Overt Injury Research Articles

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

Elevated concentrations of plasminogen activator inhibitor-1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, β-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40-80 and 200-400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10-15 nM in control animals to 20-40 nM in hPAI-1-overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment.

Serum non-coding RNAs as biomarkers for osteoarthritis progression after ACL injury

The aim of this study was to examine serum non-coding RNAs as potential biomarkers for cartilage damage associated with anterior cruciate ligament (ACL) injury. Serum was obtained from 80 patients 1 year after surgery for ACL injury and 60 normal donors without overt skeletal injury. Total serum RNA was isolated, small non-coding RNAs profiled by TaqMan array MicroRNA (miRNA) analysis and individual small RNA assays performed by quantitative TaqMan RT-PCR (qPCR). Semi-quantitative magnetic resonance imaging (MRI) analysis was performed using Whole Organ Magnetic Resonance Knee Score (WORMS) scoring for analysis of cartilage damage. Initial TaqMan array miRNA profiling showed an increased serum concentration of a small nucleolar RNA (snoRNA), U48, in five patients with cartilage damage compared with that in five patients without cartilage damage and six normal donors. Independent qPCR analysis of snoRNAs in serum from all patients and normal donors showed a strong association between the serum level of another snoRNA, U38, and cartilage damage in ACL injury patients and together with snoRNA, U48, clear distinction between ACL injury patients and normal donors. SnoRNAs U38 and U48 are significantly elevated in the serum of patients developing cartilage damage at 1 year after ACL injury. Serum levels of U38 have the potential to facilitate early diagnosis of patients with cartilage damage after ACL injury. This study suggests serum non-coding RNAs may serve as novel noninvasive biomarkers for the detection and assessment of cartilage damage after ACL injury.

Preterm infants: eyes into brain function

The brains of preterm infants at term differ from the brains of term born infants in a number of ways that have been identified by magnetic resonance imaging techniques. At term, the brain of the preterm without overt brain injury has different relative volumes for multiple regions of the brain relative to the brain of the infant born at term. These structural differences can persist into childhood, and processing of information can occur in brain regions that differ for the preterm and term brain in childhood. Another avenue into the developing brain is via the senses (eg, the maturation of hearing pathways have been compared for preterm and term infants). In this issue of The Journal, Loe et al report that ocular responses can be used to evaluate executive brain function in children 9 to 16 years of age who were born preterm and at term. They found that the children who were born preterm have comparable sensorimotor function, but measurements of eye movements identified the children who were born preterm to be more distractible and to have prolonged response preparation. This information will be particularly helpful if it can be used for prevention or treatment strategies during early childhood. The more that is learned about the preterm brain, the more compelling it becomes to avoid prematurity whenever possible. Article page 427▶ Oculomotor Assessments of Executive Function in Preterm ChildrenThe Journal of PediatricsVol. 161Issue 3PreviewTo use objective, nonverbal oculomotor tasks to assess executive function and infer the neural basis of impairments in preterm children. Full-Text PDF

Is antiepileptogenesis a realistic goal in clinical trials? Concerns and new horizons*

Any attempt to make antiepileptogenesis a realistic goal in clinical trials should be based on the experience of failures of the past. A wide variety of experimental studies and clinical trials using chronic antiseizure drug therapy during the extended post-injury period have had minimal success. The disappointing results of these studies may be due to several factors including the possibility that antiseizure drugs, despite the fact they suppress seizure activity, do not interfere in any substantial way with the “epileptogenic” process of focal epilepsies. Although the reasons for the failure are not entirely clear, it may be that the antiseizure drugs may have been tested at the wrong doses, for the wrong duration, or at the wrong time after brain injury. Surprisingly, the anti-absence drug ethosuximide has also been shown to be antiepileptogenic in several experimental models of absence epilepsy. In addition, clinical trials aimed at preventing focal post-injury epilepsy have suffered from poor enrolment and other issues related to the comorbidity of severe epilepsies that follow overt brain injury. Testing specific anti-inflammatory and immunological antiepileptogenic agents to prevent focal epilepsies, as well as prevention trials for genetic epilepsies, possibly with anti-absence drugs, may be a way to resolve the dilemma. Although more evidence is needed, there is hope on the horizon for antiepileptogenic therapy that works.

SOD1, but not SOD3, deficiency accelerates diabetic renal injury in C57BL/6-Ins2Akita diabetic mice

Superoxide dismutase (SOD) is a major defender against excessive superoxide generated under hyperglycemia. We have recently reported that renal SOD1 (cytosolic CuZn-SOD) and SOD3 (extracellular CuZn-SOD) isoenzymes are remarkably down-regulated in KK/Ta-Ins2Akita diabetic mice, which exhibit progressive diabetic nephropathy (DN), but not in DN-resistant C57BL/6- Ins2Akita (C57BL/6-Akita) diabetic mice. To determine the role of SOD1 and SOD3 in DN, we generated C57BL/6-Akita diabetic mice with deficiency of SOD1 and/or SOD3 and investigated their renal phenotype at the age of 20 weeks. Increased glomerular superoxide levels were observed in SOD1−/−SOD3+/+ and SOD1−/−SOD3−/− C57BL/6-Akita mice but not in SOD1+/+SOD3−/− C57BL/6-Akita mice. The SOD1−/−SOD3+/+ and SOD1−/−SOD3−/− C57BL/6-Akita mice exhibited higher glomerular filtration rate, increased urinary albumin levels, and advanced mesangial expansion as compared with SOD1+/+SOD3+/+ C57BL/6-Akita mice, yet the severity of DN did not differ between the SOD1−/−SOD3+/+ and SOD1−/−SOD3−/− C57BL/6-Akita groups. Increased renal mRNA expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), reduced glomerular nitric oxide (NO), and increased renal prostaglandin E2 (PGE2) production were noted in the SOD1−/−SOD3+/+ and SOD1−/−SOD3−/− C57BL/6-Akita mice. This finding indicates that such renal changes in fibrogenic cytokines, NO, and PGE2, possibly caused by superoxide excess, would contribute to the development of overt albuminuria by promoting mesangial expansion, endothelial dysfunction, and glomerular hyperfiltration. The present results demonstrate that deficiency of SOD1, but not SOD3, increases renal superoxide in the setting of diabetes and causes overt renal injury in nephropathy-resistant diabetic mice, and that SOD3 deficiency does not provide additive effects on the severity of DN in SOD1-deficient C57BL/6-Akita mice.

Toll-like receptors in the immature human brain

Toll-like receptors (TLR), found on glial and neuronal cells, are major components of the innate immune system. They act as sensors that recognise pathogen-associated or damage-associate molecular patterns. There is...

Chronic Inflammatory IFN-γ Signaling Suppresses Hepatocarcinogenesis in Mice by Sensitizing Hepatocytes for Apoptosis

Chronic liver inflammation is a critical component of hepatocarcinogenesis. Indeed, inflammatory mediators are believed to promote liver cancer by upholding compensatory proliferation of hepatocytes in response to tissue damage. However, inflammation can also mediate the depletion of malignant cells, but the difference between tumor-suppressive and tumor-promoting inflammation is not defined at the molecular level. Here, we analyzed the role of the major inflammatory mediator IFN-γ in chemical hepatocarcinogenesis of transgenic mice that overexpress IFN-γ in the liver; these mice manifest severe chronic inflammatory liver damage and lasting compensatory regeneration. We found that chronic exposure to IFN-γ suppressed chemical hepatocarcinogenesis, despite overt liver injury. Indeed, IFN-γ-transgenic mice had significantly fewer and significantly less advanced malignant lesions than nontransgenic mice. This tumor-suppressive effect of IFN-γ seemed to be mediated in part by its known immune activating function, indicated by infiltration of IFN-γ-transgenic livers with CD8 T cells, natural killer T cells, and natural killer cells. However, IFN-γ seemed to prevent carcinogenesis also by activating the cell-intrinsic p53 tumor suppressor pathway. Indeed, exposure to IFN-γ in vivo or in vitro was associated with accumulation of p53 in hepatocytes and the sensitization of hepatocytes to apoptosis induced by genotoxic stress. The IFN-γ-induced increase in apoptosis of hepatocytes seemed to be p53 dependent. Thus, chronic inflammation dominated by IFN-γ may prevent hepatocarcinogenesis, despite continued inflammatory liver injury and regeneration. Therefore, the carcinogenic potential of inflammation seems to be determined by type and composition of its mediators and manipulating the type of chronic inflammation may serve the prevention of cancer.

Plasma glial fibrillary acidic protein levels in children with sickle cell disease

To determine if glial fibrillary acidic protein (GFAP) is associated with brain injury in children with sickle cell disease (SCD), we measured plasma GFAP among cross-sectional groups of unselected children with SCD, subsets of children with SCD and normal brain MRI or MRI evidence of cerebral infarct, healthy pediatric controls, and adults with brain injury. Children with SCD had higher plasma GFAP than healthy pediatric controls (mean concentrations 0.14 ± 0.37 vs. 0.07 ± 0.08 ng/mL; P 5 0.003); also, 16.0% (16/100) of children with SCD and cerebral infarct had GFAP elevations above the 95th percentile of healthy pediatric controls (P 5 0.04). Although not statistically significant, children with SCD and cerebral infarct had more elevated GFAP levels than with SCD and no infarct (16/100, 16.0% vs. 14/168, 8.3%; P 5 0.07). Children with SCD and acute brain ischemia had a higher proportion of elevated GFAP than SCD children with normal MRI (3/6, 50% vs.8.3%; P 5 0.01). GFAP was associated with elevated systolic blood pressure in the preceding year and correlated positively with white blood cell count and negatively with age and performance IQ. Plasma GFAP is elevated among children with SCD and may be associated with subclinical brain injury.

Programmed hypertension in rats treated with a NF-κB inhibitor during nephrogenesis: renal mechanisms

Suppression of the renin-angiotensin system (RAS) during murine lactation causes progressive renal injury, indicating a physiological action of angiotensin II on nephrogenesis. The nuclear factor NF-κB system is one of the main intracellular mediators of angiotensin II. We investigated whether inhibition of this system with pyrrolidine dithiocarbamate (PDTC) during rat nephrogenesis would lead to similar hypertension and renal injury as observed with RAS suppressors. Immediately after delivery, 32 Munich-Wistar dams, each nursing 6 male pups, were divided into 2 groups: C, untreated, and PDTC, receiving PDTC, 280 mg kg(-1) day(-1) orally, during 21 days. After weaning, the offspring were followed until 10 months of age without treatment. Adult rats that received neonatal PDTC exhibited stable hypertension and myocardial injury, without albuminuria. To gain additional insight into this process, the renal expression of RAS components and sodium transporters were determined by quantitative real-time PCR (qRT-PCR) at 3 and 10 months of life. Renal renin and angiotensinogen were upregulated at 3 and downregulated at 10 months of age, suggesting a role for early local RAS activation. Likewise, there was early upregulation of the proximal sodium/glucose and sodium/bicarbonate transporters, which abated later in life, suggesting that additional factors sustained hypertension in the long run. The conclusions drawn from the findings were as follows: (1) an intact NF-κB system during nephrogenesis may be essential to normal renal and cardiovascular function in adult life; (2) neonatal PDTC represents a new model of hypertension, lacking overt structural injury or functional impairment of the kidneys; and (3) hypertension in this model seems associated with early temporary activation of renal RAS and sodium transporters.

Mechanisms of alcoholic pancreatitis

Alcoholic pancreatitis is a major complication of alcohol abuse. The risk of developing pancreatitis increases with increasing doses of alcohol, suggesting that alcohol exerts dose-related toxic effects on the pancreas. However, it is also clear that only a minority of alcoholics develop the disease, indicating that an additional trigger may be required to initiate clinically evident pancreatic injury. It is now well established that alcohol is metabolized by the pancreas via both oxidative and non-oxidative metabolites. Alcohol and its metabolites produce changes in the acinar cells, which may promote premature intracellular digestive enzyme activation thereby predisposing the gland to autodigestive injury. Pancreatic stellate cells (PSCs) are activated directly by alcohol and its metabolites and also by cytokines and growth factors released during alcohol-induced pancreatic necroinflammation. Activated PSCs are the key cells responsible for producing the fibrosis of alcoholic chronic pancreatitis. Efforts to identify clinically relevant factors that may explain the susceptibility of some alcoholics to pancreatitis have been underway for several years. An unequivocal, functionally characterized, association is yet to be identified in clinical studies, although in the experimental setting, endotoxin has been shown to trigger overt pancreatic injury and to promote disease progression in alcohol-fed animals. Thus, while the molecular effects of alcohol on the pancreas have been increasingly clarified in recent years, identification of predisposing or triggering factors remains a challenge.

The Clinical Significance of Occult Thoracic Injury in Blunt Trauma Patients

Increased use of thoracic CT (TCT) in diagnosis of blunt traumatic injury has identified many injuries previously undetected on screening chest x-ray (CXR), termed "occult injury". The optimal management of occult rib fractures, pneumothoraces (PTX), hemothoraces (HTX), and pulmonary contusions is uncertain. Our objective was to determine the current management and clinical outcome of these occult blunt thoracic injuries. A retrospective review identified patients with blunt thoracic trauma who underwent both CXR and TCT over a 2-year period at a Level I urban trauma center. Patients with acute rib fractures, PTX, HTX, or pulmonary contusion on TCT were included. Patient groups analyzed included: (1) no injury (normal CXR, normal TCT, n=1337); (2) occult injury (normal CXR, abnormal TCT, n=205); and (3) overt injury (abnormal CXR, abnormal TCT, n=227). Patients with overt injury required significantly more mechanical ventilation and had greater mortality than either occult or no injury patients. Occult and no injury patients had similar ventilator needs and mortality, but occult injury patients remained hospitalized longer. No patient with isolated occult thoracic injury required intubation or tube thoracostomy. Occult injuries, diagnosed by TCT only, have minimal clinical consequences but attract increased hospital resources.

Effects of aging on glomerular function and number in living kidney donors

To elucidate the pathophysiologic changes in the kidney due to aging, we used physiological, morphometric, and imaging techniques to quantify GFR and its determinants in a group of 24 older (≥ 55 years) compared to 33 younger (≤ 45 years) living donors. Mathematical modeling was used to estimate the glomerular filtration coefficients for the whole kidney (K(f)) and for single nephrons (SNK(f)), as well as the number of filtering glomeruli (N(FG)). Compared to younger donors, older donors had a modest (15%) but significant depression of pre-donation GFR. Mean whole-kidney K(f), renocortical volume, and derived N(FG) were also significantly decreased in older donors. In contrast, glomerular structure and SNK(f) were not different in older and younger donors. Derived N(FG) in the bottom quartile of older donors was less than 27% of median-derived N(FG) in the two kidneys of younger donors. Nevertheless, the remaining kidney of older donors exhibited adaptive hyperfiltration and renocortical hypertrophy post-donation, comparable to that of younger donors. Thus, our study found the decline of GFR in older donors is due to a reduction in K(f) attributable to glomerulopenia. We recommend careful monitoring for and control of post-donation comorbidities that could exacerbate glomerular loss.

Focus

Focus

Skeletal Muscle Injury Versus Adaptation with Aging

A growing body of data supports a view that skeletal muscle's response after mechanical loading does not always result in the classically reported "injury response." Furthermore, current evidence supports a model of muscle adaptation and/or maladaptation, distinct from overt injury, in which myofiber degeneration and inflammation do not contribute as significantly as once reported even in aged populations.

Glial Fibrillary Acidic Protein as a Plasma Biomarker of Brain Injury in Children with Sickle Cell Disease.

Abstract 1512Poster Board I-535 IntroductionIn children with sickle cell disease (SCD), silent cerebral infarct (SCI) is an independent risk factor for lower IQ, poorer school performance, and overt stroke. MRI is the only method to identify patients with SCI. A blood biomarker of SCI in patients with sickle cell disease (SCD) would fill a clinical void because blood is easy to obtain and measure, a biomarker may determine risk of or progression of neurologic injury to overt stroke, and a biomarker could benchmark current and new therapies for SCI and other subclinical forms of neurologic injury in SCD. We hypothesized that an unbiased proteomics screen of plasma from sickle cell patients would yield biomarkers of brain injury. MethodsA cross-sectional sample of children 5-14 years old with sickle cell disease (HbSS and HbSB0) who were screened for the Silent Infarct Transfusion (SIT) Trial (ClinicalTrials.gov NCT00072761) were studied (n=258) along with 60 age-matched healthy controls and 28 adults with overt brain injury. For biomarker discovery, plasma underwent depletion of hemoglobin with nickel- nitrilotriacetic acid and immunoaffinity depletion of the 12 most abundant plasma proteins. After fractionation by reverse phase liquid chromatography over a C18 column and acetonitrile gradient and trypsin digestion, peptide spectra from each fraction were obtained by LC/MS/MS (LTQ-Orbitrap) and were searched using X!Tandem and human IPI database version 3.5. Post search analysis was performed using Proteomics Analysis Software System (Integrated Analysis Inc., Bethesda, MD). An electrochemiluminescent immunoassay was developed for one of the candidate proteins identified, glial fibrillary acidic protein (GFAP), for protein validation (MesoScale Discovery). ResultsGFAP, a brain-specific intermediate filament known to be a marker of acute stroke and head trauma in adults, was identified in discovery sample plasma. Four percent of sickle cell subjects in steady-state had plasma GFAP concentrations similar to stroke and brain surgery controls (>0.45 ng/mL). Among sickle cell subjects 5-14 years old screened for the SIT Trial, 9.3% had GFAP concentrations above the 95th percentile of age-matched controls (95th percentile cutoff: 0.227 ng/mL; p=0.08). Sickle cell subjects with silent cerebral infarct (SCI) had more elevations above the normal 95th percentile (10/69; 14.5%) than those without SCI (9/131; 6.8%), although this difference was not statistically significant (p=0.08). The sensitivity of GFAP as a marker of brain injury was demonstrated in a case of an 11 year-old child with HbSS and acute stroke in which plasma GFAP was 1.52 ng/mL (6 times the normal control 95th percentile) before the stroke was clinically evident and peaked at 2.83 ng/mL.We discovered GFAP as a circulating brain protein in plasma of children with SCD. Plasma GFAP is a marker of acute stroke in sickle cell disease but discriminates only minimally between SCI and non-SCI status in the SIT Trial using baseline cross-sectional samples. Four percent of children with sickle disease 5-14 years old have plasma GFAP levels similar to controls with severe brain injury without clinical evidence of overt stroke. ConclusionsElevations in circulating brain proteins such as GFAP show promise as indicators of subacute brain injury in children with SCI, but will require longitudinal studies of plasma GFAP in children with SCI to clarify the utility of GFAP as a plasma biomarker of SCI and a predictor of neurologic risk. DisclosuresNo relevant conflicts of interest to declare.

Ovarian steroids decrease DNA fragmentation in the serotonin neurons of non-injured rhesus macaques.

We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys received Silastic implants that were empty (placebo) or containing estradiol (E), progesterone (P) or estradiol plus progesterone (E+P) for one month. Eight levels of the dorsal raphe nucleus in a rostral to caudal direction were immunostained with TUNEL (terminal deoxynucleotidyl transferase nick end labeling). Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area. A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2 and TUNEL positive cells were counted. In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL positive cells (Mann Whitney test, both treatments p=0.04) and E+P treatment reduced the number of TUNEL positive cells/cubic millimeter (Mann Whitney test, p=0.04). Double immunocytochemistry for TUNEL and TPH indicated that DNA fragmentation was prominent in serotonin neurons. These data suggest that in the absence of ovarian steroids, a cascade of gene and protein expression leads to an increase in DNA fragmentation in serotonin neurons. Conversely, ovarian steroids have a neuroprotective role in the non-injured brain and prevent DNA fragmentation and cell death in serotonin neurons of nonhuman primates.

Changes in hepatic gene expression in response to hepatoprotective levels of zinc.

Zinc (Zn) administration at non-toxic doses protects against the hepatotoxicity produced by many agents, but the underlying mechanisms remain elusive. To examine the basis of Zn-induced generalised hepatoprotective effects. Rats and mice were given Zn at known hepatoprotective levels (100 mumol ZnCl2/kg/day, s.c., for 4 days) and molecular responses were assessed. Zn treatment produced changes in 5% of the genes on custom-designed mouse liver array and Rat Toxicology-II array. Changes in gene expression were further confirmed and extended by real-time reverse transcriptase-polymerase chain reaction. Zn treatment dramatically increased the expression of the metallothionein (Mt), and modestly increased the expression of acute-phase protein genes (ceruloplasmin, Stat3, egr1, Cxc chemokines and heat-shock proteins). For genes encoding for antioxidant enzymes, some were increased (Nrf2 and Nqo1), while others remained unaltered (Cu, Zn SOD and glutathione S-transferases). Expressions of cytokine and pro-inflammatory genes were not affected, while genes related to cell proliferation (cyclin D1) were modestly upregulated. Some metabolic enzyme genes, including cytochrome P450s and UDP-glucuronosyltransferase, were modestly suppressed, perhaps to switch cellular metabolic energy to acute-phase responses. Liver Zn content was increased between 1.6- and 2.1-fold, while hepatic MT protein was increased between 50 and 200-fold. Mice typically showed greater responses than rats. Such gene expression changes, particularly the dramatic induction of MT and Nrf2 antioxidant pathway, occur in the absence of overt liver injury, and are probably important in the hepatoprotective effects of Zn against toxic insults.

Esophageal Contour Changes during Cryoablation of Atrial Fibrillation

We have observed contour changes of the barium-filled esophagus during atrial fibrillation (AF) ablation with cryo-energy delivered in direct proximity to the esophagus. To evaluate the frequency, location, and severity of esophageal contour changes during cryo-energy application close to the esophagus. We retrospectively analyzed cine-fluoroscopic images acquired during hybrid cryo-radiofrequency AF ablation in 100 consecutive patients with cryo-energy delivered only in direct proximity to the esophagus. Esophageal contour changes were observed in 28 (32%) of 89 patients (and 74 [6.2%] of 1,191 of all cryo applications). They were more frequent in the left common pulmonary vein (PV) (50%) and less so in the right common PV and the upper PVs (4-5%). The distance of the ablation catheter from the endoesophageal contour prior to cryo-energy applications associated with contour changes was 1.8 +/- 1.5 mm, which increased to 4.1 +/- 1.6 mm at the time of peak contour change (P < 0.001). The esophageal contour deformation was 2.3 +/- 0.9 mm. There were no apparent complications related to cryo-energy application for 3-4 minutes, even if associated with contour changes. Esophageal contour changes were observed in >6% of cryo applications in direct proximity to the esophagus (32% of patients) and were most frequent in the posterior aspect of the left common and right lower PV ostium when cryo-energy was delivered at a distance of <or=5 mm from the esophageal contrast silhouette. No overt esophageal injury occurred. However, the safety of cryo-energy application in direct proximity to the esophagus remains to be further confirmed.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2 +/− mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2 +/− mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2 +/− mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2 +/− mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

Changes in midline thalamic recruiting responses in the prefrontal cortex of the rat during the development of chronic limbic seizures

Mesial temporal lobe epilepsy (MTLE) is a common form of epilepsy that affects the limbic system and is associated with decreases in memory and cognitive performance. The medial prefrontal cortex (PC) in rats, which has a role in memory, is associated with and linked anatomically to the limbic system, but it is unknown if and how MTLE affects the PC. We evoked responses in vivo in the PC by electrical stimulation of the mediodorsal (MD) and reuniens (RE) nuclei of the thalamus at several time points following status epilepticus, before and after onset of spontaneous seizures. Kindled animals were used as additional controls for the effect of seizures that were independent of epilepsy. Epileptic animals had decreased response amplitudes and significantly reduced recruiting compared to controls, whereas kindled animals showed an increase in both measures. These changes were not associated with neuronal loss in the PC, although there was significant loss in both the MD and RE in the epileptic animals. There is a significant reduction in the thalamically induced evoked responses in the PCs of epileptic animals. This finding suggests that physiologic dysfunction in MTLE extends beyond primary limbic circuits into areas without overt neuronal injury.