Plant-Based Antioxidants for Prevention and Treatment of Neurodegenerative Diseases: Phytotherapeutic Potential of Laurus nobilis, Aronia melanocarpa, and Celastrol.

With the progress of medicine, especially in the last century, life expectancy increased considerably. As a result, age-related diseases also increased, especially malignancies and degenerative diseases of the central nervous system. The incidence and prevalence of neurodegenerative diseases steadily increased over the years, but despite efforts to uncover the pathophysiological processes behind these conditions, they remain elusive. Among the many theories, oxidative stress was proposed to be involved in neurodegenerative processes and to play an important role in the morbidity and progression of various neurodegenerative disorders. Accordingly, a number of studies discovered the potential of natural plant constituents to have significant antioxidant activity. This review focused on several plant-based antioxidants that showed promising results in the prevention and treatment of neurodegenerative diseases. Laurus nobilis, Aronia melanocarpa, and celastrol, a chemical compound isolated from the root extracts of Tripterygium wilfordii and T. regelii, are all known to be rich in antioxidant polyphenols.

Modeling Central Nervous System Injury In Vitro: Current Status and Promising Future Strategies.

The central nervous system (CNS) injury, which occurs because of mechanical trauma or ischemia/hypoxia, is one of the main causes of mortality and morbidity in the modern society. Until know, despite the fact that numerous preclinical and clinical studies have been undertaken, no significant neuroprotective strategies have been discovered that could be used in the brain trauma or ischemia treatment. Although there are many potential explanations for the failure of those studies, it is clear that there are questions regarding the use of experimental models, both in vivo and in vitro, when studying CNS injury and searching new therapeutics. Due to some ethical issues with the use of live animals in biomedical research, implementation of experimental strategies that prioritize the use of cells and tissues in the in vitro environment has been encouraged. In this review, we examined some of the most commonly used in vitro models and the most frequently utilized cellular platforms in the research of traumatic brain injury and cerebral ischemia. We also proposed some future strategies that could improve the usefulness of these studies for better bench-to-bedside translational outcomes.

Immunometabolic Modulatory Role of Naltrexone in BV-2 Microglia Cells.

Background: Naltrexone is an opioid receptor antagonist commonly used to treat opioid and alcohol dependence. The use of low dose naltrexone (LDN) was found to have anti-inflammatory properties for treatment of diseases such as fibromyalgia, Crohn's disease, multiple sclerosis and regional pain syndromes. Related to its anti-neuroinflammatory properties, the mechanism of action is possibly mediated via Toll-like receptor 4 antagonism, which is widely expressed on microglial cells. The aim of the present study was to assess the immunometabolic effects of naltrexone on microglia cells in in vitro conditions. METHODS: All experiments were performed in the BV-2 microglial cell line. The cells were treated with naltrexone at 100 µM concentrations corresponding to low dose for 24 h. Cell viability was assessed for every drug dose. To induce additional activation, the cells were pretreated with LPS and IFN-γ. Immunofluorescence was used to analyse the classical microglial activation markers iNOS and CD206, while Seahorse was used for real-time cellular metabolic assessments. mTOR activity measured over the expression of a major direct downstream target S6K was assessed using western blot. RESULTS: LDN induced a shift from highly activated pro-inflammatory phenotype (iNOShighCD206low) to quiescent anti-inflammatory M2 phenotype (iNOSlowCD206high) in BV-2 microglia cells. Changes in the inflammatory profile were accompanied by cellular metabolic switching based on the transition from high glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). LDN-treated cells were able to maintain a metabolically suppressive phenotype by supporting OXPHOS with high oxygen consumption, and also maintain a lower energetic state due to lower lactate production. The metabolic shift induced by transition from glycolysis to mitochondrial oxidative metabolism was more prominent in cells pretreated with immunometabolic modulators such as LPS and IFN-γ. In a dose-dependent manner, naltrexone also modulated mTOR/S6K expression, which underlies the cell metabolic phenotype regulating microglia immune properties and adaptation. CONCLUSION: By modulating the phenotypic features by metabolic switching of activated microglia, naltrexone was found to be an effective and powerful tool for immunometabolic reprogramming and could be a promising novel treatment for various neuroinflammatory conditions.

Capsaicin - Potential Solution for Chronic Pain Treatment.

Chronic pain is a painful condition defined by its duration where pain persists three months or more. Pain is connected with the high price of health care, work inability and disability. Moreover, it has significant consequences for patients and their families, working organizations and the society as a whole. The prevalence of chronic pain can range between 11.0% and 51.3% in general population. Pain is usually coherent with distress and a range of psychological symptoms such as depression, anxiety, altered attention and cognition manifesting as fear. Comprehensive pain management should always include the treatment of associated psychological symptoms. Multidisciplinary approach in treating chronic pain and its comorbidities and proper education of primary care physicians and different specialists involved in the management of chronic pain are crucial for better clinical outcomes. Topical capsaicin acts as a highly selective agonist of transient receptor potential vanilloid 1 of C and Aδ nociceptors. Repeated applications or high concentrations give rise to a long-lasting effect termed defunctionalisation. In addition, the reduction of central sensitization through reduced C-nociceptor input contributes to capsaicin's indirect mechanism of action. Capsaicin provides effective durable pain relief and reduction of intensity and area of pain in adult patients with chronic pain with a faster onset of analgesia and considerably fewer systemic adverse effects than the conventional treatment. While offering high levels of pain relief, additional improvements in sleep, fatigue, depression and quality of life have been noticed. Topical administration avoids dangerous systemic adverse effects and enables the combination with other drugs and analgesics with limited drug-drug interactions. Adding capsaicin to the standard chronic pain treatment might improve, fasten and ease the challenging path of managing chronic pain consequently providing the patient and their society with better quality of life.

Antihypertensive Drugs in Croatia: What Changes the Drug Usage Patterns?

PURPOSE: Possible factors that could influence changes in patterns of prescribing antihypertensives could be identified by monitoring national trends in hypertension treatment. The choice of pharmacologic treatment in people with hypertension has important therapeutic and financial implications, due to the fact that the financial costs associated with hypertension continue to increase. The aims of our study were to identify and analyze changes in the usage of antihypertensive drugs in Croatia from 2000 to 2016 and to identify the changes in prescribing patterns as well as mean prices per defined daily dose (DDD). METHODS: Data on consumption in Croatia were obtained from the International Medical Statistics database. According to the World Health Organization's Collaborating Center for Drugs Statistics Methodology, per-annum volumes of drugs are presented in DDD per 1000 population per day (DDD/1000), while data on financial expenditure are presented in euros. FINDINGS: The consumption of drugs for cardiovascular disease in Croatia during the period from 2000 to 2016 increased 150.81%, while financial expenditure in the same period increased 47.32%. The most frequently prescribed subgroup was agents acting on the renin-angiotensin system (RAS). Their share among antihypertensives increased from 39.13% (2000) to 53.39% (2016). The share of diuretics in the same period decreased from 20.16% in 2000 to 12.73% in 2016. IMPLICATIONS: The prescribing patterns of antihypertensive drugs in Croatia have changed, which could be a result of a combination of different factors, such as changes in laws, pharmaceutical marketing, and guidelines on hypertension therapy. The most prescribed subgroup in all of the investigated years was agents acting on the RAS, mainly because of the increased prescribing of combinations of RAS agents plus diuretics. The financial implications of legal changes and the introduction of new generic drugs led to decreased cost per DDD of antihypertensives during the investigated period, but the total expenditure on antihypertensives in Croatia increased due to increased consumption.

Decrease in Oxidative Stress Parameters after Post-Ischaemic Recombinant Human Erythropoietin Administration in the Hippocampus of Rats Exposed to Focal Cerebral Ischaemia.

Recombinant human erythropoietin (rhEpo) is a multi-functional drug with antioxidant potential. However, the underlying molecular mechanisms of its action are still unclear. The purpose of this study was to investigate the effects of rhEpo on the brain infarct volume as well as on the levels of the neuronal damage, oxidative stress parameters and active caspase-3, nuclear factor erythroid 2-related factor 2 (Nrf2) and haemeoxygenase-1 (HO-1) expressions in the hippocampi of rats exposed to the right middle cerebral artery occlusion (MCAO) for 1 hr. Ischaemic animals received either vehicle or rhEpo (5000 IU/kg, i.p.) immediately or 3 hr after the induction of ischaemia. Sham-operated, vehicle-treated animals served as the control group. Rats were killed 24 hr after the onset of the ischaemic or sham experimental procedure. MCAO caused ipsilateral brain infarction within the striatum and cortex. In the CA1 region of the hippocampi, we did not find significant neuronal loss, but a statistically significant rise in the active caspase-3 and Nrf2 protein expressions was registered. We detected also significant increases in the hippocampal levels of oxidative stress parameters (thiobarbituric acid-reactive substances, superoxide dismutase, glutathione peroxidase). Post-ischaemic administration of rhEpo significantly reduced the brain infarct volume, decreased levels of all tested oxidative stress parameters and increased the Nrf2 expression level. These findings suggest that decrease in oxidative stress parameters in the hippocampus could be an early indicator of post-ischaemic neuroprotective effect of rhEpo in rats exposed to focal cerebral ischaemia and that this effect could be attributable to additional post-ischaemic activation of Nrf2 endogenous antioxidant system.

A single dose of PPARγ agonist pioglitazone reduces cortical oxidative damage and microglial reaction following lateral fluid percussion brain injury in rats.

Neuroprotective actions of the peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been observed in various animal models of the brain injuries. In this study we examined the effects of a single dose of pioglitazone on oxidative and inflammatory parameters as well as on neurodegeneration and the edema formation in the rat parietal cortex following traumatic brain injury (TBI) induced by the lateral fluid percussion injury (LFPI) method. Pioglitazone was administered in a dose of 1mg/kg at 10min after the brain trauma. The animals of the control group were sham-operated and injected by vehicle. The rats were decapitated 24h after LFPI and their parietal cortices were analyzed by biochemical and histological methods. Cortical edema was evaluated in rats sacrificed 48h following TBI. Brain trauma caused statistically significant oxidative damage of lipids and proteins, an increase of glutathione peroxidase (GSH-Px) activity, the cyclooxygenase-2 (COX-2) overexpression, reactive astrocytosis, the microglia activation, neurodegeneration, and edema, but it did not influence the superoxide dismutase activity and the expressions of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha in the rat parietal cortex. Pioglitazone significantly decreased the cortical lipid and protein oxidative damage, increased the GSH-Px activity and reduced microglial reaction. Although a certain degree of the TBI-induced COX-2 overexpression, neurodegeneration and edema decrease was detected in pioglitazone treated rats, it was not significant. In the injured animals, cortical reactive astrocytosis was unchanged by the tested PPARγ agonist. These findings demonstrate that pioglitazone, administered only in a single dose, early following LFPI, reduced cortical oxidative damage, increased antioxidant defense and had limited anti-inflammatory effect, suggesting the need for further studies of this drug in the treatment of TBI.

Temporal and regional changes of superoxide dismutase and glutathione peroxidase activities in rats exposed to focal cerebral ischemia.

Reactive oxygen species are important cause of tissue injury during cerebral ischemia and reperfusion (I/R). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are intracellular enzymes responsible for endogenous antioxidant defense of tissues affected by I/R. The aim of this study was to examine temporal and regional changes of SOD and GSH-Px activities in animals exposed to transient focal cerebral ischemia. Male Wistar Hannover rats were subjected to the right middle cerebral artery occlusion for 2 h. The animals were sacrificed immediately, 0·5, 1, 2, 3, 6, 24, 48, 72 or 168 h after ischemic procedure. SOD and GSH-Px activities were determined spectrophotometrically in the hippocampus and parietal cortex, both unilaterally and contralaterally to the occlusion. Sham-operated animals were used as the control group. Our results indicated that transient focal cerebral ischemia causes significant changes in SOD activities in the hippocampus and parietal cortex such as in GSH-Px activities in the parietal cortex, unilaterally and contralaterally to the lesion in rats during different reperfusion periods. Statistically significant activation of GSH-Px was registered neither in the right nor in the left hippocampus of ischemic animals.

Drug usage by outpatients in Croatia during an 8-year period: Influence of changes in pricing policy.

OBJECTIVE: The aim of our study was to investigate the changes in drug usage and financial expenditure according to legal changes in Croatia during the period 2001 - 2008, especially considering pricing policy. MATERIALS: The data on outpatient drug usage during the studied period was obtained from the Croatian National Health Insurance (CNHI). CNHI maintains a database on drugs prescribed by primary health care physicians and dispensed by pharmacies. METHODS: The data was calculated and presented in defined daily doses (DDD) per inhabitant per year for antibiotics and in DDD/1,000 inhabitants/day for other drugs. The data is also presented in Euro/DDD and the financial expenditures are presented in Euros. RESULT: During the investigated period drug usage increased 81.33%, while financial expenditure increased 77.23%. While total DDD/1,000 increased ~ 10% every year, financial expenditure increased 10 - 20% annually until 2006, but since then there have been no significant changes. CONCLUSION: Pricing policy changes could influence drug financial expenditure considerably in the short-term, but it is also important to apply a combination of measures for drug expenditure control. Numerous interventions from authorities from different countries all over the world, prove that there is still no so called "gold standard" which could restrain growing usage and expenditure of drugs. Clinical pharmacologists and clinical pharmacists should be included in these processes.

Oxidative stress parameters in different brain structures following lateral fluid percussion injury in the rat.

Free radicals mediated damage of phospholipids, proteins and nucleic acids results in subsequent neuronal degeneration and cell loss. Aim of this study was to evaluate the existence of lipid and protein oxidative damage and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in various rat brain structures 24 h after lateral fluid percussion brain injury (LFPI). Parietal cortex, hippocampus, thalamus, entorhinal cortex, and cerebellum from the ipsilateral hemisphere were processed for analyses of the thiobarbituric acid reactive substances (TBARS) and oxidized protein levels as well as for the SOD and GSH-Px activities. Immunohistochemical detection of oxidized proteins was also performed. Results of our study showed that LFPI caused significant oxidative stress in the parietal cortex and hippocampus while other brain regions tested in this study were not oxidatively altered by LFPI. GSH-Px activities were significantly increased in the parietal cortex and hippocampus, while the SOD activities remained unchanged following LFPI in all regions investigated.

Activation of ERK and JNK MAP kinases in optic nerves of rats exposed to global cerebral ischemia.

OBJECTIVES: To determine the influence of global cerebral ischemia on the activation of extracellular-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in optic nerves of rats exposed to different reperfusion periods. MATERIALS AND METHODS: Transient global cerebral ischemia (20-min duration) was induced by the four-vessel occlusion method. After different reperfusion periods (5 and 10 min; 1; 6 and 12 h after ischemia), optic nerves were extracted and ERK and JNK activation signals were determined by Western immunoblot analyses. RESULTS: The activation signals of ERK and JNK were detected within first 10 min of reperfusion, but striking activation for both enzymes was found 1 h after ischemia. After a transient decrease, the activation of ERK returned to peak level after 12 h of reperfusion in the second wave of kinase activation. In that period, a slight increase of JNK activation was registered. CONCLUSION: Our results demonstrated for the first time that ERK and JNK were activated in rat optic nerves during early and later periods of reperfusion, suggesting their potential active role in the response of cerebral white matter tissue to ischemic injury.

Characteristics of blood-pressure control in treated hypertensive patients in Croatia.

The aim of our study was to investigate blood pressure (BP) control and different factors with possible influence on BP control in Croatian hypertensive patients. In this cross-sectional investigation, a representative sample of target populations (primary care physicians and patients) from different parts of Croatia was included according to the study protocol. During December 2003 and January 2004, we included, according to correctly completed questionnaires, 141 physicians and 814 hypertensive patients. A controlled BP (BP < 140/90 mmHg) in this hypertensive population treated with antihypertensive drugs was in 23% of patients. The analysis of BP control according to risk factors showed that significantly related with higher levels of systolic or diastolic BP were the age (poorer systolic BP control in patients older than 60 years), left ventricular hypertrophy, changes of the eye retina, smoking and diabetes mellitus. Furthermore, patients from towns closer to the hospital, from urban centers, with higher education and employed had significantly lower average BP. According to our results of hypertension control in Croatia, there is a need and a possibility for the improvement of the quality of hypertension care. The relationship between demographic and cardiovascular risk factors with poor BP control should be taken into account when treating patients.

Effects of the hyperbaric oxygen treatment on the Na+,K+ -ATPase and superoxide dismutase activities in the optic nerves of global cerebral ischemia-exposed rats.

The effects of hyperbaric oxygen (HBO) treatment on the Na+,K+ -ATPase and superoxide dismutase (SOD) activities were examined in the optic nerves of the rats exposed to global cerebral ischemia. Animals were exposed to global cerebral ischemia of 20-min duration and were either sacrificed or exposed to the first HBO treatment immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na+,K+ -ATPase activities measurement) or 2, 24, 48 or 168 h after ischemia (for SOD activities measurement). HBO procedure was repeated for 7 consecutive days. It was found that global cerebral ischemia induced a statistically significant decrease in the Na+,K+ -ATPase activity of the optic nerves, starting from 0.5 to 168 h of reperfusion. Maximal enzymatic inhibition was registered 24 h after the ischemic damage. The decline in the Na+,K+ -ATPase activity was prevented in the animals exposed to HBO treatment within the first 6 h of reperfusion. The results of the presented experiments demonstrated also a statistically significant increase in the SOD activity after 24, 48 and 168 h of reperfusion in the optic nerves of non-HBO-treated ischemic animals as well as in the ischemic animals treated with HBO. Our results indicate that global cerebral ischemia induced a significant alterations in the Na+,K+ -ATPase and SOD activities in the optic nerves during different periods of reperfusion. HBO treatment, started within the first 6 h of reperfusion, prevented ischemia-induced changes in the Na+,K+ -ATPase activity, while the level of the SOD activity in the ischemic animals was not changed after HBO administration.

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+,K+-ATPase activities in global cerebral ischemia-exposed rats.

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na(+),K(+)-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na(+),K(+)-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na(+),K(+)-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na(+),K(+)-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na(+),K(+)-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na(+),K(+)-ATPase activity within particular periods of reperfusion, could be indicators of a possible beneficial role of HBO treatment in severe brain ischemia.

The influence of MK-801 on the hippocampal free arachidonic acid level and Na+,K+-ATPase activity in global cerebral ischemia-exposed rats.

The influence of 20 min global cerebral ischemia on the free arachidonic acid (FAA) level and Na+,K+-ATPase activity in the rat hippocampus at different time points after ischemia was examined. In addition, the effect of MK-801 on mentioned parameters was studied. Animals were exposed to 20 min global cerebral ischemia and were sacrificed immediately, 0.5, 1, 2, 6, 24, 48, 72, and 168 h after ischemic procedure. The level of the FAA and the Na+,K+-ATPase activity was measured during all reperfusion periods examined. Various doses of MK-801 (0.3, 1.0, 3.0, and 5.0 mg/kg) had been injected 30 min before ischemic procedure started. It was found that 20 min global cerebral ischemia induces a statistically significant increase of the FAA level immediately after ischemia and during the first 0.5 h of reperfusion. After a transient decrease, the level of FAA level increased again after 24 and 168 h of recirculation. Treatment with 3.0 mg/kg of MK-801 significantly prevented the FAA accumulation immediately and 0.5 h after ischemic insult while application of 5.0 mg/kg of MK-801 exerted a protective effect during the first 24 h. Global cerebral ischemia induces the significant decline in the Na+,K+-ATPase activity in the hippocampus starting from 1 to 168 h of reperfusion. Maximal inhibition was obtained 24 h after the ischemic damage. Application of 3.0 mg/kg of MK-801 exerted statistically significant protection during the first 24 h while the treatment with 5.0 mg/kg of MK-801 prevented fall in enzymatic activity during all reperfusion periods examined. Our results suggest that, in spite of different and complex pathophysiological mechanisms involved in the increase of FAA level and the decrease of the Na+,K+-ATPase activity, blockade of NMDA receptor subtype provides a very important strategy for the treatment of the postischemic excitotoxicity.