Tenecteplase in the treatment of acute pulmonary thrombo-embolism.

This is a retrospective study documenting the use of tenecteplase in 41 cases of suspected or confirmed pulmonary embolism receiving in-hospital tenecteplase as per weight-adjusted dosing in addition to standard heparin and oral anticoagulant therapy. The presenting symptoms of dyspnoea, chest pain, hemoptysis and syncope were found in 40 (97.56%), 19 (46.34%), 6 (14.63%) and 9 (21.95%) patients, respectively. There was one case of mortality who was a 26 yrs old female of postpartum pulmonary thrombo-embolism with severe hypotension, cyanosis, bilateral crepitations in lungs and pulmonary hypertension. In the 40 survived patients, there was alleviation of dyspnoea and hemoptysis in all patients. Significant reduction in tachycardia (P < 0.0001) and increase in the oxygen saturation (SaO2) (P < 0.0001) were seen at discharge as compared to at the time of presentation. Eighteen patients had hypotension which recovered in all patients till the time of discharge (P < 0.0001). There was a significant reduction in right ventricular systolic pressure in all 18 patients who underwent 2-D echocardiography both before and after the tenecteplase therapy. Resolution of pulmonary embolism on CT pulmonary angiography was documented in only two patients. No bleeding events or any other adverse events were reported during this study. The present study suggests favourable efficacy of tenecteplase in patients with suspected or confirmed acute pulmonary embolism. Although no major adverse events were noted, a large prospective study on the use of tenecteplase in pulmonary embolism is suggested.

Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction.

Increased expression of heat shock protein 70 (HSP70) in the brain has been extensively documented in association with a variety of insults, including ischemia, and is suggested to play a role in cell survival and recovery after ischemic injury. To more directly assess the protective role of HSP70 during ischemic brain damage, we used transgenic mice overexpressing the rat HSP70 (HSP70tg mice). In contrast to wild-type (wt) littermates, high levels of HSP70 messenger RNA and protein were detected in brains of HSP70tg mice under normal conditions, and immunohistochemical analysis revealed primarily neuronal expression of HSP70. Heterozygous HSP70tg mice and their wt littermates were subjected to permanent focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Cerebral infarction after 6 hours of ischemia, as evaluated by Nissl staining, was significantly less in HSP70tg mice compared with wt mice. This reduction in infarction volume in HSP70tg mice was not attributable to an altered cardiovascular anatomy or to initial differences in body temperature or hemodynamic parameters. The HSP70tg mice were still protected against cerebral infarction 24 hours after permanent focal ischemia. The data suggest that HSP70 can markedly protect the brain against ischemic damage and that approaches aimed at inducing HSP70 may lead to new therapeutic interventions in cerebrovascular injuries.

Fluoxetine prevents PCP- and MK801-induced HSP70 expression in injured limbic cortical neurons of rats.

BACKGROUND: N-Methyl-D-aspartate (NMDA) receptor antagonists, including phencyclidine (PCP) and dizocilpine (MK801), cause schizophrenialike psychosis in humans, and produce vacuolated neurons in the cingulate and retrosplenial cortices of the rat brain. Since psychotically depressed patients and schizophrenic depressed patients may require treatment with selective serotonin reuptake inhibitors (SSRIs), it is of interest to examine the relationship between SSRIs and NMDA antagonist neurotoxicity. METHODS: The neurotoxicity of PCP and MK801 was assessed using heat shock protein (HSP70) immunocytochemistry and HSP70 Western blots because HSP70 is expressed in the injured, vacuolated neurons. Female rats were given fluoxetine (0, 5, 10, and 20 mg/kg IP) followed 1 hour later by MK801 (1 mg/kg IP) or PCP (50 mg/kg IP). RESULTS: Pretreatment with fluoxetine (20 mg/kg IP) 1 hour before MK801 prevented the induction of HSP70 by MK801 in the cingulate and retrosplenial cortices. Pretreatment with fluoxetine (10 or 20 mg/kg IP) 1 hour before PCP also prevented the HSP70 induction by PCP. CONCLUSIONS: Fluoxetine prevents the neurotoxicity of NMDA receptor antagonists in rat brain. This suggests the possibility that SSRIs could modulate psychosis, and may provide a model for examining the link between the hallucinogenic properties of PCP and lysergic acid diethylamide.

Stress proteins as molecular markers of neurotoxicity.

In response to many environmental and pathophysiologic stressful stimuli, cells undergo a stress response characterized by induction of a variety of proteins, including the heat shock protein family. The inducible heat shock protein 70 (hsp70) is believed to participate in an array of cellular activities, including cytoprotection. Normal brain cells have little detectable hsp70 RNA or protein. However, following a stressful condition hsp70 mRNA and protein are induced in different cell types depending on the severity and the nature of the stimulus. The induction of hsp70 protein correlates with the regional and cellular vulnerability to a particular injury as identified by standard histologic methods. The pattern of hsp70 expression differs in response to various neurotoxic stimuli, including hyperthermia, ischemia, seizures, hemorrhage, and N-methyl-D-aspartate receptor antagonist administration. Hsp70 expression is a useful marker of cellular injury and may help to identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus. Hsp70 may also play a neuroprotective role in the brain.

Acute phencyclidine neurotoxicity in rat forebrain: induction of haem oxygenase-1 and attenuation by the antioxidant dimethylthiourea.

Phencyclidine and other N-methyl-D-aspartate receptor antagonists are toxic to pyramidal neurons in the posterior cingulate/retrosplenial cortex of rat brain. Previous studies have shown induction of heat shock protein 70 in affected neurons. In this study, expression of haem oxygenase-1, a heat shock protein induced by oxidative stress, was examined in rat forebrain after administration of a single intraperitoneal dose of phencyclidine (50 mg/kg). Northern and Western blot analyses of brain tissue extracts from phencyclidine-treated rats revealed a marked induction of haem oxygenase-1 mRNA and protein, respectively. Immunohistochemistry studies revealed that phencyclidine increased haem oxygenase-1 immunoreactivity primarily in posterior cingulate/retrosplenial, piriform and entorhinal cortices, striatum and hippocampus. Haem oxygenase-1 protein was induced in non-neuronal cells, mainly astrocytes. Some microglia expressing haem oxygenase-1 protein were also found in the posterior cingulate/retrosplenial cortex. Haem oxygenase-1 immunoreactive astrocytes and microglia were present in close proximity to the heat shock protein 70-positive neurons in the posterior cingulate/retrosplenial cortex following phencyclidine. Pretreatment of rats with 1,3-dimethylthiourea, an antioxidant, significantly reduced haem oxygenase-1 protein induction by phencyclidine. Thus, induction of haem oxygenase-1 in glia by phencyclidine appears to be mediated mostly by oxidative stress. Experiments with the amino cupric silver stain for neuronal degeneration revealed phencyclidine-induced neurotoxicity in the posterior cingulate/retrosplenial cortex. The number of affected neurons was significantly reduced after 1,3-dimethylthiourea pretreatment. This suggests that the neurotoxicity of N-methyl-D-aspartate antagonists is due in part to the oxidative stress and may be amenable to therapeutic interventions.

NMDA receptor-mediated neurotoxicity: a paradoxical requirement for extracellular Mg2+ in Na+/Ca2+-free solutions in rat cortical neurons in vitro.

Accumulation of intracellular Ca2+ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2+ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2+ content independently of Ca2+ to investigate the potential role of Mg2+ in excitotoxicity in rat cortical neurons in vitro. In Ca2+-free solutions in which the Na+ was replaced by N-methyl-D-glucamine or Tris (but not choline), which also contained 9 mM Mg2+, exposure to 100 microM glutamate or 200 microM NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2+ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2+-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg2+ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.

Calcium influx but not pH or ATP level mediates glutamate-induced changes in intracellular magnesium in cortical neurons.

1. We have recently shown that glutamate increases [Mg2+]i in cultured rat cortical neurons. However, the mechanism of this increase in [Mg2+]i is not well understood. We used fluorescence microscopic methods to measure [Mg2+]i, [Ca2+]i, and pHi in single neurons. Intracellular ATP analysis was performed by high-performance liquid chromatography (HPLC). 2. A 25-mM NH4Cl pulse followed by Na(+)-free wash rapidly acidified the cytosol. In 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded neurons, the pHi was reduced by 2.46 units, and in magfura-2-loaded neurons the [Mg2+]i was increased by 0.62 mM. Five-minute treatment with 100 microM glutamate, on the other hand, reduced the cytosolic pH by 0.73 units and increased the [Mg2+]i by 7.24 mM in rat cortical neurons. These results indicate that change in pHi does not play a significant role in the glutamate-induced [Mg2+]i elevation. 3. The metabolic inhibition (5 mM KCN and 1 mM iodoacetate) for 30 min significantly reduced the intracellular ATP levels. However, 5-min treatment with 100 microM glutamate did not significantly deplete intracellular ATP in cultured cortical neurons. When tested under similar conditions in magfura-2-loaded neurons, glutamate increased [Mg2+]i to a significantly larger extent than metabolic inhibition. This suggests that ATP depletion and subsequent release of Mg2+ from Mg(2+)-ATP complex is not the primary source of [Mg2+]i elevation observed during glutamate stimulation. 4. To further study the role of glutamate-induced Ca2+ influx in subsequent [Mg2+]i elevation, extracellular Ca2+ was elevated from 1.4 to 3.0 mM during glutamate application in magfura-2-loaded neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclothiazide modulates AMPA receptor-mediated increases in intracellular free Ca2+ and Mg2+ in cultured neurons from rat brain.

We investigated the modulation of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca2+]i were increased by 0.3-100 microM cyclothiazide, with an EC50 value of 2.40 microM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N-methyl-D-aspartate (NMDA) receptor antagonists were also potentiated by 10 microM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na(+)-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 microM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.

Glutamate-induced intracellular calcium changes and neurotoxicity in cortical neurons in vitro: effect of chemical ischemia.

To study the role of calcium in neuronal death during ischemia, we examined the characteristics of intracellular Ca2+ ([Ca2+]i) changes in single rat forebrain neurons exposed for 5 min to glutamate (3 microM + 1 microM glycine), NMDA (30 microM + 1 microM glycine), kainate (100 microM) or high K+ (50 mM), under both normal and ischemic conditions. The parameters of [Ca2+]i change measured included peak [Ca2+]i level, plateau [Ca2+]i level, area under the [Ca2+]i response curve and time taken by [Ca2+]i to recover to 10% of the peak response. Under normal conditions, all the agonists studied produced similar [Ca2+]i changes. Chemical ischemia simulated by application of 5 mM KCN in glucose-free buffer had no effect on the basal level of [Ca2+]i, but significantly enhanced and prolonged the [Ca2+]i changes produced by all the agonists. However, in toxicity studies, chemical ischemia significantly potentiated the toxicity of only glutamate and N-methyl-D-aspartate. In correlation studies, all the neurons which died displayed an irreversible secondary [Ca2+]i load prior to loss of viability. These studies suggest that while Ca2+ entry may play a critical role in neuronal death, the magnitude of initial [Ca2+]i change does not predict the toxicity of an agonist in cortical neurons.

Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons.

We describe here the properties of calcium green-5N (CG5N), a novel Ca(2+)-sensitive fluorescent probe. CG5N binds Ca2+ with an affinity of 4.29 +/- 0.67 microM. Ca2+ binding is associated with a 14.7-fold increase in fluorescence. Mg2+ binds to CG5N with an affinity of 5 mM but produces a very small change in fluorescence. Using CG5N [Ca2+]i was recorded in single cultured neurons from fetal rat forebrain. Glutamate together with glycine increased [Ca2+]i by 1.17 +/- 0.15 microM. Pretreating cells with 5 mM potassium cyanide significantly potentiated the response to glutamate, resulting in a mean increase of 8.8 +/- 2 microM. These results demonstrate that CG5N is a useful indicator for measuring very large changes in [Ca2+]i in neurons.

Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons.

Intracellular free Mg2+ concentrations ([Mg2+]i) in single rat brain neurons were measured with the Mg(2+)-sensitive fluorescent dye magfura-2. Addition of glutamate with glycine raised [Mg2+]i from 1 to more than 11 mM compared with the resting concentration of 0.5 mM, an effect mediated by N-methyl-D-aspartate receptors. Most of the increase in [Mg2+]i was independent of extracellular Mg2+, but was dependent on extracellular Ca2+. The second component of the increase induced by glutamate was independent of extracellular Ca2+, but required extracellular Mg2+ and was amplified by extracellular Na+ removal. These results indicate that regulation of [Mg2+]i by neurotransmitters such as glutamate may be important in controlling neuronal excitability.

Effects of pH on the actions of dizocilpine at the N-methyl-D-aspartate receptor complex.

1. The effects of varying pH from 6.5 to 7.4 and 8.0 on the actions of dizocilpine (MK801) on the N-methyl-D-aspartate (NMDA) receptor were investigated by use of a [3H]-dizocilpine binding assay and NMDA-induced intracellular free Ca2+ ([Ca2+]i) increases in cultured forebrain neurones. 2. Increasing pH from 6.5 to 8.0 significantly increased the rate of association and dissociation of [3H]-dizocilpine. The association process was better described by two rate constants under each condition, while only dissociation of [3H]-dizocilpine at pH 8.0 required two rate constants adequately to describe the curve. Equilibrium affinity of [3H]-dizocilpine was not altered by changing pH from 6.5 to 8.0. 3. NMDA and glycine together increased [Ca2+]i measured by fura-2 microspectrofluorimetry in single cultured neurones from rat forebrain. Compared to control response measured at pH 7.4, the combined effects of NMDA and glycine were reduced to 38.9% of control values by lowering pH to 6.5 and increased to 148.9% by raising pH to 8.0. 4. Dizocilpine (200 nM) effectively reversed increases in [Ca2+]i produced by NMDA together with glycine. The rate of reversal produced by this concentration of dizocilpine was considerably slower than that required for cells to recover to baseline following agonist removal. The rate at which dizocilpine reversed NMDA- and glycine-induced [Ca2+]i increases was not altered by raising pH from 6.5 to 8.0. 5. These data support the hypothesis that the rates associated with [3H]-dizocilpine binding are controlled by the level of activation of the receptor. In addition, these data confirm previous findings that NMDA responses are sensitive to small changes in pH.However, the lack of sensitivity to pH changes of dizocilpine inhibition of NMDA responses show that other factors are also important in controlling the action of channel-blocking NMDA receptor antagonists.

[125I]thienylphencyclidine, a novel ligand for the NMDA receptor.

We have monitored the binding of [125I]thienylphencyclidine ([125I]TCP), a novel high affinity radioiodinated ligand that specifically recognizes the NMDA (N-methyl-D-aspartate) receptor in rat brain membranes. [125I]TCP binds with an affinity of about 30 nM, and recognizes a similar number of binding sites to previously employed ligands for this receptor. [125I]TCP binding is characterized by slow association and dissociation rates, and the latter can be modified by the addition of Mg2+ or Zn2+, as previously described for [3H]dizocilpine ([3H]MK801). Other phencyclidine-like ligands displaced [125I]TCP binding with the order of potency dizocilpine greater than thienylphencyclidine greater than ITCP greater than phencyclidine greater than ketamine. The binding of [125I]TCP was also increased by NMDA and glycine-site agonists and inhibited by antagonists of these sites. Surprisingly, however, the polyamines spermidine and spermine did not increase [125I]TCP, even though the polyamine antagonist arcaine was an effective inhibitor of binding. These results show that [125I]TCP is a useful ligand for the NMDA receptor complex that binds to the receptor in a manner that is qualitatively distinct from previously described ligands.

Effects of monovalent and divalent cations on 3-(+)[125I]iododizocilpine binding to the N-methyl-D-aspartate receptor of rat brain membranes.

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.

The effect of acute and chronic treatment of verapamil on pressor response to angiotensin II in rats.

The effect of verapamil on pressor response to Angiotensin II (A II) was investigated in rats. The responses to A II (10, 20, 40 ng, iv) was reduced after verapamil (50 and 100 micrograms, iv per rat) in a dose dependent manner. Treatment for 5 day with verapamil (18 mg/100 g/day, po) let to some reduction in basal blood pressure but pressor response to A II was not changed. Treatment for 20 days significantly reduced the basal blood pressure and increased the responsiveness to A II. it is concluded that Ca++ plays a major role in the pressor responses to A II and that chronic treatment with verapamil may reduce basal blood pressure.

Effect of Ca++ channel blockers and saralasin on angiotensin II induced contraction in rabbit aortic strip.

To investigate the role of calcium in angiotensin II (A II) induced contractions in rabbit aortic strip, the action of verapamil, nifedipine, cinnarizine and saralasin was studied. The cumulative dose response curves obtained with A II shifted to right with increasing concentrations of all these four agents. The antagonism was noncompetitive. The pD'2 value of saralasin was 8.49 of nifedipine, 8.15 and or verapamil 7.92. Cinnarizine which mainly acts at intracellular site had pD'2 value 5.54. The results indicate that A II induced contractions critically depend on entry of calcium through channels which appear to be closely associated with angiotensin receptors.