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The Journal of Neuroscience, January 15, 2003, 23(2):384-391

PKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

Ami P. Raval¹, Kunjan R. Dave¹, Daria Mochly-Rosen², Thomas J. Sick¹, and Miguel A. Pérez-Pinzón¹

¹ Cerebral Vascular Disease Research Center, Department of Neurology and Neuroscience, University of Miami School of Medicine, Miami, Florida 33101, and ² Division of Chemical Biology, Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C  (PKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited PKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of PKC. The possible correlation between NMDA, Ca²⁺, and PKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca²⁺ could activate the calcium-insensitive PKC isozyme. These results demonstrated that the PKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

Key words: metabolism; in vitro cultures; glutamate receptors; anoxia; tolerance; signal transduction

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Ischemic preconditioning is an intrinsic adaptive condition that results in tolerance in different organs when they are subjected to mild ischemic insults before a "lethal" ischemic insult. The exact mechanism underlying ischemic preconditioning (IPC)-induced tolerance remains unclear, although a number of possible induction pathways have been investigated, including, among others, neuroactive cytokines (Nawashiro et al., 1996; Ohtsuki et al., 1996), activation of glutamate receptors (Best et al., 1996; Pringle et al., 1996), adenosine receptors, the ATP-sensitive potassium channel (K⁺_ATP) (Heurteaux et al., 1995; Perez-Pinzon et al., 1996, 1999; Riepe et al., 1997; Blondeau et al., 2000), nitric oxide (Caggiano and Kraig, 1998; Centeno et al., 1999; Gonzalez-Zulueta et al., 2000), oxidative stress (Ohtsuki et al., 1992), hypothermia (Nishio et al., 2000), and hyperthermia (Chopp et al., 1989; Rordorff et al., 1991).

It now appears that almost any sublethal stress may render cells tolerant against lethal stress. For example, it has been established that overstimulation of glutamate receptors promotes neuronal cell death (Rothman and Olney, 1986; Albers et al., 1992; Choi, 1995; Pellegrini-Giampietro et al., 1999), whereas blocking NMDA receptors during ischemia appears to be neuroprotective (Simon et al., 1984; Goldberg et al., 1987; Ozyurt et al., 1988; Steinberg et al., 1988; Swan and Meldrum, 1990; Rao et al., 2000; Bruno et al., 2001; Liniger et al., 2001; Nishizawa, 2001). However, MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], an NMDA antagonist, administered during sublethal ischemia blocked the development of ischemic tolerance in gerbils (Kato et al., 1992a). In vitro studies also supported the role of NMDA receptors during IPC but not kainate or AMPA receptors (Bond et al., 1999; Grabb and Choi, 1999).

Subsequent increases of cytosolic calcium result from NMDA receptor activation during IPC, and this Ca²⁺ increase may promote a signal transduction cascade. It has been suggested that a putative neuroprotective pathway may involve a calcium-induced activation of PKC, because PKC translocation and phosphorylation of several membrane proteins are mediated by NMDA receptors through calcium influx (Vaccarino et al., 1991). Strong evidence exists of the involvement of PKC in the induction of IPC tolerance in the heart (Downey et al., 1994). In brain, however, different preconditioning models have shown contradictory results (Perez-Pinzon and Born, 1999; Tauskela et al., 1999; Reshef et al., 2000).

We reported recently that sublethal in vitro ischemia in organotypic hippocampal slice cultures protects against neuronal cell death produced by lethal in vitro ischemia (Xu et al., 2002). The present study, using the organotypic slice cultures, investigates three issues concerning the mechanism of IPC: (1) whether the NMDA receptors are involved in the triggering phase of IPC via calcium, (2) whether the PKC isozymes are involved in induction of neuroprotection, and (3) whether PKC is involved in the signaling pathway of IPC neuroprotection as shown in the heart (Souroujon and Mochly-Rosen, 1998).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Preparation of cultures

All of the protocols were approved by the University of Miami Animal Care and Use Committee. Organotypic slice cultures of the hippocampus were made according to the methods described by Bergold and Casaccia-Bonnefil (1997). Sprague Dawley neonatal rats (9-11 d old) were anesthetized by intraperitoneal injections of ketamine (1.0 mg/pup). The pups were decapitated, and the hippocampi were dissected out and sliced transversely (400 µm) on a McIlwain tissue chopper. Slices were placed in Gey's balanced salt solution (Invitrogen, San Diego, CA) supplemented with 6.5 mg/ml glucose (Sigma, St. Louis, MO) for 1 hr at 4°C. They were then transferred onto 30-mm-diameter membrane inserts (Millicell-CM; Millipore, Bedford, MA). Each insert had two slices obtained from two different pups. The inserts were placed into six-well culture trays with 1 ml of slice culture medium per well. The slice culture medium consisted of 50% minimum essential medium (Invitrogen), 25% HBSS (Invitrogen), and 25% heat-inactivated horse serum (Invitrogen) supplemented with 6.5 mg/ml glucose and glutamine (1 mM). The cultures were maintained at 36°C in an incubator (CF autoflow; NuAire, Plymouth, MN) with an atmosphere of 100% humidity and 5% CO₂. The slice culture medium was changed twice per week. Slices were kept in culture for 14-15 d before experiments.

Oxygen-glucose deprivation

We defined the ischemia and preconditioning protocols in a previous study (Xu et al., 2002). The organotypic cultures have been used to study mechanisms underlying neuronal death induced by hypoxia-aglycemia (Pringle et al., 1997a) and excitotoxins (Sakaguchi et al., 1997). To model ischemic events, organotypic cultures were exposed to oxygen-glucose deprivation (OGD) using an anaerobic chamber. Cimarosti et al. (2001) and Laake et al. (1999) suggested the suitability of this model for the study of ischemic lesions and neuroprotective drugs. They observed that the lesions induced by OGD were similar to those shown by animals submitted to transient cerebral ischemia. We corroborated recently that, like global cerebral ischemia, OGD promotes selective cell death in the CA1 subregion of the hippocampus (Xu et al., 2002). The slices were washed three times with glucose-free HBSS, pH 7.4, containing the following (in mM): 1.26 CaCl₂ · 2 H₂O, 5.37 KCl, 0.44 KH₂PO₄, 0.49 MgCl₂, 0.41 MgSO₄ · 7 H₂O, 136.9 NaCl, 4.17 NaHCO₃, 0.34 Na₂HPO₄ · 7 H₂O, and 15 sucrose (all from Sigma). The slices were then placed into an airtight chamber, and 95% N₂-5% CO₂ gas, preheated to 36°C, was blown through the chamber for 5 min (4 l/min) to achieve anoxic conditions. After 5 min, the chamber was sealed and placed in the incubator, in which the temperature was maintained at 36°C, for 10 min (for a total of 15 min for preconditioning) or 35 min (for a total of 40 min for ischemic insult). After OGD, the slices were placed back into the incubator in normal culture medium.

Assessment of cell death by image analysis using propidium iodide

Propidium iodide (PI) (Sigma) was used for identifying dead cells. Before experimental treatment (OGD or preconditioning), slices were incubated in culture medium supplemented with 2 µg/ml PI for 1 hr and then removed and replaced by regular media. The slices were studied using an inverted fluorescence microscope (Olympus IX 50; Olympus Optical, Tokyo, Japan). Fluorescence digital pictures were taken using a SPOT charge-coupled device (CCD) camera (Diagnostic Instruments, Sterling Heights, MI) and SPOT advanced software. The bright-field (0.254 sec exposure time) and PI (1.902 sec exposure time, red filter) images of cultured slices were taken before the experiment, followed by preconditioning treatment. After preconditioning, the slices were reperfused for 48 hr followed by 40 min of "test" ischemia. The PI fluorescence was taken 24 hr after test ischemic insult, and then NMDA was added onto the slices (100 µM) (Sigma) for 1 hr. The last image was taken 24 hr after NMDA treatment. The bright-field image was used to align the slice in the same position during subsequent measurements. The intensity of PI fluorescence in the CA1 subfield of the hippocampal slices was used as an index of cell death.

For quantification purposes, a set of fluorescence images taken for one slice was opened in the following sequence: (1) 24 hr after NMDA treatment, (2) 24 hr after test ischemia, and (3) obtained at the onset of the experiment. They were then stacked using Scion Image software (Windows version) (Scion, Frederick, MD). The region of interest (ROI) was selected from the final image depicting total neuronal cell death, which remained constant for a particular slice. The ROI was subsequently superimposed on the fluorescence images taken 24 hr after test ischemia and at the beginning of the experiment. Relative cell death was calculated from each ROI as follows: relative percentage cell death = (F_exp  F_min)/(F_max  F_min) × 100, where F_exp is the fluorescence of the test condition, F_max is maximum fluorescence (100 µM NMDA treatment for 1 hr), and F_min is background fluorescence (before preconditioning or OGD). In all groups, experiments (except Western blot analysis) were terminated by superfusing slices with an overdose of NMDA 24 hr after the end of the experiments to determine total number of cells using the PI technique (F_max above).

Cell fractionation and Western blot analysis

To determine whether the PKC isozyme was translocated after IPC in organotypic slices, we used Western blot analysis. This method is adapted from one described previously (Mackay and Mochly-Rosen, 2001). Hippocampal organotypic slices were frozen in liquid nitrogen at different time intervals and stored at 80°C until the analysis. At the time of Western blot analysis, the hippocampal organotypic slice cultures were separated from the supporting membrane. For one sample, ~32 slices were pooled together. The separated pooled slices were washed twice with cold PBS. Slices were pelleted at 1000 × g, and PBS was removed. The pellet was resuspended in 400 µl of cell lysis buffer (4 mM ATP, 100 mM KCl, 10 mM imidazole, 2 mM EGTA, 1 mM MgCl₂, 20% glycerol, 0.05% Triton X-100, 17 µg/ml PMSF, 20 µg/ml soybean trypsin inhibitor, 25 µg/ml leupeptin, and 25 µg/ml aprotinin). The suspended slices were homogenized using an all-glass homogenizer. The homogenate was then centrifuged at 4°C at 1000 × g for 10 min. The supernatant (soluble fraction) was carefully taken off and recentrifuged at 16,000 × g for 15 min to exclude any contaminating pellet material. The initial pellet was resuspended in 250 µl of cell lysis buffer containing 1% Triton X-100 and was extracted on ice for 60 min. Samples were centrifuged at 16,000 × g for 15 min. The supernatant is the particulate fraction. Both fractions were analyzed for protein content by the Bradford assay, and 40 µg of protein from each fraction was separated by 12% SDS-PAGE. One soluble and one particulate fraction of each group were run on the same gel and analyzed at the same time. Each group consisted of three samples. Protein was transferred to Immobilon-P (Millipore) membrane and incubated with the primary antibody anti-PKC (Calbiochem, La Jolla, CA) (1:500). In the case of phorbol myristate acetate (PMA), the translocation of two additional isozymes of PKC (i.e., PKC and PKC) was also determined by blotting the membrane with primary antibody anti-PKC (1:1000) and anti-PKC (1:500) (Calbiochem). Immunoreactivity was detected using enhanced chemiluminescence (ECL Western blotting detection kit; Amersham Biosciences, Little Chalfont, UK). Autoradiographic images were digitized at eight-bit precision by means of a CCD-based camera (8-12 bits) (Xillix Technologies, Vancouver, British Columbia, Canada) equipped with a 55 mm Micro-Nikkor lens (Nikon, Tokyo, Japan). The camera was interfaced to an advanced image-analysis system (MCID model M2; Imaging Research, St. Catherines, Ontario, Canada). The digitized immunoblots were subjected to densitometric analysis using MCID software.

Experimental design

The organotypic slices were divided into five major groups (Fig. 1), as follows.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Description of different experimental groups. Group-I, Sham; Group-II, ischemia with any other treatment; Group-III, preconditioning experiments; Group-IV, pharmacological preconditioning (PPC) with NMDA, PMA, RACK, and OAG; Group-V, experimental design of pharmacological blockade preconditioning (PC) using MK-801, BAPTA AM, BAPTA, chelerythrine chloride, and V1-2.

Group I: sham

These slices were incubated for 15 min with HBSS supplied with equimolar concentrations of glucose instead of sucrose (sham OGD). Subsequently, slices were transferred back to regular media. After 48 hr, the slices were exposed for 40 min of sham OGD.

Group II: ischemia

Slices were exposed to sham IPC (group I), and, 48 hr later, test ischemia (40 min of OGD) was induced.

Group III: IPC

Slices were exposed to IPC (15 min of OGD) 48 hr before test ischemia.

Group IV: pharmacological emulation of IPC

NMDA treatment. NMDA at 1 µM (Sigma) was administered onto slices for 15, 30, and 60 min. Subsequently, the drug was washed out, and, after 48 hr, test ischemia was induced.

PMA-induced translocation of PKCs. PMA at 1 µM (Sigma) was administered onto slices for 30 min. Subsequently, the drug was washed out, and, after 48 hr, test ischemia was induced.

Pharmacological preconditioning using RACK (a PKC agonist). In this group, RACK (receptor for activated C kinase) was administered onto slices for 15 min using different concentrations (0.02, 0.2, 0.6, and 1 µM). The drug was washed out, and, after 48 hr, test ischemia was induced.

Pharmacological preconditioning with oleoylacetyl glycerol. Oleoylacetyl glycerol (OAG) (10 nM; Sigma), an analog of diacylglycerol (DAG), was administered onto slices for 15 min. Subsequently, the drug was washed out, and, after 48 hr, test ischemia was induced.

Group V: pharmacological blockade of IPC

Blocking NMDA receptors with MK-801 maleate. Slices were bathed with MK-801 (10 µM; Sigma) either during IPC alone or for 30 min before and during IPC (to ensure that the NMDA receptor was blocked during IPC). The drug was washed out at the end of IPC, and, after 48 hr, test ischemia was induced.

Treatment with calcium chelators. Slices were divided into three subgroups: (1) slices were bathed with the cytoplasmic calcium chelator BAPTA AM (100 µM; Sigma) for 45 min and during IPC; (2) slices were treated in a similar manner as in 1 but using the extracellular BAPTA (10 µM; Sigma) instead of the cytoplasmic calcium chelator; and (3) slices were bathed with both intracellular and cytoplasmic calcium chelators BAPTA AM for 45 min and during IPC. Subsequently, the drug was washed out, and, after 48 hr, test ischemia was induced.

Calcium chelators and NMDA preconditioning. In this group, slices were bathed with both BAPTA AM (100 µM) and BAPTA (10 µM) for 45 min. Subsequently, slices were bathed with 1 µM NMDA in the presence of both calcium chelators for 1 hr. The drugs were washed out, and, after 48 hr, test ischemia was induced.

Blocking of PKC activation by chelerythrine chloride. Slices were bathed with chelerythrine (10 µM) during IPC. The drug was washed out at the end of IPC, and, after 48 hr, test ischemia was induced.

Blocking of PKC isozyme activation with V1-2. Slices were bathed with V1-2 (0.02, 0.2, 0.6, and 1 µM) during IPC. The drug was washed out at the end of IPC, and, after 48 hr, test ischemia was induced.

NMDA preconditioning along with blockade of PKC. The slices were preconditioned for 60 min with NMDA (1 µM) along with V1-2 (0.02 µM). The drug was washed out at the end of NMDA preconditioning, and, after 48 hr, test ischemia was induced.

OAG preconditioning along with blockade of PKC. The slices were bathed for 15 min with OAG (10 nM) in the presence of V1-2 (0.02 µM).

To provide better controls in these experiments, all six-well plates used contained at least one well for the sham, IPC, and ischemic groups. In addition, for statistical purposes, each insert had two slices obtained from two different pups. Thus, n = 1 (one slice) represents a different animal.

Peptide preparation and delivery

The V1-2 [PKC inhibitor, amino acids 14-21 (EAVSLKPT)] (Gray et al., 1997) and RACK [PKC activator, amino acids 85-92 (HDAPIGYD)] (Dorn et al., 1999) peptides were synthesized at Stanford's Protein and Nucleic Acid facility and conjugated to Tat [carrier peptide, amino acids 47-57 (YGRKKRRQRRR)] (Schwarze et al., 1999) via a cysteine-cysteine bond at their N termini, as described previously (Chen et al., 2001).

In the past, peptides have been delivered initially into the cells by transient permeabilization of the cells or by microinjection. However, an alternative to this method was used recently in which the peptides are conjugated via disulfide bonds to short cell-permeable peptides (Souroujon and Mochly-Rosen, 1998). Once the peptide crosses the cell membrane, the disulfide bond breaks, the peptide cargo is trapped in the cell, and the peptide carrier can be washed out. These studies already demonstrated that the peptides have highly selective effects; they inhibit or induce translocation of their corresponding isozymes but not that of others (Gray et al., 1997; Dorn et al., 1999). In addition, at concentrations up to ~10 µM, the peptides or the carrier have no toxic effects on the cell. Finally, peptides are introduced to all of the cells at a final concentration that does not exceed 10% of that applied (Souroujon and Mochly-Rosen, 1998).

Statistical analysis

The results are expressed as mean ± SD. Statistical significance was determined with an ANOVA test, followed by a Bonferroni's post hoc test.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In the present study, we confirm our previous findings in which IPC conferred protection against test ischemia in hippocampal organotypic slices (Fig. 2). The hippocampal organotypic slices that were preconditioned by 15 min of sublethal ischemia 48 hr before 40 min of test ischemia showed 34.13 ± 19.25% (n = 20) of PI fluorescence in the CA1 region of the hippocampus compared with sham (5.45 ± 2.85%; n = 8; p < 0.01). In contrast, 40 min of test ischemia led to 59.64 ± 20.82% (n = 24) of PI fluorescence (Fig. 3). Thus, IPC significantly lowered cell death by ~42% (p < 0.01).

View larger version (124K): [in this window] [in a new window]   Figure 2.   Typical images of hippocampal slice cultures. A, C, Bright-field images of ischemic and IPC groups, respectively. B, D, PI fluorescence images showing neuronal death in the ischemic and IPC groups taken 24 hr after the test ischemic insult. Scale bars, 2.25 mm.

View larger version (16K): [in this window] [in a new window]   Figure 3.   PI fluorescence values measured 24 hr after test ischemia in seven experimental groups: (1) sham, (2) ischemia, (3) IPC, (4) NMDA-induced preconditioning, (5) IPC with MK-801, (6) calcium chelators with NMDA preconditioning, and (7) calcium chelators with IPC. Significance compared with sham (Ap < 0.01; ap < 0.001), ischemia (Bp < 0.01; bp < 0.001), or IPC (Cp < 0.01; cp < 0.001).

One of the suggested mechanisms by which IPC promotes ischemic tolerance in other animal models of cerebral ischemia is by activation of the NMDA receptor. We tested this hypothesis in the organotypic cultures by bathing slices with NMDA (1 µM) alone for 15 min at 48 hr before the test ischemic insult, which resulted in 42.67 ± 17.05% (n = 16) of PI fluorescence. This level of PI fluorescence was significantly lower than in test ischemia (p < 0.05). Increasing the time of NMDA exposure to 30 or 60 min further reduced percentage PI fluorescence by ~34% (n = 8) and 69% (n = 5), respectively, compared with test ischemia (p < 0.001).

To further characterize the role of the NMDA receptor during IPC, the NMDA receptor was blocked (MK-801, 10 µM) during the preconditioning insult. This treatment significantly eliminated IPC-induced neuroprotection. The PI fluorescence values were 87.25 ± 12.43% (n = 7) and 59.64 ± 20.82% for MK-801-treated and ischemic groups, respectively. Similar results were obtained when the NMDA receptor was blocked before and during the IPC insult (Fig. 3). In control experiments, when MK-801 was administered to slices without any other treatment, percentage PI fluorescence was 55.43 ± 13.65% (n = 5) of PI fluorescence, similar to that of ischemia.

A suggested pathway by which NMDA promotes ischemic tolerance is by influx of Ca²⁺ into the cytoplasm. To determine whether influx of calcium plays a role in preconditioning-induced neuroprotection in the organotypic slice, we removed both extracellular and cytoplasmic calcium using calcium chelators. Both extracellular and intracellular calcium chelators (BAPTA and BAPTA AM) administered before and during IPC blocked the protection afforded by IPC (p < 0.001) (Fig. 3). Furthermore, chelation of either extracellular or cytoplasmic calcium alone before and during IPC followed by OGD 48 hr later resulted in 62.09 ± 13.65% (n = 8) or 75.01 ± 10.43% (n = 8) of PI fluorescence values, respectively, which were significantly higher than in the IPC group (p < 0.01 and p < 0.001, respectively) (Fig. 3). Chelation of either extracellular or cytoplasmic calcium alone followed by OGD 48 hr later resulted in 54.84 ± 5.96% (n = 4) and 60.68 ± 7.10% (n = 4) of PI fluorescence, respectively.

In control experiments, extracellular or cytoplasmic calcium chelation without any OGD insult resulted in 3.24 ± 1.13% (n = 5) and 4.86 ± 0.39% (n = 5) of PI fluorescence, respectively. No significant increases in PI fluorescence were observed with these treatments compared with sham values.

To examine whether NMDA-induced preconditioning also occurred via calcium influx, we chelated calcium during NMDA preconditioning. The ischemic tolerance afforded by NMDA pretreatment was abolished by bathing hippocampal slices with calcium chelators before and during NMDA-induced preconditioning (p < 0.001) (Fig. 3). The PI fluorescence values of calcium-chelated NMDA-preconditioned and ischemic groups were 72.14 ± 9.71% (n = 11) and 59.64 ± 20.85%, respectively (Fig. 3).

An increase in cytosolic calcium by itself can directly and indirectly activate PKC isozymes (Downey et al., 1994). Thus, we examined whether hippocampal organotypic slices could be preconditioned by PMA (1 µM), a nonselective PKC isozyme translocator. The percentage cell death (PI fluorescence) in the CA1 region of the hippocampus was 87.34 ± 15.43% (n = 10), 67.66 ± 30.77% (n = 7), 28.90 ± 17.35% (n = 10), and 5.45 ± 2.85% for the PMA-treated, ischemia, IPC, and sham groups, respectively. The ischemia and PMA-treated groups were significantly higher than both the sham and IPC groups (p < 0.001) (Fig. 4), demonstrating that this nonselective PKC translocation did not emulate IPC. Next, PKC activity was blocked with chelerythrine (nonselective inhibitor of PKC) (10 µM) during the preconditioning insult. IPC-induced neuroprotection was partially reduced by this treatment (Fig. 4). The PI fluorescence values were 49.03 ± 11.08% (n = 14) and 67.66 ± 30.77% for the chelerythrine-treated and ischemia groups, respectively (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Figure 4.   PI fluorescence values measured 24 hr after test ischemia in five experimental groups: (1) sham, (2) ischemia, (3) IPC, (4) PMA, and (5) chelerythrine (CHL) plus IPC. Significance compared with sham (Ap < 0.01; ap < 0.001), ischemia (Bp < 0.01; bp < 0.001), or IPC (Cp < 0.01; cp < 0.001).

The PMA and chelerythrine results were in contradiction to each other. A plausible explanation was that different PKC isozymes played different roles during IPC. Thus, we explored the possible role of PKC isozyme that is key for ischemic tolerance in the heart (Souroujon and Mochly-Rosen, 1998). Slices were bathed with the specific blocker of PKC (V1-2 peptide at different concentrations, i.e., 0.02, 0.2, 0.6, and 1 µM) during IPC. This treatment significantly blocked IPC-induced neuronal protection at all of the concentrations used (p < 0.01). The PI fluorescence was 64.85 ± 15.46% (n = 8) with V1-2 peptide (0.02 µM) treatment compared with IPC (28.90 ± 17.35%; n = 10) (Fig. 5).

View larger version (12K): [in this window] [in a new window]   Figure 5.   PI fluorescence values measured 24 hr after test ischemia in five experimental groups: (1) sham, (2) ischemia, (3) IPC, (4) V1-2 (0.02 µM) treatment during IPC, and (5) preconditioning with PKC activator peptide RACK (0.02 µM). Significance compared with sham (Ap < 0.01; ap < 0.001), ischemia (Bp < 0.01; bp < 0.001), or IPC (Cp < 0.01; cp < 0.001).

To further corroborate the role of PKC, we emulated IPC with the RACK peptide (agonist of PKC) for 15 min at different concentrations (i.e., 0.02, 0.2, 0.6, and 1 µM). All of these treatments resulted in significant neuroprotection compared with the ischemia group (p < 0.001). In control experiments, the Tat carrier peptide administered for 15 min at 48 hr before OGD at different concentrations did not promote any significant protection compared with the ischemia group. The PI fluorescence values of Tat peptide treatment at 0.02, 0.2, 0.6, and 1 µM concentrations were 61.44 ± 11.15% (n = 5), 63.24 ± 8.38% (n = 4), 65.48 ± 5.24% (n = 4), and 60.84 ± 7.76% (n = 6), respectively.

To determine whether PKC was also involved during NMDA-induced preconditioning, the PKC inhibitor (V1-2 peptide, 0.02 µM) was administered during the NMDA preconditioning treatment. This treatment resulted in significant inhibition of protection (p < 0.001) compared with IPC and NMDA preconditioning. The PI fluorescence values of NMDA preconditioning along with the V1-2 peptide group and IPC- and NMDA-preconditioned groups were 82.09 ± 7.91% (n = 7), 28.90 ± 17.35% (n = 9), and 21.44 ± 10.97% (n = 6), respectively (Fig. 6).

View larger version (15K): [in this window] [in a new window]   Figure 6.   PI fluorescence values measured 24 hr after test ischemia in seven experimental groups: (1) sham, (2) ischemia, (3) IPC, (4) NMDA (1 µM) preconditioning for 60 min, (5) NMDA preconditioning with V1-2 (0.02 µM), (6) OAG treatment for 15 min, and (7) OAG with V1-2. Significance compared with sham (Ap < 0.01; ap < 0.001), ischemia (Bp < 0.01; bp < 0.001), IPC (Cp < 0.01; cp < 0.001), or NMDA (Dp < 0.01).

Activation of PKC is associated with translocation from the soluble to the particulate fraction of cells (Kraft and Anderson, 1983; Dorn et al., 1999). Therefore, to confirm that IPC translocates PKC isozyme in hippocampal organotypic slices, the effect of IPC on subcellular localization of PKC isozyme was determined by cell fractionation and Western blot analysis. The subcellular fraction method was used to separate proteins into a soluble and a particulate fraction. PKC isozyme translocated in response to PMA (1 mM, 30 min) from the soluble to the particulate fraction (Fig. 7C). Relative to the control group, PKC decreased in the soluble fraction by 43%, with a corresponding increase in the particulate fraction. We also confirmed that incubation with PMA (1 mM, 30 min) translocated other PKC isozymes, such as PKC and PKC isozymes. We found that the PKC and PKC isozymes also translocated from the soluble to the particulate fraction. PKC and PKC isozymes decreased in the soluble fraction by 25 and 35%, respectively, with a corresponding increase in the particulate fraction (Fig. 7C). The PKC isozyme translocated from the soluble to the particulate fraction by 7, 28 (p < 0.05), 24, and 8% after 15, 30, 60, and 180 min of reperfusion after IPC, respectively, with a corresponding increase in the particulate fraction. The PKC isozyme concentration remained the same in the soluble and particulate fractions after 60 min of reperfusion when IPC was performed in presence of chelerythrine. Administration of the specific PKC antagonist V1-2 during IPC inhibited translocation of PKC (Fig. 7).

View larger version (28K): [in this window] [in a new window]   Figure 7.   A, Immunoblot of PKC isozyme in control baseline (untreated) and 15 min, 30 min, 1 hr, and 3 hr after IPC. Organotypic slices underwent subcellular fractionation and Western blot analysis, probed with antibodies detecting the PKC isozyme. The soluble and particulate fractions are shown. One experiment representative of three is shown. B, Immunoblots as represented in A were subjected to densitometric analysis, and percentage translocation from soluble to particulate fraction was determined. Results are expressed as percentage of total PKC in soluble fraction for each sample, which was then expressed as percentage of control. For a decrease in soluble fraction, there is a corresponding increase in particulate (data not shown). Results are expressed as mean ± SD of three experiments. Ap < 0.05 versus control. C, Immunoblots blotted with anti-PKC, anti-PKC, and anti-PKC of soluble and particulate fraction of organotypic slices harvested 30 min after exposure to PMA were subjected to densitometric analysis, and percentage translocation from soluble to particulate fraction was determined. Results are expressed as percentage of total PKC, PKC, and PKC in soluble fraction after PMA treatment, which was then expressed as percentage of control. For a decrease in soluble fraction, there is a corresponding increase in particulate (data not shown). Results are expressed as mean ± SD of three experiments.

PKC belongs to the Ca²⁺-independent type of PKC isozymes, and NMDA-induced tolerance was blocked by the analog of PKC; thus, we tested the hypothesis that NMDA plus PKC was indirectly linked by DAG, which is produced by Ca²⁺-stimulated phospholipase C activation (Nishizuka, 1992). Slices were preconditioned with the DAG analog OAG. OAG pretreatment provided protection from ischemia similar to that afforded by IPC. PI fluorescence values for OAG and IPC were 12.25 ± 5.77% (n = 5) and 28.90 ± 17.35% (n = 9), respectively. The PKC inhibitor V1-2 blocked protection afforded by OAG. The PI fluorescence was 67.99 ± 22.11% (n = 5) when OAG and V1-2 were administered together 48 hr before test ischemia.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In this study, we demonstrated that IPC 48 hr before the test ischemic insult induced neuroprotection in the CA1 region of the hippocampal organotypic slices. We also showed that preconditioning by activation of sublethal NMDA receptor was highly neuroprotective, whereas antagonizing the NMDA receptor by MK-801 blocked the protection. We further demonstrated that calcium chelation prevented both IPC- and NMDA-mediated neuroprotection, suggesting that increased cytosolic calcium plays a key role in the induction of IPC and NMDA neuroprotection. Pharmacological preconditioning with the PKC activator PMA could not emulate IPC neuroprotection, whereas chelerythrine blocked the preconditioning effect, suggesting that different PKC isozymes play different roles. This was corroborated by our findings that we could emulate IPC neuroprotection with the agonist of PKC (RACK) and block it with its antagonist V1-2. This result demonstrates that the preconditioning-induced neuroprotective effect of the RACK is caused by selective activation of PKC. In addition, we found that pharmacological preconditioning using the DAG analog OAG also promoted neuroprotection, suggesting a link between the calcium pathways with the Ca²⁺-independent PKC isozyme (PKC) (Nishizuka, 1992).

A number of studies have shown that glutamate and glutamate receptors play a central role in acute neurodegeneration after cerebral ischemia (Rothman and Olney, 1986; Albers et al., 1992; Choi, 1995; Pellegrini-Giampietro et al., 1999). However, mild (nonlethal) depolarization of brain cells before ischemia, such as that produced by spreading cortical depression, promoted protection against such insults in vivo and in vitro (Kobayashi et al., 1995; Matsushima et al., 1996; Kawahara et al., 1997; Plumier et al., 1997). These studies suggested the possible role of glutamate receptors in the induction phase of IPC. This hypothesis was supported by studies in vivo in which inhibition of NMDA receptors with MK-801 during IPC resulted in blockade of ischemic tolerance by IPC in gerbils (Kato et al., 1992b) and in vitro (Pringle et al., 1997b). Gonzalez-Zulueta et al. (2000) showed that, during OGD preconditioning in neuronal cell cultures, the signaling cascade was initiated by activation of the NMDA receptors, calcium influx, and production of nitric oxide in an RAS-extracellular signal-regulated protein kinase (ERK)-dependent manner, leading to the development of neuronal tolerance to ischemia.

The results from the present study support the hypothesis that implicates NMDA receptors mediating IPC. A previous study by Pringle et al. (1997b) in organotypic slice culture demonstrated that administration with 1 µM NMDA for 3 hr significantly reduced the neuronal damage produced by either 45 or 60 min of ischemia. In contrast to that study, our data show robust neuroprotection when slices were exposed to NMDA for only 1 hr. However, the time of ischemia in the present study was only 40 min. The role of the NMDA receptor in inducing tolerance is also supported by our findings that blockade of the NMDA receptor before and during IPC significantly ameliorates IPC- and NMDA-induced neuroprotection. Because the longest duration of NMDA exposure (60 min) was more efficacious than that observed with 15 and 30 min of NMDA preconditioning, we can surmise that continuous stimulation of the NMDA receptor was necessary to build a critical level of intracellular calcium to promote tolerance.

The hypothesis that calcium plays a key role in the induction of tolerance by IPC- and NMDA-induced preconditioning is supported by our findings that removal of cytoplasmic and extracellular calcium before and during IPC blocks neuroprotection. Furthermore, the extracellular and intracellular calcium chelation was also efficacious in blocking the induction of tolerance by NMDA preconditioning. We conjecture that a critical level of calcium is necessary to stimulate calcium-dependent enzymes, including PKC (Huang et al., 1999; Katsura et al., 1999) and nitric oxide synthase (Gonzalez-Zulueta et al., 2000; Nandagopal et al., 2001), that will lead to a signal transduction pathway that could ultimately play a role in neuroprotection.

Recently, the role of PKC during IPC in brain was investigated. Tauskela et al. (1999) found that neuroprotection by IPC was maintained despite pharmacological inhibition of PKC, suggesting that PKC does not play a role during IPC. In contrast, Reshef et al. (2000) showed that the preconditioning-induced neuroprotection initiated by adenosine receptors was mediated through activation of PKC in primary rat neuronal cultures. Because results from these studies contradicted each other, we surmised that differences between these studies might be a result of the different roles of PKC isozymes. In both heart (Brooks and Hearse, 1996) and brain (Savolainen et al., 1995; Sieber et al., 1998; Tan et al., 1998; Koponen et al., 2000), the role of PKC is controversial, probably because of the use of non-isozyme-specific tools. We also found contradictory data using the general PKC activator PMA and non-isozyme-specific inhibitor of PKC chelerythrine in organotypic slice culture. In the present study, we also show that the PMA translocated PKC and PKC along with translocation of PKC. The lack of protection with PMA (emulating IPC) and the inhibition of IPC produced by chelerythrine may be because of activation of more than one PKC isozyme that may have different roles in neurons. It has been reported that PKC and  PKC isozymes have opposite effects to ischemia in the heart, in which PKC promoted pathology, whereas PKC was protective (Chen et al., 2001). It is likely that, in brain as well, different PKC isozymes have opposite effects. For example, activation of PKC exacerbates damage during ischemia in three different models: isolated myocytes, intact heart ex vivo, and intact heart in vivo (Chen et al., 2001). Furthermore, overexpression of a catalytic fragment of PKC in PC12 cell leads to apoptosis (Bharti et al., 1998; Anantharam et al., 2002). PKC is a brain-specific PKC isozyme, and its translocation has been correlated with ischemic cell death, whereas its downregulation by ischemic preconditioning resulted in neuroprotection, thus suggesting that this isozyme may play a negative role after cerebral ischemia (Shamloo and Wieloch, 1999; Katsura et al., 2001). Thus, it is possible that there might be a dual involvement of PKC isozymes after PMA superfusion.

We demonstrate in the present study that, like in cardiac preconditioning, PKC is a key player in the induction of IPC neuroprotection. To the best of our knowledge, these results are the first evidence that a specific PKC isozyme plays a key role in the induction of tolerance by IPC in the brain.

Because NMDA-induced preconditioning neuroprotection was also blocked by the PKC blocker V1-2, activation of this isozyme by calcium had to occur indirectly. In fact, a transient increase in [Ca²⁺]_i activates phospholipase C-induced release of 1,4,5-inositol triphosphate and DAG, which can stimulate Ca²⁺-independent isozymes of PKC, such as PKC and PKC (Nishizuka, 1992). In the heart, transient increase in calcium activates diacylglycerol (Wang and Ashraf, 1999), which subsequently activates both classic (, I, II, and ) and novel (, , , and ) PKC isozymes (Guang et al., 1999). Our data demonstrate that we could emulate preconditioning neuroprotection with the DAG analog OAG and that this protection could be blocked by addition of the PKC antagonist, indicating that PKC is activated via DAG.

The signal transduction pathway that ensues after PKC activation remains undefined. Other candidates in the signal transduction pathway after IPC include the RAS-ERK pathway (Gonzalez-Zulueta et al., 2000). It remains to be determined whether PKC and the RAS-ERK pathway are linked together or are two different pathways that promote tolerance against cerebral ischemia. Finally, the similarity in the signal transduction pathways in the heart and brain is a significant finding, because it is possible that a novel therapeutic approach could emerge that would protect both organs from global ischemic conditions.

	FOOTNOTES

Received May 14, 2002; revised Oct. 23, 2002; accepted Oct. 28, 2002.

This work was supported by Public Health Service Grants NS34773, NS05820, NS38276, and AHA 0225227B.

Correspondence should be addressed to Dr. Miguel A. Pérez-Pinzón, Department of Neurology (D4-5), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101. E-mail: perezpinzon{at}miami.edu.

	References

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

• Albers GW, Goldberg MP, Choi DW (1992) Do NMDA antagonists prevent neuronal injury? Yes. Arch Neurol 49:418-420[Abstract/Free Full Text].
• Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy AG (2002) Caspase-3-dependent proteolytic cleavage of protein kinase C delta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J Neurosci 22:1738-1751[Abstract/Free Full Text].
• Bergold PJ, Casaccia-Bonnefil P (1997) Preparation of organotypic hippocampal slice cultures using the membrane filter method. Methods Mol Biol 72:15-22[Medline].
• Best N, Sunsstrom LE, Mitchell J, Wheal HV (1996) Pre-exposure to subtoxic levels prevents kainic acid lesions in organotypic hippocampal slice cultures: effects of kainic acid on parvalbumin-immunoreactive neurons and expression of heat shock protein-72 following induction of tolerance. Eur J Neurosci 8:1209-1219[Web of Science][Medline].
• Bharti A, Kraeft SK, Gounder M, Pandey P, Jin S, Yuan ZM, Lees-Miller SP, Weichselbaum R, Weaver D, Chen LB, Kufe D, Kharbanda S (1998) Inactivation of DNA-dependent protein kinase by protein kinase C delta: implications for apoptosis. Mol Cell Biol 18:6719-6728[Abstract/Free Full Text].
• Blondeau N, Plamondon H, Richelme C, Heurteaux C, Lazdunski M (2000) K(ATP) channel openers, adenosine agonists and epileptic preconditioning are stress signals inducing hippocampal neuroprotection. Neuroscience 100:465-474[Web of Science][Medline].
• Bond A, Lodge D, Hicks CA, Ward MA, O'Neill MJ (1999) NMDA receptor antagonism, but not AMPA receptor antagonism attenuates induced ischaemic tolerance in the gerbil hippocampus. Eur J Pharmacol 380:91-99[Web of Science][Medline].
• Brooks G, Hearse DJ (1996) Role of protein kinase C in ischemic preconditioning: player or spectator? Circ Res 79:627-630.
• Bruno V, Battaglia G, Copani A, D'Onofrio M, Di Iorio P, De Blasi A, Melchiorri D, Flor PJ, Nicoletti F (2001) Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab 21:1013-1033[Web of Science][Medline].
• Caggiano AO, Kraig RP (1998) Neuronal nitric oxide synthase expression is induced in neocortical astrocytes after spreading depression. J Cereb Blood Flow Metab 18:75-87[Web of Science][Medline].
• Centeno JM, Orti M, Salom JB, Sick TJ, Perez-Pinzon MA (1999) Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices. Brain Res 836:62-69[Web of Science][Medline].
• Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW, Mochly-Rosen II D (2001) Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci USA 98:11114-11119[Abstract/Free Full Text].
• Choi DW (1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 18:58-60[Web of Science][Medline].
• Chopp M, Chen H, Ho KL, Dereski MO, Brown E, Hetzel FW, Welch KM (1989) Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neurology 39:1396-1398[Abstract/Free Full Text].
• Cimarosti H, Rodnight R, Tavares A, Paiva R, Valentim L, Rocha E, Salbego C (2001) An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci Lett 315:33-36[Web of Science][Medline].
• Dorn II GW, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G, Lorenz JN, Mochly-Rosen D (1999) Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation. Proc Natl Acad Sci USA 96:12798-12803[Abstract/Free Full Text].
• Downey JM, Cohen MV, Ytrehus K, Liu Y (1994) Cellular mechanisms in ischemic preconditioning: the role of adenosine and protein kinase C. Ann NY Acad Sci 723:82-98[Web of Science][Medline].
• Goldberg MP, Pham PC, Choi DW (1987) Dextrorphan and dextromethorphan attenuate hypoxic injury in neuronal culture. Neurosci Lett 80:11-15[Web of Science][Medline].
• Gonzalez-Zulueta M, Feldman AB, Klesse LJ, Kalb RG, Dillman JF, Parada LF, Dawson TM, Dawson VL (2000) Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning. Proc Natl Acad Sci USA 97:436-441[Abstract/Free Full Text].
• Grabb MC, Choi DW (1999) Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J Neurosci 19:1657-1662[Abstract/Free Full Text].
• Gray MO, Karliner JS, Mochly-Rosen D (1997) A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272:30945-30951[Abstract/Free Full Text].
• Guang SL, Cohen MV, Mochly-Rosen D, Downey JM (1999) Protein kinase C- is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol 31:1937-1948[Web of Science][Medline].
• Heurteaux C, Lauritzen I, Widmann C, Lazdunski M (1995) Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K⁺ channels in cerebral ischemic preconditioning. Proc Natl Acad Sci USA 92:4666-4670[Abstract/Free Full Text].
• Huang HM, Weng CH, Ou SC, Hwang T (1999) Selective subcellular redistributions of protein kinase C isoforms by chemical hypoxia. J Neurosci Res 56:668-678[Web of Science][Medline].
• Kato H, Araki T, Murase K, Kogure K (1992a) Induction of tolerance to ischemia: alterations in second-messenger systems in the gerbil hippocampus. Brain Res Bull 29:559-565[Web of Science][Medline].
• Kato H, Liu Y, Araki T, Kogure K (1992b) MK-801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus. Neurosci Lett 139:118-121[Web of Science][Medline].
• Katsura K, Kurihara J, Siesjo BK, Wieloch T (1999) Acidosis enhances translocation of protein kinase C but not Ca²⁺/calmodulin-dependent protein kinase II to cell membranes during complete cerebral ischemia. Brain Res 849:119-127[Web of Science][Medline].
• Katsura KI, Kurihara J, Kato H, Katayama Y (2001) Ischemic pre-conditioning affects the subcellular distribution of protein kinase C and calcium/calmodulin-dependent protein kinase II in the gerbil hippocampal CA1 neurons. Neurol Res 23:751-754[Web of Science][Medline].
• Kawahara N, Croll SD, Wiegand SJ, Klatzo I (1997) Cortical spreading depression induces long-term alterations of BDNF levels in cortex and hippocampus distinct from lesion effects: implications for ischemic tolerance. Neurosci Res 29:37-47[Web of Science][Medline].
• Kobayashi S, Harris VA, Welsh FA (1995) Spreading depression induces tolerance of cortical neurons to ischemia in rat brain. J Cereb Blood Flow Metab 15:721-727[Web of Science][Medline].
• Koponen S, Goldsteins G, Keinanen R, Koistinaho J (2000) Induction of protein kinase Cdelta subspecies in neurons and microglia after transient global brain ischemia. J Cereb Blood Flow Metab 20:93-102[Web of Science][Medline].
• Kraft AS, Anderson WB (1983) Phorbol esters increase the amount of Ca²⁺, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301:621-623[Medline].
• Laake JH, Haug FM, Wieloch T, Ottersen OP (1999) A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Brain Res Protoc 4:173-184[Medline].
• Liniger R, Popovic R, Sullivan B, Gregory G, Bickler PE (2001) Effects of neuroprotective cocktails on hippocampal neuron death in an in vitro model of cerebral ischemia. J Neurosurg Anesthesiol 13:19-25[Web of Science][Medline].
• Mackay K, Mochly-Rosen D (2001) Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C. Cardiovasc Res 50:65-74[Abstract/Free Full Text].
• Matsushima K, Hogan MJ, Hakim AM (1996) Cortical spreading depression protects against subsequent focal cerebral ischemia in rats. J Cereb Blood Flow Metab 16:221-226[Web of Science][Medline].
• Nandagopal K, Dawson TM, Dawson VL (2001) Critical role for nitric oxide signaling in cardiac and neuronal ischemic preconditioning and tolerance. J Pharmacol Exp Ther 297:474-478[Abstract/Free Full Text].
• Nawashiro H, Tasaki K, Ruetzler CA, Hallenbeck JM (1996) TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice. J Cereb Blood Flow Metab 17:483-490[Web of Science].
• Nishio S, Yunoki M, Chen ZF, Anzivino MJ, Lee KS (2000) Ischemic tolerance in the rat neocortex following hypothermic preconditioning. J Neurosurg 93:845-851[Web of Science][Medline].
• Nishizawa Y (2001) Glutamate release and neuronal damage in ischemia. Life Sci 69:369-381[Web of Science][Medline].
• Nishizuka Y (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607-614[Abstract/Free Full Text].
• Ohtsuki T, Matsumoto M, Kuwabara K, Kitagawa K, Suzuki K, Taniguchi N, Kamada T (1992) Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons. Brain Res 599:246-252[Web of Science][Medline].
• Ohtsuki T, Ruetzler CA, Tasaki K, Hallenbeck JM (1996) Interleukin-1 mediates induction of tolerance to global ischemia in gerbil hippocampal CA1 neurons. J Cereb Blood Flow Metab 16:1137-1142[Web of Science][Medline].
• Ozyurt E, Graham DI, Woodruff GN, McCulloch J (1988) Protective effect of the glutamate antagonist, MK-801 in focal cerebral ischemia in the cat. J Cereb Blood Flow Metab 8:138-143[Web of Science][Medline].
• Pellegrini-Giampietro DE, Peruginelli F, Meli E, Cozzi A, Albani-Torregrossa S, Pellicciari R, Moroni F (1999) Protection with metabotropic glutamate 1 receptor antagonists in models of ischemic neuronal death: time-course and mechanisms. Neuropharmacology 38:1607-1619[Web of Science][Medline].
• Perez-Pinzon MA, Born JG (1999) Rapid preconditioning neuroprotection following anoxia in hippocampal slices: role of the K⁺_ATP channel and protein kinase C. Neuroscience 89:453-459[Web of Science][Medline].
• Perez-Pinzon MA, Mumford PL, Rosenthal M, Sick TJ (1996) Anoxic preconditioning in hippocampal slices: role of adenosine. Neuroscience 75:687-694[Web of Science][Medline].
• Perez-Pinzon MA, Born JG, Centeno JM (1999) Calcium and increase excitability promote tolerance against anoxia in hippocampal slices. Brain Res 833:20-26[Web of Science][Medline].
• Plumier JC, Krueger AM, Currie RW, Kontoyiannis D, Kollias G, Pagoulatos GN (1997) Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones 2:162-167[Web of Science][Medline].
• Pringle AK, Benham CD, Sim L, Kennedy J, Iannotti F, Sundstrom LE (1996) Selective N-type calcium channel antagonist omega conotoxin MVIIA is neuroprotective against hypoxic neurodegeneration in organotypic hippocampal-slice cultures. Stroke 27:2124-2130[Abstract/Free Full Text].
• Pringle AK, Angunawela R, Wilde GJ, Mepham JA, Sundstrom LE, Iannotti F (1997a) Induction of 72 kDa heat-shock protein following sub-lethal oxygen deprivation in organotypic hippocampal slice cultures. Neuropathol Appl Neurobiol 23:289-298[Web of Science][Medline].
• Pringle AK, Iannotti F, Wilde GJ, Chad JE, Seeley PJ, Sundstrom LE (1997b) Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia. Brain Res 755:36-46[Web of Science][Medline].
• Rao AM, Hatcher JF, Dempsey RJ (2000) Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil. Neurosci Lett 293:1-4[Web of Science][Medline].
• Reshef A, Sperling O, Zoref-Shani E (2000) Role of K(ATP) channels in the induction of ischemic tolerance by the "adenosine mechanism" in neuronal cultures. Adv Exp Med Biol 486:217-221[Web of Science][Medline].
• Riepe MW, Esclaire F, Kasischke K, Schreiber S, Nakase H, Kempski O, Ludolph AC, Dirnagl U, Hugon J (1997) Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: "chemical preconditioning." J Cereb Blood Flow Metab 17:257-264[Web of Science][Medline].
• Rordorff G, Koroshetz WJ, Bonventre JV (1991) Heat shock protects cultured neurons from glutamate toxicity. Neuron 7:1043-1051[Web of Science][Medline].
• Rothman SM, Olney JW (1986) Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann Neurol 19:105-111[Web of Science][Medline].
• Sakaguchi T, Okada M, Kuno M, Kawasaki K (1997) Dual mode of N-methyl-D-aspartate-induced neuronal death in hippocampal slice cultures in relation to N-methyl-D-aspartate receptor properties. Neuroscience 76:411-423[Web of Science][Medline].
• Savolainen KM, Loikkanen J, Naarala J (1995) Amplification of glutamate-induced oxidative stress. Toxicol Lett 82-83:399-405[Medline].
• Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569-1572[Abstract/Free Full Text].
• Shamloo M, Wieloch T (1999) Rapid decline in protein kinase C gamma levels in the synaptosomal fraction of rat hippocampus after ischemic preconditioning. NeuroReport 10:931-935[Web of Science][Medline].
• Sieber FE, Traystman RJ, Brown PR, Martin LJ (1998) Protein kinase C expression and activity after cerebral incomplete cerebral ischemia in dogs. Stroke 29:1445-1453[Abstract/Free Full Text].
• Simon RP, Swan JH, Griffiths T, Meldrum BS (1984) Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 226:850-852[Abstract/Free Full Text].
• Souroujon MC, Mochly-Rosen D (1998) Peptide modulators of protein-protein interactions in intracellular signaling. Nat Biotechnol 16:919-924[Web of Science][Medline].
• Steinberg GK, George CP, DeLaPaz R, Shibata DK, Gross T (1988) Dextromethorphan protects against cerebral injury following transient focal ischemia in rabbits. Stroke 19:1112-1118[Abstract/Free Full Text].
• Swan JH, Meldrum BS (1990) Protection by NMDA antagonists against selective cell loss following transient ischaemia. J Cereb Blood Flow Metab 10:343-351[Web of Science][Medline].
• Tan S, Wood M, Maher P (1998) Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. J Neurochem 71:95-105[Web of Science][Medline].
• Tauskela JS, Chakravarthy BR, Murray CL, Wang Y, Comas T, Hogan M, Hakim A, Morley P (1999) Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart. Brain Res 827:143-151[Web of Science][Medline].
• Vaccarino FM, Liljequist S, Tallman JF (1991) Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons. J Neurochem 57:391-396[Web of Science][Medline].
• Wang Y, Ashraf M (1999) Role of protein kinase C in mitochondrial K_ATP channel-mediated protection against Ca⁺ overload injury in rat myocardium. Circ Res 84:1156-1165[Abstract/Free Full Text].
• Xu G-P, Dave KR, Vivero R, Schmidt-Kastner R, Sick TJ, Perez-Pinzon MA (2002) Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures. Brain Res 952:153-158[Web of Science][Medline].

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				T. Shimohata, H. Zhao, and G. K. Steinberg {epsilon}PKC May Contribute to the Protective Effect of Hypothermia in a Rat Focal Cerebral Ischemia Model Stroke, February 1, 2007; 38(2): 375 - 380. [Abstract] [Full Text] [PDF]

				F. X. Soriano, S. Papadia, F. Hofmann, N. R. Hardingham, H. Bading, and G. E. Hardingham Preconditioning doses of NMDA promote neuroprotection by enhancing neuronal excitability. J. Neurosci., April 26, 2006; 26(17): 4509 - 4518. [Abstract] [Full Text] [PDF]

				R. Bright and D. Mochly-Rosen The Role of Protein Kinase C in Cerebral Ischemic and Reperfusion Injury Stroke, December 1, 2005; 36(12): 2781 - 2790. [Abstract] [Full Text] [PDF]

				M. S. Hedrick, C. S. Fahlman, and P. E. Bickler Intracellular calcium and survival of tadpole forebrain cells in anoxia J. Exp. Biol., February 15, 2005; 208(4): 681 - 686. [Abstract] [Full Text] [PDF]

				B. Lee, G. Q. Butcher, K. R. Hoyt, S. Impey, and K. Obrietan Activity-Dependent Neuroprotection and cAMP Response Element-Binding Protein (CREB): Kinase Coupling, Stimulus Intensity, and Temporal Regulation of CREB Phosphorylation at Serine 133 J. Neurosci., February 2, 2005; 25(5): 1137 - 1148. [Abstract] [Full Text] [PDF]

				R. Bright, A. P. Raval, J. M. Dembner, M. A. Perez-Pinzon, G. K. Steinberg, M. A. Yenari, and D. Mochly-Rosen Protein Kinase C {delta} Mediates Cerebral Reperfusion Injury In Vivo J. Neurosci., August 4, 2004; 24(31): 6880 - 6888. [Abstract] [Full Text] [PDF]

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