Pretreatment with intraventricular aurintricarboxylic acid decreases infarct size by inhibiting apoptosis following transient global ischemia in gerbils

The goal of this study was to determine whether aurintricarboxylic acid (ATA), an endonuclease inhibitor known to inhibit apoptosis, could ameliorate cell damage in a gerbil model of transient ischemia. Transient ischemia was induced in gerbils by bilateral carotid artery occlusion for a period of 5 minutes. Four micrograms of ATA was administered intraventricularly 1 hour before ischemia, and the brains were assessed histologically 1 week later to quantitate cell loss in the vulnerable CA-1 subsector of the hippocampus. In a separate set of experiments, 4 microg of ATA was administered intraventricularly 1 hour before ischemia and the brains were assessed for evidence of DNA fragmentation by the TUNEL method. There was only a 16% cell loss compared with nonischemic controls in animals pretreated with ATA that was significantly less (p < 0.05) than the 48% cell loss in animals pretreated with saline alone. TUNEL-positive cells were first evident at 3 days and were still present at 7 days subsequent to ischemia. Maximal staining occurred at 4 days. Pretreatment with ATA virtually eliminated TUNEL staining at 4 days. These results support the hypothesis that the delayed cell death secondary to transient ischemia is, in part, apoptotic. Furthermore, ATA afforded significant neuronal protection and prevented DNA fragmentation.     

Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils

Neurogenesis in the dentate gyrus of adult rodents is regulated by NMDA receptors, adrenal steroids, environmental stimuli, and seizures. To determine whether ischemia affects neurogenesis, newly divided cells in the dentate gyrus were examined after transient global ischemia in adult gerbils. 5-Bromo-2'-deoxyuridine-5'-monophosphate (BrdU) immunohistochemistry demonstrated a 12-fold increase in cell birth in the dentate subgranular zone 1-2 weeks after 10 min bilateral common carotid artery occlusions. Two minutes of ischemia did not significantly increase BrdU incorporation. Confocal microscopy demonstrated that BrdU immunoreactive cells in the granule cell layer colocalized with neuron-specific markers for neuronal nuclear antigen, microtubule-associated protein-2, and calbindin D28k, indicating that the newly divided cells migrated from the subgranular zone into the granule cell layer and matured into neurons. Newborn cells with a neuronal phenotype were first seen 26 d after ischemia, survived for at least 7 months, were located only in the granule cell layer, and comprised approximately 60% of BrdU-labeled cells in the granule cell layer 6 weeks after ischemia. The increased neurogenesis was not attributable to entorhinal cortical lesions, because no cell loss was detected in this region. Ischemic preconditioning for 2 min, which protects CA1 neurons against subsequent ischemic damage, did not prevent increased neurogenesis in the granule cell layer after a subsequent severe ischemic challenge. Thus, ischemia-induced dentate neurogenesis is not attributable to CA1 neuronal loss. Enhanced neurogenesis in the dentate gyrus may be a compensatory adaptive response to ischemia-associated injury and could promote functional recovery after ischemic hippocampal injury.     

Late-onset lipid peroxidation and neuronal cell death following transient forebrain ischemia in rat brain

The receptor encoded by the W (c-kit) locus is expressed on the membrane of mouse primordial germ cells, whereas its ligand termed stem cell factor (SCF), encoded by the Sl locus, is expressed on the membrane of somatic cells associated with both the primordial germ cell migratory pathways and homing sites. Using an in vitro short time assay which allows a quantitative measure of adhesion between cells, in the present paper we show that SCF/c-kit interaction can modulate primordial germ cell adhesion to somatic cells. We report that the adhesiveness of 11.5 dpc primordial germ cells to four types of somatic cells in culture (TM4 cells, STO fibroblasts, bone marrow stromal cells and gonadal somatic cells) is significantly reduced by antibodies directed against c-kit receptor or SCF, as well by soluble SCF. This SCF/c-kit mediated adhesion seems independent of SCF-induced tyrosine autophosphorylation of c-kit receptor. Moreover, primordial germ cells showed a poor ability to adhere to a bone marrow stromal cell line carrying the Sl(d) mutation (unable to synthesize membrane-bound SCF). This adhesiveness was not further impaired by anti-c-kit antibody. These results demonstrate that SCF/c-kit interaction contributes to the adhesion of primordial germ cells to somatic cells in culture and suggest that the role played by SCF in promoting survival, proliferation and migration of these cells in vitro and in vivo, demonstrated by several studies, might depend on the ability of the membrane-bound form of this cytokine to directly mediate primordial germ cell adhesion to the surrounding somatic cells.     

Alterations in [3H]L-N(G)-nitroarginine binding in brain after transient global or transient focal ischemia in gerbils and rats

Adverse physiological effects can often interfere with the use of nitric oxide (NO) as a therapeutic agent, especially when it is systemically generated from prodrugs. NO which is generated and delivered site-specifically by transdermal donors may be useful in the treatment of parasitic, bacterial or viral skin infections without causing systemic side effects. Three diazeniumdiolates (formerly "NONOate"), including two water soluble compounds, (Z)-1-[N-2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1- ium-1,2-diolate (DETA-NO) and (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate (DPTA-NO), and one insoluble compound, DPTA-NO grafted to dextran microspheres (DPTA-NO-g-dextran) were used to transdermally deliver NO to rats. Dextran microspheres were obtained by simultaneously grafting DPTA-NO to dextran and cross-linking dextran with CNBr in an oil-water emulsion. Suspended in hydrogel, DETA-NO, DPTA-NO, and DPTA-NO-g-dextran were applied three times to depilated rats at 4 day intervals. Results show that metabolic urinary nitrate levels increase with time (24-48 h), reach a maximum, and return to baseline by the fourth day. DPTA-NO applications produced an average maximum nitrate level of 94.2 mumol/day +/- 34.2 mumol S.D. compared to the average maximum nitrate level of 39.8 mumol/day +/- 8.6 mumol S.D. obtained from treatment with DETA-NO. These results suggest that DPTA-NO delivered NO more efficiently than DETA-NO. When DPTA-NO-g-dextran microspheres were used as the NO donor, results comparable to DPTA-NO were observed with an average maximum nitrate level of 14.9 mumol/day +/- 3.0 mumol S.D. These and other conclusive data indicate that, via these diazeniumdiolates, NO can be delivered effectively through rat skin.     

Selective acceleration of arachidonic acid reincorporation into brain membrane phospholipid following transient ischemia in awake gerbil

Awake gerbils were subjected to 5 min of forebrain ischemia by clamping the carotid arteries for 5 min and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 min at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl-CoA pool was independently determined for these fatty acids in control gerbils and following 5 min of ischemia and 5 min of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl-CoAs following recirculation, it was shown that reperfusion after 5 min of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes.     

Concanavalin A binding by the hippocampal neurons following brief cerebral ischemia in gerbils

Immunoglobulin (Ig) fractions from the plasma of a group of newly diagnosed insulin-dependent diabetes mellitus (type 1) patients and set of control subjects were assessed for their effects on isolated mouse islet function. It was found that Igs from type 1 patients caused a significant inhibitory effect on insulin secretion when incubated with mouse islets as compared with controls (25.6 +/- 2.9 pg islet-1 h-1 vs 44.7 +/- 7.7 pg islet-1 h-1, P < 0.05). The plasma samples from which the Igs were obtained were then tested for the presence of antibodies to the mouse islet cell surface (ICSA). Four of the nine patients were positive for ICSA, and plasma samples from eight control subjects were all negative. ICSA-positive samples appeared to have the greatest inhibitory effect on insulin secretion when compared with their respective controls (53.3 +/- 7.0 pg insulin islet -1 min-1 vs 30.9 +/- 3.7 pg insulin islet -1 min-1, (P < 0.05). In contrast, it was also found that ICSA-positive Ig fractions had no significant effect on glucose oxidation when co-incubated with mouse islets as compared with the controls (11.3 +/- 2.3 pmol islet-1 h-1 vs 11.2 +/- 2.9 pmol islet-1 h-1). These studies suggest that Igs from newly diagnosed type 1 patients containing ICSA may impair insulin secretion from isolated mouse islets by mechanisms which do not involve the inhibition of B-cell glucose metabolism.     

The stimulus-evoked release of glutamate and GABA from brain subregions following transient forebrain ischemia in the rat

The release of glutamate and GABA in response to K+ depolarization was determined for tissue prisms prepared from brain subregions removed from rats following 30 min of forebrain ischemia or recirculation periods up to 24 h. There were statistically significant effects of this treatment on release of both amino acids from samples of the dorsolateral striatum, an area developing selective neuronal degeneration. However, for at least the first 3 h of recirculation the calcium-dependent and calcium-independent release of both amino acids in this region were similar to pre-ischemic values. Differences were observed under some conditions at longer recirculation times. In particular there was a decrease in calcium-dependent GABA release at 24 h of recirculation and a trend towards increased release of glutamate at 6 h of recirculation and beyond. No statistically significant differences were seen in samples from the paramedian neocortex, a region resistant to post-ischemic damage. These results suggest that changes in the ability to release glutamate and GABA in response to stimulation are not necessary for the development of neurodegeneration in the striatum but rather that release of these amino acids may be modified as a result of the degenerative process.     

GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils

Repetitive cerebral ischemia produces more severe damage than a similar single duration insult. We have previously shown that, in gerbils, damage in the substantia nigra reticulata (SNr) is seen with repetitive insults rather than a single insult. We have also shown that there is a progressive decrease in the extracellular GABA in the striatum in the days preceding such damage, speculating that a loss of GABA may be in part responsible for this damage. This study evaluates the GABA levels in the SNr in animals exposed to repetitive ischemic insults. Each animal received a total of three ischemic insults of 3-min duration at hourly intervals. In vivo microdialysis was carried out to analyze the GABA and glutamate dialysate levels on Days 1, 3, 5, 7, and 14 following the ischemic insult. In the control and treated (ischemic) animals, there was a significant increase in the GABA levels with the introduction of nipecotic acid on Days 1, 3, 5, and 14. However, on Day 7 there was a significant attenuation in the GABA response to nipecotic acid in the treated animals in comparison to the controls. The glutamate levels in the treated animals were similar to the control animals on Days 1, 3, 5, and 7. However, on Day 14 the glutamate levels were significantly lower than on previous days. Our experiments for the first time measure extracellular glutamate and GABA responses in the SNr in animals exposed to repetitive ischemic insults. Our experiments show that there is a significant decrease in the GABA concentrations at a time when ischemic damage is developing in this region. This confirms our hypothesis that a decrease in GABA may be one factor contributing to neuronal damage during the period following repetitive ischemic insults. Further, the rebound increase in GABA levels on Day 14 with a concomitant fall in glutamate levels would indicate that reparative processes are still active in the 2 weeks following the insult.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.     

Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.     

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

OBJECTIVES: We studied the triggering mechanism for neurally mediated syncope. BACKGROUND: Although increased transient sympathetic tone is thought to be necessary for the development of neurally mediated syncope, little is known about the triggering mechanism for neurally mediated syncope. METHODS: Plasma epinephrine (EP) and norepinephrine (NE) levels were assessed in 20 syncope patients during tilt test (80 degrees, 15 min) with and without isoproterenol (ISP, 0.01, 0.02 microg/kg/min). If syncope occurred, propranolol (0.1 mg/kg) was injected. RESULTS: Eight patients experienced syncope during tilting alone, and 9 patients required ISP for syncope. In the negative response without ISP, NE showed a small statistical 1.7-fold increase at end of tilting and EP did not change during tilting. When syncope occurred during tilting alone, a significant 11.7-fold increase in EP at syncope was registered concomitant with a small 2.5-fold increase in NE. When patients experienced syncope during tilting with ISP, a significant 5.0-fold increase in EP at syncope was registered concomitant with a small 1.7-fold increase in NE. In patients without ISP, propranolol did not interrupt syncope. In patients with ISP, six of eight receiving propranolol responded to tilting negatively. CONCLUSIONS: An increase of NE levels may result in inhibition of syncope and an EP surge may be a triggering mechanism for neurally mediated syncope. Comparatively low levels of EP may be enough to induce syncope during tilting with ISP compared with tilting alone. Propranolol is not effective in patients without ISP, but it frequently inhibits syncope in patients with ISP. Propranolol (0.1 mg/kg) may be insufficient to block the actions of high levels of circulating EP.     

Attenuated glutamate release during ischemia in ethanol-administered gerbils

Previous studies have found an association between prior ethanol consumption and aggravated stroke outcome. Gerbils were intermittently given ethanol injections (s.c.) for 21 days at doses of 1 and 4 g/kg. After cessation of injections and appropriate weight gain, subjects underwent bilateral carotid occlusion while amino acid neurotransmitter levels in the hippocampus were monitored. Both the low and high dose ethanol groups demonstrated significantly decreased glutamate release compared with saline-treated controls during ischemia (p < 0.05). These results are consistent with a long-lasting ethanol-induced decrease in synaptic density in the hippocampus. That no intergroup differences on histological or neurobehavioral measures was found may suggest a functional dissociation of glutaminergic involvement in the pathogenesis of aggravated stroke outcome with alcoholism.     

Delayed recovery of auditory cortical evoked potentials is correlated with cortical neuronal death after transient cerebral ischemia in awake gerbils

1. Spermatozoa in semen samples from 8 individual male domestic fowls were shown to have a differential and characteristic ability to hydrolyse holes in the inner perivitelline layer from laid eggs in an in vitro assay. 2. The number of holes produced by samples of spermatozoa per unit area of inner perivitelline layer in vitro was linearly correlated with sperm ATP content (r = 0.85) and motility (r = 0.76). 3. The number of holes formed in the inner perivitelline layer in vitro was also linearly correlated with the numbers of holes formed in the inner perivitelline layer of eggs fertilised in vivo, in inseminated hens (r = 0.90); and was correlated logarithmically with the proportion of fertile eggs laid by these hens.     

Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat

The immunosuppressant drug cyclosporin A (CsA) is considered to be inherently protective in conditions of ischemia, e.g. in hepatic and cardiac tissue. However, investigations of effects of CsA on neuronal tissue have been contradictory, probably because the blood-brain barrier (BBB) is virtually impermeable to CsA. In the present study, we exploited the finding that the insertion of a syringe needle into brain parenchyma obviously disrupts the BBB and allows influx of CsA, and explored whether CsA, given as intraperitoneal injections daily for 1 week before and 1 week after forebrain ischemia of 7 or 10 min duration, ameliorates the damage incurred to the hippocampal CA 1 sector. In other experiments, the needle insertion and the first i.p. injection of CsA were made 30 min after the start of recirculation, with continued daily administration of CsA during the postinsult week. In animals which were injected with CsA in daily doses of 10 mg kg-1, but in which no needle was inserted, the drug failed to ameliorate CA1 damage, whether the ischemia had a duration of 7 or 10 min. Likewise, needle insertion had no effect on CA1 damage if CsA was not administered. In contrast, when CsA was given to animals with a needle insertion, CA1 damage was dramatically ameliorated, whether treatment was initiated 1 week before ischemia, or 30 min after the start of recirculation. The effect of CsA seemed larger than that of any other drug proposed to have an anti-ischemic effect in forebrain/global ischemia. Injection of tritiated CsA in one animal with BBB disruption lead to detectable radioactivity throughout the ventricular system, suggesting a generalised increase of the entry of CsA across the BBB. The results demonstrate that immunosuppressants of the type represented by CsA markedly ameliorate delayed neuronal damage after transient forebrain ischemia, provided that they can pass the BBB. It is discussed whether the effect of the drug is one involving calcineurin, a protein phosphatase, or if CsA counteracts a permeability transition of the inner mitochondrial membrane, assumed to occur in response to adverse conditions, e.g. gradual accumulation of Ca2+ in the mitochondria in the postischemic period.     

Aging and ischemia in gerbils impair spatial memory performance

Conventional gravimetric (weight loss) calibration of jet nebulizers overestimates their aerosol output by up to 80% due to unaccounted evaporative loss. We examined two methods of measuring true aerosol output from jet nebulizers. A new adaptation of a widely available clinical assay for lithium (determined by flame photometry, LiCl method) was compared to an existing electrochemical method based on fluoride detection (NaF method). The agreement between the two methods and the repeatability of each method were examined. Ten Mefar jet nebulizers were studied using a Mefar MK3 inhalation dosimeter. There was no significant difference between the two methods (p=0.76) with mean aerosol output of the 10 nebulizers being 7.40 mg x s(-1) (SD 1.06; range 5.86-9.36 mg x s(-1)) for the NaF method and 7.27 mg x s(-1) (SD 0.82; range 5.52-8.26 mg x s(-1)) for the LiCl method. The LiCl method had a coefficient of repeatability of 13 mg x s(-1) compared with 3.7 mg x s(-1) for the NaF method. The LiCl method accurately measured true aerosol output and was considerably easier to use. It was also more repeatable, and hence more precise, than the NaF method. Because the LiCl method uses an assay that is routinely available from hospital biochemistry laboratories, it is easy to use and, thus, can readily be adopted by busy respiratory function departments.     

Neuronal damage following transient cerebral ischemia and its restoration by neural transplant

The middle cerebral artery (mca) was intraluminally occluded for one hour prior to reperfusion in the rat. Neuronal damage as well as motor imbalance were assessed in both acute and chronic stages with or without neural transplant in the striatum. In acute stage, argyrophil III staining demonstrated "collapsed" dark neurons in the ipsilateral striatum, cortex, reticular thalamus, amygdala and sometimes in the hippocampus. They had shrunken somata and corkscrew-like dendrites. In accordance with the appearance of dark neurons, the immunoreactivity for calpain of endogenous inactive form decreased or disappeared in ischemic areas. In chronic stage, ischemic core area (striatum and cortex) got into porencephaly, and animals made rotations following methamphetamine injection. Neural transplant (fetal striatal cells) was made during 2 to 4 weeks after the ischemia. Once the transplant survived and grew in the striatum, the methamphetamine rotations were attenuated. Using mca ischemic model rats we report here pathophysiological processes that lead to neuronal damage and infarct. Neural transplants into these animals brought partial restoration in motor disturbance, offering a valuable information concerning therapeutic possibility.     

Induction of ischemic tolerance following brief focal ischemia in rat brain

The objective of this study was to determine whether brief focal ischemia induces ischemic tolerance in rat brain. Focal ischemia was produced in Wistar rats by occluding the middle cerebral artery (MCA) for 20 min at a distal site. Following recovery for 24 h, the animals were subjected to a 10-min episode of forebrain ischemia using a combination of bilateral carotid artery occlusion and systemic hypotension. Histologic injury, assessed after a survival period of 3-4 days, consisted of selective neuronal necrosis bilaterally in cerebral cortex, striatum, hippocampus, and thalamus superimposed upon a small cortical infarct adjacent to the site of MCA occlusion. However, the intensity of neuronal necrosis in the MCA territory of the neocortex ipsilateral to MCA occlusion was markedly less than that in the contralateral MCA cortex. In contrast, the extent of neuronal necrosis in subcortical structures was similar in both hemispheres. Unexpectedly, animals in which the MCA was manipulated, but not occluded, also exhibited a marked reduction of neuronal necrosis in the ipsilateral MCA neocortex following forebrain ischemia. However, in animals with craniotomy alone, forebrain ischemia caused a similar extent of neuronal necrosis in the MCA neocortex of both hemispheres. Transient occlusion of the MCA induced the focal expression of the 72-kDa heat-shock protein (hsp72) in the MCA territory of the neocortex. Limited expression of hsp72 was also detected following sham occlusion, but not after craniotomy alone. These results demonstrate focal induction of ischemic tolerance in rat neocortex that may be related to expression of heat-shock proteins.     

Motor activity changes following cerebral ischemia in gerbils are correlated with the degree of neuronal degeneration in hippocampus.

Cerebral ischemia was induced in Mongolian gerbils by bilateral occlusion of the carotid arteries. Subsequent histological assessment revealed neuronal degeneration in the CA1 area of the hippocampus. A functional behavioral change was reflected in an elevation of motor activity compared with sham-operated animals. The degree of hippocampal damage was positively correlated with the increase in motor activity. It is concluded that alterations in both measures result from the interruption of blood flow to the brain but may be brought about by different mechanisms. (PsycINFO Database Record (c) 2010 APA, all rights reserved)