Brain Poster Session: Ischemic Preconditioning

79. The brain revascularization for the patients with bicarotid atherosclerotic diseases

V. Byvaltsev and E. Belykh

State Establishment Scientific Center of Reconstructive and Restorative Surgery of East Siberian Scientific Center Siberian Branch of Russian Academy of Medical Sciences, Irkutsk, Russia

Background and aims: There are 9 million people suffering from brain vascular diseases in the world. The main place belongs to the stroke of more than 6.6 million people. Despite the previous conclusions about the inefficiency of surgical brain revascularization under the conditions of ishemic stroke (1988), the latest research works prove their clinical expediency.

Purpose: The purpose of work was the analysis of treatment results of patients with the chronic cerebral ishemia caused by plural impacts of the main arteries of a brain.

Methods: Are presented by 18 patients with plural stenosis and occlusion effects of carotids which had 20 extra-intracranial microvascular anastamoses (EC-IC bypass). The median age of the patients was 54 years. The estimation of total neurological deficiency of the patients was carried out with the use of the scale of ishemic stroke NIHSS consequences. The complex tool research included: TCDG with defining cerebrovascular reserves, duplex scanning of extra-cranial arteries, CTA, MRA and in some cases a DSA (angiography). Operations were performed under the general anesthesia with the use of an operational microscope. All patients had a two-sided effect of the carotid pool. In 16 cases (88.9%) there were post-thrombotic occlusion of the internal carotid from one side in the combination with a hemodynamically significant stenosis or a pathological deformation from the opposite side. For these patients an EC-IC bypass on the side of occlusion was performed. In two cases (11.1%) post-thrombotic occlusions of both carotids was diagnosed which required the creation EC-IC bypass from both sides.

Results: There were the recourse of attributes of a cerebral ishemia according to the scale NIHSS, and also authentic increase of the total brain blood-flow and normalization of autoregulation parameters of cerebral hemodynamics (15 patients—83.3%). There were complications of surgical treatment in two cases (11,1%). One patient had epidural hematoma which required its emergency removal. In another case there was an increase of neurological deficiency during the operation. During the control inspections the thrombosis of microanastomosis (inconsistency) was revealed in one case (catamnesis—5 years).

Conclusions: The creation of EC-IC bypass of the bicarotid is an effective revasculation operation in cases of its performance on the side of clinical symptomatic prevalence, provide good condition of cerebral reserves and well passable external carotid.

195. CBF effects of brain preconditioning produced by cortical lesions

L. Zhao and T. Nowak

Department of Neurology, University of Tennessee Health Science Center, Memphis, Tennessee, USA

Aims: Diverse phenomena of brain preconditioning have been described, including effects of brief focal ischemia or discrete cortical lesions to reduce infarct volume following subsequent middle cerebral artery (MCA) occlusion. Ischemic preconditioning has been associated with early CBF recovery in penumbra during the initial hours of subsequent permanent MCA occlusion that predicted eventual infarct volume (Zhao and Nowak, J Cereb Blood Flow Metab 2006; 26:1128–1140). The current study examined CBF responses and peri-infarct depolarizations in brain preconditioned by a prior cortical cold lesion (CL).

Methods: Small cortical lesions were produced in male Spontaneously Hypertensive Rats (SHR) under isoflurane anesthesia by application of a 2 mm diameter liquid nitrogen-chilled metal probe to the thinned skull overlying MCA territory. Sham animals experienced surgical preparation without freezing. The following day rats were subjected to permanent focal occlusion of the MCA and ipsilateral common carotid artery. Infarct volumes were determined at 24 h in hematoxylin-eosin stained frozen sections. CBF was also monitored by C14-iodoantipyrine autoradiography at various time points after occlusion, or throughout the initial hours of occlusion by speckle contrast perfusion imaging with simultaneous electrophysiological recording.

Results: Edema-corrected infarct volumes were 116±14, 113±11 and 85±14 cubic mm in Naïve, Sham and CL groups (mean±SD; n = 9, 10, and 10), respectively, demonstrating the efficacy of preconditioning by prior lesions. The CBF deficit at 15 mins after occlusion was unaffected by CL, but volumes of severely ischemic territory (CBF less than 30 mL/100 g/mins) at 3 h after occlusion were 130±8, 113±10 and 67±19 cubic mm (n = 5, 4, and 7) in these groups, establishing a phenomenon of early CBF improvement identical to that previously observed after ischemic preconditioning. The magnitude of protection was independent of CL size in the range 2–8 cubic mm. Peri-infarct depolarizations were associated with propagated waves of hyperemia detected by perfusion imaging. These events were completely eliminated in animals with CL larger than 5 cubic mm. However, their incidence was also decreased in animals with smaller, still protective lesions to an extent comparable to that observed after sham surgery. Excluding rats with large lesions, median depolarization numbers during 4 h of occlusion in Naïve, Sham and CL groups were 7 (range 5–11, n = 5), 4 (range 1 to 5, n = 6) and 4 (range 3 to 5, n = 7), respectively.

Conclusions: Cortical lesions produce preconditioning mechanistically identical to that induced by prior ischemia, since both are associated with early CBF recovery. CL also reduces the number of Peri-infarct depolarizations, although sham surgery had a comparable effect, indicating that such attenuation is not by itself sufficient for protection. The potential contribution of decreased depolarization number to subsequent CBF recovery and infarct volume reduction remains to be established.

600. Hypoxic preconditioning reduces hypoxia-ischemia-induced cell proliferation in the rat subventricular zone

N. Jones¹, L. Kardashyan² and P. Beart²

¹Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW; ²Howard Florey Institute, The University of Melbourne, Melbourne, VIC, Australia

Objectives: Preconditioning with mild hypoxia and the hypoxia-mimetic compounds desferrioxamine (DFX) and cobalt chloride (CoCl₂) have been shown to have protective actions against a subsequent hypoxic-ischaemic (HI) brain insult. These treatments alone are not sufficient to cause neuronal injury, but can induce changes in gene expression and intracellular signalling pathways. The expression of the hypoxia-inducible transcription factor (HIF-1) ¹ and several HIF-1 target genes ² are up-regulated after preconditioning with hypoxia and mimetics in neonatal rat brain. Interestingly, many HIF-1 target genes (including erythropoietin and VEGF) have been shown to increase cell proliferation in the brain. ³ We have investigated whether preconditioning treatments that can increase HIF-1 have long lasting neuroprotective actions and alter cell proliferation up to 5 weeks after an HI insult.

Methods: Sprague Dawley rats (postnatal day 6 (p6)) were exposed to preconditioning with hypoxia (3 h, 8% oxygen), normoxia (3 h, room air) or injected with DFX (200 mg/kg, i.p.) or CoCl₂ (60 mg/kg, i.p.). On p7, all pups were subjected to HI or sham surgery. Bromodeoxyuridine (BrdU, 50 mg/kg, i.p.) was injected twice daily for 3 days following surgery and was used to examine cell proliferation. At 5 weeks after HI insult, brains were collected for imunohistochemical and histological processing.

Results: Hypoxia, DFX and CoCl₂ preconditioning all significantly reduced the extent of brain injury after HI insult as measured using Nissl staining and MCID analysis. Cell proliferation was examined using immunohistochemistry for BrdU and cell counts were obtained from the subventricular zone (SVZ). HI alone significantly increased (by 106%) the number of BrdU positive cells in SVZ compared to sham operated controls. Preconditioning with hypoxia and DFX performed 24 h prior to HI, reduced cell proliferation in SVZ by 28% and 33%, respectively, compared to animals which received HI alone. CoCl2 preconditioning had no effect on HI-induced cell proliferation in the SVZ. Interestingly, treatment with DFX alone, significantly increased the number of BrdU positive cells in SVZ compared to controls by 172%.

Conclusions: Modulation of HIF-1 and its target genes is likely to be involved in the brain plasticity and neuroprotective effects conferred by preconditioning with hypoxia, DFX and CoCl₂ in neonatal rat brain.

604. Longitudinal MRI characterization of stroke in ischemia tolerant rats

J. Artmann¹, M. Weller², E. Wong³ and S. Wegener²

¹Neurology, University and ETH Zurich; ²Neurology, University Zurich, Zürich, Switzerland; ³Radiology and Psychiatry, University California San Diego, La Jolla, California, USA

Objectives: The individual vulnerability to ischemia has a significant impact on tissue damage and outcome in stroke patients. Therapeutic interventions could be much better tailored to the patient if information about the individual resistance to ischemic injury was available. Tolerance to ischemia can be induced experimentally by ‘preconditioning’. One preconditioning model that has been used to render the brain more tolerant to ischemia is the injection of 3-nitroproprionic acid (3NPA), a respiratory chain inhibitor. We used longitudinal MRI in adult rats to characterize how preconditioning with 3NPA affects the development of a stroke lesion and if a typical ‘MRI signature’ of preconditioned tissue can be extracted from these data.

Methods: Adult Wistar rats received i.p. injections of 20 mg/kg 3NPA or saline and were subjected to transient (60 min) middle cerebral artery occlusion (MCAO) three days later. MR images were acquired during the occlusion (day 0) and after reperfusion (days 1, 4, 14). Quantitative CBF maps were obtained using flow-sensitive alternating inversion recovery (FAIR) arterial spin labeling with the QUIPSSII modification. In addition, diffusion-weighted (DWI) images, T1 and T2 maps and anatomical T2w images were obtained. Functional deficits were assessed at each time point using a modified neurologic deficit scale. Brains were analyzed using immunofluorescence and immunohistochemical techniques.

Results: At 15 mins after insertion of the occluding device, the extent of the initial lesion on apparent diffusion coefficient (ADC) maps was similar between groups. Over the next 30 min of MCAO, however, there was a remarkable ADC recovery in the 3NPA group (Figure 1). During MCAO, preconditioned animals had fewer voxels with severely compromised CBF than controls. CBF on the unaffected side was lower in the 3NPA group during ischemia. A similarly low CBF was also found in 3NPA- preconditioned animals without stroke (data not shown). On day 14, infarcts were smaller and functional deficits less severe in the 3NPA group. However, rescue of neurons on the ischemic side was incomplete in 3NPA animals. There were no signs of tissue necrosis similar to controls, but in the ipsilateral striatum and layers II-III of the somatosensory cortex, selective neuronal death, gliosis and a mild inflammatory infiltrate were detected.

Figure 1

(A) Masks of the ischemic lesion from individual animals of the control or NPA-preconditioned group generated from ADC maps acquired 15 (upper panel) or 45 (lower panel) minutes after the onset of MCAO. Masks are overlaid onto anatomical (T2w) images. Regions of overlaps between individual masks are color coded. While the ischemic area is similar 15 and 45 mins after onset of the vascular occlusion in controls, there is a dramatic recovery in the NPA group. (B and C) Number of voxels with a significantly reduced ADC in both groups (mean +/−s.e. of mean). While the lesion depicted on ADC maps is of similar size 15 mins after start of the occlusion (B), it is much smaller in NPA treated animals at 45 mins.

Conclusions: Even with a large initial lesion on ADC maps, tissue recovery was substantial in 3NPA-preconditioned animals. This was associated with a better functional outcome. On MRI, the 3NPA group showed higher intra-ischemic residual CBF, rapid intra-ischemic ADC recovery, less structural damage over time (depicted on fractional anisotropy, T2-w and T1-w images) and lower CBF values on the unaffected hemisphere. The latter could indicate vascular/metabolic adjustments of ischemia tolerance. Thus, MRI might have the potential to discriminate ischemia tolerant from vulnerable brains.

777. The anti-inflammatory chemokine CXCL12, and its receptor CXCR4, in hypoxic preconditioning: expression changes and cell-specific roles in establishing ischemic tolerance

A. Stowe¹, A. Freie¹, R. Hu¹, R. Klein² and J. Gidday¹

¹Neurological Surgery; ²Internal Medicine, Washington University, School of Medicine, St Louis, Missouri, USA

Objectives: Preconditioning to mild systemic hypoxia two days prior to transient middle cerebral artery occlusion (tMCAo) significantly reduces injury. ¹ We found that a series of repetitive hypoxic preconditioning (RHP) stimuli extends the duration of ischemic tolerance for many weeks, concomitant with reductions in post-stroke leukocyte-endothelial adherence. ² As chemokines regulate inflammatory neutrophil chemotaxis, the present study investigated chemokine participation—specifically, the role of the anti-inflammatory chemokine CXCL12 (SDF-1) ³ —in establishing the ischemia-tolerant phenotype. We examined spatio-temporal responses of CXCL12α/β (two splice variants identified at the mRNA level only) and one of its receptors, CXCR4, to single hypoxic preconditioning (SHP) and RHP, and to tMCAo with and without prior RHP.

Methods: Adult, male SW/ND4 mice were sacrificed 6, 12, or 24 h, 2 d, or 2 wks after SHP (4 h, 8%O₂) or RHP (9 exposures over 2 wks; 8% or 11%O₂; 2 or 4 h). Additional mice were sacrificed 24 h after 60-mins tMCAo 2 d, or 2 or 4 wks after RHP (n = 6/group). Cortical whole brain homogenates and capillary-rich fractions (isolated by differential centrifugation) were analyzed for CXCL12/CXCR4 message and protein expression by rtPCR (n = 8/group) and immunoblot (n = 4/group), or brains were sectioned for cellular localization by immunohistochemistry (n = 3/group). One-way ANOVA determined significance (P<0.05).

Results: CXCL12α mRNA increased four-fold (P<0.05), and CXCR4 mRNA doubled (P<0.01), in whole brain 12 h after SHP, returning to baseline at 2 d, a time when capillary expression of both CXCL12α and CXCR4 mRNA transiently doubled (P<0.05). Neither whole brain nor capillary expression of CXCL12α or CXCR4 mRNA was elevated following RHP. SHP doubled CXCL12β mRNA (P<0.01) by 2d in brain, but not capillaries; following RHP, this elevated expression in whole brain was maintained through 2wks (P<0.05). RHP elevated brain CXCL12 protein at 2d and 2wks. CXCL12 and CXCR4 co-localized to neurons and endothelial cells, with strong peri-endothelial CXCL12 immunoreactivity after RHP. From 2d to 4wks following tMCAo, capillary CXCL12β mRNA, but not CXCL12α or CXCR4 mRNA, declined by half (P<0.05) in mice with RHP relative to untreated ischemic controls.

Conclusions: The tightly-coupled, transient upregulation of CXCL12α/CXCR4 mRNA expression ⁴ after SHP may initiate growth factor- and angiogenesis-related gene expression changes ³ that likely contribute to ischemic tolerance; lack of a similar upregulation following RHP could reflect tachyphylaxis. The sustained increases in CXCL12β mRNA and protein in whole brain, elevated through 2wks after RHP and localized to the peri-vascular space, may strengthen the blood-brain barrier prior to stroke. ⁵ Post-stroke reductions in capillary CXCL12β expression are consistent with an anti-inflammatory phenotype at the endothelial-blood interface, lowering leukocyte recruitment and endothelial adherence in preconditioned animals. ⁴ Thus, these changes support roles for this chemokine ligand/receptor pair in establishing and maintaining the anti-inflammatory, ischemia-tolerant phenotype. Causal studies with pharmacologic inhibitors and in conditional knockout mice are underway.

873. Preservation of blood-brain barrier integrity in ischemic tolerance by microvascular sphingosine kinase 2

B. Wacker, T. Park and J. Gidday

Neurosurgery, Washington University School of Medicine, St Louis, Missouri, USA

Background: Hypoxic preconditioning (HPC) provides protection against cerebral ischemia-induced neurovascular dysfunction and cell death in mice, ¹ allowing for the study of endogenous pathways of ischemic protection. Studies in isolated mouse hearts have shown that the ischemia-protective preconditioning mechanisms depend on sphingosine kinase (SphK)-1. ² Recent work in the brain documented a heterogeneous distribution of SphK isoform activity in normal and ischemic brain suggesting bioactive lipid signaling may differ from other organs. ³ Our preliminary studies indicate that, in the cerebral microvasculature, HPC leads to increased SphK2 protein expression, peaking 2 to 4 h after preconditioning, without a concomitant change in the protein levels of the SphK1 isoform.

Objectives: The present study was undertaken to document that increased microvascular SphK2 activity in response to HPC is vital to the mechanism of HPC-induced tolerance to focal cerebral ischemia. We also sought to confirm SphK2-dependent protection of the blood-brain barrier in the HPC-induced ischemia-tolerant brain.

Methods: Temporal changes in microvascular SphK activity were measured following HPC using an established assay. ⁴ The nonselective SphK inhibitor dimethylsphingosine (DMS, 0.33 mg/kg, i.v.) was administered to adult male Swiss-Webster ND4 mice by retroorbital injection 30 min prior to HPC (4 h of 8% oxygen, systemic). Two days later, mice were subjected to a 60-min transient middle cerebral artery occlusion (tMCAO) under halothane anesthesia by the intraluminal suture method. After 24 h of reperfusion, neurological deficit was determined, and infarct volume was measured using TTC staining in conjunction with ipsilateral hemispheric swelling. In a separate set of mice, neurological deficit and vasogenic edema (brain water content) were assessed at 24 h of reperfusion.

Results: Microvascular SphK2 activity increased following HPC, while SphK1 was unchanged (which paralleled the microvascular protein expression levels of these respective isoforms we observed earlier). Reductions in infarct volume, neurological deficit, hemispheric swelling, and vasogenic edema in HPC-treated mice were abrogated when SphK was inhibited during HPC. Administering the SphK2 agonist FTY720 (0.24 mg/kg, i.p.) as an HPC mimic did not promote ischemic tolerance alone, but when combined with HPC, provided even greater (DMS-reversible) protection of the neurovascular unit, suggesting that HPC-induced increases in SphK2 activity, not additional substrate, are needed as an induction signal for ischemic tolerance.

Conclusions: These findings indicate that sphingosine-1-phosphate, formed by hypoxia-sensitive increases in SphK2 activity, serves as a proximal signaling mediator that ultimately leads to alterations in gene expression and the promotion of an ischemia-tolerant vasculoprotective phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the blood-brain barrier and neurovascular unit in stroke.

881. Hypothermic preconditioning induces rapid tolerance to hypoxia in hippocampal slices: potential mediation by erythropoietin

N. Kreisman¹, L. Wooliscroft², C. Campbell³ and A. Scanduro⁴

¹Department of Physiology; ²Neuroscience Program; ³Department of Pharmacology; ⁴Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA

Objectives: Preconditioning is a procedure where exposure to a mild insult renders tissue less vulnerable to injury following a subsequent, more severe insult. Hypothermia is a conditioning procedure that has minimal risk of injury to brain tissue. This investigation had two major objectives:

• (1)

  determine whether preconditioning hippocampal slices with hypothermia could induce rapid tolerance to a subsequent episode of severe hypoxia, and

• (2)

  determine whether rapid tolerance to severe hypoxia is mediated by erythropoietin (EPO) and its downstream signaling cascade.

Methods: Sprague-Dawley rats were anesthetized with ether, decapitated, and the brain was placed in buffered saline at 0°C for 10 mins. 400 μm slices of hippocampus were cut with a manual chopper and slices were either placed in an interface recording chamber at 35°C to 36°C (normothermia) or in a preincubation chamber at 22°C to 23°C (hypothermia). Orthodromic population spikes evoked in CA1 were used to verify slice viability. Thereafter, the recording electrode was moved to the inner blade of the dentate gyrus to monitor the extracellular potential. Light transmittance (LT) was measured as an index of tissue swelling/depolarization either in the whole slice, using a dissecting microscope with a digital video camera attached to one eyepiece (Kreisman et al, 2000), or in the dentate gyrus with a photodiode. After two hours of incubation in 95% O₂+5% CO₂ (normoxia) normothermic slices were exposed to 10 min of hypoxia (95% N₂+5% CO₂). Normothermic slices were removed from the recording chamber and replaced with hypothermic slices, which were then incubated for 60 min at 35°C–36°C before being exposed to 10 min of hypoxia. Comparisons were made of the LT index of hypoxic swelling in normothermic slices versus slices preconditioned with hypothermia.

Results: Slices preconditioned with hypothermia (n = 11) had a 46.9% decrease in the LT index of swelling in response to 10 min of hypoxia, compared to normothermic slices (n = 12). Swelling was prevented completely in the CA3 region and parts of the dentate gyrus. The tolerance mediated by hypothermia was mimicked by bathing normothermic slices (n = 12) in recombinant rat EPO (10 ng/ml), which decreased the LT index of hypoxic swelling by 38.8% (P<0.01 by Student's T test). Wortmannin (0.1 μmol/L), which blocks activation of Akt by PI3 kinase (downstream of the EPO receptor) reduced the tolerance mediated by hypothermic conditioning, as indicated by a decrease in the LT index of hypoxic swelling from 44.7% to 17.1% (n = 10).

Conclusions:

• (1)

  Hypothermic preconditioning produces rapid tolerance to subsequent hypoxic injury in hippocampal slices.

• (2)

  EPO may mediate hypothermic tolerance.

• (3)

  EPO's putative protective effect involves signaling via Akt.

961. Dietary virgin olive oil reduce ischemia-reperfusion injury in rat brain in vivo

F. Mohagheghi¹, M. Bigdeli¹, B. Rasoulian² and A.A. Zeinallou³

¹Biological Science, Shahid Beheshti, Tehran; ²Lorestan University (Medical Sciences), Razi Herbal Medicines Research Center, Lorestan; ³Agricultural Science, Seed and Plant Instrument Institut, Karaj, Iran

Background: Recent studies suggest that dietary virgin olive oil results in ischemic tolerance to reduce brain ischemia injury in rat brain slices. In this research, we attempted to investigate effects of virgin olive oil pretreatment against ischemia- reperfusion injury to the rat brain tissue.

Methods: Experimental rats were treated orally with virgin olive oil for 30 days at 0.25, 0.5 and 0.75 ml/kg/day (doses A, B and C, respectively). Control group treated with saline for 30 days. At the end of day 30, each group was exposed to middle cerebral artery occlusion for 60 min. After 24 h reperfusion, neurologic deficit scores and infarct volume were measured in all groups.

Results: Median neurologic deficit scores (NDS) were reduced by virgin olive oil, being 2, 1, 1 and 2 in doses 0.25, 0.5, 0.75 and control group, respectively. Virgin olive oil reduced infarct volume (dose A 13%, dose B 41% and dose C 45% versus control group). Neurologic deficit scores and infarct volume were reduced in doses B and C in comparison to control group significantly.

Conclusion: virgin olive oil induces ischemic preconditioning via effects against ischemic injury in the brain tissue partly by monounsaturated fatty acids and antioxidant agent found in virgin olive oil.

973. Ischemic preconditioning mediated Cyclooxygenase-2 expression via nuclear factor Kappa-B activation: an in vitro study

E.J. Kim, A. Raval, N. Hirsch and M. Perez-Pinzon

Department of Neurology, University of Miami, Miami, Florida, USA

Objectives: Ischemic preconditioning (IPC) is an endogenous protective mechanism invoked by a brief, sublethal ischemic insult prior to a subsequent lethal ischemic insult. Nuclear Factor-kappaB (NF-kappaB) activation occurs following IPC in brain. ¹ However, the upstream signaling messengers and down-stream targets of NF-kappaB required for induction of IPC remain undefined. Previously, we demonstrated that epsilon protein kinase c (ePKC) is the key mediator of IPC- and ePKC-induced cyclooygenase-2 (COX-2) expression in the in vitro models. ² Here, we hypothesized that IPC-mediated COX-2 expression was regulated by NF-kappaB.

Method: Mixed cortical neuron/astrocyte cell cultures were prepared from rat embryo/neonatal rat (18-19/1-2 days old), respectively. To simulate preconditioning or ischemia, cell cultures were exposed to 1 h or 4 h of oxygen-glucose deprivation (OGD), respectively. Epsilon PKC agonist peptide (100 nmol/L) was applied to cell culture for 1 h. Cell lysates were isolated at different time points after IPC or ePKC agonist treatment for immunoblotting with antibody against p65 and p50 subunits of NF-kappaB. To determine assessment of cell death, cytotoxicity was measured by lactate dehydrogenase (LDH) released for 48 h into culture medium. Maximal neuronal LDH release was measured in neuronal cultures exposed to NMDA (500 μmol/L; 48 h; maximal neuronal death). LDH release was measured by absorbance at 340 nm using a microplate reader (Molecular Devices, CA). Values were expressed relative to LDH measurement from maximal neuronal LDH. Results are expressed as mean±SEM. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.

Results: Translocation of p65 and p50 subunit of NF-kappaB to the nucleus occurred at 15 min after IPC and lasted for 2 h. To verify whether NF-kappaB activation was required for IPC-induced neuroprotection, NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC, 10 μmol/L), was applied during IPC. Forty-eight hours later, cells were exposed to OGD (4 h) and neuronal cell death was measured. Results showed that IPC reduced the neuronal death by 30% as compared to ischemic injury (45%±2% versus 75%±1.8% of maximal cell death; IPC versus OGD, P<0.05, n = 20) and PDTC treatment during IPC significantly abolished IPC-induced neuroprotection (45%±2% versus 65.7%±2.5% of maximal cell death: IPC versus IPC+PDTC+OGD, P<0.05, n = 20). In parallel, inhibition of either ePKC or the ERK 1/2 pathway reduced IPC-induced NF-kappaB activation. To determine whether NF-kappaB activation was involved in IPC-induced COX-2 expression in neurons, cells were treated with NF-kappaB inhibitor during IPC. Results demonstrated that NF-kappaB inhibition significantly reduced IPC-induced COX-2 expression (0.6±0.2 versus 0.15±0.02 optical density: IPC versus IPC+PDTC, P<0.05 compared with IPC, n = 5).

Conclusion: We demonstrated that IPC-signaling activates NF-kappaB via ePKC and ERK1/2 pathway and translocation of NF-kappaB to nucleus mediates COX-2 expression, resulting in neuroprotection in an in vitro model.

Grant support: PHS grants NS34773, NS054147, NS045676, NS05820.

1040. Preconditioning with ozone/oxygen mixture presented improved histopathological protection compared to hyperbaric oxygen treatment in a rat global cerebral ischemia model

S. Oter¹, E. Oztas², M. Seyrek³, B. Uysal¹, B. Duz⁴, M. Kaplan^4,5, A. Korkmaz¹, R. Ogur⁶ and S. Kahraman⁴

¹Physiology; ²Histology and Embryology; ³Pharmacology; ⁴Neurosurgery, Gulhane Military Medical Academy, Ankara; ⁵Neurosurgery, Firat University, Medical School, Elazig; ⁶Public Health, Gulhane Military Medical Academy, Ankara, Turkey

Objective: Hyperbaric oxygen (HBO) therapy is an accepted treatment alternative for preconditioning in both focal ¹ and global ² cerebral ischemia. Medical ozone therapy, a new therapeutic approach, has been reported similar molecular mechanisms to HBO in ischemic conditions. ³ One mechanism of the protective effects of HBO against central nervous system ischemia was explained with heme oxygenase-1 (HO-1) induction. ⁴ Interestingly, HO-1 is also known to play role in ozone/oxygen applications ³ and seems to be an intersection point among these two treatment modalities. ⁵ In the present study we compared HBO and ozone/oxygen pretreatments in the 2-VO rat model. ⁶

Methods: Sprague-Dawley rats were divided into 4 groups: control, global ischemia, HBO and ozone/oxygen. HBO was administered one time a day at 2,5 atmosphere/1,5 h sessions. Daily intraperitoneally ozone/oxygen (5%/95%) injections at a dose estimated to supply 0.7 mg/kg-BW of ozone. Twentyfour hours after the third applications of HBO or ozone/oxygen, global cerebral ischemia (GI) was induced via bilateral carotid artery occlusion for 10 min. Prior to sacrificing, the neurological deficit was evaluated by a blinded expert usind the Garcia score. ⁷ The rats were then sacrificed 24 h following GI induction and their brains were taken for for histopathological evaluation, namely Nissl and Tunel staining, and determination of HO-1 mRNA expression.

Figure 1

Representative photos of Nissl-stained slices.

Results: Functional evaluation according to Garcia's method resulted with comparable scores; only a slight but insignificant improvement was observed. HO-1 was found to be depressed in GI group, whereas both HBO and ozone/oxygen treatments induced increased transcripton of HO-1 mRNA with nearly the same rates.

In the Nissl-stained slices, neuronal tissue loss was apparent in the hippocampal and cortical regions of GI animals. Pretreatment with HBO was able to reduce this injury, whereas in ozone/oxygen treated animals more protection was observed. Strong TUNEL-positive staining appeared in the hippocampus and cortex after GI. Both HBO and ozone/oxygen pretreatment reduced TUNEL-positive staining in neuronal tissue, of which ozone/oxygen mixture was significantly more effective.

Conclusion: The present findings suggest that pretreatment with ozone/oxygen mixture, at least from the histopathological viewpoint, may exert more satisfying outcome than HBO. Therefore, this new treatment modality have to be taken into consideration and further detailed studies have to be performed on this area.

Grant: This study was supported by the Gulhane Military Medical Academy Research and Progress Center with the grant Nr.AR-2003/56.