THE PROBLEMS OF CRIMINAL LIABILITY OF PHARMACEUTICAL EMPLOYEES IN THE CONTEXT OF CERTAIN FORMS OF COLLABORATIVE ACTIVITIES.

OBJECTIVE: The aim: Based on the specifics of the criminal legislation, which provides liability for collaborative activity, it is necessary to offer an adequate understanding of the limits of the criminal liability of pharmaceutical employees for these criminal offenses, as well as to determine the characteristics of the components of these criminal offenses related to the pharmaceutical activities carried out by pharmaceutical employees. PATIENTS AND METHODS: Materials and methods: The conducted research is based on the analysis of the provisions of the criminal legislation of Ukraine and Georgia. CONCLUSION: Conclusions: The main problems of criminal liability for collaboration activities by pharmaceutical employees are connected to the following: without clari-fication of the aforementioned criminal offenses, it is impossible to specify the limits of criminal liability.

ANALYSIS OF DEFECTS IN THE FORENSIC MEDICAL ASSESSMENT OF CHANGES DISCOVERED DURING THE EXAMINATION OF PERSONS WHO DIED FROM TRAUMATIC BRAIN INJURY.

OBJECTIVE: The aim: To identify the main groups of expert defects that arise during the forensic assessment of changes detected during the examination of persons who died from a traumatic brain injury (TBI). PATIENTS AND METHODS: Materials and methods: A total of 102 repeated commission forensic medical examinations with changed conclusions in corpses with TBI were analyzed. Data processing and analysis were conducted using statistical methods. RESULTS: Results: The examined forensic medical assessments of TBI with changed conclusions in corpses were categorized into the following groups: defects in estab¬lishing the diagnosis of TBI - 17.65±7.4%; defects in establishing the mechanism of TBI - 35.3±9.3%; defects in establishing the duration of TBI - 39.22±9.5%: sober - 20±12.4%; with alcohol intoxication - 80±12.4%. A combination of defects was found in 7.83±5.2% of cases. Defects that directly affected the experts' incorrect establishment of the diagnosis, mechanism, and duration of TBI were also identified. CONCLUSION: Conclusions: The largest number of changed conclusions during the forensic medical examination of corpses in cases of TBI was due to the wrongly established duration of the trauma, accounting for 39.2±9.5%, with the vast majority of cases (80±12.4%) observed against the background of alcohol intoxication. The mechanism of trauma accounted for 35.3±9.3% of the changed conclusions. The main defects were incomplete collection of material for histological examination (90.2±5.8%) and incorrect interpretation of the results of histological examination (76.48±8.2%), along with the violation of the method of sectional examination (68.6±9.0%). Different groups of expert defects predominated in the cases with an incorrectly established diagnosis of TBI, duration of trauma, and mechanism.

ANALYSIS OF DEFECTS IN THE DIAGNOSIS OF TRAUMATIC BRAIN INJURY FOR THE DECEASED WITHIN 24 HOURS FOLLOWING ADMISSION.

OBJECTIVE: The aim: To conduct the analysis of medical records with the diagnosis of traumatic brain injury for the deceased within 24 hours following admission to Clinical Emergency Hospital. PATIENTS AND METHODS: Materials and methods: The study was aimed at a retrospective analysis of 102 cases of the lethality of the deceased within 24 hours following admission to Clinical Emergency Hospital for 2012-2019 in cases of traumatic brain injury. Medical histories of the deceased and data from the forensic autopsy had been analyzed. RESULTS: Results: There were 62 cases (60.8%) of isolated traumatic brain injury, and 40 cases (39.2%) of combined traumatic brain injury. The following defects were identified in the diagnosis: absence of a complete description of the local status with external injuries on the head, absence of a complete and qualitative assess¬ment and objectification of hemodynamics and the function of external breathing using laboratory indicators and electrocardiography, absence of neuroimaging. CONCLUSION: Conclusions: The percentage of diagnostic defects prevailed among traumatic brain injury patients who died from acute blood loss. The maximum number of diagnostic defects for the patients with traumatic brain injury was observed in the polytrauma department, and the minimum - in the neurological department. The maximum number of defects of a diagnostic nature as a whole fall on those patients who were admitted to the hospital in the interval I - 6:00 a.m. - 9:59 a.m. and in the interval IV - 6:00 p.m. - 9:59 p.m.

THE PROBLEMS OF DEFINITION OF THE ABETTING IN THE COMISSION OF THE OFFENCES INVOLVING THREATS TO PUBLIC HEALTH (PART 1 OF ARTICLE 9 OF THE COUNCIL OF EUROPE CONVENTION ON THE COUNTERFEITING OF MEDICAL PRODUCTS AND SIMILAR CRIMES INVOLVING THREATS TO PUBLIC HEALTH).

OBJECTIVE: The aim: based on the features of the notion of "abetting the commission of crimes established in accordance with the Convention" provided for in Part 1 of Art. 9 of the Medicrime Convention, it is necessary to offer an adequate understanding of the notion of "abetting" and define the types of criminal offenses (crimes) that are the "subject" of such abetting. PATIENTS AND METHODS: Materials and methods: the research is based on an analysis of the provisions of the Medicrime Convention and the criminal law of Ukraine. The following methods were used: dialectical method; hermeneutic method; systemic-and-structural method; and comparative-legal method. RESULTS: Results: at the legislative level, there is a problem of designating the relevant socio-legal phenomena with adequate concepts and interpretations of these concepts. In the current criminal legislation of Ukraine, there is no definition of the concept of "abetting", which is used in Part 1 of Art. 9 of the Medicrime Convention. Therefore, in the implementation of the requirements provided for in Part 1 of Art. 9 of the Medicrime Convention, each Party takes the necessary legislative and other measures to recognize abetting in committing any crimes, established under this Convention, as a crime, therefore we should take into account the existence of two alternative ways to explain the meaning of "abetting": 1) to recognize at the legislative level that "abetting" and "incitement" are synonyms, and therefore the meaning of the term "abetting" can be explained by using the term "inclination"; 2) to recognize at the legislative level that the concept of "abetting" has a meaning different from the concept of "incitement", and covers not only "inclination", but also "coercion", "motivation" and "encouragement". CONCLUSION: Conclusions: the main disadvantage of using the concept of "abetting" in the text of the Ukrainian translation of the Medicrime Convention is that without an independent explanation of this concept at the legislative level, its content should be determined depending on the meaning of the term "inciter" under Part 4 of Art. 27 of the Criminal Code of Ukraine), and means inciting a person to commit any of the crimes specified in the Medicrime Convention.

THE PROBLEMS OF CRIMINALIZATION OF THE SIMILAR CRIMES INVOLVING THREATS TO PUBLIC HEALTH (ARTICLE 8 OF THE COUNCIL OF EUROPE CONVENTION ON THE COUNTERFEITING OF MEDICAL PRODUCTS AND SIMILAR CRIMES INVOLVING THREATS TO PUBLIC HEALTH).

OBJECTIVE: The aim: Medicrime Convention is a first international treaty against counterfeit medical products and similar crimes involving threats to public health. There are problems in criminalization of those acts that are listed in Art. 8 of the Medicrime Convention because the term of "similar crimes" is absent in the current criminal legislation of Ukraine. PATIENTS AND METHODS: Materials and methods: The conducted study is based on the analysis of the provisions of the Medicrime Convention, the criminal legislation of Ukraine. The following methods: dialectical method; hermeneutic method; system-and-structural method; comparative-and-law method were used. RESULTS: Results: Comparison of the provisions of the Medicrime Convention allows to state that crimes in its Articles 5-8 that are different from those provided for in Art. 5-7 of this Convention and form independent types of actions, are at least "placing on the market" of medicinal products and medical devices provided in subparagraph "a" of paragraph 1 of Art. 8 and "commercial use of original documents" specified in subparagraph "b" of paragraph 1 of Art. 8. It's an assumption that Art. 5-8 of the Medicrime Convention provide for such independent types of crimes involving threats to public health as: 1) manufacturing of counterfeit medical products, active substances, excipients, parts, materials and accessories as well as medicinal products, medical devices, active substances and excipients; 2) the supplying, the offering to supply, the brokering, the trafficking, the keeping in stock, importing and exporting of counterfeit medical products, active substances, excipients, parts, materials and accessories; 3) the making of false documents or the act of tampering with documents; 4) placing on the market of medicinal products, medical devices; 5) the commercial use of false documents. CONCLUSION: Conclusions: The term of "similar crimes" in Art. 8 of the Medicrime Convention covers multi-ordinal intentional acts that constitute different types of independent crimes involving threats to public health, as well as special kinds of some of them. These types of crimes are not the same (identical).

S-nitrosylation therapy to improve oxygen delivery of banked blood.

From the perspectives of disease transmission and sterility maintenance, the world's blood supplies are generally safe. However, in multiple clinical settings, red blood cell (RBC) transfusions are associated with adverse cardiovascular events and multiorgan injury. Because ∼85 million units of blood are administered worldwide each year, transfusion-related morbidity and mortality is a major public health concern. Blood undergoes multiple biochemical changes during storage, but the relevance of these changes to unfavorable outcomes is unclear. Banked blood shows reduced levels of S-nitrosohemoglobin (SNO-Hb), which in turn impairs the ability of stored RBCs to effect hypoxic vasodilation. We therefore reasoned that transfusion of SNO-Hb-deficient blood may exacerbate, rather than correct, impairments in tissue oxygenation, and that restoration of SNO-Hb levels would improve transfusion efficacy. Notably in mice, administration of banked RBCs decreased skeletal muscle pO2, but infusion of renitrosylated cells maintained tissue oxygenation. In rats, hemorrhage-induced reductions in muscle pO2 were corrected by SNO-Hb-repleted RBCs, but not by control, stored RBCs. In anemic awake sheep, stored renitrosylated, but not control RBCs, produced sustained improvements in O2 delivery; in anesthetized sheep, decrements in hemodynamic status, renal blood flow, and kidney function incurred following transfusion of banked blood were also prevented by renitrosylation. Collectively, our findings lend support to the idea that transfusions may be causally linked to ischemic events and suggest a simple approach to prevention (i.e., SNO-Hb repletion). If these data are replicated in clinical trials, renitrosylation therapy could have significant therapeutic impact on the care of millions of patients.

GAT inhibition preserves cerebral blood flow and reduces oxidant damage to mitochondria in rodents exposed to extreme hyperbaric oxygen.

Oxygen breathing at elevated partial pressures (PO2's) at or more than 3 atmospheres absolute (ATA) causes a reduction in brain γ-aminobutyric acid (GABA) levels that impacts the development of central nervous system oxygen toxicity (CNS-OT). Drugs that increase brain GABA content delay the onset of CNS-OT, but it is unknown if oxidant damage is lessened because brain tissue PO2 remains elevated during hyperbaric oxygen (HBO2) exposures. Experiments were performed in rats and mice to measure brain GABA levels with or without GABA transporter inhibitors (GATs) and its influence on cerebral blood flow, oxidant damage, and aspects of mitochondrial quality control signaling (mitophagy and biogenesis). In rats pretreated with tiagabine (GAT1 inhibitor), the tachycardia, secondary rise in mean arterial blood pressure, and cerebral hyperemia were prevented during HBO2 at 5 and 6 ATA. Tiagabine and the nonselective GAT inhibitor nipecotic acid similarly extended HBO2 seizure latencies. In mice pretreated with tiagabine and exposed to HBO2 at 5 ATA, nuclear and mitochondrial DNA oxidation and astrocytosis was attenuated in the cerebellum and hippocampus. Less oxidant injury in these regions was accompanied by reduced conjugated microtubule-associated protein 1A/1B-light chain 3 (LC3-II), an index of mitophagy, and phosphorylated cAMP response element binding protein (pCREB), an initiator of mitochondrial biogenesis. We conclude that GABA prevents cerebral hyperemia and delays neuroexcitation under extreme HBO2, limiting oxidant damage in the cerebellum and hippocampus, and likely lowering mitophagy flux and initiation of pCREB-initiated mitochondrial biogenesis.

Pharmacologically augmented S-nitrosylated hemoglobin improves recovery from murine subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: S-nitrosylated hemoglobin (S-nitrosohemoglobin) has been implicated in the delivery of O(2) to tissues through the regulation of microvascular blood flow. This study tested the hypothesis that enhancement of S-nitrosylated hemoglobin by ethyl nitrite inhalation improves outcome after experimental subarachnoid hemorrhage (SAH). METHODS: A preliminary dosing study identified 20 ppm ethyl nitrite as a concentration that produced a 4-fold increase in S-nitrosylated hemoglobin concentration with no increase in methemoglobin. Mice were subjected to endovascular perforation of the right anterior cerebral artery and were treated with 20 ppm ethyl nitrite in air, or air alone for 72 hours, after which neurologic function, cerebral vessel diameter, brain water content, cortical tissue Po(2), and parenchymal red blood cell flow velocity were measured. RESULTS: At 72 hours after hemorrhage, air- and ethyl nitrite-exposed mice had similarly sized blood clots. Ethyl nitrite improved neurologic score and rotarod performance; abated SAH-induced constrictions in the ipsilateral anterior, middle cerebral, and internal carotid arteries; and prevented an increase in ipsilateral brain water content. Ethyl nitrite inhalation increased red blood cell flow velocity and cortical tissue Po(2) in the ipsilateral cortex with no effect on systemic blood pressure. CONCLUSIONS: Targeted S-nitrosylation of hemoglobin improved outcome parameters, including vessel diameter, tissue blood flow, cortical tissue Po(2), and neurologic function in a murine SAH model. Augmenting endogenous Po(2)-dependent delivery of NO bioactivity to selectively dilate the compromised cerebral vasculature has significant clinical potential in the treatment of SAH.

Autonomic activation links CNS oxygen toxicity to acute cardiogenic pulmonary injury.

Breathing hyperbaric oxygen (HBO2), particularly at pressures above 3 atmospheres absolute, can cause acute pulmonary injury that is more severe if signs of central nervous system toxicity occur. This is consistent with the activation of an autonomic link between the brain and the lung, leading to acute pulmonary oxygen toxicity. This pulmonary damage is characterized by leakage of fluid, protein, and red blood cells into the alveoli, compatible with hydrostatic injury due to pulmonary hypertension, left atrial hypertension, or both. Until now, however, central hemodynamic parameters and autonomic activity have not been studied concurrently in HBO2, so any hypothetical connections between the two have remained untested. Therefore, we performed experiments using rats in which cerebral blood flow, electroencephalographic activity, cardiopulmonary hemodynamics, and autonomic traffic were measured in HBO2 at 5 and 6 atmospheres absolute. In some animals, autonomic pathways were disrupted pharmacologically or surgically. Our findings indicate that pulmonary damage in HBO2 is caused by an abrupt and significant increase in pulmonary vascular pressure, sufficient to produce barotrauma in capillaries. Specifically, extreme HBO2 exposures produce massive sympathetic outflow from the central nervous system that depresses left ventricular function, resulting in acute left atrial and pulmonary hypertension. We attribute these effects on the heart and on the pulmonary vasculature to HBO2-mediated central sympathetic excitation and catecholamine release that disturbs the normal equilibrium between excitatory and inhibitory activity in the autonomic nervous system.

Impact of the European Union on access to medicines in low- and middle-income countries: A scoping review.

This Scoping Review synthesises evidence of the impacts of European Union (EU) law, regulation, and policy on access to medicines in in non-EU low- and middle-income countries (LMICs), and the mechanisms and nature of those impacts. We searched eight scholarly databases and grey literature published between 1995-2021 in four languages. The EU exerts global influence on pharmaceuticals in LMICs in three ways: explicit agreements between EU-LMICs (ex. accession, trade, and economic agreements); LMICs' reliance on EU internal regulation, standards, or methods (ex. market authorisation); 'soft' forms of EU influence (ex. research funding, capacity building). This study illustrates that EU policy makers adopt measures with the potential to influence medicines in LMICs despite limited evidence of their positive and/or negative impact(s). The EU's fragmented internal and external actions in fields related to pharmaceuticals reveal the need for principles for global equitable access to medicines to guide EU policy.

Esta revisión exploratoria sintetiza la evidencia disponible sobre el impacto que ejercen las leyes, las políticas y las regulaciones de la Unión Europea (UE) sobre el acceso a los medicamentos en países de bajo y mediano ingreso (PBMI) que no pertenecen a la UE. La búsqueda se realizó en ocho bases de datos académicas, incluyendo literatura gris. Se incluyeron publicaciones en cuatro idiomas entre 1995 y 2021. Como resultado principal se encontró que la UE ejerce su influencia sobre los productos farmacéuticos en los PBMI a través de tres mecanismos principales: i) acuerdos explícitos entre la UE y los PBMI, por ejemplo, acuerdos de ascensión a la UE o tratados comerciales, ii) utilización de la normativa, estándares o métodos de la UE por parte de los PBMI (reliance) para, por ejemplo, autorizar el ingreso de nuevos medicamentos a partir de la autorización previa por parte de la UE) y, iii) formas blandas de influencia de la UE, por ejemplo, a través de financiación a la investigación o al desarrollo de capacidades locales. Esta revisión revela que los tomadores de decisión de la UE adoptan medidas que, a pesar de la escasa evidencia que sustenta su impacto, positivo o negativo, tienen el potencial de influir en el acceso a los medicamentos de los PBMI. El accionar fragmentado de la UE respecto a los productos farmacéuticos, tanto a nivel interno como externo, son una clara muestra de la necesidad de crear principios que guíen las políticas de la UE frente al acceso equitativo a los medicamentos a nivel global.

Essential Medicines in Universal Health Coverage: A Scoping Review of Public Health Law Interventions and How They Are Measured in Five Middle-Income Countries.

Very few studies exist of legal interventions (national laws) for essential medicines as part of universal health coverage in middle-income countries, or how the effect of these laws is measured. This study aims to critically assess whether laws related to universal health coverage use five objectives of public health law to promote medicines affordability and financing, and to understand how access to medicines achieved through these laws is measured. This comparative case study of five middle-income countries (Ecuador, Ghana, Philippines, South Africa, Ukraine) uses a public health law framework to guide the content analysis of national laws and the scoping review of empirical evidence for measuring access to medicines. Sixty laws were included. All countries write into national law: (a) health equity objectives, (b) remedies for users/patients and sanctions for some stakeholders, (c) economic policies and regulatory objectives for financing (except South Africa), pricing, and benefits selection (except South Africa), (d) information dissemination objectives (ex. for medicines prices (except Ghana)), and (e) public health infrastructure. The 17 studies included in the scoping review evaluate laws with economic policy and regulatory objectives (n = 14 articles), health equity (n = 10), information dissemination (n = 3), infrastructure (n = 2), and sanctions (n = 1) (not mutually exclusive). Cross-sectional descriptive designs (n = 8 articles) and time series analyses (n = 5) were the most frequent designs. Change in patients' spending on medicines was the most frequent outcome measure (n = 5). Although legal interventions for pharmaceuticals in middle-income countries commonly use all objectives of public health law, the intended and unintended effects of economic policies and regulation are most frequently investigated.

Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism.

The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of mitochondrial biogenesis in the hypoxia-sensitive subcortex of wild-type (Wt) and neuronal NOS (nNOS) and endothelial NOS (eNOS)-deficient mice. Mice were exposed to hypobaric hypoxia for 6 h, and changes in immediate hypoxic transcriptional regulation of mitochondrial biogenesis was assessed in relation to mitochondrial DNA (mtDNA) content and mitochondrial density. There were no differences in cerebral blood flow or hippocampal PO2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1 alpha), nuclear respiratory factor-1, and mitochondrial transcription factor A. After 24 h, new mitochondria, localized in reporter mice expressing mitochondrial green fluorescence protein, were seen primarily in hippocampal neurons. eNOS-/- mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS-/- mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS-/- mice but not in nNOS-/- mice. Hypoxia stimulated PGC-1alpha protein expression and phosphorylation of protein kinase A and cAMP response element binding (CREB) protein in Wt mice, but CREB only was activated in eNOS-/- mice and not in nNOS-/- mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1alpha and CREB.

Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity.

Recent investigations have elucidated some of the diverse roles played by reactive oxygen and nitrogen species in events that lead to oxygen toxicity and defend against it. The focus of this review is on toxic and protective mechanisms in hyperoxia that have been investigated in our laboratories, with an emphasis on interactions of nitric oxide (NO) with other endogenous chemical species and with different physiological systems. It is now emerging from these studies that the anatomical localization of NO release, which depends, in part, on whether the oxygen exposure is normobaric or hyperbaric, strongly influences whether toxicity emerges and what form it takes, for example, acute lung injury, central nervous system excitation, or both. Spatial effects also contribute to differences in the susceptibility of different cells in organs at risk from hyperoxia, especially in the brain and lungs. As additional nodes are identified in this interactive network of toxic and protective responses, future advances may open up the possibility of novel pharmacological interventions to extend both the time and partial pressures of oxygen exposures that can be safely tolerated. The implications of a better understanding of the mechanisms by which NO contributes to central nervous system oxygen toxicity may include new insights into the pathogenesis of seizures of diverse etiologies. Likewise, improved knowledge of NO-based mechanisms of pulmonary oxygen toxicity may enhance our understanding of other types of lung injury associated with oxidative or nitrosative stress.

Phosphodiesterase-5 inhibitors oppose hyperoxic vasoconstriction and accelerate seizure development in rats exposed to hyperbaric oxygen.

Oxygen is a potent cerebral vasoconstrictor, but excessive exposure to hyperbaric oxygen (HBO(2)) can reverse this vasoconstriction by stimulating brain nitric oxide (NO) production, which increases cerebral blood flow (CBF)-a predictor of O(2) convulsions. We tested the hypothesis that phosphodiesterase (PDE)-5 blockers, specifically sildenafil and tadalafil, increase CBF in HBO(2) and accelerate seizure development. To estimate changes in cerebrovascular responses to hyperoxia, CBF was measured by hydrogen clearance in anesthetized rats, either control animals or those pretreated with one of these blockers, with the NO inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME), with the NO donor S-nitroso-N-acetylpenicillamine (SNAP), or with a blocker combined with l-NAME. Animals were exposed to 30% O(2) at 1 atm absolute (ATA) ("air") or to 100% O(2) at 4 or 6 ATA. EEG spikes indicated central nervous system CNS O(2) toxicity. The effects of PDE-5 blockade varied as a positive function of ambient Po(2). In air, CBF did not increase significantly, except after pretreatment with SNAP. However, at 6 ATA O(2), mean values for CBF increased and values for seizure latency decreased, both significantly; pretreatment with l-NAME abolished these effects. Conscious rats treated with sildenafil before HBO(2) were also more susceptible to CNS O(2) toxicity, as demonstrated by significantly shortened convulsive latency. Decreases in regional CBF reflect net vasoconstriction in the brain regions studied, since mean arterial pressures remained constant or increased throughout. Thus PDE-5 blockers oppose the protective vasoconstriction that is the initial response to hyperbaric hyperoxia, decreasing the safety of HBO(2) by hastening onset of CNS O(2) toxicity.

Increased Antiseizure Effectiveness with Tiagabine Combined with Sodium Channel Antagonists in Mice Exposed to Hyperbaric Oxygen.

Hyperbaric oxygen (HBO2) is acutely toxic to the central nervous system, culminating in EEG spikes and tonic-clonic convulsions. GABA enhancers and sodium channel antagonists improve seizure latencies in HBO2 when administered individually, while combining antiepileptic drugs from different functional classes can provide greater seizure latency. We examined the combined effectiveness of GABA enhancers (tiagabine and gabapentin) with sodium channel antagonists (carbamazepine and lamotrigine) in delaying HBO2-induced seizures. A series of experiments in C57BL/6 mice exposed to 100% oxygen at 5 atmospheres absolute (ATA) were performed. We predicted equally effective doses from individual drug-dose response curves, and the combinations of tiagabine + carbamazepine or lamotrigine were tested to determine the maximally effective combined doses to be used in subsequent experiments designed to identify the type of pharmacodynamic interaction for three fixed-ratio combinations (1:3, 1:1, and 3:1) using isobolographic analysis. For both combinations, the maximally effective combined doses increased seizure latency over controls > 5-fold and were determined to interact synergistically for fixed ratios 1:1 and 3:1, additive for 1:3. These results led us to explore whether the benefits of these drug combinations could be extended to the lungs, since a centrally mediated mechanism is believed to mediate hyperoxic-induced cardiogenic lung injury. Indeed, both combinations attenuated bronchoalveolar lavage protein content by ~ 50%. Combining tiagabine with carbamazepine or lamotrigine not only affords greater antiseizure protection in HBO2 but also allows for lower doses to be used, minimizing side effects, and attenuating acute lung injury.

Adrenoceptor blockade modifies regional cerebral blood flow responses to hyperbaric hyperoxia: protection against CNS oxygen toxicity.

Exposure to extreme hyperbaric oxygen (HBO2) >5-6 atmospheres absolute (ATA) produces baroreflex impairment, sympathetic hyperactivation, hypertension, tachycardia, and cerebral hyperemia, known as phase II, culminating in seizures. We hypothesized that attenuation of the effects of high sympathetic outflow would preserve regional cerebral blood flow (rCBF) and protect against HBO2-induced seizures. To explore this possibility, we tested four adrenoceptor antagonists in conscious and anesthetized rats exposed to HBO2 at 5 and 6 ATA, respectively: phentolamine (nonselective α1 and α2), prazosin (selective α1), propranolol (nonselective ß1 and ß2), and atenolol (selective ß1). In conscious rats, four drug doses were administered to rats before HBO2 exposures, and seizure latencies were recorded. Drug doses that provided similar protection against seizures were administered before HBO2 exposures in anesthetized rats to determine the effects of adrenoceptor blockade on mean arterial pressure, heart rate, rCBF, and EEG spikes. All four drugs modified cardiovascular and rCBF responses in HBO2 that aligned with epileptiform discharges, but only phentolamine and propranolol effectively increased EEG spike latencies by ~20 and 36 min, respectively. When phentolamine and propranolol were delivered during HBO2 at the onset of phase II, only propranolol led to sustained reductions in heart rate and rCBF, preventing the appearance of epileptiform discharges. The enhanced effectiveness of propranolol may extend beyond ß-adrenoceptor blockade, i.e., membrane stability and reduced metabolic activity. These results indicate that adrenoceptor drug pretreatment will minimize the effects of excessive sympathetic outflow on rCBF and extend HBO2 exposure time.NEW & NOTEWORTHY Blocking adrenergic receptors with phentolamine (nonselective α1 and α2), prazosin (selective α1), propranolol (nonselective ß1 and ß2), and atenolol (selective ß1) modified cardiovascular and regional cerebral blood flow (rCBF) responses in hyperbaric oxygen (HBO2) at 6 atmospheres absolute (ATA); however, only phentolamine and propranolol extended EEG spike latencies. When these two agents were delivered at the onset of sympathetic hyperactivation, only propranolol reduced heart rate and rCBF throughout the exposure and prevented epileptiform discharges. These data validate the strong role of adrenergic control of cardiovascular function and rCBF in extreme HBO2 and the potential use of antiadrenergic drugs to prevent seizures.

S-nitrosylation of GAD65 is implicated in decreased GAD activity and oxygen-induced seizures.

Breathing oxygen at partial pressures ≥2.5 atmospheres absolute, which can occur in diving and hyperbaric oxygen (HBO2) therapy, can rapidly become toxic to the central nervous system (CNS). This neurotoxicity culminates in generalized EEG epileptiform discharges, tonic-clonic convulsions and ultimately death. Increased production of neuronal nitric oxide (NO) has been implicated in eliciting hyperoxic seizures by altering the equilibrium between glutamatergic and GABAergic synaptic transmission. Inhibition of glutamic acid decarboxylase (GAD) activity in HBO2 promotes this imbalance; however, the mechanisms by which this occurs is unknown. Therefore, we conducted a series of experiments using mice, a species that is highly susceptible to CNS oxygen toxicity, to explore the possibility that NO modulates GABA metabolism. Mice were exposed to 100% oxygen at 4 ATA for various durations, and brain GAD and GABA transaminase (GABA-T) activity, as well as S-nitrosylation of GAD65 and GAD67 were determined. HBO2 inhibited GAD activity by 50% and this was negatively correlated with S-nitrosylation of GAD65, whereas GABA-T activity and S-nitrosylation of GAD67 were unaltered. These results suggest a new mechanism by which NO alters GABA metabolism, leading to neuroexcitation and seizures in HBO2.

Antiepileptic drugs prevent seizures in hyperbaric oxygen: A novel model of epileptiform activity.

Breathing oxygen at sufficiently elevated pressures can trigger epileptiform seizures. Therefore, we tested the hypothesis that pre-treatment with FDA-approved antiepileptic drugs could prevent seizure onset in hyperoxia at 5 atmospheres absolute. We selected drugs from two putative functional categories, Na+-channel antagonists and GABA enhancers, each administered intraperitoneally at four doses in separate groups of C57BL/6 mice. The drugs varied in efficacy at the doses used. Of the five tested Na+-channel antagonists, carbamazepine and lamotrigine more than tripled seizure latency compared to values seen in vehicle controls. Primidone, zonisamide and oxcarbazepine were less effective. Of the four GABA reuptake inhibitors, tiagabine and vigabatrin also increased seizure latency by more than three times control values; valproic acid was less effective, and the GABA synthesis promoter gabapentin was intermediate in effectiveness. We infer that Na+-channel function and GABA neurotransmission may be critical targets in the pathophysiology of CNS O2 toxicity. Because these essential components of neuronal excitation and inhibition are also implicated in the pathogenesis of other seizure disorders, including generalized epilepsy, we propose that, at some level, common pathways are involved in these pathologies, although the initiating insults differ. Furthermore, hyperoxic exposures are not known to cause the spontaneously-recurring seizures that characterize true clinical epilepsy. Nonetheless, experimental studies of hyperbaric oxygen toxicity could provide new insights into molecular mechanisms of seizure disorders of various etiologies. In addition, the neuropathology of hyperbaric oxygen is particularly relevant to the hypothesis held by some investigators that oxidative stress is an etiological factor in clinical epilepsies.

Regulation of the brain's vascular responses to oxygen.

The mechanism of oxygen-induced cerebral vasoconstriction has been sought for more than a century. Using genetically altered mice to enhance or disrupt extracellular superoxide dismutase (EC-SOD, SOD3), we tested the hypothesis that this enzyme plays a critical role in the physiological response to oxygen in the brain by regulating nitric oxide (NO*) availability. Cerebral blood flow responses in these genetically altered mice to changes in PO2 demonstrate that SOD3 regulates equilibrium between superoxide (*O2-) and NO*, thereby controlling vascular tone and reactivity in the brain. That SOD3 opposes inactivation of NO* is shown by absence of vasoconstriction in response to PO2 in the hyperbaric range in SOD3+/+ mice, whereas NO-dependent relaxation is attenuated in SOD3-/- mutants. Thus, EC-SOD promotes NO* vasodilation by scavenging *O2- while hyperoxia opposes NO* and promotes constriction by enhancing endogenous *O2- generation and decreasing basal vasodilator effects of NO*.

Cerebral blood flow and brain oxygenation in rats breathing oxygen under pressure.

Hyperbaric oxygen (HBO(2)) increases oxygen tension (PO(2)) in blood but reduces blood flow by means of O(2)-induced vasoconstriction. Here we report the first quantitative evaluation of these opposing effects on tissue PO(2) in brain, using anesthetized rats exposed to HBO(2) at 2 to 6 atmospheres absolute (ATA). We assessed the contribution of regional cerebral blood flow (rCBF) to brain PO(2) as inspired PO(2) (PiO(2)) exceeds 1 ATA. We measured rCBF and local PO(2) simultaneously in striatum using collocated platinum electrodes. Cerebral blood flow was computed from H(2) clearance curves in vivo and PO(2) from electrodes calibrated in vitro, before and after insertion. Arterial PCO(2) was controlled, and body temperature, blood pressure, and EEG were monitored. Scatter plots of rCBF versus PO(2) were nonlinear (R(2)=0.75) for rats breathing room air but nearly linear (R(2)=0.88-0.91) for O(2) at 2 to 6 ATA. The contribution of rCBF to brain PO(2) was estimated at constant inspired PO(2), by increasing rCBF with acetazolamide (AZA) or decreasing it with N-nitro-L-arginine methyl ester (L-NAME). At basal rCBF (78 mL/100 g min), local PO(2) increased 7- to 33-fold at 2 to 6 ATA, compared with room air. A doubling of rCBF increased striatal PO(2) not quite two-fold in rats breathing room air but 13- to 64-fold in those breathing HBO(2) at 2 to 6 ATA. These findings support our hypothesis that HBO(2) increases PO(2) in brain in direct proportion to rCBF.