Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.     

Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: An objective of therapeutic intervention after cerebral ischemia is to promote improved functional outcome. Improved outcome may be associated with a reduction of the volume of cerebral infarction and the promotion of cerebral plasticity. In the developing brain, neuronal growth is concomitant with expression of particular proteins, including microtubule-associated protein 2 (MAP-2), growth-associated protein 43 (GAP-43), and cyclin D1. In the present study we measured the expression of select proteins associated with neurite damage and plasticity (MAP-2 and GAP-43) as well as cell cycle (cyclin D1) after induction of focal cerebral ischemia in the rat. METHODS: Brains from rats (n=28) subjected to 2 hours of middle cerebral artery occlusion and 6 hours, 12 hours, and 2, 7, 14, 21, and 28 days (n=4 per time point) of reperfusion and control sham-operated (n=3) and normal (n=2) rats were processed by immunohistochemistry with antibodies raised against MAP-2, GAP-43, and cyclin D1. Double staining of these proteins for cellular colocalization was also performed. RESULTS: Loss of immunoreactivity of both MAP-2 and GAP-43 was observed in most damaged neurons in the ischemic core. In contrast, MAP-2, GAP-43, and cyclin D1 were selectively increased in morphologically intact or altered neurons localized to the ischemic core at an early stage (eg, 6 hours) of reperfusion and in the boundary zone to the ischemic core (penumbra) during longer reperfusion times. CONCLUSIONS: The selective expressions of the neuronal structural proteins (MAP-2 in dendrites and GAP-43 in axons) and the cyclin D1 cell cycle protein in neurons observed in the boundary zone to the ischemic core are suggestive of compensatory and repair mechanisms in ischemia-damaged neurons after transient focal cerebral ischemia.     

Effects of ischemia on cerebral arteriolar dilation to arterial hypoxia in piglets

BACKGROUND AND PURPOSE: Arterial hypoxia mediates cerebral arteriolar dilation primarily via mechanisms involving activation of ATP-sensitive K+ channels (K[ATP]), which we have shown to be sensitive to ischemic stress. In this study, we determined whether ischemia/reperfusion alters cerebral arteriolar responses to arterial hypoxia in anesthetized piglets. Since adenosine plays an important role in cerebrovascular responses to hypoxia, we also determined whether adenosine-induced arteriolar dilation is affected by ischemic stress. We tested the hypothesis that reductions in cerebral arteriolar dilator responses after ischemia would be proportional to the contribution of K(ATP) to hypoxia and adenosine. METHODS: Pial arteriolar diameters were measured using a cranial window and intravital microscopy. We examined arteriolar responses to arterial hypoxia (inhalation of 8.5% and 7.5% O2), to topical adenosine (10[-5] and 10[-4] mol/L) and to arterial hypercapnia (inhalation of 5% and 10% CO2 in air) before and after 10 minutes of global ischemia. Ischemia was achieved by increasing intracranial pressure. Arterial hypercapnia was used as a positive control for the effectiveness of the ischemic insult. In addition, we evaluated cerebral arteriolar responses to 10(-5) and 10(-4) mol/L adenosine applied topically with or without glibenclamide, a selective inhibitor of K(ATP) (10[-5] and 10[-6] mol/L). Finally, we administered theophylline (20 mg/kg, i.v.) to assess the contribution of adenosine to cerebral arteriolar dilation to arterial hypoxia. RESULTS: Before ischemia, cerebral arterioles dilated by 19+/-3% to moderate and 29+/-4% to severe hypoxia (n=7; P<.05); 13+/-2% to 10(-5) and 20+/-1% to 10(-4) mol/L adenosine (n=9; P<.05); and by 17+/-2% to moderate and 28+/-3% to severe hypercapnia (n=6; P<.05). After ischemia, cerebral arteriolar responses to hypoxia and adenosine were unchanged. In contrast, cerebral arteriolar dilation to hypercapnia was impaired by ischemia (1+/-1% and 2+/-1% at 1 hour; n=6). Glibenclamide reduced cerebral arteriolar dilation to adenosine by approximately one half (n= 7). In addition, blockade of adenosine receptors by theophylline (20 mg/kg, i.v.) almost totally suppressed cerebral arteriolar dilation to arterial hypoxia (n = 6). CONCLUSIONS: Cerebrovascular responsiveness is selectively affected by anoxic stress. In addition, cerebral arteriolar dilation to hypoxia and adenosine is maintained after ischemia despite the expected impairment in K(ATP) function.     

Temporal characteristics of the protective effect of aminoguanidine on cerebral ischemic damage

We investigated the temporal profile of the reduction in focal cerebral ischemic damage exerted by aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS). In anesthetized spontaneously hypertensive rats, the middle cerebral artery (MCA) was occluded distal to the origin of the lenticulostriate arteries. Rats were treated with vehicle (saline) or AG (100 mg kg-1, i.p.) immediately after MCA occlusion and, thereafter, two times per day. Rats were sacrificed 1(n = 7), 2(n = 8), 3 (n = 6) or 4 days (n = 5) after MCA occlusion. Injury volume (mm3) was determined in thionin-stained sections using an image analyzer. Volumes were corrected for ischemic swelling. Administration of AG up to 2 days after MCA occlusion did not reduce cerebral ischemic damage (p < 0.05 from vehicle; t-test). Treatment for a longer period decreased injury volume, the reduction averaging 21 +/- 5% at 3 days (p < 0.05) and 30 +/- 9% at 4 days (p < 0.05). Aminoguanidine did not affect ischemic brain swelling (p > 0.05). Administration of AG did not substantially modify arterial pressure, arterial blood gases, pH, hematocrit, plasma glucose and rectal temperature. We conclude that the protective effect of AG is time-dependent and occurs only when the drug is administered for longer than 2 days, starting after induction of ischemia. Because iNOS enzymatic activity develops more than 24 h after MCA occlusion [C. Iadecola, X. Xu, F. Zhang, E.E. El-Fakahany, M.E. Ross, Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia, J. Cereb. Blood Flow, Metab. 14 (1995) 52-59; C. Iadecola, F. Zhang, X. Xu, R. Casey, M.E. Ross, Inducible nitric oxide synthase gene expression in brain following cerebral ischemia, J. Cereb. Blood Flow Metab. 15 (1995) 378-384.], the data support the hypothesis that the protective effect of AG is medicated by inhibition of iNOS in the post-ischemic brain.     

The early and late phases of ischemic preconditioning: a comparative analysis of their effects on infarct size, myocardial stunning, and arrhythmias in conscious pigs undergoing a 40-minute coronary occlusion

The effectiveness of the late phase of ischemic preconditioning (PC) in protecting against myocardial infarction and the concomitant contractile dysfunction after sustained ischemia remains unclear. The early and late phases of PC have not been compared using the same protocol in the same experimental model; furthermore, the late phase of PC has not been assessed in the conscious state in a large animal preparation. The goal of this study was to directly compare the effects of early and late PC on myocardial infarct size and postischemic dysfunction in chronically instrumented, conscious pigs. Four groups of pigs were subjected to a 40-minute coronary occlusion followed by 3 days of reperfusion. Group 1 (n=7) served as control. Group 2 (n=6) was subjected to ten 2-minute occlusion/2-minute reperfusion cycles 25 minutes before the 40-minute occlusion (early PC). Groups 3 (n=7) and 4 (n=4) were subjected to 10 and 25 cycles, respectively, of 2-minute occlusion/2-minute reperfusion 24 hours before the 40-minute occlusion (late PC). Infarct size averaged 45.1+/-5.9% of the region at risk in control pigs, was reduced by 79% (to 9.4+/-3.2%) in group 2, but did not differ in groups 3 (33.3+/-4.8%) and 4 (38.8+/-8.2%) versus group 1. Power analysis demonstrated that there was an 80% probability of detecting a 40% decrease in infarct size in groups 3 and 4 versus group 1. The recovery of systolic wall thickening (measured with ultrasonic crystals) after the 40-minute occlusion was poor in groups 1, 3, and 4 but markedly enhanced in group 2 throughout the 3 days of reperfusion; this beneficial effect could have been due to limitation of infarct size, alleviation of stunning, or both. Thus, a series of ten 2-minute coronary occlusions had a profound (approximately 80%) early infarct-limiting effect, which was associated with a marked functional benefit. This protection, however, disappeared 24 hours later and could not be reinstituted by increasing the number of PC coronary occlusions to 25. The incidence and duration of ventricular tachycardia after reperfusion was not changed by either early or late PC; no conclusions could be drawn regarding ventricular fibrillation or ischemia-induced ventricular tachycardia, since these arrhythmias did not occur in control animals. Taken together, the present results demonstrate striking differences between the early and late effects of PC: In conscious swine subjected to a sustained coronary occlusion, a PC protocol that induces powerful protection during the early phase of PC fails to induce any protection during the late phase, indicating either that a late protective effect of PC does not exist or that, if it exists, it must be weaker than the early protective effect.     

Ischemic preconditioning does not protect against contractile dysfunction in the presence of residual flow: studies in the isolated, blood-perfused rat heart

BACKGROUND: We have previously demonstrated that ischemic preconditioning (PC) does not protect when oxygen deprivation is accompanied by a high level of perfusion (hypoxia). Since clinical ischemia can vary from mild to severe, we wished to determine whether PC could protect against injury arising from low-flow ischemia. METHODS AND RESULTS: Functional recovery after 30 minutes of reperfusion was assessed in isolated, blood-perfused rat hearts (n=6 per group) subjected to (A) 30 minutes of zero-flow ischemia, (B) 30 minutes of zero-flow ischemia preceded by 3xPC (PC=5 minutes of ischemia+5 minutes of reperfusion), (C) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.31+/-0.02 mL/min per gram wet wt), (D) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.29+/-0.02 mL/min per gram wet wt) preceded by 3xPC. PC significantly protected against injury resulting from zero-flow ischemia (developed pressure recovered to 67+/-6% versus 31+/-12% in B and A, respectively; P<.05) but not resulting from low-flow ischemia (recovery of developed pressure was 40+/-8% versus 37+/-7% in C and D, respectively). Protein kinase C (PKC) is widely considered to be involved in the mechanism of PC such that prior activation and translocation of PKC by the PC protocol allows phosphorylation of the end-effector protein early during the subsequent ischemic insult, before loss of adenosine triphosphate occurs. However, because adenosine triphosphate content falls slowly during low-flow ischemia, PKC may be activated and translocated early enough to be active during this insult. If so, inhibition of PKC should decrease functional recovery in the control group. However, functional recovery in control groups was not decreased in the presence of the PKC inhibitor polymyxin B (50+/-6%), suggesting that if activation of PKC occurred during low-flow ischemia, it was not protective. CONCLUSIONS: PC does not protect against contractile dysfunction in the rat when a low level (10% of baseline flow) of ischemic perfusion remains during the prolonged insult.     

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.     

The relationship between rhombomeres and vestibular neuron populations as assessed in quail-chicken chimeras

The mRNA expression pattern for four different immediate early genes was examined dynamically in rat brain after administration of phencyclidine (PCP; 0.86 or 8.6 mg/kg) or MK801 (0.1 or 1.0 mg/kg). Following each treatment, the expression of cfos, cjun, junB, and zif268 mRNA changed distinctively and dynamically between 1 and 48 hours. cfos mRNA was induced in cortical areas at early times after either dose of PCP or of MK801; the change was especially prominent in cingulate and auditory cortices. zif268 mRNA showed an early (1 hour) activation and a delayed (24-48 hour) suppression after PCP and MK801 in neocortical areas. PCP also caused cjun and junB mRNA induction in cortical areas at early times, with a distribution and time course similar to its effects on cfos mRNA. No alterations in cfos, cjun, or junB mRNA were found in neocortical or hippocampal areas at any delayed time (>6 hours) after PCP treatment, whereas suppression of zif268 expression was prominent even at 48 hours post-treatment. CPP, a competitive NMDA antagonist, showed a similar pattern of effects on cfos and zif268 mRNA expression. These functional consequences of a PCP- or MK801-induced reduction in NMDA-sensitive glutamate transmission may be relevant to an understanding of animal NMDA pharmacology and/or to clinical psychotomimetic side effects of antiglutamatergic treatments.     

Nitric oxide scavenging by hemoglobin or nitric oxide synthase inhibition by N-nitro-L-arginine induces cortical spreading ischemia when K+ is increased in the subarachnoid space

We investigated the combined effect of increased brain topical K+ concentration and reduction of the nitric oxide (NO.) level caused by nitric oxide scavenging or nitric oxide synthase (NOS) inhibition on regional cerebral blood flow and subarachnoid direct current (DC) potential. Using thiopental-anesthetized male Wistar rats with a closed cranial window preparation, brain topical superfusion of a combination of the NO. scavenger hemoglobin (Hb; 2 mmol/L) and increased K+ concentration in the artificial cerebrospinal fluid ([K+]ACSF) at 35 mmol/L led to sudden spontaneous transient ischemic events with a decrease of CBF to 14+/-7% (n=4) compared with the baseline (100%). The ischemic events lasted for 53+/-17 minutes and were associated with a negative subarachnoid DC shift of -7.3+/-0.6 mV of 49+/-12 minutes' duration. The combination of the NOS inhibitor N-nitro-L-arginine (L-NA, 1 mmol/L) with [K+]ACSF at 35 mmol/L caused similar spontaneous transient ischemic events in 13 rats. When cortical spreading depression was induced by KCl at a 5-mm distance, a typical cortical spreading hyperemia (CSH) and negative DC shift were measured at the closed cranial window during brain topical superfusion with either physiologic artificial CSF (n=5), or artificial CSF containing increased [K+]ACSF at 20 mmol/L (n=4), [K+]ACSF at 3 mmol/L combined with L-NA (n=10), [K+]ACSF at 10 mmol/L combined with L-NA (five of six animals) or [K+]ACSF at 3 mmol/L combined with Hb (three of four animals). Cortical spreading depression induced longlasting transient ischemia instead of CSH, when brain was superfused with either [K+]ACSF at 20 mmol/L combined with Hb (CBF decrease to 20+/-20% duration 25+/-21 minutes, n=4), or [K+]ACSF at 20 mmol/L combined with L-NA (n=19). Transient ischemia induced by NOS inhibition and [K],ACSF at 20 mmol/L propagated at a speed of 3.4+/-0.6 mm/min, indicating cortical spreading ischemia (CSI). Although CSH did not change oxygen free radical production, as measured on-line by in vivo lucigenin-enhanced chemiluminescence, CSI resulted in the typical radical production pattern of ischemia and reperfusion suggestive of brain damage (n=4). Nimodipine (2 microg/kg body weight/min intravenously) transformed CSI back to CSH (n=4). Vehicle had no effect on CSI (n=4). Our data suggest that the combination of decreased NO. levels and increased subarachnoid K+ levels induces spreading depression with acute ischemic CBF response. Thus, a disturbed coupling of metabolism and CBF can cause ischemia. We speculate that CSI may be related to delayed ischemic deficits after subarachnoid hemorrhage, a clinical condition in which the release of Hb and K+ from erythrocytes creates a microenvironment similar to the one investigated here.     

Incomplete infarct and delayed neuronal death after transient middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: The clinical syndrome of transient ischemic attacks is accompanied in a significant percentage of patients by brain lesions or neuroimaging abnormalities whose structural counterparts have not been defined. The objective of this study was to analyze, in an experimental model of short-term (< 25 minutes) focal ischemia and long-term (< or = 28 days) reperfusion, the extent and nature of the structural abnormalities affecting neurons and glia located within the territory of the transiently occluded artery. METHODS: Adult Wistar rats (n = 121) had the origin of one middle cerebral artery (MCA) occluded with a nylon monofilament for periods of 10 to 25 minutes. Experiments of transient MCA occlusion were terminated at variable periods ranging from 1 day to 4 weeks. Control experiments consisted of (1) MCA occlusion without reperfusion (n = 7) lasting 7 to 14 days and (2) sham operations (n = 2) followed by 1- to 4-day survival. After in situ fixation, brain specimens were serially sectioned and subjected to detailed morphometric evaluations utilizing light and electron microscopes. The statistical method used to evaluate the results was based on ANOVA followed by Bonferroni's corrected t test and Student's t test comparisons. RESULTS: Brain lesions were not detectable in the sham-operated controls. All brains with permanent MCA occlusion (7 to 14 days) had large infarctions with abundant macrophage infiltration and early cavitation. Forty-five (37%) of the experiments involving transient MCA occlusion had no detectable brain lesions after 4 weeks. Selective neuronal necrosis was found in 76 of 121 rats (63%) with transient MCA occlusion. Neuronal necrosis always involved the striatum, and in 29% of the brains with ischemic injury, necrosis also included a short segment of the cortex. In the striatum, the length of the arterial occlusion was the main determinant of the number of necrotic neurons (20 minutes [22.6 +/- 19] is worse than 10 minutes [4.9 +/- 7]) (P < .0001). In the cortex, the length of reperfusion determined the number of necrotic neurons appearing in layer 3. Experiments with reperfusion of 4 to 7 days' duration yielded more necrotic neurons per microscopic field (2.02 +/- 3) than those lasting fewer days (0.04 +/- 0.1) (P < .05). The histological features of these lesions underwent continuous change until the end of the fourth week, at which time necrotic neurons were still visible both in the striatum and in the cortex. CONCLUSIONS: Arterial occlusions of short duration (< 25 minutes) produced, in 76 of 121 experiments (63%), brain lesions characterized by selective neuronal necrosis and various glial responses (or incomplete infarction). This lesion is entirely different from the pannecrosis/cavitation typical of an infarction that appears 3 to 4 days after a prolonged arterial occlusion. Delayed neuronal necrosis, secondary to a transient arterial occlusion or increasing numbers of necrotic neurons in experiments with variable periods of reperfusion, was a response observed only at a predictable segment of the frontoparietal cortex.     

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size

In order to examine the relationship between local adenosine concentrations before, during, and after ischemia and the extent of ischemic myocardial damage, measurements of interstitial fluid (ISF) nucleosides were made using microdialysis probes implanted in the ischemic region of isoflurane anesthetized Micropigs undergoing 60' coronary artery occlusion (CAO) and 3 h of reperfusion (REP). Nucleoside concentrations in the dialysate collected from the microdialysis probes were used as an index of ISF levels. Dialysate nucleoside concentrations (ADO, inosine and hypoxanthine), myocardial infarct size, and myocardial blood flow (MBF) were determined in control animals (n = 6), animals preconditioned with a single 10' cycle of CAO and REP (PC, n = 6), and those treated with the adenosine deaminase inhibitor pentostatin (n = 6, 0.2 mg/Kg i.v. 30' prior to CAO). The brief PC occlusion resulted in a transient but significant increase in dialysate ADO (6.7 +/- 1.8 microM vs. 0.67 +/- 0.1 microM at baseline). Pentostatin administration had no significant effect on either dialysate nucleosides or MBF at baseline. During the 60' CAO, dialysate ADO increased in control animals. In PC animals, however, dialysate ADO during CAO was lower than control. Pretreatment with pentostatin resulted in a six-fold augmentation in dialysate ADO during the 60 min CAO when compared to the control values (110.62 +/- 30.2 microM vs. 16.31 +/- 2.1 microM at 60 min of ischemia). Pentostatin also resulted in a significant reduction in the accumulation of inosine and hypoxanthine, indicating inhibition of adenosine deaminase activity. There were no significant differences in MBF between groups at any time point. Following 3 h REP, infarct size was 35.4 +/- 5.5%, 8.1 +/- 1.5% and 8.3 +/- 1.8% of the region at risk in control, PC, and pentostatin groups, respectively. These data suggest that marked increase in ISF ADO during CAO, may be as effective in reducing INF as a modest increase in ISF ADO prior to prolonged CAO.     

Terikalant, an inward-rectifier potassium channel blocker, does not abolish the cardioprotection induced by ischemic preconditioning in the rat

Recent results have shown that the sulfonylurea receptor couples to several types of inward-rectifier potassium (KIR) channels, which suggests that sensitivity to blockade of a pathophysiological phenomenon such as ischemic preconditioning (PC) by glibenclamide may not be the result of this compound selectively blocking the ATP-sensitive potassium (KATP) channel. Therefore, to address this possibility, a role for myocardial KIR v KATP channels in ischemic PC was evaluated in the rat. To test this hypothesis, anesthetized, open-chest, male Wistar rats were assigned to one of seven experimental protocols. Animals assigned to group I (control) received 30 min of occlusion and 2 h of reperfusion. Ischemic PC was produced by 3x5-min occlusion and 2-h reperfusion periods (group II). Terikalant (TK), an inward-rectifier potassium channel blocker, was used to test the role of other K+ channels, most notably the KIR, in the cardioprotective effect of ischemic PC in the rat. TK was given at a dose of 3 mg/kg, i.v., 15 min before the prolonged occlusion and reperfusion periods (group III). In groups IV, V, and VI terikalant (1, 3 and 6 mg/kg, i.v.) was given 15 min before ischemic PC (lowTK+PC, medTK+PC and hiTK+PC, respectively). Group VII consisted of glibenclamide (0.3 mg/kg, i.v.) given 30 min prior to ischemic PC (GLY+PC). Infarct size (IS) as a percent of the area at risk (AAR) was measured using the histochemical stain, 2,3, 5-triphenyltetrazolium chloride. The average IS/AAR for the control was 49.9+/-2.1%. Ischemic PC markedly reduced infarct size (8.6+/-1. 8%; * P<0.05 v control). Terikalant (TK; 1, 3 and 6 mg/kg, i.v.) did not abolish the cardioprotective effect of ischemic PC at any dose (15.5+/-6.4, 16.4+/-5.2 and 8.8+/-1.6%, respectively; * P<0.05 v control). TK itself had no effect on infarct size. GLY completely abolished the cardioprotective effect of ischemic PC (48.2+/-6.4%). In addition, the high dose of TK significantly (P<0.05) increased the action potential duration at 50% repolarization from 48+/-3 to 64+/-4 ms and 30 microM of TK, a concentration which produced a 39% decrease in the inward-rectifier potassium channel current in isolated guinea-pig ventricular myocytes in the whole-cell patch-clamp mode did not block the increase in K ATP current produced by the KATP opener bimakalim (3 microM). These results demonstrate that although the myocardial KATP channel belongs to the K IR superfamily, the endogenous myocardial KIR channel does not mediate ischemic PC in the rat heart; however, the K ATP channel does mediate its cardioprotective effect.     

Visualization of ischemic insult in caudate putamen with beta-CIT

Dopamine (DA) has been considered to play an important role in the development of ischemic neuronal injury in the caudate putamen (CPu). The goal of this study was to examine the change in the dopamine transporter (DAT) after ischemic insult in CPu. METHODS: Male Mongolian gerbils (n = 10) were exposed to 10-min forebrain ischemia. Animals were decapitated 24 hr (n = 5) and 96 hr (n = 5) after ischemia. The change in the amount of DAT binding sites in CPu was evaluated by in vitro autoradiography with [125I]-beta-CIT (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester). In addition, the expression of DAT mRNA in CPu and the substantia nigra pars compacta (SNC) was examined. Results: Iodine-125-beta-CIT specific binding was significantly increased in dorsolateral CPu with ischemic damage both 24 hr and 96 hr after ischemia, with greater increase at 96 hr. DAT mRNA in SNC was also significantly increased 96 hr after ischemia, which corresponded with the increase of [125I] beta-CIT binding. However, DAT mRNA in SNC was decreased 24 hr after ischemia. In the ischemic lesion in CPu, no expression of DAT mRNA could be detected both 24 hr and 96 hr after ischemia. CONCLUSION: The change in DAT after ischemic insult is clarified with [125I] beta-CIT. This increase of [125I] beta-CIT binding does not come from de novo expression of DAT in glial cells in the damaged area in CPu. This increase of beta-CIT binding reflects increase of DAT synthesis in DA neurons in SNC (96 hr) or other factors such as the impairment of the degradation of DAT in the damaged area in CPu.     

Complement activation in the central nervous system following blood-brain barrier damage in man

The central nervous system (CNS) is virtually isolated from circulating immunological factors such as complement (C), an important mediator of humoral immunity and inflammation. In circulation, C is constantly inhibited to prevent attack on host cells. Since a host of diseases produce an abnormal blood-brain/cerebrospinal fluid (blood-brain/CSF) permeability allowing C protein extravasation, we investigated if C activation occurs in CSF in vitro and in CNS in vivo during subarachnoid hemorrhage (SAH) or brain infarction. After SAH (n = 15), the terminal complement complex (TCC) concentration on days 0 to 2 was higher in the CSF, 210 +/- 61 ng/ml, than in the plasma, 63 +/- 17 ng/ml, but null in the CSF of controls (n = 8) or patients with an ischemic stroke (n = 7). TCC was eliminated from the CSF after SAH (24 +/- 10 ng/ml on days 7 to 10). Incubation of normal human CSF with serum in vitro also activated the terminal C pathway. In 10 fatal ischemic brain infarctions, immunohistochemical techniques demonstrated neuronal fragment-associated deposition of C9 accompanied by neutrophil infiltration. We conclude that the C system becomes activated intrathecally in SAH and focally in the brain parenchyma in ischemic stroke. By promoting chemotaxis and vascular perturbation, C activation may instigate nonimmune inflammation and aggravate CNS damage in diseases associated with plasma extravasation.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.     

Fastigial stimulation increases ischemic blood flow and reduces brain damage after focal ischemia

Electrical stimulation of the cerebellar fastigial nucleus (FN) increases CBF and reduces brain damage after focal ischemia. We studied whether FN stimulation "protects" the brain from ischemic damage by increasing blood flow to the ischemic territory. Sprague-Dawley rats were anesthetized (halothane 1-3%) and artificially ventilated through a tracheal cannula inserted transorally. CBF was monitored by a laser-Doppler probe placed over the convexity at a site corresponding to the area spared from infarction by FN stimulation. Arterial pressure (AP), blood gases, and body temperature were controlled, and the electroencephalogram (EEG) was monitored. The stem of the middle cerebral artery (MCA) was occluded. After occlusion, the FN was stimulated for 60 min (100 microA; 50 Hz; 1 s on-1 s off) while AP was maintained at 97 +/- 11 mm Hg (mean +/- SD) by controlled hemorrhage. Rats were then allowed to recover, and infarct volume was determined 24 h later in thionin-stained sections. In unstimulated rats (n = 7), proximal MCA occlusion reduced CBF and the amplitude of the EEG. One day later, these rats had infarcts involving neocortex and striatum. FN stimulation after MCA occlusion (n = 12) enhanced CBF and EEG recovery [61 +/- 34 and 73 +/- 43%, respectively at 60 min; p < 0.05 vs. unstimulated group; analysis of variance (ANOVA)] and reduced the volume of the cortical infarct by 48% (p < 0.05). In contrast, hypercapnia (PCO2 = 64 +/- 4; n = 7) did not affect CBF and EEG recovery or infarct volume (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of halothane and isoflurane on Purkinje fibers in the infarcted canine heart: conduction, regional refractoriness, and reentry

The actions of halothane (HAL) and isoflurane (ISO) on conduction and regional refractoriness were studied in infarcted canine hearts to compare their effects on reentry in vitro. In two anesthetic groups of 8 hearts, high and low dose effects were assessed using action potentials recorded from Purkinje fibers located in the nonischemic and ischemic regions. An extrastimulus technique was used to determine the relationship between delay of conduction of premature impulses into the more refractory ischemic region and induction of reentrant responses. At high doses (HAL 0.60 mM and ISO 0.64 mM, approximately 2.3 minimum alveolar anesthetic concentration [MAC]) both anesthetics decreased (P < or = 0.05) the effective refractory period for direct intracellular stimulation of nonischemic fibers (local ERP, initial control: 294 +/- 8 ms); the decrease with HAL (-29 +/- 6 ms) was smaller (P < or = 0.05) than with ISO (-50 +/- 7 ms). HAL and ISO also decreased (P < or = 0.05) the coupling interval of the earliest premature impulse which conducted into the infarct (system effective refractory period [SERP], control: 301 +/- 7 ms) by -31 +/- 11 and -44 +/- 8 ms, respectively. In contrast, the functional refractory period (FRP) in the ischemic region (control:354 +/- 4 ms) was increased by HAL (26 +/- 8 ms; P < or = 0.05) but decreased by ISO (-14 +/- 4 ms, P < or = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapeutic time window for the 21-aminosteroid, U-74389G, in global cerebral ischemia

A novel 21-aminosteroid (U-74389G), a new potent antioxidant, was evaluated for its protective effect on transient global cerebral ischemia. Ischemia was induced by 20 minutes of four-vessel occlusion in adult male Wistar rats. Injection of 21-aminosteroid (U-74389G, 5 mg/kg intraperitoneally injected) was repeated three times. The second injection was performed 30 minutes after the first injection, and the third injection was performed 210 minutes after that. Experimental animals were divided into five groups according to the time drug administration was initiated. Group I (n = 8) began vehicle administration 30 minutes before occlusion. Group II (n = 9) started 21-aminosteroid administration 30 minutes before occlusion. Drug administration in Group III (n = 9) began at the time of reperfusion, in Group IV (n = 8), 30 minutes after reperfusion, and in Group V (n = 6), 60 minutes after reperfusion. Animals in the control group (n = 5) underwent sham operations. One week after ischemia, the number of viable pyramidal neurons was counted in the hippocampal CA1 subfield. The results were as follows: the number of living neurons in Group I was 18.8 +/- 8.7; in Group II, was 44.7 +/- 9.5; in Group III, was 46.4 +/- 9.4; in Group IV, was 40.3 +/- 6.6; in Group V, was 10.2 +/- 2.5; and in the control group was 131 +/- 3.3. Groups II, III, and IV demonstrated significantly higher numbers of living neurons compared with Group I (P < 0.05). The present study revealed that U-74389G attenuated delayed neuronal death in global cerebral ischemia when it was administered before or soon after the ischemic episode.