significant effects were established in urinary parameters during the short period of this study. Thus an increase in CO(2) is associated with stimulated pituitary hormone release. The effect on the neurohypophysial hormones may account for the fluid retention and hyponatraemia seen in COPD and hence provide a rationale for treatment.

Chicken keel cartilage as a source of chondroitin sulfate.

Chondroitin sulfate is used extensively as a treatment for osteoarthritis. This study was conducted to evaluate whether chondroitin sulfate could be isolated from chicken keel cartilage in sufficient quantities and of requisite quality to make it a feasible source of chondroitin sulfate. Proteoglycans were extracted from chicken keel cartilage obtained immediately after slaughter by using 3 M MgCl2 at room temperature. The extract was then dialyzed and digested with papain to remove proteins. Glycosaminoglycans were obtained by ethanol precipitation, lyophilized, and characterized by using gel filtration on Sepharose CL-6B columns. Guanidine-HCI extraction was also used as a control to investigate the efficiency of extraction using MgCl2. Results showed that, from every gram of wet or non-lyophilized keel cartilage, 32.9 +/- 4.8 mg (dry weight) of glycosaminoglycans could be obtained following MgCl2 extraction. Analyses revealed that 75.5 +/- 4.2% of these glycosaminoglycans were chondroitin sulfate. Chromatographic analyses showed a single symmetrical peak, which could be almost entirely removed by prior digestion with chondroitinase ABC, indicating that the material in the peak was in fact chondroitin sulfate. The average molecular weight (also called relative molecular mass, Mr) of the glycosaminoglycans was also estimated (Mr 48,500). Characterization using polyacrylamide or agarose gel electrophoresis showed diffuse bands containing chondroitin sulfate, which could be entirely removed by prior digestion with chondroitinase ABC. This study shows that chicken keel cartilage is a readily available source of chondroitin sulfate.

Collagen X expression in oviduct tissue during the different stages of the egg laying cycle.

The purpose of this investigation was to study the expression of type X collagen in the hen's oviduct. Type X collagen is a short-chain collagen that is present in the fibers of eggshell membranes, and there is evidence to suggest that it contributes to structural integrity. In situ hybridization and Northern blot analysis were used to study the expression of this important matrix constituent. The results demonstrated that gene expression was predominantly in the tubular gland cells of the isthmus segment of the oviduct. In contrast to observations with other matrix proteins, such as parathyroid hormone-related peptide and osteopontin, gene expression did not fluctuate with the position of the egg in the oviduct.

Critical illness in systemic lupus erythematosus and the antiphospholipid syndrome.

OBJECTIVES: To investigate the causes, course, and outcome of critical illness requiring emergency admission to the intensive care unit (ICU) in patients with systemic lupus erythematosus (SLE) or the antiphospholipid syndrome (APS), or both. METHODS: Critically ill patients with SLE or APS, or both, admitted to a London teaching hospital ICU over a 15 year period were studied. Demographic, diagnostic, physiological, laboratory, and survival data were analysed. Kaplan-Meier survival curves were constructed by age, time from first diagnosis of SLE, and time from first ICU admission. The log rank test and a backwards stepwise Cox regression were used to identify factors associated with reduced survival. RESULTS: Sixty one patients with SLE alone (39%) and/or APS (61%) required 76 emergency admissions to the ICU. Patients had high severity of illness scores (median APACHE II 22 (range 8-45)) and multiorgan dysfunction. The primary diagnoses for patients admitted were infection in 31/76 (41%), renal disease in 16/76 (21%), cardiovascular disease in 12/76 (16%), and coagulopathies in 11/76 (14%). The commonest secondary diagnosis was renal dysfunction (49%). Factors associated with an increased risk of death were cyclophosphamide before admission, low white cell count, and high severity of illness score. Before adjustment for these factors renal disease had a strong adverse effect on long term survival (analysis by age at diagnosis p=0.005, analysis by time since first ICU admission, p=0.07). After adjustment, infection at admission to ICU was associated with an increased ICU mortality (p=0.02) and was the cause of death in 13/17 patients who died in the ICU. Similarly, after adjustment, APS was associated with reduced ICU survival (p=0.1) and reduced long term (p=0.03) survival. Seventeen patients (28%) died in the ICU, and 31 patients (51%) had died by the last follow up. Median time from ICU admission to death was four years. Overall five year survival from the first ICU admission was 43%. CONCLUSION: Critical illness requiring ICU admission may occur in patients with SLE and APS. In this study, ICU survival was better than previously described, but long term survival was poor. Cyclophosphamide administration, low white cell count, and high severity of illness score were associated with reduced survival. Before adjustment for these factors, only renal disease had an adverse effect on outcome but after adjustment, infection and APS reduced survival.

The pulmonary physician in critical care * 2: oxygen delivery and consumption in the critically ill.

Early detection and correction of tissue hypoxia is essential if progressive organ dysfunction and death are to be avoided. However, hypoxia in individual tissues or organs caused by disordered regional distribution of oxygen delivery or disruption of the processes of cellular oxygen uptake and utilisation cannot be identified from global measurements. Regional oxygen transport and cellular utilisation have an important role in maintaining tissue function. When tissue hypoxia is recognised, treatment must be aimed at the primary cause. Supplemental oxygen may be life saving in some situations but cannot correct inadequate oxygen delivery caused by a low cardiac output or impaired ventilation. Recent innovations include artificial oxygen carrying proteins and "haemoglobin" molecules designed to improve tissue blood flow by reducing viscosity. Regulating cell metabolism using different substrates or drugs has so far been poorly explored but is an exciting area for further research. A minimum level of global oxygen delivery and perfusion pressure must be maintained in the critically ill patient with established "shock", but advances in the understanding and control of regional distribution and other "downstream" factors in the oxygen cascade are needed to improve outcome in these patients.

Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor.

1. The mechanisms responsible for sensing hypoxia and initiating hypoxic pulmonary vasoconstriction (HPV) are unclear. We therefore examined the roles of the mitochondrial electron transport chain (ETC) and glycolysis in HPV of rat small intrapulmonary arteries (IPAs). 2. HPV demonstrated a transient constriction (phase 1) superimposed on a sustained constriction (phase 2). Inhibition of complex I of the ETC with rotenone (100 nM) or complex III with myxothiazol (100 nM) did not cause vasoconstriction in normoxia, but abolished both phases of HPV. Rotenone inhibited the hypoxia-induced rise in intracellular Ca(2+) ([Ca(2+)](i)). Succinate (5 mM), a substrate for complex II, reversed the effects of rotenone but not myxothiazol on HPV, but did not affect the rise in NAD(P)H fluorescence induced by hypoxia or rotenone. Inhibition of cytochrome oxidase with cyanide (100 microM) potentiated phase 2 constriction. 3. Phase 2 of HPV, but not phase 1, was highly correlated with glucose concentration, being potentiated by 15 mM but abolished in its absence, or following inhibition of glycolysis by iodoacetate or 2-deoxyglucose. Glucose concentration did not affect the rise in [Ca(2+)](i) during HPV. 4. Depolarisation-induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by approximately 50 %. Depolarisation-induced constriction was depressed by rotenone during hypoxia by 23 +/- 4 %; cyanide was without effect. 5. Hypoxia increased 2-deoxy-[(3)H]glucose uptake in endothelium-denuded IPAs by 235 +/- 32 %, and in mesenteric arteries by 218 +/- 38 %. 6. We conclude that complex III of the mitochondrial ETC acts as the hypoxic sensor in HPV, and initiates the rise in smooth muscle [Ca(2+)](i) by a mechanism unrelated to changes in cytosolic redox state per se, but more probably by increased production of superoxide. Additionally, glucose and glycolysis are essential for development of the sustained phase 2 of HPV, and support an endothelium-dependent Ca(2+)-sensitisation pathway rather than the rise in [Ca(2+)](i).

Continuous venovenous haemofiltration for the treatment of theophylline toxicity.

In patients with severe theophylline toxicity charcoal haemoperfusion is the recommended method for rapid reduction of serum theophylline levels. However, access to this technique is limited in most hospitals. This case report shows that continuous venovenous haemofiltration, a technique available in most hospitals, is an effective alternative to charcoal haemoperfusion.

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate.

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.

The use of growth factors in the proliferation of avian articular chondrocytes in a serum-free culture system.

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor beta (TGF-beta) significantly stimulated H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-beta, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.

Gene expression and tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal deformity associated with rapid growth in a number of avian species. The disease is the result of a disruption in the cascade of events that occur in the epiphyseal growth plate. Whereas the incidence of TD is susceptible to genetic selection, no specific genetic defect has been identified. Although there are extensive data describing the morphological and biochemical characteristics of the lesion, the mechanism of lesion formation is unknown. However, naturally occurring or induced genetic mutations in other species can provide important clues to possible mechanisms responsible for lesion development. Disruption of normal chondrocyte differentiation by constitutive activation of the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, inactivation of the fibroblast growth factor receptor-3 (FGFR-3) receptor, and blocking vascular endothelial growth factor (VEGF) signaling all result in lesions that resemble TD. Impairment of vascular penetration due to the ablation of matrix metalloproteinase-9 (MMP-9) or tartrate-resistant acid phosphatase (TRAP) activity also results in similar cartilage abnormalities. We have integrated these observations with our current knowledge of TD to describe a hypothesis for the sequence of events responsible for the development of tibial dyschondroplastic lesions.

Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries.

To assess effects of smooth muscle energy state and intracellular pH (pH(i)) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, P(i), and pH(i) by (31)P-NMR spectroscopy and isometric tension in phenylephrine-contracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and pH(i) decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower pH(i) under baseline conditions and did not exhibit late contraction or recovery of energy state and pH(i) during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and pH(i), but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0. 1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and pH(i) at 10 mM. These results suggest that 1) regulation of tone, energy state, and pH(i) differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or pH(i) due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and pH(i) in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and pH(i) for transduction in smooth muscle or extracellular glucose for production and release by endothelium.