Cytotoxic Hypoxia Research Articles

A Novel Fluorescent Probe for Nitroreductase Detection and Imaging of cancer Cells under Hypoxia Conditions.

Nitroreductase (NTR) is to be pivotal in the biodegradation of nitroaromatics. NTR is produced in tumor tissues under hypoxic conditions, which is one of the markers for early tumor diagnosis. In this study, a novel probe FD-NTR was developed for NTR detection. Probe FD-NTR can exhibit a specific reaction with NTR in the presence of NADH. The probe displayed satisfactory selectivity and sensitivity towards NTR with a calculated detection limit of 12 ng/mL. Under the conditions of low cytotoxic hypoxia, the FD-NTR probe has shown successful application in imaging both MCF-7 cells and tumor tissues, which indicated that the FD-NTR probe holds promising application prospects for detecting NTR in tumors.

Peripheral chemoreflex activation induces expiratory but not inspiratory excitation of C1 pre-sympathetic neurones of rats.

Stimulation of peripheral chemoreceptors, as during hypoxia, increases breathing and respiratory-related sympathetic bursting. Activation of catecholaminergic C1 neurones induces sympathoexcitation, while its ablation reduces the chemoreflex sympathoexcitatory response. However, no study has determined the respiratory phase(s) in which the pre-sympathetic C1 neurones are recruited by peripheral chemoreceptor and whether C1 neurone activation affects all phases of respiratory modulation of sympathetic activity. We addressed these unknowns by testing the hypothesis that peripheral chemoreceptor activation excites pre-sympathetic C1 neurones during inspiration and expiration. Using the in situ preparation of rat, we made intracellular recordings from baroreceptive pre-sympathetic C1 neurones during peripheral chemoreflex stimulation. We optogenetically activated C1 neurones selectively and compared any respiratory-phase-related increases in sympathetic activity with that which occurs following stimulation of the peripheral chemoreflex. Activation of peripheral chemoreceptors using cytotoxic hypoxia (potassium cyanide) increased the firing frequency of C1 neurones and both the frequency and amplitude of their excitatory post-synaptic currents during the phase of expiration only. In contrast, optogenetic stimulation of C1 neurones activates inspiratory neurones, which secondarily inhibit expiratory neurones, but produced comparable increases in sympathetic activity across all phases of respiration. Our data reveal that the peripheral chemoreceptor-mediated expiratory-related sympathoexcitation is mediated through excitation of expiratory neurones antecedent to C1 pre-sympathetic neurones; these may be found in the Kölliker-Fuse nucleus. Despite peripheral chemoreceptor excitation of inspiratory neurones, these do not trigger C1 neurone-mediated increases in sympathetic activity. These studies provide compelling novel insights into the functional organization of respiratory-sympathetic neural networks.

Особливості цитокінової регуляції при мультирезистентному туберкульозі залежно від ступеня ендогенної інтоксикації

Актуальність. Туберкульоз (ТБ) відноситься до інтерлейкінзалежних імунодефіцитів із вираженими змінами в цитокіновій системі. Незважаючи на значні успіхи в досягненні контролю над ТБ, залишається невизначеною роль окремих цитокінів у формуванні резистентних його форм, не вирішені питання ідентифікації інтегральних маркерів прогресування патологічного процесу при мультирезистентному туберкульозі легень (МРТБ), а також відсутнє прогностичне визначення їх ролі в забезпеченні ефективності стандартних програм протитуберкульозної хіміотерапії. У зв’язку із вищезазначеним закономірно виникає необхідність аналізу показників окремих цитокінів у сироватці крові пацієнтів із МРТБ залежно від профілю резистентності, що може відображати ступінь вираженості місцевого та системного характеру специфічного запалення, особливості імуноцитокінової регуляції з метою удосконалення програми лікування. ТБ відноситься до захворювань, які супроводжуються ендогенною інтоксикацією, тобто накопиченням в організмі кінцевих та проміжних продуктів метаболізму при порушеному обміні внаслідок запальної реакції. Шляхами формування ендогенної інтоксикації при ТБ є цитотоксична гіпоксія, порушення внутрішньоклітинного гомеостазу, масивне утворення та подальша резорбція продуктів тканинного розпаду, імуноцитокіновий та гормональний дисбаланс тощо. Мета дослідження — оцінити показники ендогенної інтоксикації, встановити особливості цитокінової регуляції у хворих на мультирезистетний туберкульоз легень залежно від профілю резистентності мікобактерій та визначити їх роль у формуванні системної запальної реакції. Матеріали та методи. Об’єктом дослідження стали 116 пацієнтів із діагнозом вперше діагностованого туберкульозу легень із збереженою чутливістю до антимікобактеріальних препаратів; із МРТБ із діагностованою стійкістю як мінімум до двох препаратів I ряду; хворі на туберкульоз із розширеною резистентністю. Використано клінічні, рентгенологічні, біохімічні, мікроскопічні, мікробіологічні, імуноферментні та статистичні методи дослідження. Результати. Проведений кореляційний аналіз за Пірсоном між про- і протизапальними цитокінами показав, що у хворих на МРТБ коефіцієнт кореляції негативний, слабкої сили між показниками інтерлейкіну (ІЛ)-6 та ІЛ-10, між ІЛ-18 та ІЛ-10 (–0,22, р < 0,001; –0,16, р < 0,001). Продукція ІЛ-6 та ІЛ-10 у групах хворих на ТБ незалежно від резистентності зростає у відповідь на підвищення синтезу ендотоксинів мікобактерій туберкульозу, наростання ендогенної інтоксикації та цитотоксичної гіпоксії створює передумови для розвитку їх резистентності. Дисбаланс співвідношення ІЛ-18 та ІЛ-10 у цих хворих характеризує збільшення ступеня тяжкості стану пацієнта, поширеність запального процесу в легенях і формування резистентності; відзначається істотна перевага лімфоцитів Тх 2-го типу (гуморального), що вказує на розвиток дефекту клітинно-опосередкованого імунного захисту та перевагу неефективної протизапальної імунної активації. Висновки. Комплексна оцінка показників інтегральних індексів ендогенної інтоксикації та рівня окремих про- та протизапальних цитокінів у плазмі крові хворих на МРТБ свідчить про помірно виражену ендогенну інтоксикацію, виснажені клітинні ланки імунної реактивності за рахунок формування умов розвитку резистентності мікобактерій туберкульозу із подальшим наростанням цитотоксичної гіпоксії та активації синдрому системної запальної відповіді. Аналіз вмісту ІЛ-6, ІЛ-10 і ІЛ-18 у плазмі крові хворих із мультирезистентністю довів, що їх рівень залежить від характеру резистентності мікобактерій туберкульозу.

Etiopathogenesis of Central Nervous System Gliomas

There is much evidence that gliomas are caused by progressive ischemia, followed by increasing and prolonged hypoxia very close to the subventricular zone. In this way, hypoxia on immature glial cells is the primary cause in gliomas genesis. The ischemic process caused by atherosclerosis, anatomical variants of the arteries in the affected area and associated with carcinogenic agents are the harmful factors. While cytotoxic hypoxia triggers a reactive oxygen species (ROS) overproduction in the glial progenitor cells. Consequently, cellular impairment caused by ROS alters the cell cycle phases and provokes genesis of low-grade glioma. Subsequently, its progression causes angiogenesis and higher-grade cancer cells. Then, the vascular recanalization through aspirin is the indicated therapy to reduce the ischemic process by increasing blood flow and oxygen in the affected zone. Therefore, we must fight against the genesis of atherosclerosis and thus decrease the incidence of “neurodegenerative” diseases and cancer.

Chemoreflex and baroreflex alterations in Parkinsonism induced by 6-OHDA in unanesthetized rats

Parkinson’s disease (PD) is mainly characterized by motor signals. However, non-motor signals also affect and decrease the quality of life of PD patients. Among these non-motor signs are cardiovascular disorders as orthostatic hypotension, postprandial hypotension and cardiac arrhythmias, which may be due to the involvement of both central nervous system and peripheral autonomic nervous system. In the present study we investigated the cardiovascular function, evaluating cardiovascular reflexes (chemoreflex and baroreflex), in an animal model of Parkinsonism induced by bilateral infusion of the toxin 6-hydroxydopamine (6-OHDA), in the substantia nigra pars compacta (SNpc). The results showed that the animals induced to Parkinsonism had lower arterial pressure (AP) and heart rate HR) compared to control animals. We showed that after activation of the baroreceptors by phenylephrine (Phe) and sodium nitroprusside (SNP), the baroreflex sensitivity index was not changed between the groups. However, there was a greater increase in the AP when stimulated with Phe and greater tachycardia when stimulated with SNP in 6-OHDA animals. After activation of the peripheral chemoreceptors through KCN injection (cytotoxic hypoxia), there was a higher increase in pressor and bradycardic response in injured animals with bilateral 6-OHDA. These changes in the cardiovascular reflexes may be important adjustments mechanisms to maintain the cerebral blood flow in those animals, and may be a result of denervation supersensitivity to catecholamines in autonomic targets.

Evidence of a suffocation alarm system sensitive to clinically-effective treatments with the panicolytics clonazepam and fluoxetine.

Dyspnea, 'hunger for air', and the urge to flee are the cardinal symptoms of respiratory-type panic attacks. Patients also show baseline respiratory abnormalities and a higher rate of comorbid and antecedent respiratory diseases. Panic attacks are also precipitated by both the infusion of 0.5 M sodium lactate and the inhalation of 5-7% carbon dioxide (CO2) in predisposed patients, but not in healthy volunteers nor patients without panic disorder. Further studies show that patients with panic are also hyper-responsive to hypoxia. These and other observations led Klein (1993) to suggest that clinical panic is the misfiring of a suffocation alarm system. In rats, cytotoxic hypoxia of chemoreceptor cells by intravenous injection of potassium cyanide (KCN) produces short-lasting flight behaviors reminiscent of panic attacks. KCN-induced flight behaviors are blocked both by denervation of chemoreceptor cells and lesion of dorsal periaqueductal gray matter, a likely substrate of panic. Herein, we show that KCN-evoked flight behaviors are also attenuated by both acute and chronic treatment with clonazepam (0.01-0.3 mg/kg, intraperitoneally (i.p.)) and fluoxetine (1-4 mg/kg/day, i.p. for 21 days), respectively. Attenuation of KCN-evoked panic-like behaviors by clinically-effective treatment with panicolytics adds fresh evidence to the false suffocation alarm theory of panic disorder.

Effect of sodium cyanide on the activities of some oxidative enzymes and metabolites in Clarias gariepinus

-1 ) of sodium cyanide. Fish showed a gradual decrease in respiratory rate, increase in LDH and decrease in SDH levels, and lactic acid followed a similar trend with pyruvate in an eight-day trial. The changes in the levels of these enzyme activities may be as a result of impaired oxidative metabolism and cellular damage, which had effect on the release of these enzymes. Elevation in the level of lactic acid and decrease in pyruvic acid was due to shift from aerobic to anaerobic metabolism which resulted to a severe drop in the respiratory rate of the fish. It may be as a result of blockage of electron transfer from cytochrome-c oxidase to molecular oxygen which might lead to cellular hypoxia even in the presence of normal oxygenation of hemoglobin. Thus, inhibition of oxidative metabolism by sodium cyanide in C. garipenus was reconfirmed. Behavioral changes caused by sodium cyanide exposure in the fish were probably due to the combination of lactate acidosis with cytotoxic hypoxia, which might depress the central nervous system.

Sodium cyanide-induced modulations in the activities of some oxidative enzymes and metabolites in the fingerlings of Cyprinus carpio (Linnaeus)

Carp fingerlings exposed to a sublethal concentration (0.5 mg L−1) of sodium cyanide showed a steady decrement over a 7-day period in respiratory rate, rise in lactate dehydrogenase (LDH), and fall in succinate dehydrogenase (SDH) activities followed by variations in lactic and pyruvate levels. Changes in these enzyme activities might be due to impaired oxidative metabolism and severe cellular damage leading to the release of these enzymes. Decline in the activities of SDH and LDH clearly represents a shift from aerobic to anaerobic metabolism as evidenced by elevated lactate and decline in pyruvate levels. The shift to anaerobic metabolism is also reflected by severe drop in the respiratory rate of the fish. This may be a consequence of the blockage of electron transfer from cytochrome c oxidase to molecular oxygen, thus ceasing cellular respiration and it can lead to cellular hypoxia even in the presence of normal hemoglobin oxygenation. Hence, we indirectly reconfirm the inhibition of oxidative metabolism by sodium cyanide. Alterations in behavioral pattern induced by sublethal sodium cyanide exposure may be due to the combination of cytotoxic hypoxia with lactate acidosis, which depresses the central nervous system (CNS); as the brain is the most sensitive site to anoxia, it results in impaired CNS function.

Effect of Hyperbaric Oxygen Therapy on whole blood cyanide concentrations in carbon monoxide intoxicated patients from fire accidents

BackgroundHydrogen cyanide (HCN) and carbon monoxide (CO) may be important components of smoke from fire accidents. Accordingly, patients admitted to hospital from fire accidents may have been exposed to both HCN and CO. Cyanide (CN) intoxication results in cytotoxic hypoxia leading to organ dysfunction and possibly death. While several reports support the use of hyperbaric oxygen therapy (HBO) for the treatment of severe CO poisoning, limited data exist on the effect of HBO during CN poisoning. HBO increases the elimination rate of CO haemoglobin in proportion to the increased oxygen partial pressure and animal experiments have shown that in rats exposed to CN intoxication, HBO can increase the concentration of CN in whole blood.ObjectiveThe purpose of the present study was to determine whole blood CN concentrations in fire victims before and after HBO treatment.Materials and methodsThe patients included were those admitted to the hospital because of CO intoxication, either as fire victims with smoke inhalation injuries or from other exposures to CO. In thirty-seven of these patients we measured CN concentrations in blood samples, using a Conway/microdiffusion technique, before and after HBO. The blood samples consisted of the remaining 2 mL from the arterial blood gas analysis. CN concentration in blood from fire victims was compared to 12 patients from non-fire accidents but otherwise also exposed to CO intoxication.ResultsThe mean WB-CN concentration before patients received HBO did not differ significantly between the two groups of patients (p = 0.42). The difference between WB-CN before and after HBO did not differ significantly between the two groups of patients (p = 0.7). Lactate in plasma before and after did not differ significantly between the two groups of patients. Twelve of the 25 fire patients and one of the non-fire patients had been given a dose of hydroxycobalamin before HBO.Discussion and ConclusionCN concentrations in blood from patients admitted to hospital with CO intoxication and smoke inhalation exposure did not differ significantly from controls. Accordingly, we were not able to detect any changes in CN concentrations in blood after treatment with HBO.Trial RegistrationClinicalTrials.gov identifier: NCT00280579

Respiratory distress and behavioral changes induced by sodium cyanide in the fresh water teleost, Cyprinus carpio (Linnaeus)

An acute toxicity (LC50) test using a static renewal bioassay method was conducted to determine the toxicity of sodium cyanide in freshwater exotic carp, Cyprinus carpio exposed for 96 h to different concentrations of sodium cyanide. The acute toxicity value was found to be 1 mg/L; one third of the LC50 (0.33 mg/L) was selected as the sublethal concentration for subacute studies. Behavioral patterns were observed in lethal (1, 2, 3, and 4 d) and sublethal concentrations (1, 5, 10, and 15 d). Cyprinus carpio in toxic media exhibited irregular, erratic, and darting swimming movements, hyperexcitability, loss of equilibrium, and sinking to the bottom, which might be due to inhibition of cytochrome c oxidase activity and decreased blood pH. The combination of cytotoxic hypoxia with lactate acidosis depresses the central nervous system and myocardium, the most sensitive critical sites for anoxia, resulting in respiratory arrest and death. A decrease in oxygen consumption (-28.36 to -78.28%; -14.22 to -47.25%) was observed at both lethal and sublethal concentrations of sodium cyanide respectively. Fish at the sublethal concentration were found under stress, but that was not fatal.

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia by D'Agostino D, Mazza E, and Neubauer JA

the effect of hypoxia to stimulate breathing and the cardiovascular system has been well known for more than a century (for a review, see Ref. [11][1]). In 1938, Heymans was awarded the Nobel Prize for Physiology or Medicine for demonstrating the importance of carotid body chemoreceptors. However,

N-PEP-12 – a novel peptide compound that protects cortical neurons in culture against different age and disease associated lesions

The neuroprotective potency of N-PEP-12, a novel, proprietary compound consisting of biopeptides and amino acids was investigated. Lesion models have been applied in neuronal cultures of embryonic chicken cortex, pre-treated with N-PEP-12 from the first day onwards. On day 8 in vitro neurons were lesioned and cell viability was measured 24 and 48 hours later. To simulate acute brain ischemia, cytotoxic hypoxia was induced by sodium cyanide or by iodoacetate and excitotoxicity by L-glutamate. Ionomycin for up to 48 hours induced calcium overload. The cytoskeleton was disrupted by addition of colchicine. N-PEP-12 shows dose-dependent neuroprotection in all different models. The effect size depends on the recovery time but also on the extent of the lesion. In cases of mild to moderate lesion pronounced dose-dependent effects could be demonstrated. This indicates that chronic exposure to N-PEP-12 is able to prevent neuronal cell death associated to conditions occurring during normal aging and neurological disorders like ischemic stroke, hypoxia, brain trauma, or AD.

Methylmercaptan Poisoning: Usefulness Of Monitoring Jugular Venous Oxygenation

A 47-year-old man admitted for a confused state and severe metabolic acidosis shortly after exposure to methylmercaptan. To treat methylmercaptan-induced cytotoxic hypoxia and resultant confusion status, mechanical ventilation was performed to maintain an adequate oxygen supply to organs, especially to the brain. Sodium nitrite (3%, total 40ml), which caused methemoglobinemia (maximally 22%), was given intravenously to remove methylmercaptan from the cytochrome oxidase complex. Oxygen content in arterial blood was maintained at over 15ml/dl throughout the time course in our intensive care unit. Jugular venous oxyhemoglobin (Hbo2) decreased to 60%; Sjvo2 (oxygen saturation in the jugular venous blood) was maintained at over 75%. His level of consciousness improved on the next day. The treatment for methylmercaptan poisoning is similar to that for hydrogen sulfide poisoning. Excessive sodium nitrite treatment might cause hypoxic hypoxia due to induced methemoglobinemia. Jugular venous Hbo2 may indicate the adequate degree of induced methemoglobinemia at a point of the balance between oxygen delivery and consumption in the brain.

Cardiovascular responses to chemoreflex activation with potassium cyanide or hypoxic hypoxia in awake rats

Although intravenous (iv) injection of potassium cyanide (KCN) activates the arterial chemoreflex, it has been questioned whether cytotoxic hypoxia reproduces a physiological stimulus such as hypoxic hypoxia (low inspired O 2 tension). Thus, the goal of the present study was to compare the cardiovascular responses elicited by intravenous injection of KCN to those caused by hypoxic hypoxia in awake rats before and after bilateral ligature of carotid body arteries. We tested the hypothesis that hypoxic hypoxia activates the cardiovascular chemoreflex just as KCN does, causing an increase in arterial pressure and bradycardia. Intact adult Wistar rats received an intravenous injection of KCN (160 μg/kg) and were exposed to hypoxic hypoxia (7–5% O 2 breathing) for 10–15 s at random while mean arterial pressure (MAP) and heart rate (HR) were measured. After the experiments, the animals were submitted to bilateral ligature of carotid body arteries or sham operation and the protocol was repeated on the subsequent day. Before surgery, all rats showed an abrupt rise in arterial pressure accompanied by a marked bradycardia in response to KCN or hypoxic hypoxia, with a very similar pattern. After surgery, these responses persisted only in the sham-operated group and were totally abolished in the ligature group. In conclusion, our data show that KCN is an appropriate stimulus to activate arterial chemoreflex because its cardiovascular responses are comparable to those induced by hypoxic hypoxia. Thus, the use of KCN as a tool to evaluate different aspects of the complex pattern of cardiovascular, respiratory, and behavioural responses to chemoreflex activation seems to be physiologically acceptable.

In vitro models of brain ischemia: the peptidergic drug cerebrolysin protects cultured chick cortical neurons from cell death.

Glutamate (1 mM), iodoacetate (0.01 mM) and ionomycin (0.25 micro M) are reported to induce several characteristics of ischemia and neuronal degeneration in vitro, e.g. glutamate and ionomycin lesion result in a disturbance of Ca(2+) homeostasis, iodoacetate impairment leads to an inhibition of energy metabolism, suppression of protein synthesis and generation of oxygen free radicals. In this study these three lesion models were used to investigate the effects of the nootropic drug Cerebrolysin (Cere) on the survival of cortical neurons in culture and on the occurrence of apoptosis. The viability of the cells was evaluated with the colorimetric MTT-reduction assay. Apoptosis was detected with Bisbenzimide (Hoechst:33258), a fluorescent DNA stain. Administration of Cere resulted in dose dependent neuroprotection independent from the kind of lesion. In the glutamate model the drug almost doubled neuronal viability compared to lesioned controls. After acute glutamate exposure Cere reduced the number of apoptotic cells significantly. In spite of the protective efficacy after cytotoxic hypoxia induced by iodoacetate, the drug significantly increased the number of apoptotic neurons, indicating a shift from necrosis to apoptosis. In contrast to previous studies investigating acute ionomycin lesions, the chronic Ca(2+)-overload used here did not increase the abundance of apoptosis compared to the unlesioned control. Summarizing the findings it can be suggested that Cere is able to stabilize Ca(2+) homeostasis, to protect protein synthesis and to counteract neuronal death in different in vitro medels of ischemia.

Effects of NaCN and ionomycin on neuronal viability and on the abundance of microtubule-associated proteins MAP1, MAP2, and tau in isolated chick cortical neurons.

The effects of two toxins, sodium cyanide (NaCN) and ionomycin (IM), on neuronal viability and on the expression of the microtubule-associated proteins MAP1, MAP2, and tau were studied in isolated chick cortical neurons. Cytotoxic hypoxia due to NaCN treatment was performed to mimic acute neuronal damage, whereas long-term IM treatment was used as a model for chronic neuronal impairment. After 5 days in vitro, a cytotoxic lesion was induced either by addition of NaCN (0.01-10 mM) or IM (0.01-10 microM). The NaCN solution was aspirated after 30 min and cells were allowed to regenerate for 6 h, 24 h, 48 h, or 72 h; whereas the permanent IM lesions were left undisturbed during the same periods of time. Neuronal viability was assessed by MTT assay. The abundance of MAP1, MAP2, and tau was evaluated by immunoblotting and, for MAP2, by immunohistochemistry also. Results showed that NaCN and IM lesions dose-dependently decreased viability. Irreversible cell damage occurred after impairment with 10 mM NaCN and 1 microm or 10 microm IM, while neurons lesioned with lower concentrations regenerated partially or adapted to the toxic environment. However, the same level of viability as of untreated cells was never reached. Furthermore abundance of MAPs was changed after both lesions. But while after extended recovery from NaCN lesion protein expression was normalizing (MAP2) or at least still detectable (MAP1A, tau), the consequences of a permanent IM lesion were more severe, since neurons were not able to maintain or even restore their MAP expression. Immunohistochemical experiments for MAP2 revealed that, compared with controls, NaCN and, to a much higher extent, IM treatment resulted in a loss of immunoreactivity in neurites due to progressing cell death.

DIFFERENT RESPONSE OF CEREBRAL AND NON-CEREBRAL ENDOTHELIAL CELLS TO CYTOTOXIC HYPOXIA

The present study investigated the effect of cytotoxic hypoxia on cerebral and non-cerebral endothelial cells. Hypoxia was induced by inhibiting the cellular respiratory chain with 1 mM sodium cyanide. Cerebral endothelial cells were damaged after 2 h of hypoxia as assessed by a decrease in cell viability by 25% and by a 2.7-fold higher lactate dehydrogenase release compared to controls. Additional glucose deprivation did not significantly exacerbate hypoxic injury. In addition, we found after 2 h of hypoxia an increase in the release of lactate of 1.02 and 0.42 mg/mg protein compared to 0.27 and 0.07 mg/mg protein in controls in the presence and absence of glucose, respectively. While the activity of ALP of cerebral endothelial cells was maintained at the control level, we found a significant decrease in the γ-GT activity from 3.8 ± 1.3 to 1.09 ± 0.3 U/mg protein after 3 h of hypoxia in the presence as well as in the absence of glucose. The paracellular permeability of the cell monolayer decreased after 1 h and returned to control level after 3 h of hypoxia in the presence of glucose. Non-cerebral endothelial cells remained 98% viable with no change in the release of lactate dehydrogenase and lactate after 2 h of hypoxia in the presence and absence of glucose. The activities of ALP and γ-GT in non-cerebral endothelial cells were 10 and 3 times lower and remained unchanged during hypoxia. We conclude from our experiments that sodium cyanide is useful to study hypoxic injury and that cerebral endothelial cells are more sensitive than non-cerebral endothelial cells to cytotoxic hypoxia. © 1997 Elsevier Science Ltd

Determination of intracellular Ca 2+ concentration can be a useful tool to predict neuronal damage and neuroprotective properties of drugs

The purpose of the present study was to examine the relationship between elevation in intracellular Ca 2+ concentration ([Ca 2+] i) and development of neuronal damage after cytotoxic hypoxia in vitro. Chick telencephalic neurons were exposed to NaCN 1 mM for up to 2 h. [Ca 2+] i was assessed by means of fura-2 based microfluorometry and viability was measured by means of trypan blue exclusion on the same relocated cells for a period of 24 h after initiation of hypoxia. Exposure to sodium cyanide resulted in an up to 10 fold increase in [Ca 2+] i and led to subsequent neuronal damage. According to [Ca 2+] i and viability neurons in four different stages could be revealed. The percentage of neurons showing elevated [Ca 2+] i paralleled exactly the percentage of neuronal damage. The elevation in [Ca 2+] i clearly preceded neuronal damage suggesting a time window for pharmacological intervention. The NMDA antagonists dizocilpine, memantine and amantadine were capable of reducing the percentage of neurons showing elevated [Ca 2+] i and attenuated neuronal damage. Dizocilpine proved to be the most potent and amantadine to be the weakest antagonist whereas the α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline (NBQX) was ineffective. Under our experimental conditions, measurement of [Ca 2+] i was able to predict the extent of neuronal damage as well as the neuroprotective potency of drugs.

Effects of dopaminergic blockade upon carotid chemosensory activity and its hypoxia-induced excitation

The effects of domperidone, antagonist of D 2 receptors, on arterial chemoreceptor activity were studied in spontaneously breathing and pentobarbitone anesthetized cats, in which recordings of chemosensory impulse activity were obtained simultaneously from both cut carotid (sinus) nerves. Intravenous injections of domperidone 50 μg/kg produced a maintained increase in the basal frequency of chemosensory discharges, after which hyperoxic tests (breathing 100% O 2 for 30 s) evoked larger falls in the rate of chemosensory impulses. Chemosensory responses evoked by hypoxic hypoxia (100% N 2 tests) and by cytotoxic hypoxia (i.v. injections of NaCN) reached higher impulse rates after domperidone treatment. The effects of domperidone reveal that a resting release of dopamine from glomus cells maintains a low level of basal chemosensory activity under normoxic conditions. Domperidone turns off such restraining dopaminergic control and enhances the transient chemosensory responses to hypoxic stimuli. Present data support a modulatory role for dopamine within the chemoreceptor process, but not its participation as excitatory transmitter between glomus cells and sensory nerve endings.

Protective effects of 5-HT1A receptor agonists against neuronal damage demonstrated in vivo and in vitro.

The aim of the present study was to evaluate the neuroprotective effect of the 5-hydroxytryptamine1A (5-HT1A) agonists, CM 57493 and urapidil, in vivo and in vitro, respectively. In vivo permanent occlusion of the middle cerebral artery (MCA) was performed in male Wistar rats. Forty-eight hours after electrocoagulation of the MCA the infarct volume was determined. Pretreatment of the rat with the 5-HT1A agonist urapidil significantly reduced infarct development. The neuroprotective effect of the agent was restricted to the cortical area; the striatal damage was not influenced. As the stimulation of the 5-HT1A receptor by serotonin is supposed to induce inhibitory, hyperpolarizing effects by opening of a Ca(2+)-independent neuronal K+ ionophore, the efficacy of agonistic drugs directly on the neuron was investigated in vitro. Cyanide-induced cytotoxic hypoxia as well as glutamate-induced excitotoxicity were performed using primary neuronal cell cultures from chick embryo cerebral hemispheres. Treatment with the 5-HT1A agonists urapidil and CM 57493 significantly increased protein content of hypoxic cultures. CM 57493 added to the culture medium (1-10 microM) during and up to 24 h after glutamate exposure ameliorated viability of the neurons. The results demonstrate neuroprotective potency of the 5-HT1A agonists, urapidil and CM 57493, when applied under hypoxic, excitotoxic and ischemic conditions in vivo and in vitro, respectively. Both, presynaptically induced inhibition of glutamate release as well as postsynaptically induced inhibition of neuronal excitability could be discussed as possible mechanisms of action of the 5-HT1A receptor agonism.