Chryssa Pourzitaki

Pharmacological Aspects and Potential New Clinical Applications of Ketamine: Reevaluation of an Old Drug

Ketamine, the phencyclidine derivative described in 1965, is an intravenous anesthetic with a variety of applications. The enthusiasm following its initial release subsided due to side effects from the central nervous system. New anesthetics limited the role of ketamine in anesthetic practice. However, its hemodynamically stable profile, along with its beneficial respiratory properties and analgesic potency, rendered the drug invaluable in battlefield medicine, sedation of the uncooperative child, analgesia, and sedation in burn units. Reevaluation, though, of analgesic properties of ketamine resulted in new interest regarding its use in perioperative and chronic pain management. Moreover, recent studies in the effects of the substance on intracranial pressure and cerebral blood flow led to revising the recommendation against its use in brain injury. Furthermore, the bronchodilating effects of the substance led to increasing interest for potential use in asthma treatment. In addition, separation of the 2 enantiomers and subsequent separate studies indicated beneficial results of the S(+) one. Thus, new controlled multicentered clinical trials are to be conducted to justify approval for new uses of ketamine and take advantage of its unique range of applications.

PDE5 Inhibitors: In Vitro and In Vivo Pharmacological Profile

PDE5 inhibitors have been clearly established as first-line therapy for the treatment of erectile dysfunction (ED). Three PDE5 inhibitors--sildenafil (Viagra), vardenafil (Levitra) and tadalafil (Cialis)--are currently approved by the FDA and the EMEA for use in ED, whereas sildenafil is also marketed under a different proprietary name (Revatio) for the treatment of pulmonary arterial hypertension (PAH). A forth PDE5 inhibitor, udenafil (Zydena), is currently marketed. In the present review the molecular basis and the mechanism of action of PDE5 inhibitors is discussed. In addition experimental and clinical data concerning their effects on different tissues, organs and systems is systematically reviewed and their possible beneficial action in numerous disorders is presented.

Ropivacaine myotoxicity after single intramuscular injection in rats

Background and objective: We conducted this study in order to evaluate the potential myotoxic effects of ropivacaine after single injection in rats and the time‐course of the possible damage. Methods: One hundred and twenty‐eight male Wistar rats were allocated to four different groups. The first three groups received intramuscular injections with ropivacaine 0.75%, ropivacaine 0.5% and normal saline, respectively, into the right tibialis anterior muscle. The fourth group received needle puncture without injection. Eight rats from each group were sacrificed 2, 4, 7 and 30 days after injection. Samples were blindly examined under light microscope for evidence of myotoxicity, scored as 0 = no damage to 3 = myonecrosis and statistically analysed. Samples obtained 7 days after injection were also examined under transmission electron microscope. Results: Ropivacaine 0.75% and ropivacaine 0.5% caused extensive destruction to muscles fibres, compared to saline or needle on days 2, 4 and 7. Statistically significant differences were found in muscle damage by drug injections among all groups except for saline vs. needle groups. Thirty days after injections all sample appearances had returned to normal. Conclusions: Ropivacaine after single intramuscular injection caused reversible muscle damage in a dose‐dependent manner.

Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Oxygen–Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen–glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Baroreceptors discharge due to bilateral aortic denervation evokes acute neuronal damage in rat brain

Deep hypothermic circulatory arrest in cardiothoracic surgery evokes severe brain damages. On the other hand, blood pressure stimuli discontinuation to the brain has been found to induce alterations in neurotransmitter release, including glutamate, in numerous brain regions. Furthermore, it is well established that excessive glutamate release can induce neuronal injury, a process called excitotoxicity. Aim of the present study was the evaluation of possible acute neuronal damage after bilateral aortic denervation (bAD), imitating the baroreceptors discharge during circulatory arrest. Male, Wistar rats underwent either bAD or Sham operation under continuous hemodynamic monitoring. Two hours after completion of the procedure, rats were sacrificed and the brains were dissected and cut in specific levels corresponding to selective brain regions, based on either their participation in neuronal circuits, regulating blood pressure, or their vulnerability, after deep hypothermic circulatory arrest. Slices were stained and examined under light microscope using morphometric techniques. Increased number of necrotic neurons were found among bAD rats in amygdaloid complex (p=0.005), motor cortex (p=0.001), CA1 and CA3 (p=0.02 and 0.015) but not in posterior hypothalamic nucleus and Purkinje cell. Higher ratios of necrotic neurons were found in amygdaloid complex (p=0.002), motor layer (p=0.003 and p=0.000) and the hippocampal CA1 region (p=0.027) of bAD rats. The present study shows that baroreceptors discharge due to bAD may induce acute neuronal loss in brain regions involved in blood pressure regulation. Neuronal loss might be attributed to excitotoxic phenomena and it is following the same topographic distribution seen in deep hypothermic circulatory arrest, revealing a concurrent to hypoxia/ischemia mechanism of brain damage.

Furosemide modifies heart hypertrophy and glycosaminoglycan myocardium content in a rat model of neurogenic hypertension

Hypertension is a major risk factor for atherogenesis and heart hypertrophy, both of which are associated with specific morphological and functional changes of the myocardium. Glycosaminoglycans (GAGs) are complex molecules involved both in tissue morphology and function. In the present study, we investigated the effects of neurogenic hypertension and subsequent antihypertensive treatment with furosemide, on heart hypertrophy and the content of GAGs in the myocardium. Neurogenic hypertension was achieved in male Wistar rats by bilateral aortic denervation (bAD). At days 2, 7 and 15 after surgery, animals were sacrificed and the hearts were dissected away, weighted, and homogenized. Total GAGs were assessed by measuring the uronic acid content colorimetrically and individual GAGs were isolated and characterized by enzymatic treatment, with GAG-degrading enzymes, using electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes. In bAD-animals blood pressure, blood pressure lability, heart rate and heart weight were significantly increased 15 days postoperatively. These effects were prevented by treatment with furosemide. Major GAGs identified in the heart were chondroitin sulphates, heparin (H), heparan sulphate (HS) and hyaluronic acid. The content of uronic and the relative content of H and HS in the heart in bAD animals significantly decreased from day 2 to day 15 postoperatively. Furosemide prevented the bAD induced decrease in GAG content. Considering that H and HS are potent inhibitors of cardiomyocyte hypertrophy, our results indicate that heart hypertrophy induced by neurogenic hypertension may be associated with decreases in the relative content of heparin and heparan sulphate in the heart.

Chronic aortic denervation decreases anxiety and impairs social memory in rats.

AIMS The present study investigates anxiety-like behaviour and social cognitive performance in rats with chronic aortic denervation. MAIN METHODS The aortic depressor nerve was bilaterally transected in Wistar rats, causing an almost complete disruption of baroreceptors. Bilateral aortic denervated (bAD), sham-operated (SHAM), and intact (CTRL) rats performed an elevated plus-maze test and an olfactory social memory test, one and three months after operation. Blood pressure and heart rate were monitored in all animals. KEY FINDINGS Systolic blood pressure, blood pressure lability and heart rate were elevated in bAD rats compared to SHAM and CTRL rats. In the elevated plus-maze test, bAD rats spent clearly more time in investigating open arms and performed more open arm entries than SHAM and CTRL rats during both testing sessions. The olfactory social memory test revealed that acquisition time during first contact with a juvenile rat did not differ between the groups of rats. The recognition time spent by SHAM and CTRL group of rats was distinctly decreased in comparison to the acquisition time, an indication of social memory. bAD rats investigated the juvenile rat during the second contact to a similar extent than during the first contact, both one and three months after denervation. SIGNIFICANCE These results suggest that bilateral aortic denervation induces chronic neurogenic hypertension and elevated blood pressure lability, decreases anxiety-like behaviour and deteriorates social memory in rats while acquiring of social information is not affected.

Nonmedical Use of Prescription Medications Among Medical Students in Greece: Prevalence of and Motivation for Use

ABSTRACT Background: Non-medical use of prescription medications has risen to unprecedented levels over the past decade worldwide; however, studies assessing misuse across medical students are sparse. Objectives: The purpose of this study was to1) estimate the lifetime and the past-year prevalence of non-medical use of prescription medications among medical students in Greece 2) identify the motivation for use. Methods: 591 medical students completed an anonymous, self-administered, web-based survey assessing lifetime and past-year prevalence of non-medical use of four classes of prescription drugs (opioid painkillers, tranquillizers, sleeping and stimulant medications). According to the motivation to use the responders were classified into three subtypes (selftreatment, recreational, and mixed). Results: The prevalence of lifetime use was 10.7% for at least one of the four prescription drug classes and 9% of the respondents reported lifetime misuse of multiple categories of prescription drugs. The past-year prevalence was approximately 7.7% for at least one of the four prescription drug classes, while the majority misused the drugs “1-2 times per year”. Senior students used tranquilizers more than junior students. Self-treatment and mixed subtypes of tranquillizers misuse was more prevalent among women than men while the self-treatment subtype was the most prevalent subtype in all the four drug classes. Conclusions: This is the first study investigating non-medical use of prescription medications among Greek medical students and indicates a high prevalence of misuse of some categories of prescription drugs, mostly for self-treatment purposes.

Calpain-Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide

Abstract Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.