Double Insult Research Articles

Association of the hOGG1 Ser326Cys polymorphism with sporadic amyotrophic lateral sclerosis

Amyotropic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease causing the loss of motoneurons of the brain and the spinal cord. The etiology of ALS is still uncertain, but males are at increased risk for the disease than females. Several studies have suggested that motoneurons in ALS might be subjected to the double insult of increased DNA oxidative damage and deficiencies in DNA repair systems. Particularly, increased levels of 8-oxoguanine and impairments of the DNA base excision repair system have been observed in neurons of ALS patients. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. To evaluate the role of the hOGG1 Ser326Cys polymorphism in sporadic ALS (sALS), we screened 136 patients and 129 matched controls. In the total population, we observed association between both the Cys326 allele (p=0.02) and the combined Ser326Cys+Cys326Cys genotype (OR=1.65, 95% CI=1.06-2.88) and increased risk of disease. After stratification by gender, the Cys326 allele (p=0.01), both the Ser326Cys genotype (OR=2.14, 95% CI=1.09-4.19) and the combined Ser326Cys+Cys326Cys genotype (OR=2.15, 95% CI=1.16-4.01) were associated with sALS risk only in males. No significant association between the Ser326Cys polymorphism and disease phenotype, including age and site of onset and disease progression, was observed. Present results suggest a possible involvement of the hOGG1 Ser326Cys polymorphism in sALS pathogenesis.

Characterization of a thromboembolic photochemical model of repeated stroke in mice

Many stroke research groups utilize the model of middle cerebral artery occlusion induced by insertion of an intraluminal thread, owing to its pragmatism and reliability of cerebral infarct generation. However, 75% of stroke cases result from a thromboembolic event and 10% from occlusive atherothrombosis in situ. Here, we characterize a mouse model of repeated thromboembolic stroke, which closely mimics the intravascular pathophysiology of arterial thrombus generation from an atherosclerotic plaque, and subsequent release of a thrombus into the cerebral circulation as an embolus. Common carotid artery thrombosis (CCAT) was induced photochemically leading to non-occlusive platelet aggregation in C57/BL6 male mice (n=35), and was followed by mechanical assistance to facilitate release of the thrombus (MRT) and thus promote embolism. Six experimental groups, differing by changes in the surgical protocol, were used for the purpose of determining which such procedure yielded the most reliable and consistent brain infarct volumes with the lowest mortality at 3 days after surgery. The group which best satisfied these conditions was a double insult group which consisted of animals that underwent CCAT for 2min by means of argon laser irradiation (514.5nm) at an intensity of ca. 130W/cm2, with concomitant injection of erythrosin B (EB) (35mg/kg infused over those same 2min), followed by MRT 1min later; the entire procedure was repeated 24h later. This group showed a percent of brain lesion volume of 15±4% (mean±S.D.) with no associated 3-day mortality. Compared to a single insult group which sustained a percent brain lesion volume of 7±3%, there was a statistically significant (p<0.05) increase in the volume of infarction in the double-insult group.

Acute Intermittent Porphyria, Rasmussen Encephalitis, or Both?

A case of a young woman who suffers from refractory epilepsy in the form of Rasmussen encephalitis and acute intermittent porphyria is presented. The patient developed refractory partial seizures with progressive hemispheric atrophy in the first decade. Both her serum and cerebrospinal fluid contained significantly elevated levels of anti-GluR3B antibodies. Her serum also contained anti-NR2A antibodies (directed against the N-methyl-D-aspartate receptor). Seven years later, acute intermittent porphyria was diagnosed as she developed an acute episode of abdominal pain, dark urine, and hyponatremia. For several years, all attempts to discontinue porphyrinogenic antiepileptic drugs such as phenobarbital and valproate resulted in seizure worsening. During a major acute intermittent porphyria crisis, brain edema and coma developed, allowing the discontinuation of phenobarbital. On recovery, atrophy of the right hemisphere ensued. Several etiologic hypotheses are presented. Double insults, porphyria, and an autoimmune process are suggested for the development of Rasmussen encephalitis in this patient. The authors recommend testing for porphyria in cases of Rasmussen encephalitis and other intractable seizures.

A Ser326Cys polymorphism in the DNA repair gene hOGG1 is not associated with sporadic Alzheimer's disease

Oxidative damage accumulates in the DNA of the human brain over time, and is supposed to play a critical role in the pathogenesis of Alzheimer's disease (AD). It has been suggested that the brain in AD might be subjected to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for the removal of oxidized bases. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the base excision repair (BER) pathway, and a decrease in BER activity was observed in post-mortem brain regions of AD individuals, especially in the activity of 8-oxoguanine DNA glycosylase. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 ( hOGG1) gene is associated with a reduced DNA repair activity. However, although a deficient BER was proposed in the etiology of AD by several authors, polymorphisms of BER genes have not been studied in AD yet. We performed a case-control study including 178 patients with sporadic AD (sAD) and 146 matched controls to evaluate the role of the Ser326Cys polymorphism as a risk factor for sAD. In the present study we failed to find any association between allele ( χ 2 = 0.03, p = 0.86) or genotype ( χ 2 = 0.25, p = 0.882) frequencies of hOGG1 Ser326Cys and the risk of sAD. Present results suggest that the Ser326Cys polymorphism of the hOGG1 gene is not an independent risk factor for sAD.

Differential expression of calcitonin gene-related peptide (CGRP) and choline acetyltransferase (ChAT) in the axotomized motoneurons of normoxic and hypoxic rats

We employed a double injury model (axotomy along with hypoxia) to determine how nerve injury and hypoxic insult would affect the expression of calcitonin gene-related peptide (CGRP) and choline acetyltransferase (ChAT) in the hypoglossal nucleus (HN) and nucleus ambiguus (NA). Adult rats were subjected to unilateral vagus and hypoglossal nerve transection, following which half of the animals were kept in an altitude chamber ( PO 2 = 380 Torr). The immunoexpression of CGRP and ChAT (CGRP-IR/ChAT-IR) were examined by quantitative immunohistochemistry at 3, 7, 14, 30 and 60 days post-axotomy. The results revealed that CGRP-IR in the HN was increased at 3 days but decreased to basal levels at 7 days following nerve injury. The decline was followed by a second rise in CGRP-IR at 30 days post-axotomy, followed again by a return to basal levels at 60 days. In the NA, CGRP-IR was up-regulated at 3 days and remained increased for up to 60 days after nerve injury. Animals treated with a double injury showed a greater CGRP-IR than normoxic group in both nuclei at all post-axtomized periods. In contrast to CGRP, ChAT-IR was markedly reduced in the HN and NA at 3 days reaching its nadir at 14 days following nerve injury. Hypoxic animals showed a stronger reduction of ChAT-IR in both nuclei at all post-axtomized periods. Results of cell counting showed that neuronal loss was somewhat obvious in hypoxic HN than that of normoxic ones. The present results suggest that up-regulation of CGRP-IR may exert its trophic effects while down-regulation of ChAT-IR may correlate with the poor neurotransmission within the injured neurons. It is speculated that the enhanced expression of CGRP-IR and the pronounced reduction of ChAT-IR in hypoxic rats may result from a drastic shift of intracellular metabolic pathways, which in turn could lead to more metabolic loading to the severely damaged neurons following the double insult.

Diverse regulation of NF-kappaB and peroxisome proliferator-activated receptors in murine nonalcoholic fatty liver.

Fatty liver is highly sensitive to inflammatory activation. Peroxisome proliferator-activated receptors (PPAR) have anti-inflammatory effects and regulate lipid metabolism in the fatty liver. We hypothesized that fatty liver leads to endotoxin sensitivity through an imbalance between pro- and anti-inflammatory signals. Leptin-deficient, ob/ob mice and their lean littermates were challenged with single or double insults and pro- and anti-inflammatory pathways were tested on cytokine production and activation of nuclear regulatory factors NF-kappaB and peroxisome proliferator receptor element (PPRE). Ob/ob mice produced significantly higher serum tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 6 and showed increased hepatic NF-kappaB activation compared to lean littermates after stimulation with a single dose of lipopolysaccharide (LPS) or alcohol. In ob/ob mice, double insults with alcohol and LPS augmented proinflammatory responses mediated by increased degradation of inhibitory kappaB (IkappaB)-alpha and IkappaB-beta and preferential induction of the p65/p50 NF-kappaB heterodimer. In lean mice, in contrast, acute alcohol attenuated LPS-induced TNF-alpha, IL-6 production, and NF-kappaB activation through reduced IkappaB-alpha degradation and induction of p50/p50 homodimers. PPRE binding was increased in fatty but not in lean livers after alcohol or LPS stimulation. However, cotreatment with alcohol and LPS reduced both PPRE binding and nuclear levels of PPAR-alpha in fatty livers but increased those in lean livers. In conclusion, our results show opposite PPRE and NF-kappaB activation in fatty and lean livers. PPAR activation may represent an anti-inflammatory mechanism that fails in the fatty liver on increased proinflammatory pressure. Thus, an imbalance between PPAR-mediated anti-inflammatory and NF-kappaB-mediated proinflammatory signals may contribute to increased inflammation in the fatty liver.

Temporal profile of enhanced vulnerability of the postthrombotic brain to secondary embolic events.

Patients with vascular or cardiac disease may experience recurrent thrombosis and embolization to the cerebral vasculature. Transient distal platelet accumulation after common carotid artery thrombosis (CCAT) leads to hemodynamic, metabolic, and molecular events that may influence the response of the postthromboembolic brain to secondary emboli. We investigated the effect of repeated embolic episodes on histopathological outcome at various time intervals using a clinically relevant model of embolic stroke. Six groups of rats underwent either photochemically induced CCAT followed by sham surgery or 2 episodes of CCAT separated by 10 minutes or 1, 3, 5, or 7 days. Outcome measures included routine histopathological analysis and determination of the number of infarct loci and their total volume. Rats that underwent a second CCAT at 1, 3, or 5 days after the first insult had 20 to 30 times larger infarct volumes than rats in the single-CCAT group (P<0.05). In addition, rats in the 10-minute and 1-, 3-, and 5-day groups had 2 to 3 times as many infarcts as those in the single-CCAT group (P<0.05). Infarcts produced by double insults commonly extended through the neuraxis and were necrotic, edematous, and sometimes hemorrhagic. A prior thromboembolic event puts the brain at risk for severe infarction after a second embolic event. These findings cannot be explained solely by a greater number of infarcts. Elucidating pathomechanisms responsible for the vulnerability of the postthromboembolic brain may provide targets for new treatment strategies to prevent the severe consequences of embolic stroke.

Heat shock proteins, inflammation, and cardiovascular disease.

Atherosclerosis and its associated complications, such as coronary artery disease, peripheral vascular disease, and stroke, are the leading causes of morbidity and mortality in all racial groups of most westernized societies. A better understanding of the events that lead to the induction and progression of atherosclerosis and the development of strategies that control these processes would have a significant impact on human health. Atherosclerosis is a chronic inflammatory condition that usually begins at an early age in the absence of lipid accumulation, with fatty streaks composed of lipid-laden macrophages (foam cells) developing at later stages. T lymphocytes, predominantly of the CD4+ phenotype, are associated with the intima layer of the vessel. The inflammatory process at atherogenic sites leads to the production of cytokines and other inflammatory mediators, resulting in cell migration, proliferation, extracellular matrix production, and plaque development.1–3⇓⇓ Monocytes and macrophages appear intimately involved with the development of atherosclerosis; mice with the osteopetrotic ( op ) mutation in the macrophage colony-stimulating factor (M-CSF) gene, which results in a complete absence of M-CSF in the serum and tissues and a marked reduction in the number of circulating monocytes, exhibit significantly less atherosclerosis than control littermates when bred into an apolipoprotein (apo) E–deficient background,.4 The precise role of T and B cells in atherosclerosis remains unclear. Although activated T cells are a dominant feature of atherosclerotic lesions,5,6⇓ studies demonstrating that atherosclerosis can be induced by hypercholesterolemia in mice deficient in T and B cells suggest that these cells are not essential.7–9⇓⇓ However, the cholesterol levels in these studies far exceeded those present in human subjects without any genetic defect, and the findings should therefore be interpreted with caution. Indeed, other evidence indicates that T cells may attenuate atherogenesis by some means, because the elimination of …

Increased DNA oxidation and decreased levels of repair products in Alzheimer's disease ventricular CSF.

One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solid-phase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations (p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples (p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.

Heat shock protein and the double insult theory for the development of insulin dependent diabetes.

Heat shock proteins (HSP) are the most widely conserved group of proteins in phylogeny and play an important role in infection and autoimmunity. HSP65 has been suggested as the primary antigen in insulin dependent diabetes while an alternative antigen glutamic acid decarboxylase (GAD), has similar amino acid sequences. A 'double insult theory' for the development of insulin dependent diabetes is suggested whereby a bacterial infection leads to the production of HSP antibody. If during a 'window of opportunity' this is followed by a viral infection of the islet cells this could, in certain histocompatibility locus antigen (HLA) groups only, lead to the production of HSPs on the cell surface and a destructive autoimmune reaction.

Experimental cardiac anaphylaxis: Physiologic, pharmacologic and biochemical aspects of immune reactions in the isolated heart

Experimental cardiac anaphylaxis is initiated by a specific reaction between antigen and antibody, one of which has to be fixed to susceptible cells in the cardiac tissue. The biochemical steps initiated by the reaction appear eventually to release a variety of agents which, either by their direct effect or by their indirect effect as enzymes acting on plasma substrates, exert pharmacologic actions on the heart, producing a variety of irregularities. In most animals, and particularly in the guinea pig, histamine is the principal agent liberated. It is especially abundant in the mast cells of the right atrium, and its release accounts for most of the increased rate and amplitude of contraction, coronary constriction and atrioventricular block observed in the perfused heart. The chronotropic and inotropic responses in anaphylaxis are demonstrated equally well in the isolated atria and they are indistinguishable, electrophysiologically, from similar changes produced by histamine. The intensity of the reaction, as demonstrated in experiments utilizing passive sensitization, depends on the load of immunologic reactant fixed to the tissue and the concentration of the agent, either antigen or antibody, used to evoke it. The character of the vasoactive agent released is, additionally, determined by the electrophoretic class of antibody used for sensitization. Finally, bacterial toxins such as streptolysin can simultaneously produce a double insult to the heart of an immunized animal: They evoke anaphylaxis by the specific reaction of the antigenic determinant with sensitized cells at the same time that they produce damage to other regions of the heart through the toxic group of the molecule.