Ultrasound-guided percutaneous cryoneurolysis for management of acute sternal fracture pain.

Background: Establishing adequate analgesia for rib and sternal fractures remains a challenge due to the prolonged nature of the associated pain. Historically, cryoneurolysis has demonstrated beneficial in treating chronic pain, and the recent development of hand-held devices has allowed its functionality to expand into the management of acute pain. Case: We present a polytrauma patient with sternal and multiple rib fractures that underwent ultrasound-guided intercostal cryoneurolysis at bedside, resulting in significant analgesia lasting several weeks and improving mobilization. This is the first report of the utilization of cryoneurolysis to treat acute sternal fracture pain. Conclusion: The most common sternal fracture pattern is transverse which only requires treatment of four intercostal nerves, making cryoneurolysis feasible in trauma centers. This portable, minimally invasive, and low risk technique has the added benefits of reducing opioid requirements, decreasing length of hospital stay, and improving mobility in polytrauma patients.

Late-intervention study with ebselen in an experimental model of type 1 diabetic nephropathy.

BACKGROUND AND AIM: Previous studies have shown that preventive treatment with the antioxidant, ebselen, in experimental models of type 1 diabetic nephropathy resulted in an attenuation of structural and functional damage in the kidney. However, evidence for the effectiveness of ebselen in late-intervention studies is lacking. Thus, we aimed to investigate the effects of ebselen in attenuating established renal injury in type 1 diabetic nephropathy using the Akita mouse model. METHODS: Baseline blood glucose and albumin-to-creatinine ratio (ACR) were measured in wild-type (WT) and heterozygous Akita mice at 9 weeks of age. At 10 weeks of age, WT and Akita mice were randomized to receive either vehicle (5% carboxymethyl cellulose) or ebselen by oral gavage at 10mg/kg twice daily. Kidney and urine were collected after 16 weeks of treatment with ebselen for histological and functional analyses. RESULTS: At 9 weeks of age, Akita mice displayed well-established renal dysfunction with significant increases in ACR and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels when compared with WT controls. After 16 weeks of treatment with ebselen, oxidative stress, as measured by nitrotyrosine immunostaining and urinary 8-OHdG levels, was significantly reduced in the Akita mice. Furthermore, gene expression of the major reactive oxygen species-producing nicotinamide adenine dinucleotide phosphate enzyme, Nox4, was also reduced by ebselen. However, ebselen had no effect on ACR and glomerulosclerosis. CONCLUSION: Chronic treatment with ebselen significantly reduced oxidative stress in the Akita mice. However, ebselen failed to attenuate functional or structural kidney damage in this late-intervention study using the Akita mouse model.

Phagocyte-derived reactive oxygen species do not influence the progression of murine blood-stage malaria infections.

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91(phox-/-) mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91(phox-/-) or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91(phox-/-) mice and wild-type mice were not significantly different when they were treated with l-N(G)-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.

An altered antioxidant balance occurs in Down syndrome fetal organs: implications for the "gene dosage effect" hypothesis.

Down syndrome (DS) is the congenital birth defect responsible for the greatest number of individuals with mental retardation. It arises due to trisomy of human chromosome 21 (HSA21) or part thereof. To date there have been limited studies of HSA21 gene expression in trisomy 21 conceptuses. In this study we investigate the expression of the HSA21 antioxidant gene, Cu/Zn-superoxide dismutase-1 (SOD1) in various organs of control and DS aborted conceptuses. We show that SOD1 mRNA levels are elevated in DS brain, lung, heart and thymus. DS livers show decreased SOD1 mRNA expression compared with controls. Since non-HSA21 antioxidant genes are reported to be concomitantly upregulated in certain DS tissues, we examined the expression of glutathione peroxidase-1 (GPX1) in control and DS fetal organs. Interestingly, GPX1 expression was unchanged in the majority of DS organs and decreased in DS livers. We examined the SOD1 to GPX1 mRNA ratio in individual organs, as both enzymes form part of the body's defense against oxidative stress, and because a disproportionate increase of SOD1 to GPX1 results in noxious hydroxyl radical damage. All organs investigated show an approximately 2-fold increase in the SOD1 to GPX1 mRNA ratio. We propose that it is the altered antioxidant ratio that contributes to certain aspects of the DS phenotype.

Oxidative stress and neural dysfunction in Down syndrome.

Total or partial trisomy of chromosome 21 occurs with relatively high frequency and is responsible for the occurrence of Down syndrome. Phenotypically, individuals with Down syndrome display characteristic morphological features and a variety of clinical disorders. One of the challenges for researchers in this field has been to ascertain and understand the relationship between the Down syndrome phenotype with the gene dosage effect resulting from trisomy of chromosome 21. Much attention therefore, has been given towards investigating the consequences of overexpressing chromosome 21-linked genes. In particular, an extensive analysis of SOD1 and APP have provided important insights as to how perturbations in the expression of their respective genes may contribute to the Down syndrome phenotype. In this review we will highlight studies which support a key role for SOD1 and APP in the pathogenesis of neural abnormalities observed in individuals with Down syndrome. Central to this relationship is how the redox state of the cell is affected and its consequences to neural function and integrity.

Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide.

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (-/-) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H2O2. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (-/-) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg.kg-1 (approximately (1)/(7) the LD50 of wild-type controls). The effects of paraquat were dose-related. In the 30 mg.kg-1-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulates Gpx1 in normal cells, reinforcing a role for Gpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (-/-) mice are more susceptible to H2O2; 30% of neurons from Gpx1 (-/-) mice were killed when exposed to 65 microM H2O2, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H2O2. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest that Gpx1 (-/-) mice may be valuable for studying the role of H2O2 in neurodegenerative disorders.

Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis?

Hydrogen peroxide has been shown to induce either apoptosis or features of senescence in different cultured cell lines. We now show that both processes can be induced in the same culture of primary human diploid fibroblasts and that the outcome of apoptosis or the senescence-like phenotype is determined by the H2O2 concentrations. At 50 and 100 microM, H2O2 predominantly induced the senescence-like state, characterized by a reduced rate of proliferation, an increased number of cells in G0-G1, typically enlarged and flattened morphology, and increased CIP1 and fibronectin expression. At 300 and 400 microM, H2O2 mainly triggered apoptosis. At the intermediate 200 microM H2O2, features of both senescence and apoptosis were observed in the same culture. Thus, the higher the H2O2 concentration, the higher the proportion of cells undergoing apoptosis, suggesting a key role of the level of damage in the choice of a cell population to enter apoptosis and/or the senescence-like state. Before the induction of one or the other process, cells entered a transient "shock state" characterized by a typical morphological change, cell cycle arrest in G0-G1, and the induction of CIP1 and BCL-2.

Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide.

Although reactive oxygen species have been proposed to play a major role in the aging process, the exact molecular mechanisms remain elusive. In this study we investigate the effects of a perturbation in the ratio of Cu/Zn-superoxide dismutase activity (Sod1 dismutases .O2-to H2O2) to glutathione peroxidase activity (Gpx1 catalyses H2O2 conversion to H2O) on cell growth and development. Our data demonstrate that Sod1 transfected cell lines that have an elevation in the ratio of Sod1 activity to Gpx1 activity produce higher levels of H2O2 and exhibit well characterised markers of cellular senescence viz. slower proliferation and altered morphology. On the contrary, Sod1 transfected cell lines that have an unaltered ratio in the activity of these two enzymes, have unaltered levels of H2O2 and fail to show characteristics of senescence. Furthermore, fibroblasts established from individuals with Down syndrome have an increase in the ratio of Sod1 to Gpx1 activity compared with corresponding controls and senesce earlier. Interestingly, cells treated with H2O2 also show features of senescence and/or senesce earlier. We also show that Cip1 mRNA levels are elevated in Down syndrome cells, Sod1 transfectants with an altered Sod1 to Gpx1 activity ratio and those treated with H2O2, thus suggesting that the slow proliferation may be mediated by Cip1. Furthermore, our data demonstrate that Cip1 mRNA levels are induced by exposure of cells to H2O2. These data give valuable insight into possible molecular mechanisms that contribute tribute to cellular senescence and may be useful in the evolution of therapeutic strategies for aging.

Changes in the levels of enzymes which modulate the antioxidant balance occur during aging and correlate with cellular damage.

Oxidative metabolism produces a flux of superoxide anions that must be removed from the cellular environment if the cell is to survive. The levels of antioxidant enzyme involved in the elimination of superoxide anions and/or hydrogen peroxide were investigated in an attempt to correlate any changes in the levels of these enzymes during aging with changes in free radical mediated cellular damage. Cu/Zn superoxide dismutase (Sod1), glutathione peroxidase (Gpx1) and catalase levels were measured in a number of organs during murine aging. Sod1 enzyme activity rose during aging in all organs studied, while the levels of both Gpx1 and catalase showed organ specific profiles. Both organs in which lipid peroxidation damage (which was used as a marker of free radical mediated damage) increased with age, namely the brain and small intestine, also showed a significant increase in the ratio of Sod1 to Gpx1 enzyme activity. In organs where either the ratio of Sod1/Gpx1 activity or Sod1/catalase levels (in the lung only) ratios were maintained during aging, no increased lipid peroxidation damage was detected. In the lung where Sod1/Gpx1 ratio did increase, Sod1/catalase remained constant and this was able to provide protection during aging. Thus our data shows that alterations in the balance between first and second steps of the antioxidant pathway correlate with cellular damage, and that this may contribute to the aging changes seen in some organs.

Cu/Zn-superoxide dismutase and glutathione peroxidase during aging.

During oxidative metabolism harmful reactive oxygen species (ROS) are generated. These species are neutralized by antioxidant enzymes. Firstly, superoxide dismutase (Sod) converts superoxide radicals (.O2-) to hydrogen peroxide (H2O2). Thereafter catalase (Cat) and glutathione peroxidase (Gpx) independently convert this to water. An imbalance in the ratio of Sod to Gpx and Cat results in the accumulation of H2O2 which may participate in the Fenton reaction, resulting in the formation of noxious hydroxyl radicals. These ROS are highly reactive and cause damage to macromolecules such as DNA, protein and lipids. We propose that it is the balance in the activity of the Sod to Gpx plus Cat ratio (Sod/(Gpx plus Cat)) that is an important determinant of cellular aging. This is based on our observation that an altered Cu/Zn-superoxide dismutase (Sod1)/(Gpx1 plus Cat) ratio exists in the brain of aging mice and that this correlates with increased lipid damage. Conversely, aging liver and kidney have an unaffected Sod1/(Gpx1 plus Cat) ratio and lipid damage is not increased with aging. We also examine the Sod1 to Gpx1 ratio in Down syndrome tissue and show that all organs have an altered ratio. This may contribute to the premature aging seen in these individuals. We show that binding of a p50/p65 complex to an NF-kappa B consensus sequence is enhanced by H2O2 treatment in NIH3T3 cells. Thus an altered Sod1/(Gpx1 plus Cat) ratio may also affect gene expression by altering the binding and/or availability of transcription factors to DNA.

Expression of copper/zinc superoxide dismutase and glutathione peroxidase in organs of developing mouse embryos, fetuses, and neonates.

The rise in antioxidant enzyme activity in the lungs of late-gestation fetuses is thought to be caused by the preparation of the pulmonary antioxidant system for birth. However, recent data have shown that such a rise also occurs in the livers of late-gestation fetuses. Consequently, this surge cannot solely be ascribed to the preparation of the pulmonary antioxidant system for birth. In this study we examine the expression of copper/zinc superoxide dismutase (Sod1) and glutathione peroxidase (Gpx1) in various organs of late-gestational mouse fetuses. Furthermore, we compare the expression of these genes in organs of fetuses, neonates, and adult mice. These studies were carried out to investigate whether the change in mRNA levels for these two genes is related to a developmental change in oxidant stress. Our data demonstrate that an increase in both Sod1 and Gpx1 mRNA occurs in lungs and livers of late-gestational mouse fetuses. The brain demonstrates an increase in Sod1 expression at or around the time of birth, the kidney shows an elevation in Gpx1 mRNA levels, and the heart fails to demonstrate a surge in both Sod1 and Gpx1 mRNA levels. Our data show that the liver is the organ with the highest levels of Sod1 and Gpx1 mRNA in embryos and neonates (immediately after birth). In the adult, the liver has the highest levels of Sod1 mRNA and the spleen the highest level of Gpx1 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cu/Zn superoxide dismutase mRNA and enzyme activity, and susceptibility to lipid peroxidation, increases with aging in murine brains.

To protect against reactive oxygen species, prokaryotic and eukaryotic cells have developed an antioxidant defence mechanism where O2- is converted to H2O2 by superoxide dismutase (Sod), and in a second step, H2O2 is converted to H2O by catalase (Cat) and/or glutathione peroxidase (Gpx). If Sod levels are increased without a concomitant Gpx increase, then the intermediate H2O2 accumulates. This intermediate could undergo the Fenton's reaction, generating hydroxyl radicals which may lead to lipid peroxidation in cells. In this study, we investigate the expression of Sod1, Gpx1 and susceptibility to lipid peroxidation during the aging process in mouse brains. We demonstrate that the mRNA levels and enzyme activity of Sod1 are higher in brains from adult mice compared to neonatal mice. Furthermore, we show that a linear increase in Sod1 mRNA and enzyme activity occurs with aging (1-100 weeks). On the contrary, we find that the mRNA and enzyme activity for Gpx1 does not increase with aging in mouse brains. In addition, our results demonstrate that the susceptibility of murine brains to lipid peroxidation increases with aging. The data in this study are consistent with the notion that reactive oxygen species may contribute to the aging process in mammalian brains. These results are discussed in relation to the normal aging process in mammals, and to the premature aging and mental retardation in Down syndrome.

Localization of four human chromosome 21 genes--SOD1, ETS2, IFNAR, and CBR--to two different chromosomes in the marsupial species Macropus eugenii.

We have mapped the chromosomal location of four genes previously assigned to human chromosome 21--Cu/Zn superoxide dismutase (SOD1), the protooncogene ETS2, the interferon alpha/beta receptor gene (IFNAR), and the carbonyl reductase gene (CBR)--in the tammar, Macropus eugenii. The genes are localized on two separate autosomes: SOD1 and CBR map to chromosome 7 and ETS2 and IFNAR map to chromosome 3 or 4. These results provide the first example of asynteny between SOD1/CBR and ETS2/IFNAR in a mammalian species. The results suggest that either this synteny group has been disrupted in the marsupial lineage, or, alternatively, the genes located on human chromosome 21 may have been joined after the marsupials diverged from the eutherian mammals some 130-150 million years ago.

Effect of thymidine analogs on tyrosinase activity and mRNA accumulation in mouse melanoma cells.

The thymidine analog 5-bromodeoxyuridine (BrdU) has been found to inhibit terminal differentiation of a variety of cell types without significantly affecting the growth of these cells. We have compared the effect of BrdU with two other thymidine analogs, 5-iododeoxyuridine and 5-fluorodeoxyuridine, on the growth, tyrosinase activity, and tyrosinase-mRNA accumulation in BL-6 mouse melanoma cells. We show that all three analogs inhibit growth and tyrosinase activity, but only BrdU significantly inhibits the accumulation of tyrosinase mRNA. We consider these results in the light of current understanding of BrdU action.

Differential effects of DNA synthesis inhibitors on DNA methylation in normal and transformed cells.

1-beta-D-Arabinofuranosylcytosine (ara-C) and aphidicolin are well known inhibitors of DNA synthesis, each one acting through a different mechanism. We have examined the effects of these compounds on DNA methylation in normal human embryonic lung fibroblasts (WI-38) as well as in their Simian Virus 40 (SVWI-38) and gamma radiation (CT-1) transformed counterparts. Analysis of the methylation status of the total genomic DNA in WI-38 cells revealed that hydroxyurea was the only drug which resulted in a significant increase in the 5-methylcytosine content under conditions where greater than 98% inhibition of DNA synthesis was achieved. Under the same conditions, all three drugs were capable of inducing hypermethylation in the SVWI-38 cells, whereas none of them showed any effect on the methylation status of the DNA in the CT-1 cells. In cells where a limited degree of replication was allowed to occur at drug concentrations resulting in 50% inhibition of DNA synthesis, a different pattern emerged. Under these conditions, DNA which was synthesized in the presence of either ara-C or aphidicolin was significantly hypermethylated in both the transformed cell lines, whereas hydroxyurea had no effect. In the normal WI-38 cells however, hydroxyurea was still the only drug which caused any significant hypermethylation. Different cells thus responded differently to these three agents, and the mere slowing down of DNA synthesis did not ipso facto lead to increased DNA methylation.

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts.

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.

Effects of sodium butyrate on the synthesis and methylation of DNA in normal cells and their transformed counterparts.

The effects of various concentrations of sodium butyrate were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40-transformed line (SVWI-38) and a cell line transformed by gamma-irradiation (CT-1). The rate of thymidine incorporation into DNA was inhibited by 60-80% in the WI-38 cells, even at butyrate concentrations as low as 5 mM. The two transformed cell lines showed no inhibition of DNA synthesis, even at concentrations of 75 mM butyrate. Analysis of RNA and protein synthesis revealed that the former was inhibited by +/- 20% at 5-10 mM butyrate in the normal WI-38 cell line, while protein synthesis was not inhibited at these concentrations. The inhibition of RNA synthesis was not dose dependent up to butyrate concentrations of 20 mM, and protein synthesis was inhibited less than 15% at this concentration. None of these inhibitory effects was observed in the case of the SVWI-38 or CT-1 cell lines. Analysis of the 5-methylcytosine content of DNA that was labeled either prior to or during treatment with butyrate revealed an increased content of methylcytosine when compared with control cells. Both preexisting and newly synthesized DNAs were thus subject to hypermethylation. Although all three cell lines showed a dose-dependent hypermethylation of DNA, the extent of this methylation differed in the normal and transformed lines, as preexisting DNA was more methylated in WI-38 cells compared with SVWI-38 and CT-1 cells, while methylation of newly synthesized DNA occurred to a greater extent in the SVWI-38 cells. These studies show that sodium butyrate affects major macromolecular synthetic processes as well as DNA methylation quite differently in normal and transformed cells.