Dual-energy micro-CT of the rodent lung.

The purpose of this work is to investigate the use of dual-energy micro-computed tomography (CT) for the estimation of vascular, tissue, and air fractions in rodent lungs using a postreconstruction three material decomposition method. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact rat lungs were carefully removed from the thorax, injected with an iodine-based contrast agent, and then inflated with different volumes of air (n = 2). Finally, we performed in vivo imaging studies in C57BL/6 mice (n = 5) using fast prospective respiratory gating in end inspiration and end expiration for three different levels of positive end expiratory pressure (PEEP). Before imaging, mice were injected with a liposomal blood pool contrast agent. The three-dimensional air, tissue, and blood fraction maps were computed and analyzed. The results indicate that separation and volume estimation of the three material components of the lungs are possible. The mean accuracy values for air, blood, and tissue were 93, 93, and 90%, respectively. The absolute accuracy in determining all fraction materials was 91.6%. The coefficient of variation was small (2.5%) indicating good repeatability. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end expiration but no significant changes at end inspiration. Our method has applicability in preclinical pulmonary studies where changes in lung structure and gas volume as a result of lung injury, environmental exposures, or drug bioactivity would have important physiological implications.

Underutilization of echocardiography for patent foramen ovale in divers with serious decompression sickness.

The presence of a patent foramen ovale (PFO) in compressed gas diving has been considered a risk factor for serious decompression illness (DCS) for more than 20 years. We conducted a ten year retrospective chart review aimed at determining if physicians treating DCS in a university medical center setting used echocardiography to assess PFO in patients with severe DCS, and if so whether PFO is over-represented in that population. Over the ten-year period, 113 divers underwent recompression therapy for decompression sickness. Of these patients, 48 had serious DCS defined by at least one objective neurological finding. We reviewed medical records for the presence of agitated saline contrast echocardiogram testing and whether or not PFO was present. Only 12 of 48 patients with serious DCS underwent transthoracic agitated saline contrast echocardiogram testing. Of these 12 patients, 6 (50%) had a resting PFO. Binomial proportion testing yielded 95% confidence limits of 21% and 79%. Given 27% PFO prevalence in the general population, PFO may be over-represented in our group of most seriously injured DCS patients yet 75% of patients with objective neurological signs did not undergo echocardiography.

Hyperbaric oxygen treatment and bisphosphonate-induced osteonecrosis of the jaw: a case series.

PURPOSE: Bisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) is an emerging problem with few therapeutic options. Our pilot study of BP-ONJ investigated a possible role for hyperbaric oxygen (HBO(2)) therapy. PATIENTS AND METHODS: A total of 16 patients, ranging in age from 43 to 78 years, with BP-ONJ were treated with adjunctive HBO(2) between July 2003 and April 2006. Staging was based on the size and number of oral lesions. Clinical response after treatment and at distant follow-up; the odds of remission, stabilization, or relapse; and time to failure analysis were calculated. RESULTS: The median time on BP therapy before appearance of ONJ symptoms was 18 months, and that from symptom onset to HBO(2) therapy was 12 months. Fourteen of 16 patients (87.5%) improved in stage. The size and number of ONJ lesions were decreased after HBO(2) therapy (P < .001 and P = .008, respectively; Wilcoxon signed-rank test). Immediately after HBO(2) therapy, 7 of 16 patients (44%) were in remission and 8 (50%) had stabilized; however, stabilization without remission was sustained in only 2 patients. At follow-up, 10 of the patients (62.5%) were still in remission or had stabilized. The 7 patients who continued on BP treatment during HBO(2) therapy had a shorter time to failure (8.5 months; 95% confidence interval [CI] = 7.1 to 9.8) than those who discontinued the drug (20.1 months; 95% CI = 17.5 to 23.9; P = .006 by the log-rank test). Clinical response was not associated with cancer type or malignancy remission status. CONCLUSIONS: Adjunctive HBO(2) therapy may benefit patients with BP-ONJ; however, the outcome is improved with cessation of BP administration.

Protecting the permeability pore and mitochondrial biogenesis.

Recent evidence links the pathogenesis of multiple organ dysfunction syndrome (MODS) in sepsis to mitochondrial damage. Our hypothesis is that cellular mechanisms maintaining mitochondrial function must be protected in order to prevent MODS. Recent animal experiments indicate that host defences which target and kill microbes, in part via reactive oxygen and nitrogen production, also injure mitochondria, thus activating mitochondrial cell death pathways. To limit such collateral damage, the cell up-regulates and imports into mitochondria several nuclear-encoded proteins for antioxidant defence and mitochondrial DNA (mtDNA) replication. Fully integrated responses lead to mitochondrial biogenesis, which may alter cellular phenotype to avoid mitochondrial permeability transition, apoptosis, or energy failure. Key to the cell's vulnerability to oxidant generation by the innate immune response is the mtDNA content. MtDNA depletion is opposed by oxidation reduction (redox) signals that communicate the extent of mitochondrial damage to the nucleus. Molecular studies suggest that redox mechanisms activate two biogenic transcription factors, nuclear respiratory factors 1 and 2, which forestall a deterioration of oxidative phosphorylation during infection. Biogenic failure or an intrinsic biogenic arrest could hasten degradation of mitochondrial function and drive the cell to apoptosis or necrosis. By implication, novel protective strategies for biogenesis hold promise for the prevention of MODS.

Metabolic capacity regulates iron homeostasis in endothelial cells.

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.

Nitric oxide amplifies the excitatory to inhibitory neurotransmitter imbalance accelerating oxygen seizures.

CNS O2 toxicity is manifested most profoundly by generalized motor convulsions. The hypothesis was tested that HBO2 triggers seizures by an excitatory to inhibitory neurotransmitter imbalance produced by neuronal nitric oxide (NO) activity. Anesthetized rats were exposed to 5 ATA HBO2 for 75 min with or without prior inhibition of nNOS. Interstitial NO and amino acids: aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were determined in the striatum by microdialysis coupled with HPLC. Blood flow and EEG in the same striatal region were measured simultaneously. Rats treated with 7-NI showed no EEG spikes of O2 toxicity, while seizure latency for untreated rats was 63 +/- 7 min. Significant increases in NO metabolites and blood flow were observed in control rats before seizures. HBO2 did not change Glu significantly and increased Asp slightly whereas GABA decreased progressively by 37 +/- 7%. Pretreatment with 7-NI led to a significantly smaller decline in GABA. Overall, the simplified excitotoxicity index Glu/GABA increased significantly after 60 min of HBO2 in control but fell in rats treated with 7-NI. We conclude that HBO2-stimulated neuronal NO production promotes an imbalance between glutamatergic and GABAergic synaptic function implicated in the genesis of oxygen-induced seizures.

Oxygen-induced mitochondrial biogenesis in the rat hippocampus.

The hypothesis that damage to mitochondrial DNA by reactive oxygen species increases the activity of nuclear and mitochondrial transcription factors for mitochondrial DNA replication was tested in the in vivo rat brain. Mitochondrial reactive oxygen species generation was stimulated using pre-convulsive doses of hyperbaric oxygen and hippocampal mitochondrial DNA content and neuronal and mitochondrial morphology and cell proliferation were evaluated at 1, 5 and 10 days. Gene expression was subsequently evaluated to assess nuclear and mitochondrial-encoded respiratory genes, mitochondrial transcription factor A, and nuclear respiratory transcription factors-1 and -2. After 1 day, a mitochondrial DNA deletion emerged involving Complex I and IV subunit-encoding regions that was independent of overt neurological or cytological O(2) toxicity, and resolved before the onset of cell proliferation. This damage was attenuated by blockade of neuronal nitric oxide synthase. Compensatory responses were found in nuclear gene expression for manganese superoxide dismutase, mitochondrial transcription factor A, and nuclear respiratory transcription factor-2. Enhanced nuclear respiratory transcription factor-2 binding activity in hippocampus was accompanied by a nearly three-fold boost in mitochondrial DNA content over 5 days. The finding that O(2) activates regional mitochondrial DNA transcription, replication, and mitochondrial biogenesis in the hippocampus may have important implications for maintaining neuronal viability after brain injury.

Reduced inspiratory flow attenuates IL-8 release and MAPK activation of lung overstretch.

Lung overstretch involves mechanical factors, including large tidal volumes (VT), which induce inflammatory responses. The current authors hypothesised that inspiratory flow contributes to ventilator-induced inflammation. Buffer-perfused rabbit lungs were ventilated for 2 h with 21%, O2+5%, CO2, positive end-expiratory pressure of 2-3 cmH2O and randomly assigned to either: 1) normal VT (6 mL x kg(-1)) at respiratory rate (RR) 30, inspiration:expiration time ratio (I:E) 1:1, low inspiratory flow 6 mL x kg(-1) x s(-1); 2) large VT (12 mL x kg(-1)) at RR 30, I:E 1:1, high inspiratory flow 12 mL x kg(-1) x s(-1) (HRHF); 3) large VT at RR 15, I:E 1:1, low inspiratory flow 6 mL x kg(-1) x s(-1) (LRLF); or 4) large VT at RR 15, I:E 1:2.3, high inspiratory flow 10 mL x kg(-1) x s(-1) (LRHF). Physiological parameters, tumour necrosis factor (TNF)-alpha, interleukin (IL)-8 and activation of mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK)1/2, p38 and stress-activated protein kinase (SAPK)/ c-Jun N-terminal kinase (JNK)) were measured. HRHF increased weight gain, perfusate IL-8 and phosphorylation of ERK1/2, p38 and SAPK/JNK. These responses were absent during LRLF but present during LRHF. Changes in TNF-alpha were small. Tissue IL-8 and phospho-ERK1/2 staining was localised primarily to smooth muscle, adventitia and bronchial epithelium within larger bronchioles and arterioles. These results indicate that mild overstretch of perfused lungs during high inspiratory flow enhances inflammatory signalling by cells in lung regions most affected by strong turbulent airflow.

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Internal carotid artery dissection in stroke from SCUBA diving: a case report.

Although diving with compressed air is generally safe, neurological problems resulting from infarction in SCUBA diving are well known, including arterial gas embolism and decompression sickness (caisson's disease, bends) involving the brain and spinal cord. While air gas embolism forms the overwhelming majority of causes for stroke in divers, internal carotid artery (ICA) dissection is another potential mechanism for central nervous system infarction in the setting of SCUBA diving. A 38 year-old female, who presented with complaints of headache, nausea, vomiting, and left sided hemiparesis after rapid ascent to the surface from a depth of 120 feet of seawater was initially treated for decompression illness in a hyperbaric chamber. Further neurological workup revealed a right ICA dissection. This case demonstrates the dangers of ICA dissection following rapid ascent to the surface from underwater and emphasizes an interesting presentation of stroke associated with SCUBA diving.

Tissue factor in experimental acute lung injury.

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.

Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons.

Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 x 10(9) CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 x 10(10) CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.

Nitric oxide production is enhanced in rat brain before oxygen-induced convulsions.

Central nervous system oxygen toxicity (CNS O2 toxicity) is preceded by release of hyperoxic vasoconstriction, which increases regional cerebral blood flow (rCBF). These increases in rCBF precede the onset of O2-induced convulsions. We have tested the hypothesis that hyperbaric oxygen (HBO2) stimulates NO* production in the brain that leads to hyperemia and anticipates electrical signs of neurotoxicity. We measured rCBF and EEG responses in rats exposed at 4 to 6 atmospheres (ATA) of HBO2 and correlated them with brain interstitial NO* metabolites (NO(x)) as an index of NO* production. During exposures to hyperbaric oxygen rCBF decreased at 4 ATA, decreased for the initial 30 min at 5 ATA then gradually increased, and increased within 30 min at 6 ATA. Changes in rCBF correlated positively with NO(x) production; increases in rCBF during HBO2 exposure were associated with large increases in NO(x) at 5 and 6 ATA and always preceded EEG discharges as a sign of CNS O2 toxicity. In rats pretreated with L-NAME, rCBF remained maximally decreased throughout 75 min of HBO2 at 4, 5 and 6 ATA. These data provide the first direct evidence that increased NO* production during prolonged HBO2 exposure is responsible for escape from hyperoxic vasoconstriction. The finding suggests that NO* overproduction initiates CNS O2 toxicity by increasing rCBF, which allows excessive O2 to be delivered to the brain.

Inhaled carbon monoxide and hyperoxic lung injury in rats.

Because carbon monoxide (CO) has been proposed to have anti-inflammatory properties, we sought protective effects of CO in pulmonary O(2) toxicity, which leads rapidly to lung inflammation and respiratory failure. Based on published studies, we hypothesized that CO protects the lung against O(2) by selectively increasing expression of antioxidant enzymes, thereby decreasing oxidative injury and inflammation. Rats exposed to O(2) with or without CO [50-500 parts/million (ppm)] for 60 h were compared for lung wet-to-dry weight ratio (W/D), pleural fluid volume, myeloperoxidase (MPO) activity, histology, expression of heme oxygenase-1 (HO-1), and manganese superoxide dismutase (Mn SOD) proteins. The brains were evaluated for histological evidence of damage from CO. In O(2)-exposed animals, lung W/D increased from 4.8 in normal rats to 6.3; however, only CO at 200 and 500 ppm decreased W/D significantly (to 5.9) during O(2) exposure. Large volumes of pleural fluid accumulated in all rats, with no significant CO treatment effect. Lung MPO values increased after O(2) and were not attenuated by CO treatment. CO did not enhance lung expression of oxidant-responsive proteins Mn SOD and HO-1. Animals receiving O(2) and CO at 200 or 500 ppm showed significant apoptotic cell death in the cortex and hippocampus by immunochemical staining. Thus significant protection by CO against O(2)-induced lung injury could not be confirmed in rats, even at CO concentrations associated with apoptosis in the brain.

Antibody to intercellular adhesion molecule 1 (CD54) decreases survival and not lung injury in baboons with sepsis.

Neutrophil influx into the lung is an important event in the pathogenesis of acute lung injury in gram-negative sepsis. We hypothesized that administration of a monoclonal antibody to intercellular adhesion molecule 1 (ICAM-1, CD54), a molecule mediating neutrophil adhesion to endothelial cells, would decrease neutrophil sequestration and transmigration in the lung and attenuate lung injury in Escherichia coli sepsis. Sepsis was induced in 12 baboons primed with heat-killed E. coli (1 x 10(9) CFU/kg) 12 h before infusion of live bacteria (1 x 10(10) CFU/kg). Six animals received monoclonal antibody to CD54 (1 mg/kg) intravenously at the time of live E. coli infusion. After 48 h or when blood pressure could not be maintained, tissues were harvested and bronchoalveolar lavage (BAL) samples were obtained. Median survival time was decreased in anti-CD54-treated animals. This group also had decreased mean arterial pressure, increased metabolic acidosis, and decreased urine output. Measures of lung injury including gas exchange, lung lavage protein and lactate dehydrogenase (LDH), lung thiobarbituric acid-reactive species, and lung histology, including alveolar neutrophil volumes, were unaffected by treatment. The effect of anti-CD54 on neutrophil influx into tissues as measured by myeloperoxidase was organ specific. These data show that monoclonal antibody to CD54 does not ameliorate acute lung injury in E. coli sepsis, and septic primates given anti-CD54 have worsened metabolic parameters and decreased survival.

Vascular release of nonheme iron in perfused rabbit lungs.

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.