Guidelines for conducting epidemiological studies of blast injury.

Blast injuries are often caused by more than one mechanism, do not occur in isolation, and typically elicit a secondary multi-system response. Research efforts often do not separate blast injuries caused by blast waves from those caused by blunt force trauma and other mechanisms. 15 experts from nine different NATO nations developed in the HFM Research Task Group (RTG; HFM-234 (RTG)) 'Environmental Toxicology of Blast Exposures: Injury Metrics, Modelling, Methods and Standards' Guidelines for Conducting Epidemiological Studies of Blast Injury. This paper describes these guidelines, which are intended to provide blast injury researchers and clinicians with a basic set of recommendations for blast injury epidemiological study design and data collection that need to be considered and described when conducting prospective longitudinal studies of blast injury.

Guidelines for using animal models in blast injury research.

Blast injury is a very complex phenomenon and frequently results in multiple injuries. One method to investigate the consequences of blast injuries is with the use of living systems (animal models). The use of animals allows the examination and evaluation of injury mechanisms in a more controlled manner, allowing variables such as primary or secondary blast injury for example, to be isolated and manipulated as required. To ensure a degree of standardisation across the blast research community a set of guidelines which helps researchers navigate challenges of modelling blast injuries in animals is required. This paper describes the guidelines for Using Animal Models in Blast Injury Research developed by the NATO Health Factors and Medicine (HFM) Research Task Group 234.

Molecular characterization of atrazine resistance in common ragweed (Ambrosia artemisiifolia L.).

Common ragweed (Ambrosia artemisiifolia L.) is the most frequent weed in the Carpathian Basin and is spreading fast in other parts of Europe. In recent years, besides the wild type, a mutant genotype resistant to atrazine herbicides has evolved and is now widespread in many areas. The present study demonstrates that the atrazine resistance of ragweed is maternally inherited, and is caused by a point mutation in the psbA chloroplast gene. The promoter 5'-untranslated region and the open reading frame regions of the gene were analysed, and a homology search was performed. Both the atrazine-resistant and susceptible types of cpDNA were present in atrazine-resistant plants, while the mixed presence of both genotypes in the same plant, known as heteroplasmy, was not unequivocally detectable in susceptible plants.

Analysis of the applicability of molecular markers linked to the PVY extreme resistance gene Rysto, and the identification of new markers.

In this study molecular markers linked to the Rysto gene, which originates from the wild potato species Solanum stoloniferum and confers extreme resistance against PVY, were identified and the applicability of recently published Rysto, markers was analyzed. Three RAPD markers covering a total distance of 8.60 cM were detected in this experiment. The closest of these markers was located 0.53 cM from the gene. From among the published markers only one had diagnostic value in the experimental plant material, and mapped 2.95 cM from the gene, on the side opposite the RAPD markers developed in the present study. All the markers analyzed were present in Solanum stoloniferum accessions, irrespective of their resistance, indicating that these sequences are linked to the locus and not exclusively to the dominant allele of the Rysto gene in the wild species. The inapplicability of several published markers indicates that the genetic background is decisive in this tetraploid and highly heterozygous species. This means that it may be necessary to develop markers from the breeding material itself, until the resistance gene is not cloned and cannot be used as a selection marker in marker-assisted selection.

The temporal profile of edema formation differs between male and female rats following diffuse traumatic brain injury.

Although female hormones are known to influence edema formation following traumatic brain injury (TBI), no studies have actually compared the temporal profile of edema formation in both male and female rats following diffuse TBI. In this study, male, female, and female ovariectomized rats were injured using the 2 m impact acceleration model of diffuse TBI. The temporal profile of brain water content was assessed over 1 week post-trauma. Male animals demonstrated increased (p < 0.05) edema at 5 hours, 24 hours, 3 days, 4 days, and 5 days after TBI with a peak at 5 hours post-injury. This time point was associated with increased blood-brain barrier (BBB) permeability. In contrast, intact females showed increased levels of edema (p < 0.05) at 5 hours, 24 hours, 3 days, and 4 days post-TBI, with a peak at 24 hours. No BBB opening was present in intact females at 5 hours. Female animals demonstrated more edema than male animals at 24 hours, but less at 5 hours, 3 days, and 5 days. Ovariectomy produced an edema profile that was similar to that observed in males. The temporal profile of edema formation after TBI seems to depend on endogenous hormone levels, a difference which may have an influence on clinical management.

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways.

Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.

Regulation of intracellular free magnesium in central nervous system injury.

Traumatic injury to the central nervous system (CNS) initiates an autodestructive cascade of biochemical and pathophysiological changes that ultimately results in irreversible tissue damage. Known as secondary injury, this delayed injury process is multifactorial in nature and it is generally thought that the simultaneous attenuation of a number of the secondary injury factors will be required for interventional therapies to have a significant beneficial effect on outcome. This review summarizes the growing body of evidence that suggests that magnesium plays a pivotal role in the secondary injury process following CNS trauma, affecting a number of secondary injury factors including neurotransmitter release and activity, ion changes, oxidative stress, protein synthesis, and energy metabolism. By having effects on such a range of secondary injury factors following trauma, pharmacological studies have shown that magnesium may be an effective therapy following neurotrauma, improving survival, motor outcome and alleviating cognitive deficits.

Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats.

Traumatic brain injury results in an increased brain energy demand that is associated with profound changes in brain glycolysis and energy metabolism. Increased glycolysis must be met by increasing glucose supply that, in brain, is primarily mediated by two members of the facilitative glucose transporter family, Glut1 and Glut3. Glut1 is expressed in endothelial cells of the blood-brain barrier (BBB) and also in glia, while Glut3 is the primary glucose transporter expressed in neurons. However, few studies have investigated the changes in glucose transporter expression following traumatic brain injury, and in particular, the neuronal and glial glucose transporter responses to injury. This study has therefore focussed on investigating the expression of the glial specific 45-kDa isoform of Glut1 and neuronal specific Glut3 following severe diffuse traumatic brain injury in rats. Following impact-acceleration injury, Glut3 expression was found to increase by at least 300% as early as 4 h after induction of injury and remained elevated for at least 48 h postinjury. The increase in Glut3 expression was clearly evident in both the cerebral cortex and cerebellum. In contrast, expression of the glial specific 45-kDa isoform of Glut1 did not significantly change in either the cerebral cortex or cerebellum following traumatic injury. We conclude that increased glucose uptake after traumatic brain injury is primarily accounted for by increased neuronal Glut 3 glucose transporter expression and that this increased expression after trauma is part of a neuronal stress response that may be involved in increasing neuronal glycolysis and associated energy metabolism to fuel repair processes.

Characterization of plasma magnesium concentration and oxidative stress following graded traumatic brain injury in humans.

Plasma magnesium, calcium, and oxidative status were investigated in 31 male casualties with traumatic brain injury (TBI) during a 7-day posttraumatic period. The study group consisted of eight patients with mild closed head injury (Glasgow Coma Scale score [GCS] of 13-15), 10 patients with extensive penetrating head injury (GCS 4-6), and 13 patients with blast injuries but without direct head trauma. The latter group was included since previous experimental and clinical data have confirmed the development of indirect brain trauma in patients with blast injuries. Patients with multiple injuries were not included. Significant declines in plasma divalent cations were found in GCS 4-6 patients immediately after TBI and persisting for the entire 7-day study period. Similar changes in magnesium, but not calcium, were present in the GCS 13-15 and the blast injury groups, but only up until day 3 after injury. Alterations in lipid peroxidation products and superoxide anions were also observed following TBI. Increased lipid peroxidation was noted in all three groups over the entire posttraumatic period while increases in superoxide anion generation occurred transiently immediately following TBI. Thereafter, in the GCS 13-15 and blast injury groups, superoxide anions subsequently normalized, whereas in extensive head injury (GCS 4-6), superoxide anion generation significantly declined. A negative correlation between magnesium balance and oxidative stress was observed in all patients immediately after injury persisting in GCS 4-6 patients to the end of the observation period. Our findings suggest an interrelationship between magnesium changes and blood oxidants/antioxidants after TBI, which could be of both diagnostic and prognostic value in patients with neurotrauma.

Neurogenic inflammation is associated with development of edema and functional deficits following traumatic brain injury in rats.

The present study has used capsaicin-induced neuropeptide depletion to examine the role of neurogenic inflammation in the development of edema and functional deficits following traumatic brain injury (TBI). Adult, male rats were treated with capsaicin (neuropeptide-depleted) or equal volume vehicle (controls) 14 days prior to induction of moderate/severe diffuse TBI. Injury in vehicle treated control animals resulted in acute (4-5 h) edema formation, which was confirmed as being vasogenic in origin by diffusion weighted magnetic resonance imaging and the presence of increased permeability of the blood-brain barrier (BBB) to Evans blue dye. There was also a significant decline in brain magnesium concentration, as assessed by phosphorus magnetic resonance spectroscopy, and the development of profound motor and cognitive deficits. In contrast, capsaicin pre-treatment resulted in a significant reduction in post-traumatic edema formation (p < 0.001), BBB permeability (p < 0.001), free magnesium decline (p < 0.01) and both motor and cognitive deficits (p < 0.001). We conclude that neurogenic inflammation may play an integral role in the development of edema and functional deficits following TBI, and that neuropeptides may be a novel target for development of interventional pharmacological strategies.

Consistent and reproducible slice selection in rodent brain using a novel stereotaxic device for MRI.

Typically small animal radiological images are obtained after placing the animal in the center of the imaging device using beds or platforms, and then adjusting the position after obtaining a scout image. Such a process does not permit the reproducible visualization of the same anatomical plane with repeated examinations. We have developed a device that allows stereotaxic placement of an animal in precisely the same position for repeated examinations. The instrument incorporates a full range of physiological monitoring and life support systems including temperature control, anesthesia delivery and respiratory monitoring. Using magnetic resonance imaging (MRI), the accuracy and reliability of this device is demonstrated in a rat traumatic brain injury (TBI) model.

Neuropeptide release influences brain edema formation after diffuse traumatic brain injury.

The mechanisms associated with edema formation after traumatic brain injury (TBI) have not been fully elucidated. In peripheral tissue injury, the neurogenic component of inflammation plays a significant role in increased vascular permeability and edema formation. However, few studies have examined the role of neuropeptide induced neurogenic inflammation following TBI. Adult male Sprague-Dawley rats were either left untreated, or pre-treated with capsaicin (125 mg/kg s.c.) or equal volume vehicle, and injured 14 days later using the 2-meter impact-acceleration model. Subgroups of animals were assessed for blood brain barrier (BBB) permeability (Evans Blue), brain edema (wet weight/dry weight) and functional outcome (Barnes maze and Rotarod) for up to 2 weeks post-trauma. Increased BBB permeability was present in untreated animals between 3 and 6 h after injury but not at later time-points. Edema was maximal at 5 h after trauma, declined and then significantly increased over the 5 days post-trauma. In contrast, capsaicin pre-treated, neuropeptide-depleted animals exhibited no significant increase in BBB permeability or edema compared to vehicle treated animals after injury. Notably, motor and cognitive impairments were significantly reduced in the capsaicin-pretreated animals. We conclude that neurogenic inflammation contributes to the development of edema and posttraumatic deficits after diffuse TBI.

Intraarterial perfusion of prostaglandin E1 after lumbar sympathectomy or reconstruction on femoropopliteal segment.

Out of 100 patients treated by intraarterial perfusion of prostaglandin E1 we selected 36 cases who have been treated after a lumbar sympathectomy or reconstruction on the femoro-popliteal segment. The patients were in the III and IV stage of occlusive diseases by Fontain. All patients were divided into four groups: (a) prostaglandin E1 after a lumbar sympathectomy (20); (b) prostaglandin E1 after failed femoro-popliteal bypass (8); (c) prostaglandin E1 with patent femoro-popliteal bypass and distal progression of the occlusive disease (3); (d) prostaglandin E1 with previously femoro-popliteal reconstruction and poor run off (5). After intraoperative introduction of a catheter into the superficial femoral artery, profunda femoral artery (a, b), a patent graft (c) or just implanted graft (d), a continuous intraarterial perfusion of prostaglandin E1 was applied, in doses 10 nanograms/kg body weight/minute, in total doses 3000 nanograms. The perfusion time was 48-72 h. The patients were controlled immediately after treatment as well as 1, 3, 6 and 12 months after. Our early and late results of the intraarterial perfusion of prostaglandin E1 proved as a very successful limb salvage procedure.

Experimental magnesium depletion in adult rabbits caused by blast overpressure.

The complex pressure wave (blast) generated by some explosions causes pulmonary pathological changes which resemble the histological findings of the adult respiratory distress syndrome (ARDS). The development of indirect neurotrauma following experimental pulmonary blast injury has been shown previously. The purpose of this study was to evaluate lung and brainstem total tissue magnesium concentrations in adult rabbits following pulmonary blast injury. In order to assess the interrelationship between magnesium and other secondary injury factors, total calcium and high energy phosphate (phosphocreatine, PCr; adenosine triphosphate, ATP) contents were simultaneously measured. Twenty adult male rabbits were divided into two groups. Group C (n = 10) served as control, while group B (n = 10) was subjected to a focused blast wave, generated in laboratory conditions using an air-driven shock tube. Moderate pulmonary blast injury was verified by histological examination in group B. Total tissue magnesium and calcium contents were measured by atomic absorption spectrophotometry in the lungs and brainstem of adult rabbits 30 min following blast overpressure and in their uninjured controls. Simultaneously, PCr and ATP contents were measured by fluorimetric enzymatic analyses in same structures. Lung and brainstem water contents were determined by wet weight to dry weight ratio. Blast overpressure to the lungs induced significant magnesium depletion, increased calcium and decreased the Mg/Ca ratio in lung tissue of injured animals. Increases in water content and PCr/ATP ratio were also observed. Significant correlations between these Mg/Ca and PCr/ATP and between Mg and ATP parameters confirmed the functional relationship between magnesium depletion and impaired bioenergetic state in indirect neurotrauma in adult rabbits through blast overpressure.

Alterations in magnesium and oxidative status during chronic emotional stress.

Magnesium and oxidative status were investigated in young volunteers exposed to chronic stress (political intolerance, awareness of potential military attacks, permanent stand-by duty and reduced holidays more than 10 years) or subchronic stress consisting of everyday mortal danger in military actions lasting more than 3 months. Significant decreases in plasma ionized Mg2+, total Mg and ionized Ca2+ concentrations were found in both groups. Similarly, both study groups exhibited oxidative stress as assessed by increased plasma superoxide anions and malondialdehyde and modified antioxidant defense. There were no significant differences between the two stress groups. A negative correlation between magnesium balance and oxidative stress was observed suggesting that the same etiological factor (chronic stress) initiate decreases in both free and total magnesium concentrations and simultaneously increase oxidative stress intensity. These findings support the need for magnesium supplementation with antioxidant vitamins for people living in conditions of chronic stress.

Relations among plasma prolactin, testosterone, and injury severity in war casualties.

Tissue trauma leads to a complex hormonal response of pituitary end-organ axis. This response can be recorded by determining parameters that represent the functional integrity of these systems. The concentrations of serum prolactin (PRL), serum testosterone, and plasma adrenocorticotropin (ACTH) were measured in 62 adult male casualties from the recent war in former Yugoslavia. Patients with brain injury were not included. Venous blood samples were taken as soon as possible (2-18 hours) after admission and at 1, 2, 5, and 14 days after injury. The severity of gunshot/missile wounds was assessed by the Injury Severity Score (ISS). The control group consisted of healthy blood donors. Uninjured subjects who had undergone great stress on the battlefield (explosion in the vicinity without injury) served as the sham-control group. Tissue trauma leads to a severity-dependent decrease in serum testosterone concentrations during the first 5 days following injury. Significant correlations were observed between ACTH, prolactin, and ISS during the first 18 hours after injury. A strong negative correlation between testosterone and prolactin serum concentrations was found during the first 18 hours. In patients with additional complications or unsatisfactory outcome, the prolactin concentrations remained elevated, whereas testosterone concentrations were reduced. Our results support the usefulness of recording hormonal changes for determining trauma severity and monitoring the clinical course. Such monitoring also helps assess the efficacy of therapeutic strategies. The relation between testosterone and prolactin might be helpful for predicting the clinical course and trauma outcome.

Influence of low level laser irradiation on biochemical processes in brainstem and cortex of intact rabbits.

The influence of low level laser (LLL) irradiation at wavelength at 660 and 904 nm on oxidative stress (lipid peroxidation activity-LP, production of superoxide anion radicals-NBT reduction), activity of enzymes of antioxidative defense (superoxide dismutase-SOD, glutathione reductase-GR) and functional activity of sodium pump (Na+K+ ATPase) in relation with applied wavelength of LLL was investigated. The investigation was performed at the adult rabbits (n = 21) classificated in three groups: control group (C), the group of rabbits irradiated with LLL wavelength 904 nm (CL1) and the group of rabbits irradiated with LLL wavelength 660 nm (CL2). The irradiation was performed in the upper cervical region in the anatomical projection of the brainstem. It was established that LLL induced oxidative stress in the brainstem and the cortex of treated rabbits, independently of applied wavelength of laser beams. The registrated changes in functional activity of sodium pump were dependent on the applied wavelength. The irradiation at 904 nm caused the significant increase of the substrate uptake rate of sodium pump in the brainstem tissue. The irradiation at 660 nm caused the "competitive inhibition" of the sodium pump. Decrease of norepinefrine content in the brainstem of treated rabbits pointed on the indirect mechanism of functional activity of sodium pump as well as the oxidative stress.

[The effect of indobufen on patency in femoro-popliteal/crural bypass using artificial grafts]. / Uticaj indobufena na prohodnost femoro-poplitealnog/kruralnog bypass-a ucinjenog sintetskim graftom.

The aim of this clinical prospective study was to determine the effect of indobufen upon synthetic graft patency in femoral-popliteal/crural position. 15 operated patients were observed during the three-month period. One day prior to operation patients were given 400 mg of indobufen perorally. The same daily dose was continued on the first postoperative day as well as during the following three months. Blood levels of 6-keto-prostaglandin (PG) F1alpha (stable metabolite of PGI2) and thromboxane (Tx) B2 (stable metabolite of TxA2) were determined by RIA before indobufen administration, i.e., one day and three months postoperatively. The three-month patency of grafts was achieved in 86% of cases. Plasma levels in all observed time periods showed significantly reduced TxB2, increased 6-keto-PGF1alpha, and higher PGI2 levels compared with TxA2 that could suggest the normalization of aggregation/antiaggregation process.