141. Redistribution of mRNA after global brain ischemia and reperfusion
J.T. Jamison, M.K. Lewis, J.J. Szymanski and D.J. De Gracia
Department of Physiology, Wayne State University, Detroit, Michigan, USA
Objective: Death of vulnerable neuron populations following global brain ischemia and reperfusion (I/R) correlates with irreversible translation arrest (TA), but the mechanisms of irreversible TA are not known. However, all post-ischemic neurons show a stress-induced TA, but translation recovery follows HSP70 translation in resistant neurons.
Methods: Here, we evaluated markers of translation and translation regulation including ribosomal protein S6, eIF4G, PABP, TIA-1, TTP, HuR, HSP70 by immunofluorescence histochemistry, and poly-adenylated [p(A)] mRNAs using fluorescent in situ hybridization in brain slices after [SRK1] 48h reperfusion following 10 mins global brain ischemia. We assessed different combinations of staining pairs. Additionally we used co-immunoprecipitation to evaluate protein-mRNA interactions in post-ischemic brain homogenates, specifically at 8h reperfusion, when markers of TA show the greatest divergence. Normothermic I/R were performed by bilateral carotid artery occlusion (2VO) plus hypotension, and various durations of reperfusion between 1 and 48 h. Brains were perfusion fixed and double labeled for various combinations of the above markers. Standard methods for co-immunoprecipitation were used [SRK2].
Results: We observed that p(A) mRNAs underwent redistribution from a homogeneous cytoplasmic pattern to a granular pattern during reperfusion. Granulation of the mRNA correlated precisely with TA as measured by in vivo radioactive amino acid incorporation. The ‘mRNA granules’ colocalized with eIF4G and PABP, but not S6, TIA-1 or TTP. HuR colocalized to mRNA granules at 1hr reperfusion in resistant neurons, and only after 36 hr reperfusion in vulnerable neurons. Colocalization of HuR and mRNA granules correlated with HSP70 translation. IP of PABP co-precipitated GAPDH and HSP70 mRNA, whereas IP of HuR co-precipitated only HSP70 mRNA. Co-IP results validated the microscopy studies.
Conclusions: These results indicate that mRNAs are generally sequestered from 40S subunits following brain reperfusion, and that selective translation of stress induced messages occurs early in resistant areas but is delayed in vulnerable neurons. This delay likely plays a prominent role in irreversible TA and hence cell death.
445. Effect of the resveratrol treatment on the NA+,K+-ATPase activity in the hipocampus and cortex of global cerebral ischemia-exposed rats
F. Simão, A. Matté, C. Matté, F.M.S. Soares, A.T. Wyse, C.A. Netto and C.G. Salbego
Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
Objectives: Cerebral ischemia causes an interruption of neuronal and glial oxidative metabolism and can lead to irreversible loss of brain function. Neuronal damage caused by global ischemia is associated with an imbalance in ionic.1 Na+,K+-ATPase is a membrane enzyme responsible for the active transport of sodium and potassium ions in the central nervous system, maintaining the ionic gradient necessary for neuronal excitability and regulation of neuronal cell volume.2 Resveratrol (RSV, trans-3,4,5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a wide variety of plants. RSV has been reported to elicit many cellular responses including cell cycle arrest, differentiation, and apoptosis, and it has antiinflammatory, antileukemic, neuroprotective, and antiviral properties.3 Previously we demonstrated that RSV was able to protect brain slices against lesion induced by oxygen and glucose deprivation.4 Here we investigated the effect of RSV treatment on the Na+,K+-ATPase activity in hippocampus and cerebral cortex of the rats submitted to global cerebral ischemia.
Methods: Global cerebral ischemia was induced in male wistar rats (300 to 350 g) by four vessel occlusion for 10 mins. Animal groups included in the study were: sham, sham plus RSV treatment, ischemic and ischemic plus RSV treatment (30 mg/kg i.p. for 7 days before ischemia). Neuronal injury was analyzed by Fluoro-Jade C and Nissl staining. The expression and activity of the Na+,K+-ATPase were measured in cortex and hippocampal homogenates by western blotting and by assaying the inorganic phosphate released from ATP, respectively. The effect of RSV on Na+,K+-ATPase was analyzed at 1 h, 24 h and 7days after global cerebral ischemia.
Results: Global cerebral ischemia induced a statistically significant decrease in the Na+,K+-ATPase activity in both, hippocampus and cortex, at 1 h and 24 h of reperfusion. Maximal decrease was observed 24 h after the ischemic damage. The decline in the Na+,K+-ATPase activity was prevented in the animals treated with RSV at 24 h of reperfusion. Our results indicate that global cerebral ischemia induced a significant alterations in the Na+,K+-ATPase activity in the hippocampus and cortex and RSV treatment prevented these changes in the Na+,K+-ATPase activity.
Conclusion: We suggest that the maintenance of Na+,K+-ATPase activity afforded by RSV could be related to cellular neuroprotection.
505. Perk activation following global brain ischemia is independent of unfolded proteins
T. Sanderson1, M. Deogracias1, K. Nangia1, J. Wang1 and R. Kumar1,2
1Emergency Medicine; 2Physiology, Wayne State University, Detroit, Michigan, USA
Objectives: Transient global brain ischemia results in death of neurons in the CA1 hippocampus. Following ischemia, protein synthesis is inhibited throughout the entire brain. Most neurons eventually recover translational competence, however the CA1 never recovers. Protein synthesis is inhibited by phosphorylation of eIF2α. Following endoplasmic reticulum (ER) stress, eIF2α is phosphorylated by PERK. The prevailing dogma is that accumulation of newly synthesized (unfolded) proteins cause activation of PERK, eliciting the unfolded protein response (UPR). Activated PERK then phosphorylates eIF2α. However, it has never been directly demonstrated that unfolded proteins activate PERK after brain ischemia. We hypothesized that accumulation of unfolded proteins in the ER after global brain ischemia causes PERK activation, phosphorylation of eIF2α, and subsequent inhibition of protein synthesis. To assay this, we inhibited protein synthesis before ischemia to empty the ER of newly synthesized proteins. We theorized that in the absence of unfolded proteins, PERK would not be activated by ischemia/reperfusion.
Methods: m. The sections were incubated with primary antibodies for phosphorylated PERK (p-PERK), phosphorylated eIF2α (p-eIF2α), and neuronal nuclei (NeuN). AlexaFluor conjugated secondary antibodies were used. Images were obtained on a Leica LSM510 confocal microscope, under a 63X objective. A series of 10 optical sections were taken 0.35 μm in the z-plane and stacked into z-stacks of 3.5 μm. Protein synthesis was inhibited by intrahippocampal injection of anisomycin 90 mins prior to ischemia. Anisom ycin injection was verified with methylene blue. To assay in vitro protein synthesis, hippocampal homogenates were incubated with 100 μmol/L amino acids except methionine and cysteine and 1.5 μCi[35S]-methionine/cysteine. Long Evans rats were subjected to 8 mins of global brain ischemia utilizing bilateral carotid occlusion/hypotension. After 10 mins of reperfusion, rat brains were fixed, cryoprotected, frozen and cryosectioned at 8
Results: Tracer dye showed that anisomycin was injected into the CA1 hippocampus. Anisomycin-injected hippocampal homogenates had a 99% inhibition of protein synthesis compared to vehicle-injected homogenates. Triple-label immunofluoresence in sham-operated animals showed minimal detectable p-PERK and p-eIF2α colocalizing with CA1 neuronal fluorescence. Ischemia and 10 mins reperfusion resulted in an increase in p-PERK and p-eIF2α in CA1 neurons. Treatment of sham-operated control animals with anisomycin showed results similar to sham-operated controls. Interestingly, ischemia/reperfusion plus anisomycin had a similar increase in p-PERK and p-eIF2α to untreated ischemia/reperfused animals.
Conclusions: Protein synthesis was effectively inhibited with anisomycin. The average ER translocation time of proteins in CA1 hippocampal neurons is under 30 mins. Therefore, administration of anisomycin 90 min before the onset of reperfusion results in an ER devoid of proteins undergoing folding. If unfolded proteins were involved in PERK activation, we would expect no PERK phosphorylation when the ER is emptied of unfolded proteins. Interestingly, we found that PERK was phosphorylated in the CA1 region even with anisomycin treatment. This suggests that the prevailing dogma for PERK activation is incorrect in the setting of brain reperfusion. Specifically, unfolded proteins do not activate PERK during brain ischemia or reperfusion. Further studies will be needed to discover the mechanism of PERK activation during brain reperfusion.
613. Cilostazol improves cognitive function by enhancing vasculogenesis and neurogenesis in CA1 hippocampus in a mouse model of transient global ischemia
1Division of Meridian and Structural Medicine, School of Oriental Medicine, Pusan National University; 2MRC for Ischemic Tissue Regeneration, Pusan National University; 3Department of Obstetrics & Gynecology, School of Medicine, Pusan National University, Yangsan-si, South Korea
Objectives: Cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone) increases intracellular cyclic AMP levels by inhibiting type III phosphodiesterase. It has been recently suggested that cilostazol has a beneficial effect in the treatment of transient focal cerebral ischemic injury. Neovasculogenesis improves tissue microenvironment around the hippocampal CA1 injured ischemic area as a vascular niche and favors the proliferation and differentiation of neuronal precursor cells. We aimed to investigate whether vasculogenesis is enhanced in the hippocampus CA1 area in response to transient global ischemic injury under treatment with cilostazol, and whether reappearance of CA1 neurons is associated with recovery of deficit hippocampal-dependent spatial learning and memory.
Methods: To determine the optimal duration of bilateral common carotid arterial occlusion, C57BL/6 mice were subjected to global cerebral ischemia for various duration of time. The degree of hypoxia was assessed in the injured tissue using Hypoxyprobe™-1 kit. We chose occlusion of 20-mins duration to produce global cerebral ischemia. C57BL/6 mice were treated with cilostazol (10 mg/kg per day, intraperitoneally) and histologically evaluated at 7, 14, and 28 days after the global ischemia.
Results: 20-min of global cerebral ischemia resulted in remarkable neuronal damage in CA1 areas compared with sham-operated animals and cilostazol (10 mg/kg per day, intraperitoneally) significantly reduced neuronal damage in the CA1 region. Treatment with cilostazol increased expression of brain-derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in the brain. Stromal cell-derived factor-1α (SDF-1α) protein expression was significantly upregulated in the cilostazol-treated group at 14 days after ischemia, which was colocalized with CD31. To determine the effects of cilostazol on cell proliferation, BrdU was administered twice daily on day 1 to 3 after transient global ischemia. At 14 days after ischemia, BrdU-positive cells as well as BrdU and CD31 double-positive cells were greatly increased in cilostazol-treated groups. The escape latency was significantly increased in the vehicle group from 3 to 21 days after global ischemia when compared with the sham-operated group in Morris water maze task. The prolonged latency induced by global ischemia was significantly shortened by administration of cilostazol.
Conclusions: In summary, cilostazol showed beneficial effects promoting vascularization in the hippocampus area by upregulation of BDNF, eNOS, VEGF and SDF-1α and preserving the CA1 hippocampal region, thereby resulting in improvement of spatial learning memory in mice subjected to transient global ischemia. Enhancement of vascularization by cilostazol can be a valuable therapeutic option for the neurologic functional recovery after stroke.
This work was supported by the Korea Research Foundation Grant (KRF-2008-314-E00477, Shin) and the MRC program of MOST/KOSEF (R13-2005-009, Kim).
615. Chronic cerebral hypoperfusion model in mice
C. Kudo1,2, K. Eikermann-Haerter1, I. Yuzawa1, T. Qin1, C. Liu3, Y. Gu1, H. Niwa2, C. Waeber1, M.A. Moskowitz1, J.R. Sims1,4 and C. Ayata1,4
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, USA; 2Department of Dental Anesthesiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan; 3Martinos Center for Biomedical Imaging, Department of Radiology; 4Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, USA
Background and aims: Chronic cerebral hypoperfusion occurs in the setting of severe intracranial or cervical artery disease, or intrinsic microvasculopathies such as chronic hypertension, diabetes and amyloid angiopathy. Periventricular white matter changes are often associated. Bilateral common carotid ligation model used in rats to create chronic cerebral hypoperfusion is not suitable for mice due to severe ischemia and premature death. Recently, a bilateral carotid stenosis model has been developed by placing steel coils around common carotid arteries (Shibata M, Stroke 2004). We performed a detailed hemodynamic, histopathological, MRI, and neurological characterization of this new model in mice.
Methods: We used C57Bl/6 mice (24 to 29 g, male) due to the frequently incomplete circle of Willis. Under brief isoflurane anesthesia, steel coils (inner diameter: 0.18 mm, total length: 2.5 mm, Sawane Spring Co., Japan) were placed around both common carotid arteries. Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry (LDF) during coil placement. Absolute CBF was measured 1d or 28d after coil placement using 14C-iodoantipyrine technique (IAP). Cerebrovascular reserve was determined by measuring hypercapnic hyperemia (5% CO2) using laser speckle flowmetry. Neurological deficits were characterized by water-maze test at 28d. Ventricular enlargement was assessed by MRI. At time of sacrifice, mice were perfused with carbon black to examine circle of Willis anatomy. Brains were cryosectioned and stained by hematoxylin/eosin (routine histopathology), luxol fast blue (myelin), and glut-1 immunohistochemistry (microvascular architecture).
Purpose: Hemodynamic, histopathological, morphometric, and neurological characterization of bilateral carotid stenosis in mice.
Results: Cortical CBF decreased by ∼30% bilaterally after coil placement, as measured by LDF. A similar reduction in absolute CBF was detected in structures supplied by carotid arteries (cortex, striatum, corpus callosum, hippocampus, dorsal thalamus) but not in cerebellum or brain stem, using IAP 1d after coil placement. At 28d, absolute CBF was restored to normal levels in all brain regions. Although the proportion of mice with posterior communicating arteries (PComm) was unchanged, the diameters of existing PComm were significantly larger in coiled mice at 28d. Capillary diameters were ∼10% larger in cortex without a change in capillary length-density, while in striatum capillary length-density was decreased by ∼30% without a change in capillary diameter. Consequently, cerebrovascular reserve in cortex was normal when tested 28d after bilateral carotid stenosis. Morphometric (MRI) and histopathological analyses showed an increase in ventricular volume suggesting brain atrophy. Brain myelin content was reduced by ∼25% at 28d. Scattered microinfarcts were observed in striatum and cortex associated with neuronal loss and gliosis. Mice with bilateral carotid stenosis performed worse in spatial learning acquisition as tested by the water-maze test.
Conclusions: Our data suggest that bilateral carotid stenosis (coil) technique leads to relatively mild hypoperfusion and loss of cerebrovascular reserve that are restored within 4 weeks by posterior communicating artery and capillary remodeling. Associated myelin loss, occasional microinfarcts and gross brain atrophy are likely responsible for the neurological deficits. The model may be suitable to investigate the pathophysiology of chronic cerebral hypoperfusion implicated in intracranial or cervical artery stenosis, as well as vasculopathies.
915. Effect of donepezil on nitric oxide production in the brain during global ischemia and reperfusion in mice
Y. Asano1, T. Ohkubo2, K. Hattori2, T. Shimazu1, H. Nagoya2, M. Yamazato2, Y. Ito2, Y. Kato2 and N. Araki2
1Neurology, Saitama Neuropsychiatric Institute, Saitama-shi; 2Neurology, Saitama Medical University, Moroyama-machi, Japan
Objectives: Donepezil (DON), a selective acetylcholinesterase (AChE) inhibitor, may induce neuroprotective factors by stimulating nicotinic acetylcholine receptors (nAChRs) and protect cortical neurons by against glutamate neurotoxicity.1,2 In addition, it was reported that donepezil protects the brain ischemia induced by MCA occlusion and that this effect may be due to, at least in part, to donepezil stimulation of α7-nAChRs.3 Nitric oxide (NO) plays an important role in the pathogenesis of neuronal cell injury which is closely related to glutamate neurotoxicity. However, effect of donepezil on NO production in the brain during ischemia have not been previously determined. Therefore, we examined the effect of donepezil on generation of NO metabolites during global ischemia and reperfusion in mice and striatum using an in vivo microdialysis technique.
Methods: Male C57BL/6 mice were anesthetized with halothane. Polyethylene catheter (PE-10) was inserted into the right femoral artery for measuring blood pressure. A Microdialysis probe was inserted into left striatum. After 2 h equilibrium period, fractions were collected every 10 mins. A Laser Doppler probe was placed on the right skull surface. In doenepzil group and control group, global ischemia was produced by clipping both common carotid arteries using Zen clips for 10 mins. Donepezil (1 mg/kg or 5 mg/kg) was administered subcutaneously at 30 mins before ischemia. The levels of nitrite (NO2−) and nitrate (NO3−) in the dialysate samples were measured by the Griess reaction. After euthanasia, the brains were immunostained using a primary antibody directed against nNOS in an immunoperoxidase method and visualized by diaminobenzidine in striatum.
Results: In steady state, blood pressure was transiently increased at 10 mins and cerebral blood flow was increased at 10 (107.5%±6.9%, mean±SD), 20 (109.4±10.8) and 30 mins (109.4±10.6) after administration of 5 mg/kg doenepzil. Total NO (amount of nitrite and nitrate) levels in the dialysate from striatum were decreased significantly at 10 (3.27±0.97 μmol/L) and 20 mins (3.24±0.91) after administration of 1 mg/kg donepezil and 20 min (4.08±1.07) after administration of 5 mg/kg donepezil as compared with each baseline (3.69±0.86, 4.64±1.03) (Figure). In addition, total NO levels in 1 mg/kg donepezil group were significantly higher (99.30%±22.76%) during ischemia and significantly lower at 60 min after reperfusion (113.57±32.74) than those of control (82.65±19.50, 144.92±40.92). nNOS-positive area were decreased in 1 mg/kg (1.14%±0.39%) and 5 mg/kg donepezil group (1.24±0.54) as compared with control (1.51±0.70), but there was no significant differences between three groups.
Changes in total NO levels.
Conclusions: These data indicate that donepezil attenuates NO production in the brain and may afford neuroprotective effects against ischemia and reperfusion injury.
944. Ventricular fibrillation induced cardiac arrest in the rat as a model of global cerebral ischemia
K. Dave, D. Della-Morte, I. Saul and M. Perez-Pinzon
Neurology, University of Miami, Miami, Florida, USA
Objectives: Cardiopulmonary arrest (CA) remains one of the leading causes of death and disability in the USA. Although ventricular fibrillation (VF) models in rodents mimic the ‘square wave’ type of insult (rapid loss of pulse and pressure) commonly observed in adult humans at the onset of CA, those approaches are not popular because of complicated animal procedure, poor animal survival and thermal injury.1–3 Here we present modified, improvised, simple, reliable, ventricular fibrillation-induced rat model of CA that could be useful in studying the mechanisms of cerebral ischemia induced delayed cell death as well as efficacy of neuroprotective drugs.
Methods: CA was induced in male Sprague-Dawley rats weighing 300 to 370 g using modified method from von Planta.3 In brief, VF was induced in anesthetized, paralyzed, mechanically ventilated rats by an alternating current delivered to the entrance of the superior vena cava. The electrode was placed at the correct location using a fluoroscope. The fluoroscope enabled us to lower the VF-inducing current, and identify the exact location to place the electrode and overcome minor rat-to-rat anatomical differences. The head and body temperatures were maintained at 37°C using heating lamp and heating pad, respectively. After 5 mins 30 secs, resuscitation was initiated by administering a bolus injection of epinephrine and sodium bicarbonate followed by mechanical ventilation and manual chest compressions and counter shocked with a 10-J DC current. Neurologic deficit score (NDS) was quantified up to three days of reperfusion. Seven days after ischemia, rat brains were fixed for histopathological assessment. Hippocampal sections at the level of 3.8 mm posterior to bregma were examined for normal neurons.
Results: During the induction of CA, an immediate VF was observed followed by a sudden drop in blood pressure to close to 0 mm Hg. When the fibrillating current was stopped, asystole was observed and blood pressure remained zero mm Hg until resuscitation. Ten minutes after resuscitation, ECG appeared normal. After 10 mins of VF all rats returned to normal sinus rhythm with a heart rate of 367±22 beats per min and exhibited normal electrical heart conduction. In CA rats at 3, 24, 48, and 72 h following CA, NDS score was 63±5, 26±2, 14±2 and 6±1, respectively. The NDS remained zero at all time points studied in sham rats. The number of normal neurons in the CA1 hippocampal region in sham CA rats was 1263±43 (n = 6). The number of normal neurons was decreased by 79% (261±58, n = 5, P<0.001) in CA group as compared to the sham CA group. No signs of thermal injury were observed at the site of electrode placement.
Conclusion: The presently described ventricular fibrillation-induced model of cardiac arrest in rat as a model of whole body ischemia provides a tool to study the mechanism of cardiac arrest-induced neuronal death without compromising heart functions.
Support: PHS grants NS34773, NS05820, NS045676 and NS054147.
949. Cerebral reperfusion upregulates genes distinct from ischemia alone in the rat
B. Ander, X. Zhan, D. Liu and F. Sharp
Neurology, UC Davis MIND Institute, Sacramento, California, USA
Objectives: Ischemia and reperfusion are distinct phases of cerebral injury contributing to stroke. Previously, we have examined gene expression in the rat following permanent focal ischemia. We hypothesize that reperfusion will elicit its own change in gene expression and that this change can be measured in the blood.
Methods: Adult male Sprague Dawley rats were anaesthetized and subjected to middle cerebral artery occlusion using the intraluminal suture technique. The suture was left in place for either two hours (reperfusion group) or permanently for 24 h (ischemia group). At 24 h following the surgery, rats were anaesthetized and whole blood was collected into PAXgene blood collection tubes to stabilize RNA. Total RNA was isolated with the PAXgene system and processed using reagents from NuGEN Technologies to generate labelled cDNA for hybridization onto Affymetrix Rat Genome 230 2.0 arrays. Partek software was used to create gene lists. A false discovery rate ⩽0.05 and two-fold change cutoff were applied for each stroke group compared to normal and sham controls.
Results: A total of 344 and 31 gene transcripts were upregulated in the reperfusion and ischemia groups, respectively. Of these, a unique set of 97 gene transcripts were significantly upregulated only in the reperfusion group and common in comparisons to both normal and sham groups. These 97 transcripts represented genes with significant representation in many biogroups including enzyme regulation, binding of calcium ions, inflammation and responses to stress and wounding.
Conclusions: Cerebral reperfusion injury results in upregulation of many genes that are not induced by ischemic injury alone. These specific gene profiles are measureable in the blood.
994. Decreased hypoxic ventilatory response in aged rat following transit global ischemia
K. Xu1, M.A. Puchowicz2, X. Sun1 and J.C. Lamanna1
1Physiology and Biophysics; 2Nutrition, Case Western Reserve University, Cleveland, Ohio, USA
Introduction: Transient global brain ischemia induced by cardiac arrest and resuscitation results in reperfusion injury. There is increased incidence of cardiac arrest with aging. The age-related changes in the brain could alter the outcome of diseases as well as the potential therapeutic strategies. In this study we investigated the effects of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) in the aged rats following cardiac arrest and resuscitation.
Methods: Male Fischer 344 rats (Young: 6-month old; Aged: 24-month old) underwent cardiac arrest and resuscitation, and 4-day overall survival rats were determined. Brainstem function was assessed by measuring hypoxic ventilatory response (HVR; ratio of minute volume, hypoxia versus normoxia). For the PBN-treated rats, PBN (100 mg/kg) was infused intravenously immediately after resuscitation for 60 min. The untreated rats were given normal saline instead.
Results: With aging the 4-day overall survival rates were decreased; with greater mortality during the first 2 days of recovery. The overall survival rate in the aged untreated rats was significantly less than that of the young rats (38.5%, 5/13 total versus 69.2%, 9/13 total); however, PBN treatment significantly improved the overall survival rate by 28%. Compared to the young rats (3.0±0.3, n = 5), the aged group had significantly lower pre-arrest HVR values (2.2±0.4, n = 15), with similar normoxic ventilation (minute volume, mL, 206±33 versus 205±35) but lower hypoxic ventilation (minute volume, mL, 455±74 versus 600±117). During the first 4 days of recovery, the untreated aged rats have significantly lower HVR compared to their pre-arrest values; but the PBN-treated rats had similar ventilation performance in response to hypoxia (see Figure, * indicates significance compared to the untreated group).
Hypoxic ventilatory response in the aged rats.
Conclusion: These data suggest increased mortality in the aged may be associated with brainstem dysfunction. The antioxidant treatment with PBN improved recovery following cardiac arrest and resuscitation in the aged.
1054. Effectiveness of hyperbaric oxygen and medical ozone pretreatment against oxidative stress after global cerebral ischemia in rats
S. Oter1, M. Seyrek2, S. Sadir1, B. Duz3, T. Topal1, M. Kaplan3,4, A. Korkmaz1, S. Kahraman3 and S. Obut1
1Physiology; 2Pharmacology; 3Neurosurgery, Gulhane Military Medical Academy, Ankara; 4Neurosurgery, Firat University, Medical School, Elazig, Turkey
Objective: Albeit a considerable number of experimental work reporting beneficial effects of hyperbaric oxygen (HBO) therapy in ischemic brain incidents exist, the overall outcome is insufficient to prove its effectiveness in such conditions.1 More and more studies focus on the preventive side of this therapy modality; therefore, preconditioning experiments are of particular interest. Preconditioning with HBO was shown to exert beneficial effects against ischemic brain injury by upregulation of antioxidant enzymes.2 A new therapeutic approach, medical ozone therapy, is reported to present similar actions with HBO and upregulation of antioxidant enzymes is one of their common points.3 The present study tests the efficacy of HBO and medical ozone treatments against oxidative brain injury in a rat global cerebral ischemia model.
Methods: Twenty-eight Sprague-Dawley rats were seperated into sham operated, ischemia-reperfusion, HBO- and ozone-pretreated groups. HBO was exposed as 2.5 atm for 1.5 h, once daily for a total of 3 sessions. Ozone was administered daily intraperitoneal injections for 3 days; the ozone dosis was set as 0.7 mg/kg-BW/day. Twenty-four hours after the last HBO or ozone treatments, cerebral ischemia was induced for 10 min using the 2-VO model.4 Then the animals were placed in metabolic cages to collect 24 h urine specimens and right after this period they were sacrificed. Their brain cortex and hippocampus tissues were reserved for biochemical assay. Protein carbonyl content, malondialdehyde, superoxide dismutase, glutathione peroxidase and nitrites+nitrates (NOx) were determined.
Results: Oxidative stress as well as antioxidant function indices resulted with comparable levels; only slight changes occur in the ischemia-reperfusion group, and ozone and HBO treatments presented similar levels. Data collected from NOx measurements, however, reflected interesting outcome. Cerebral ischemia-reperfusion tended to depress NOx production; in cortex and urine samples. Both HBO and ozone therapy were able to reverse this effect, of which in ozone-treated animals the values returned near to control levels.
Conclusion: The NOx results in the present study seem to be valuable for further discussion. It was reported that there are different stages of NO production, therefore decreased and increased levels at diferent time points, during global cerebral ischemia.5 Another work reported protecting effects of a NO donor against ischemic brain damage.6 Both HBO7 and ozone8 have stimulating effect on NO production. Therefore, this point seems to have particular importance and warrants further research on this matter.
Grant: This study was supported by the Gulhane Military Medical Academy Research and Progress Center with the grant Nr.AR-2003/56.
Nitrite-nitrate values (median and range)
Sham
Ischemia
HBO
Ozone
Cortex (mg/mL)
93.4 (90.9–102.1)
76.7* (55.5–93.4)
86.4 (72.1–103.1)
102.6** (88.9–136.3)
Hippocampus (mg/mL)
87.7 (73.7–99.4)
96.5 (86.7–102.1)
97.8 (83.3–109.7)
97.1 (94.1–106.1)
Urine (mg/mL)
471 (237–747)
273* (241–323)
351** (335–388)
475** (160–1252)
Urine (mg/24 h)
3592 (2373–4484)
1613* (1203–2182)
2077 (1673–2297)
4079** (1440–6447)
References
1.
SiesjoBK. Crit Care Med1988;16:954–62.
2.
EricinskaM and SilverIA. Prog Neurobiol1994;43:37–71.
