Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade.

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.

Characterization of a porcine model of post-operative pain.

BACKGROUND: Management of acute pain related to surgical intervention, termed post-operative pain or POP, continues to be a major healthcare challenge. While the rat plantar incision model provides valuable data to researchers about the mechanisms mediating POP, the development of topical and localized treatments in small animal models is limited. To help address these issues, we describe here the characterization of a large animal model of incisional pain. METHODS: Pigs underwent full-skin incision or full-skin and muscle incision and retraction (SMIR). Withdrawal thresholds were determined using the Von Frey test at baseline, 0.5-12 h post-surgery and on days 1, 2, 3, 5 and 7 post-surgery. The analgesic effects of systemic morphine [0.1 or 1.0 mg/kg intramuscular (i.m.) dose] and local anaesthetic ropivacaine were studied. Spontaneous pain-like behaviours were scored and analysed. The effects on wound healing were evaluated by gross observation and by histopathological examination. RESULTS: Pigs incurring SMIR demonstrated significantly increased mechanical hypersensitivity compared with pigs that underwent full-skin incision only (p < 0.05). Maximal analgesia was achieved with morphine (1 mg/kg i.m. dose) at 0.5 h post-treatment. Local treatment with ropivacaine was effective at increasing the withdrawal threshold to Von Frey filaments compared with saline control (p < 0.05) for a period of at least 6 h. Wounds healed normally with no signs of infection, redness or swelling. CONCLUSIONS: We propose that the pig model of incisional pain can provide an appropriate translational model for validating new topical and localized treatments for POP in humans.

The evaluation of brain CBF and mitochondrial function by a fiber optic tissue spectroscope in neurosurgical patients.

The brain of neurosurgical patients are exposed to various manipulations in the ICU or during surgery. Under such conditions brain O2 balance may become negative and as a result brain vitality and function will deteriorate. In order to evaluate brain vitality in real time it is important to measure more than one parameter. The multiparametric monitoring system used in our previous study to monitor comatose patients (Mayevsky et al., Brain Res. 740: 268-274, 1996) was changed into a "simplified" tissue spectroscope for real time monitoring of brain O2 balance. Mitochondrial function was evaluated by monitoring the NADH redox state by surface fluorometry. Microcirculatory blood flow was assessed by laser Doppler flowmetry. The combined optical probe was located on the surface of the brain during various neurosurgical procedures and the responses were recorded and presented in real time to the surgeon. A total of 32 patients were monitored during various procedures. The results could be summarized as follows: 1. Hypercapnia led to 3 different types of responses. In two patients the 'stealing' like event was recorded. In the other 7 patients the responses to high CO2 was not detectable. In the last group of 6 patients a clear CBF elevation was recorded with variable response of mitochondrial NADH. 2. Our monitoring device was able to evaluate the efficacy of the STA-MCA anastomosis during aneurysm surgery. 3. A significant correlation was recorded between CBF and NADH redox state during changes in blood pressure, papaverine injection, spontaneous drop in blood supply to the brain or during releasing of high ICP levels. We conclude that in order to evaluate the metabolic state of the brain during neurosurgical procedures it is necessary to monitor both CBF and mitochondrial NADH by using the tissue spectroscope.

Hyperbaric oxygenation affects rat brain function after carbon monoxide exposure.

The application of hyperbaric oxygenation (HBO2) has been recommended for correction of neurological injury in severely CO-poisoned patients. However, the mechanisms of HBO2 action on brain mitochondrial function under the circumstances is not yet understood completely. In the present study, the effect of HBO2 on the rat brain after CO exposure was evaluated by measuring the intramitochondrial NADH and its responses to anoxic test or repetitive induction spreading depression (SD) leading to brain activation. A unique monitoring system for bilateral monitoring of brain NADH redox state was used. Rats were exposed to 3000 ppm CO for 30 (group A) or 60 min (C). In groups B and D, after CO exposure, the rats were exposed to HBO2 (3 atm abs for 30 min). Following CO exposure in groups A and C, a definite decrease in the amplitude of the NADH response and significant increase in the number of waves of NADH was noted during induced cortical SD. Anoxic test in these two groups led to a significant decrease of maximum levels of NADH (reduction) at the end of observation. The amplitude, and the number of SD waves and magnitude of NADH deviation during anoxic test in group B after application of HBO2, was not significantly different from the values measured under the initial conditions. However, in group D, tendency of maintenance of the parameter's initial level was weaker or absent. The results obtained indicated that suppression of brain energy metabolism is a characteristic manifestation of CO poisoning in rats. Restoration of cerebral energy metabolism by adequate dosage of HBO2 may become an important factor for recovery of brain activities after CO poisoning.

Inter-relation between hemodynamic, metabolic, ionic and electrical activities during ischemia and reperfusion in the gerbil brain.

The aim of this study was to examine the inter-relation between the hemodynamic events, energy metabolism, extracellular potassium and electrical activity during the acute phase of transient ischemia in the gerbil brain. It has already been shown that partial ischemia in the gerbil brain causes changes in the blood flow, oxygen tension, electrical activity and potassium ion efflux. However, the description of the event during brain recovery from transient ischemia is not documented. In order to enable a better understanding of the pathophysiology during the ischemia as well as during reperfusion, we used the multiparametric assembly system. This system enables simultaneous and continuous monitoring of CBF, intra-mitochondrial NADH, extracellular potassium, DC potential and ECoG. Twenty anesthetized gerbils underwent reversible carotid artery occlusion procedure for 3-4 min. While monitoring the various parameters until complete recovery was reached, we found high correlation between the CBF and the NADH during occlusion as well as during the reperfusion period. However, CBF at the reperfusion period increased above the basal level while NADH returned to base line without an undershoot, suggesting that the mitochondrial need for oxygen necessary for the production of ATP is not the only factor influencing CBF during reperfusion. Furthermore, NADH returned to its normal level before extracellular potassium ion levels recovered to the baseline. This may suggest that ATP was no longer the limiting factor and ion pump activity became the factor determining and affecting the recovery processes.

Brain viability and function analyzer: multiparametric real-time monitoring in neurosurgical patients.

We have developed the Brain Viability (BVA) and Brain Function (BFA) Analyzers for monitoring the following parameters from the human cerebral cortex cerebral blood flow: (CBF), NADH redox state, Electro corticography (ECoG), brain temperature, extracellular K+, DC potential and intracranial pressure (ICP). The BVA monitors the first 4 parameters only. The Brain viability probe (BVP) and Brain function multiprobe (BFM) were used during 11 operations and in 18 ICU patients, respectively. Preliminary results from the OR showed that 5 patients exhibited a typical increase in CBF in response to changes in end-tidal CO2 without a significant change in the NADH redox state. In 4 other patients no changes in CBF and NADH were observed. Two patients exhibited a "steeling response", i.e., a decrease in CBF and an increase in NADH. In 18 comatose patients monitored in the ICU, the ICP, CBF and ECoG were measured correctly in most patients, whereas NADH and K+ were more problematic. One patient exhibited a typical response, may be due to repeated cortical spreading depression cycles and an ischemic depolarization event. Continuous realtime multiparametric monitoring in neurosurgical patients is feasible and practical in the OR and the ICU. The information provided could be used as a diagnostic tool to guide the procedures or treatment given to the patients.

Real-time multiparametric monitoring of the injured human cerebral cortex--a new approach.

Intracranial pressure (ICP) is currently the main parameter monitored following severe head injury or during the post operative period in neurosurgical patients. The normal cerebral cortex depends upon a continuous supply of O2, and direct coupling exists between adequate cerebral blood flow (O2 supply) and ion homeostasis as well as electrical activities. We have developed a new "Brain Function Analyzer-BFA" which enabled monitoring of the following parameters continuously in real time from the surface of the cortex: ICP; tissue blood flow & volume; intramitochondrial NADH redox state; DC steady potential; electrocorticography; tissue temperature. The probes were assembled in a Brain Function Multiprobe (BFM) which was connected to the brain via the burr hole procedure used for ICP monitoring. Measurements were performed in 18 comatose patients after severe head injury (GCS < or = 8) who were monitored in the ICU for 48-72 hours. The basic concept of the multiparametric monitoring approach was proven to be practical in neurosurgical patients. Clear correlations were recorded between hemodynamic, metabolic, ionic and electrical activities under various treatments administered to the patients or after pathological events. Responses similar to cortical spreading depression and ischemic depolarization were recorded from a severely head injured patient.

Multiparametric monitoring of brain oxygen balance under experimental and clinical conditions.

In order to evaluate the relationship between brain oxygen supply and demand (O2 balance) in real time, it is necessary to use a multiparametric monitoring approach. Cerebral blood flow (CBF) is a representative parameter of O2 supply. The extracellular level of K+ is a reliable indicator of O2 demand since more than 60% of the energy consumed by the brain is utilized by active transport processes. Mitochondrial NADH redox state can represent the balance between O2 supply and demand. In order to monitor the brain of experimental animals or patients, we constructed the multiparametric assembly (MPA) and the following parameters were monitored simultaneously and in real time: CBF, CBV, NADH redox state, extracellular K+, DC potential, EEG, tissue temperature and ICP. Animals were exposed to hypoxia, ischemia, hypercapnia, hyperoxia and spreading depression (SD) and the relative changes in CBF and NADH were calculated and found to be significant indicators of brain energy state. Monitoring these two parameters increases the possibility of differentiating between various pathophysiological states. Each added parameter increases the power of diagnosis and determination of the functional state of the brain. Preliminary results obtained in patients monitored in the ICU or in the OR show that the responses to hypercapnia, spreading depression or ischemia are similar to those measured in experimental animals.

Thiopental induced cerebral protection during ischemia in gerbils.

Temporary interruption or reduction of cerebral blood flow during cerebrovascular surgery may rapidly result in ischemia or cerebral infarction. Thiopental has been shown to have cerebroprotective effects. However, the cerebroprotective dose of thiopental causes burst suppression of the EEG, thus this parameter cannot be used continuously for the detection of metabolic changes in the brain during thiopental anaesthesia. This study was performed in order to examine whether the multiparametric assembly (MPA), which measures energy metabolism CBF and mitochondrial (NADH) as well as extracellular ion concentrations (K+), can shed light on the mechanism of the cerebroprotective effects of thiopental. The MPA was placed on the brain of Mongolian gerbils and burst suppression of the ECoG was induced by thiopental. Cerebral ischemia was induced by occlusion of carotid arteries after burst suppression. Burst suppression of the ECoG was accompanied by a significant decrease in cerebral blood flow. In animals that received thiopental prior to ischemia, NADH increased to a lesser degree and extracellular potassium ion concentration increased to a lesser degree than in the control animals, indicating that thiopental affords protection of the brain under ischemic conditions due to improved energy metabolism. This study also demonstrates that the MPA can monitor changes occurring in the cerebral cortex even after the ECoG can no longer be used. Those findings have a significant value in the development of a new clinical monitoring device.

Responses of rat brain to induced spreading depression following exposure to carbon monoxide.

Until recently carbon monoxide (CO) was known only for its noxious effects. Exposure to CO results in an autoregulatory increase in cerebral blood flow (CBF). Little information is available on brain energy metabolism under low CO concentrations and on the effect of CO on the stimulated brain. In this study cortical spreading depression (SD) was induced in order to cause transient brain depolarization and increased energy demand. The multisite assembly (MSA), which contains four bundles of optical fibers for monitoring the intramitochondrial NADH redox state and tissue reflectance as well as four DC electrodes enabling measurement from four consecutive points on the cerebral cortex, was used to measure energy metabolism and the propagation of SD waves during exposure to CO. CBF in the contralateral hemisphere was measured using the laser Doppler technique. Three experimental groups of animals were examined: SD was induced during exposure to 1000 ppm CO, immediately after exposure to CO and 90 min after cessation of exposure to CO. Three control groups were also examined, in which the animals underwent the same procedures but were not exposed to CO. In all animals exposure to CO was followed by a significant increase in CBF. The greatest effect was found when SD was induced immediately after cessation of exposure to CO. SD wave frequency decreased when induced immediately after exposure to CO, whereas it increased when SD was induced 90 min after exposure. The amplitude of the NADH oxidation waves and their integral were smaller during SD induced immediately after exposure to CO. The DC potential did not change, suggesting that CO did not affect the SD initiation mechanism but rather resulted in energy depletion during recovery from SD. This study demonstrates that even at a concentration of 1000 ppm CO interferes with the metabolic activity of the brain during repolarization of the SD-induced negativity.

Cortical spreading depression recorded from the human brain using a multiparametric monitoring system.

The number of parameters (i.e., EEG or ICP-intracranial pressure) routinely monitored under clinical situations is limited. The brain function analyzer described in this paper enables simultaneous, continuous on-line monitoring of cerebral blood flow (CBF) and volume (CBV), intramitochondrial NADH redox state, extracellular K+ concentrations, DC potential, electrocorticography and ICP from the cerebral cortex. Brain function of 14 patients with severe head injury (GCS < or = 8), who were hospitalized in the neurosurgical or general intensive care unit was monitored using this analyzer. Leao cortical spreading depression (SD) has been reported in many experimental animals but not in the human cerebral cortex. In one of the patients monitored, spreading depression was observed. This is the first time that spontaneous repetitive cortical SD cycles have been recorded from the cerebral cortex of a patient suffering from severe head injury. Typical SD cycles appeared 4-5 h after the beginning of monitoring this patient. During the first 3-4 cycles the responses of this patient were very similar to the responses to SD recorded in normoxic experimental animals. Electrocorticography was depressed whereas extracellular K+ levels increased. The metabolic response to spreading depression was characterized by oxidation of intramitochondrial NADH concomitant to a large increase in CBF. During brain death, an ischemic depolarization, characterized by decrease in CBF and an irreversible increase in extracellular K+, was recorded.

Effects of carbon monoxide on the brain may be mediated by nitric oxide.

Carbon monoxide (CO) is known to be a toxic molecule due to the high affinity of hemoglobin for it. However, it has recently been shown that low doses of CO may play a physiological role. The aim of the present study was to examine processes occurring in the brain during exposure to 1,000 parts per million CO that result in an increase in cerebral blood flow (CBF) but are not accompanied by changes in oxidation metabolism. This study was carried out in awake rats with the multiprobe assembly developed in this laboratory for the simultaneous continuous measurement of CBF, intramitochondrial NADH redox levels, direct current potential, and extracellular concentrations of K+, Ca2+, and H+ as well as the electrocorticogram. Exposure to 1,000 parts per million CO in air resulted in an increased CBF without any concomitant changes in any of the other metabolic or ionic parameters measured. This indicates that tissue hypoxia was not the trigger for this vasodilation. Injection of N omega-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, before exposure to CO effectively blocked the increase in CBF that was observed when the animal was exposed to CO without prior injection of L-NNA. Furthermore, electrocorticographic depression was observed after the combined treatment of L-NNA and CO. In conclusion, exposure to relatively low doses of CO apparently does not have a deleterious effect on oxidative metabolism because the increase in CBF after this exposure is sufficient to prevent changes in oxidative metabolism, as indicated by the fact that NADH levels remained constant. This protective autoregulatory effect may be mediated by nitric oxide.

Multiparametric monitoring of the awake brain exposed to carbon monoxide.

We have applied in vivo real-time techniques to monitor the physiological changes associated with exposure to a pattern of carbon monoxide (CO) known to cause brain oxidative stress. Using a multiparametric monitoring device connected to the brain, we exposed unanesthetized rats to two levels of CO, 0.1 and 0.3% in air. Energy metabolism was evaluated by the optical monitoring of relative cerebral blood flow (CBF) and intramitochondrial redox state. Ionic homeostasis was assessed by measurements of K+,Ca2+, and H+ or Na+ levels in the extracellular space. The electrical parameters monitored were the electrocorticogram and direct current steady potential. Under 1,000 ppm of CO, the CBF was increased significantly without any measurable change in the NADH redox state, suggesting that the cause for the increased CBF was not hypoxia. Exposing the awake rat to 1,000 ppm of CO (40 min) followed by 3,000 ppm of CO (20 min) led to an increase in CBF followed by episodes of spontaneous brain depolarizations characterized by changes in ionic homeostasis and blood flow. These changes were similar to those recorded under cortical spreading depression. In most animals exposed to 3,000 ppm of CO, spontaneous oscillations in CBF and NADH redox state that were negatively correlated were recorded. The results indicate that an inspired CO level of 0.1% had effects largely restricted to blood flow, whereas at a higher CO level an additional impairment in energy supply resulted in a complex pattern of effects similar to that caused by brain ischemia.