Sage Journals: Discover world-class research

Abstract

Previous work in primary cell culture has shown that TNF-α and ceramide are involved in the signaling that induces tolerance to brain ischemia (Ginis et al., 1999; Liu et al., 2000). To validate the in vitro studies, the authors administered cell permeable analogs of ceramides intracisternally or intravenously to examine their effect on neuroprotection after focal cerebral ischemia. Permanent middle cerebral artery occlusion (MCAO) was performed in spontaneously hypertensive rats. Infarct volumes were assessed at 24 hours after surgery. D-erythro-N-acetylsphingosine (C2-ceramide) or its vehicle was infused intracisternally for 1 hour before MCAO. In a second set of studies, D-erythro-N-octanoylsphingosine (C8-ceramide) or its vehicle was injected intravenously 48 or 24 hours before MCAO to mimic preconditioning (PC) and was also injected 5 minutes after MCAO. C2-ceramide infusion significantly reduced infarct volumes by approximately 14% (P < 0.05). C8-ceramide injection reduced infarct volumes approximately 17% compared with controls. This effect was constant and significant compared with controls over the time periods examined (P < 0.01). This work supports findings in primary brain cell cultures that implicate ceramide as a downstream signal that is proximate to development of tolerance to brain ischemia. Because the degree of protection represents approximately 50% of the maximal infarct reduction observed in this model, there are probably additional signaling pathways that subserve tolerance.

Keywords

Ceramides Ischemic tolerance Lipopolysaccharides Middle cerebral artery occlusion Tumor necrosis factor-α

Brain acquires ischemic tolerance after various sublethal stress stimuli. A well-established model of this tolerance is neuronal survival in the CA1 sector of the hippocampus after sublethal ischemic preconditioning (PC) (Kitagawa et al., 1990; Kirino et al., 1991). Various proteins that promote survival of brain cells have been studied during ischemic tolerance. The expression of bcl-2 family proteins (Shimazaki et al., 1994; Shimuzu et al., 1998), manganese superoxide dismutase (Kato et al., 1995), interleukin-1 receptor antagonist (Barone et al., 1998), heat shock proteins (Kitagawa et al., 1991), and ubiquitins (Kato et al., 1993) have been demonstrated in this state. However, the intracellular signaling pathways that regulate expression of these proteins during induction of tolerance to ischemia have not been fully defined. Activation of the extracellular signal-regulated protein kinase cascade (Shamloo et al., 1999; Gonzalez-Zulueta et al., 2000) and diminished activation of p53 and several of its target genes (Tomasevic et al., 1999) have been reported.

Brief ischemic preconditioning induces tolerance to focal cerebral ischemia (Matsushima and Hakim, 1995; Chen et al., 1996). The authors found that PC with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-α) induce a degree of tolerance to local ischemia that is similar to brief ischemic PC (Nawashiro et al., 1997; Tasaki et al., 1997). Lipopolysaccharides and its downstream product, TNF-α, can activate the sphingomyelin pathway for intracellular signaling (Kolesnick and Golde, 1994). Stress stimuli can cause a breakdown of sphingomyelin and generation of ceramide and phosphocholine through activation of various sphingomyelinases. Ceramides, a class of sphingolipids formed by the acylation of sphingosine, are not mere degradation products of sphingomyelin, but have been implicated broadly in cellular stress responses (Hannun, 1996). However, the role of ceramides differs in different cell types. Ceramides are known to play a role in proapoptotic signaling in a leukemia cell line (Jarvis et al., 1994), mesencephalic neurons (Brugg et al., 1996), and synovial fibroblasts (Ichinose et al., 1998). Conversely, the administration of a ceramide analog improved cognitive function after focal cerebral ischemia (Inokuchi et al., 1998) and promoted cell survival in spinal motor (Irie and Hirabayashi, 1998), sensory (Ping and Barrett, 1998), and sympathetic neurons (Ito and Horigome, 1995). Lymphoblasts from patients with Niemann-Pick disease who are deficient in acid sphingomyelinase are resistant to radiation-induced apoptosis (Santana et al., 1996). Other results support a role for ceramide administered exogenously in the survival of cerebellar Purkinje cells (Furuya et al., 1998) and hippocampal neurons (Goodman and Mattson, 1996). These contradictory results on the effects of ceramide are further complicated by the ability of cells to synthesize ceramide de novo by condensation of serine and palmitoyl-CoA. This pathway requires several hours to generate ceramides (Bose et al., 1995).

The authors recently have shown that hypoxic PC in cultured cortical neurons results in up-regulation of intracellular ceramide levels that peak at 24 hours and elicit resistance to a subsequent hypoxic insult (Liu et al., 2000). Addition of fumonisin B₁, an inhibitor of de novo synthesis of ceramides, to the cultures reduced ceramide levels and nullified the effect of hypoxic PC (Liu et al., 2000). Based on this result, the authors hypothesized that ceramides could induce tolerance to focal cerebral ischemia and, as a downstream intracellular signaling molecule, reduce the latency for its development. If ceramide was to be the single intracellular signal that regulates tolerance, the authors reasoned that exogenous ceramide should trigger a reduction of infarct size similar to that observed after PC with LPS or TNF-α.

MATERIALS AND METHODS

Reagents

Reagents were obtained from the following sources: D-erythro-N-acetylsphingosine (C2-ceramide, MW 341.5) from Sigma (St. Louis, MO, U.S.A.); D-erythro-N-octanoyl-sphingosine (C8-ceramide, MW 425) from Matreya (Pleasant Gap, PA, U.S.A.); dimethyl sulfoxide (DMSO, pH 7.0) from Sigma. The D-erythro configuration of ceramide is required for ceramide signaling activity. Dimethyl sulfoxide does not induce sphingomyelin hydrolysis (Hannun et al., 1993).

Animals

Adult male spontaneously hypertensive rats (SHR), 13 to 16 weeks, were obtained from Charles River Labs (Wilmington, MA, U.S.A.) and Taconic Farms (Germantown, NJ, U.S.A.). They were maintained in animal quarters with a standard 12-hour light/dark cycle with humidity and temperature controlled at normal levels. Water and food were allowed ad libitum. All procedures in animals were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee. Rats were fasted 16 hours before surgery, but had free access to water.

Experimental group

Experiment 1: intracisternal administration

Rats were anesthetized initially with 4% isoflurane in 30% oxygen to 70% nitrous oxide gas mixture. Atropine sulfate (0.1 mg/kg; Research Biomedical International, Natick, MA, U.S.A.) was administered intraperitoneally and the rats were intubated transorally. The isoflurane concentration was reduced to 1.0% to 1.5% after intubation and the rats were maintained on a mechanical ventilator (Harvard Rodent Ventilator Model 683; Harvard Instrument, South Natick, MA, U.S.A.). The right femoral artery was cannulated (PE-50; Clay-Adams, Parsippany, NJ, U.S.A.) for blood pressure monitoring and for serial blood gas sampling. Rats were fixed to a stereotaxic apparatus (Stoelting, Wood Dale, IL, U.S.A.). Intracisternal infusion was induced as described previously (Horinaka et al., 1997), with modification. Briefly, after opening of the atlanto-occipital membrane, a silastic catheter (0.31 mm inner diameter × 0.64 mm outer diameter; Helix Medical, Carpinteria, CA, U.S.A.) was inserted 5 mm into the left cerebellomedullary cistern for the administration of either C2-ceramide in a solution of DMSO (n = 8) or its vehicle (DMSO; n = 6) and was fixed at the back of the neck. After fifteen minutes for stabilization, intracisternal infusion was initiated. Infusion was maintained by a microinfusion pump (CMA/Microdialysis, Model CMA/100, Acton, MA, U.S.A.) at a rate of 1.26 μL/min and continued for 1 hour, followed immediately by middle cerebral artery occlusion (MCAO). In a preliminary study, Evans Blue infused in this manner at 1.26 μL/min had reached the MCAO territory. The dosages of C2-ceramide used in this study were selected on the basis of a pilot study in which 100 μg/kg C2-ceramide, but not 0.1, 1, 10 μg/kg, or 1 mg/kg C2-ceramide appeared to be optimally effective in reducing infarct size.

Controls were infused with the same volume of DMSO at the same infusion rate. Middle cerebral artery occlusion was induced as described previously (Tamura et al., 1981), with modification. Briefly, a 1.5-cm skin incision was placed between the left margin of the orbit and the tragus. The temporalis muscle was incised and retracted to expose the squamous portion of the temporal bone. A craniectomy was made at the juncture of the zygomatic process and the temporal bone. The dura was opened with a 30-gauge needle to expose the middle cerebral artery (MCA). The MCA was electrocauterized between the olfactory tubercle and the distal segment of the MCA. The olfactory branch of the MCA, which is consistently present, was preserved. The coagulated MCA segment was then transected with microscissors to verify that the occlusion was permanent. The intracisternal catheter was ligated and left in situ to avoid cerebrospinal fluid leakage. Rats were returned to their cages after surgery and allowed free access to food and water. A single dose of antibiotics (gentamicin, 2.5 mg/kg; Gibco BRL, Grand Island, NY, U.S.A.) was applied topically before closing the surgical sites, and 30% lidocaine cream (Foer's Pharmacy Bethesda, Bethesda, MD, U.S.A.) was also applied to all surgical sites as an analgesic. Laser-doppler cerebral blood flow measurements in a subset of 6 animals pre-MCAO and during the first 60 minutes after MCAO showed no differences between C2-ceramide- and DMSO-infused animals.

Experiment 2: intravenous administration

The methods of induction and maintenance of anesthesia, surgical MCAO, and postoperative care were exactly the same as for experiment 1. Animals were infused with C8-ceramide or DMSO at one of three time points, 48 or 24 hours before MCAO or 5 minutes after MCAO. A catheter (PE-50; Clay-Adams, Parsippany, NJ, U.S.A.) was inserted 2.5 cm into the right external jugular vein for administration of either C8-ceramide in a solution of DMSO or its vehicle (DMSO). The dosages of C8-ceramide used in this experiment were selected on the basis of a pilot study in which 1 mg/kg C8-ceramide, but not 1, 10, or 100 μg/kg C8-ceramide appeared to be optimally effective in reducing the infarct size. A 1 mg/kg dose of C8-ceramide was administered through the right external jugular vein over a 5-minute period at the following time points: 1) 48 hours before MCAO (n = 6 in each group), (2) 24 hours before MCAO (n = 5 in each group), and (3) 5 minutes after MCAO (n = 5 in each group). The catheter was removed after the injection and the vein was ligated. Laser-doppler cerebral blood flow measurements in a subset of 6 animals pre-MCAO and during the first 60 minutes after MCAO showed no differences between C8-ceramide- and DMSO-infused animals.

Physiologic variables

Physiologic variables were monitored to assess the physiologic status of the animals before MCAO and 30 minutes after MCAO. Mean arterial blood pressure was monitored by means of a TA 4000 transducer amplifier (Gould, Valley View, OH, U.S.A.). Pao₂, Paco₂, pH, and hematocrit were measured with a Corning pH/Blood Gas Analyzer (Model No. 288; Corning Medical, Medfield, MA, U.S.A.) by withdrawal of 100 μL of blood from the indwelling femoral arterial catheter. The same volume of pyogen-free warmed (37°C) heparinized saline (10 U/mL) was injected after each arterial blood sampling. Rectal and temporal muscle temperatures were measured and maintained at 37 ± 0.5°C with a heating blanket (K-20; American Pharmaseal, Valencia, CA, U.S.A.) during all surgical procedures and 2 hours after surgery. In experiment 1, mean arterial blood pressure and the rectal temperature were monitored during intracisternal infusion.

Assessment of infarct size

Twenty-four hours after MCAO, the animals were deeply anesthetized with pentobarbital (65 mg/kg intraperitoneal injection). In experiment 1, the rats were perfused with 200 mL heparinized phosphate-buffered saline (PBS) (10 U/mL, pH 7.4) followed immediately by 400 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.43). Their brains were left in situ overnight and then cryoprotected by transfer to 15% (w/v) sucrose in PBS at 4°C. Brains were then cut into 20-μm sections in a cryostat (Jung Frigocut 2800 E; Leica, Nussloch, Germany) at −20°C at 12 predetermined coronal levels (bregma +3.70 mm to −7.30 mm) at 1-mm intervals. Sections were stained with cresyl violet. In experiment 2, the brains were removed and suspended in PBS at 4°C for 30 minutes and sectioned coronally into eight 2-mm slices in a rodent brain matrix (RBM-4000C; Activational System, Warren, MI, U.S.A.). Slices were placed in 2% 2,3,5-triphenyltetrazolium chloride (Sigma) at room temperature for 30 minutes in the dark followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.43) overnight. Infarct size was determined by means of a digitized imaging system (NIH image, v.1.62). Volumes were calculated by integration of the sequential areas. Volumes of ischemic injury were corrected for brain swelling as previously described (Leach et al., 1993). Perfusion followed by fixation was adopted in experiment 1 to avoid brain injury by the implanted catheter during the removal of the brain. The variation in infarct volumes between experiment 1 and 2 is explained by these methodologic differences.

Statistics

Data are expressed as mean ± SD. Unpaired Student's t-test was used to analyze the differences in physiologic parameters and in infarct volumes in experiment 1. Two-way analysis of variance (ANOVA) was used to analyze the interaction between treatment group and time in experiment 2. If differences reached significance (two-tailed, P < 0.05), post hoc comparisons were made with Tukey's multiple comparison test.

RESULTS

Dimethyl sulfoxide has been reported to influence outcome from cerebral ischemia (de la Torre, 1983). The dose of DMSO used in this study neither reduced nor augmented infarct volumes compared with the same dose of PBS (DMSO: n = 6, 188 ± 8 mm³; PBS: n = 4, 198 ± 32 mm³). Based on this result, DMSO was used as the vehicle control for C2- and C8-ceramides, and PBS as a control for DMSO was not incorporated into experiments 1 and 2.

Physiologic variables

In experiment 1, intracisternal infusion did not cause any changes in mean arterial blood pressure or rectal temperature between the rats treated with C2-ceramide and those that received vehicle (Table 1). Hematocrit 30 minutes after MCAO in rats infused with vehicle was significantly lower than that in rats with C2-ceramide (P < 0.05, unpaired Student's t-test) (Table 1). However, these values were within normal limits. Physiologic variables in experiment 1 did not otherwise differ. In experiment 2, although it was within normal limits, arterial blood pH was significantly higher at 30 minutes after MCAO in the rats treated with C8-ceramide 5 minutes after MCAO than in controls (P < 0.01, unpaired Student's t test) (Table 2). There were no other significant differences in physiologic variables in experiment 2 (Table 2).

TABLE 1.

Physiologic variables in each experimental group of experiment 1 (intracisternal administration)

	Before intracisternal infusion		After intracisternal infusion		Before MCAO		30 minutes after MCAODMSO
	DMSO	C2-ceramide	DMSO	C2-ceramide	DMSO	C2-ceramide	DMSO	C2-ceramide
MABP (mm Hg)	118±12	126±11	130±19	122±18	140±7	142±11	135±13	135±18
PaO₂ (Torr)	NA	NA	NA	NA	149.5±16.1	147.0±8.6	145.1±17.9	125.9±28.2
PaCO₂ (Torr)	NA	NA	NA	NA	38.5±4.7	37.9±2.7	37.6±6.3	36.0±5.0
pH	NA	NA	NA	NA	7.42±0.03	7.41±0.02	7.41±0.03	7.41±0.03
RT (°C)	37.1±0.3	37.1±0.3	36.5±0.4	36.7±0.2	36.8±0.4	37.2±0.3	37.3±0.4	37.6±0.3
TMT (°C)	NA	NA	NA	NA	37.0±0.3	37.2±0.4	37.1±0.2	37.1±0.4
Hematocrit (%)	NA	NA	NA	NA	42.9±2.3	45.1±1.8	42.9±2.0	45.6 ±2.2**

Data are means ±SD. MCAO, middle cerebral artery occlusion; DMSO, dimethyl sulfoxide; C2-ceramide, D-erythro-N-acetylsphingosine; MABP, mean arterial blood pressure; NA, not applicable; RT, rectal temperature; TMT, temporal muscle temperature.

P < 0.05 compared with DMSO (unpaired Student's t-test).

TABLE 2.

Physiologic variables in each experimental group of experiment 2 (intravenous administration)

	48 hours before MCAO		24 hours before MCAO		5 minutes after MCAO
	DMSO	C8-ceramide	DMSO	C8-ceramide	DMSO	C8-ceramide
MABP (mm Hg)
Before MCAO	164±16	144 ±20	152 ±21	154 ±9	148 ±12	147 ±19
30 minutes after MCAO	150 ±12	139 ±23	139 ±17	145 ±11	158 ±16	148 ±21
PaO₂ (Torr)
Before MCAO	125.2 ±14.4	112.9 ±13.0	134.3 ±29.9	116.2 ±18.3	133.4 ±10.2	125.3 ±29.7
30 minutes after MCAO	127.0 ±8.9	116.3 ±17.1	136.8 ±21.1	108.5 ±22.3	136.1 ±13.6	147.0 ±21.7
PaCO₂ (Torr)
Before MCAO	37.1 ±4.4	39.8 ±4.5	39.5 ±5.8	38.5 ±1.6	41.0 ±3.6	37.2 ±3.2
30 minutes after MCAO	36.7 ±4.8	38.4 ±4.0	38.4 ±7.4	38.6 ±3.2	39.0 ±2.1	36.7 ±1.2
pH
Before MCAO	7.43 ±0.05	7.41 ±0.03	7.40 ±0.06	7.44 ±0.03	7.38 ±0.03	7.43 ±0.05
30 minutes after MCAO	7.43 ±0.04	7.42 ±0.03	7.41 ±0.05	7.43 ±0.03	7.40 ±0.01	7.43 ±0.01*
RT (°C)
Before MCAO	36.9 ±0.3	37.0 ±0.6	37.2 ±0.4	37.1 ±0.6	36.9 ±0.4	36.8 ±0.2
30 minutes after MCAO	37.2 ±0.1	37.2 ±0.5	37.4 ±0.3	37.1 ±0.4	37.3 ±0.3	37.3 ±0.2
TMT (°C)
Before MCAO	37.2 ±0.3	37.3 ±0.7	37.4 ±0.4	37.3 ±0.6	37.1 ±0.5	37.0 ±0.3
30 minutes after MCAO	37.2 ±0.2	37.2 ±0.5	37.3 ±0.1	37.1 ±0.2	37.1 ±0.5	37.1 ±0.3
Hematocrit (%)
Before MCAO	46.0 ±0.3	45.3 ±1.6	44.1 ±1.4	44.3 ±1.8	45.9 ±1.4	46.7 ±1.0
30 minutes after MCAO	45.5 ±1.1	44.1 ±1.6	43.1 ±0.7	44.1 ±1.7	43.3 ±1.6	44.8 ±2.3

Data are means ±SD. MCAO, middle cerebral artery occlusion; C8-ceramide, D-erythro-N-octanoylsphingosine; DMSO, dimethyl sulfoxide; MABP, mean arterial blood pressure; RT, rectal temperature; TMT, temporal muscle temperature.

P < 0.05 compared with DMSO (unpaired Student's t-test).

Infarct volumes in experiment 1 (intracisternal administration)

Intracisternal C2-ceramide infusion produced a significant reduction in infarct volume assessed 24 hours after MCAO as compared with the values of intracisternal vehicle infusion animals (DMSO: n = 6, 151 ± 18 mm³; C2-ceramide: n = 8, 130 ± 15 mm³; P = 0.03, unpaired Student's t-test). The reduction of the infarct volume was 14% in the C2-ceramide infused group (Fig. 1).

FIG. 1.

Bar graph showing effects of intracisternal infusion of D-erythro-N-acetylsphingosine (C2-ceramide) on infarct volumes. The values represent group averages (means ± SD). C2-ceramide infusion decreased the infarct size (*P < 0.05 compared with vehicle infusion, unpaired Student's t-test). DMSO, dimethyl sulfoxide.

Time and treatment effect on infarct volumes in experiment 2 (intravenous administration)

Time main effect (48 vs. 24 hours before MCAO vs. 5 minutes after MCAO) on infarct size was not significant (P = 0.767). Interaction of treatment with time also was not significant (P = 0.873). However, the treatment main effect was significant (P = 0.007, two-way ANOVA followed by Tukey's multiple comparison test). This means that the animals treated with C8-ceramide showed a significant decrease in infarct volumes compared with vehicle-treated animals at all time points (48 hours before MCAO: DMSO intravenously, n = 6, 189 ± 21 mm³; C8-ceramide: n = 6, 160 ± 43 mm³; 24 hours before MCAO: DMSO intravenously, n = 5, 206 ± 38 mm³; C8-ceramide: n = 5, 164 ± 23 mm³; 5 minutes after MCAO: DMSO intravenously, n = 5, 193 ± 7 mm³; C8-ceramide: n = 5, 164 ± 43 mm³) (Fig. 2). The average infarct volume reduction in C8-ceramide-treated animals in these experiments was approximately 17%. Overall, the infarct volume reduction in the C2-ceramide and C8-ceramide treatment groups was 16%.

FIG. 2.

Bar graph showing effect of D-erythro-N-octanoylsphingosine (C8-ceramide) on infarct volumes. The values represent group averages (means ± SD). Preconditioning with C8-ceramide 48 and 24 hours before middle cerebral artery occlusion (MCAO) reduced the infarct size (*P < 0.01, compared with vehicle-treated group, two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test). Similarly, administration of C8-ceramide 5 minutes after MCAO reduced the infarct size (*P < 0.01, compared with vehicle-treated group, two-way ANOVA followed by Tukey's multiple comparison test). DMSO, dimethyl sulfoxide.

DISCUSSION

The current work demonstrates that cell permeable exogenous ceramides (C2-ceramide and C8-ceramide) administered by two different routes do have a small, but statistically significant effect on reduction of infarct volumes produced by pMCAO in SHR. The overall 16% infarct volume reduction represents approximately 50% of the maximum salvage that has been achievable in this model (Morikawa et al., 1994; Dawson et al., 1999; Tasaki et al., 1997). In experiment 1, the authors adopted an intracisternal route for C2-ceramide administration to assure exposure of ischemic brain to the agent. The timing of the infusion corresponded to in vitro studies, in which ceramide needed to be elevated near the time of the hypoxic insult (Liu et al., 2000). An intraventricular route was not selected because previous studies had shown that fluorescence-conjugated ceramide is retained in the paraventricular area for 24 hours after intraventricular injection (Di Biase et al., 1991). The authors hypothesized that ceramide is a downstream signaling molecule that mediates the cytokine-induced ischemic tolerant state, and thus administration of a cell-permeable ceramide was expected to produce this tolerant state in the absence of LPS or TNF-α preconditioning. In fact, ceramide analogs bypass the receptors and mimic many of the biologic effects evoked by agonists such as TNF-α (Wiegmann et al., 1994). In experiment 2, the authors adopted the intravenous route for ceramide administration at several different time points. They found that by the time these experiments began intravenous administration of fluorescently labeled NBD-C6 ceramide (6-N-(7-nitrobenz-2 oxa-1, 3-diazol-4yl) aminocaproylsphingosine) penetrated the blood-brain barrier as early as 30 minutes after injection and became detectable in brain parenchymal cells. The fluorescent ceramide remained visible for at least 24 hours in the brain.

C2-ceramide is widely used in cell signaling studies. The structure of C2-ceramide is actually closer to sphingosine than to ceramides (Ping and Barrett, 1998). Ceramides are converted to glucosylceramides (GlcCer) by glucosyltransferase (glucosylceramide synthase) and to sphingosine by ceramidase. Sphingosine and sphingosine-1-phosphate have been noted to act in antiapoptotic processes (Cuvillier et al., 1996) and in myocardial protection after ischemia-reperfusion injury (Murohara et al., 1995). Because there was a possibility that the short-chain ceramide would mimic the activity of sphingosine rather than ceramide, the authors used C8-ceramide to minimize this possibility in experiment 2. The overall protective effect of ceramide (16% infarct volume reduction) did not reach the values obtained from the study of LPS tolerant models (≈30% reduction) at any of the time points examined. However, this effect was significant and provides useful information regarding signal transduction in cytokine-induced tolerance to subsequent ischemia. The current results suggest that ceramide plays a partial role in induction of tolerance to ischemia and that additional signaling pathways exist that contribute to the full induction of tolerance by LPS and TNF-α.

Recently, ceramide has been reported to activate the endothelial isoform of nitric oxide synthase (eNOS), which is independent of intracellular calcium concentrations ([Ca²⁺]_i) (Igarashi et al., 1999). Ceramide also reduced phenylephrine-induced vasoconstriction and inhibited elevation in [Ca²⁺]_i in vascular smooth muscle (Zheng et al., 1999). Moreover, the relevance of eNOS to LPS-induced tolerance has been reported. The endothelial isoform of NOS was up-regulated as tolerance developed after PC with LPS, and the protective effect of LPS was reduced by the NOS blocker, nitro-L-arginine methyl ester (Puisieux et al., 2000). In addition, eNOS and sphingomyelin are condensed in caveolae (Michel and Feron, 1997). These findings suggest a close relation among LPS, ceramide, and eNOS in improving cerebral perfusion in tolerant animals (Dawson et al., 1999).

In summary, the current results validate in vitro studies that have identified ceramide as a downstream signaling molecule in the LPS pathway for induction to brain ischemia. Moreover, ceramide analogs have a protective effect and do not need to be administered 24 to 48 hours before a stroke. Ceramide-induced infarct volume reduction is significant, but incomplete, compared with the effects of LPS in the previous study (Tasaki et al., 1997), which suggests that there are some additional signaling pathways mediating the tolerance to subsequent ischemia induced by LPS preconditioning.

Footnotes

Abbreviations used

Acknowledgments

The authors are indebted to Mrs. Mary W. Crawford for preparation of the manuscript. The authors also acknowledge Dr. Naoaki Horinaka, Laboratory of Cerebral Metabolism, National Institute of Mental Health, for critical advice concerning the intracisternal infusion technique.

References

Barone

White

Spera

Ellison

Currie

Wang

Feuerstein

(1998) Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke 29:1937–1951

Bose

Verheij

Haimovitz-Friedman

Scotto

Fuks

Kolesnick

(1995) Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82:405–414

Brugg

Michel

Agid

Ruberg

(1996) Ceramide induces apoptosis in cultured mesencephalic neurons. J Neurochem 66:733–739

Chen

Graham

Zhu

Simon

(1996) Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab 16:566–577

Cuvillier

Pirianov

Kleuser

Vanek

Coso

Gutkind

Spiegel

(1996) Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381:800–803

Dawson

Furuya

Gotoh

Nakao

Hallenbeck

(1999) Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion. J Cereb Blood Flow Metab 19:616–623

de la Torre

(1983) Role of dimethyl sulfoxide in prostaglandin-thromboxane and platelet systems after cerebral ischemia. Ann N Y Acad Sci 411:293–308

Di Biase

Argiolas

Confaloni

Salvati

(1991) In vivo metabolism of fluorescent ceramide in central nervous system myelin of adult rats. Neurochem Res 16:551–554

Furuya

Mitoma

Makino

Hirabayashi

(1998) Ceramide and its interconvertible metabolite sphingosine function as indispensable lipid factors involved in survival and dendritic differentiation of cerebellar Purkinje cells. J Neurochem 71:366–377

10.

Ginis

Schweizer

Brenner

Liu

Azzam

Spatz

Hallenbeck

(1999) TNF-α pretreatment prevents subsequent activation of cultured brain cells with TNF-α and hypoxia via ceramide. Am J Physiol (Cell Physiol) 276:C1171–C1183

11.

Gonzalez-Zulueta

Feldman

Klesse

Kalb

Dillman

Parada

Dawson

(2000) Requirement for nitric oxide activation of p21^ras/extracellular regulated kinase in neuronal ischemic preconditioning. Proc Natl Acad Sci U S A 97:436–441

12.

Goodman

Mattson

(1996) Ceramide protects hippocampal neurons against excitotoxic and oxidative insults, and amyloid beta-peptide toxicity. J Neurochem 66:869–872

13.

Hannun

(1996) Functions of ceramide in coordinating cellular responses to stress. Science 274:1855–1859

14.

Hannun

Obeid

Wolff

(1993) The novel second messenger ceramide: identification, mechanism of action, and cellular activity. Adv Lipid Res 25:43–64

15.

Horinaka

Kuang

Pak

Wang

Jehle

Kennedy

Sokoloff

(1997) Blockade of cerebral blood flow response to insulin-induced hypoglycemia by caffeine and glibenclamide in conscious rats. J Cereb Blood Flow Metab 17:1309–1318

16.

Ichinose

Eguchi

Migita

Kawabe

Tsukada

Koji

Abe

Aoyagi

Nakamura

Nagataki

(1998) Apoptosis induction in synovial fibroblasts by ceramide: in vitro and in vivo effects. J Lab Clin Med 131:410–416

17.

Igarashi

Thatte

Prabhakar

Golan

Michel

(1999) Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci U S A 96:12583–12588

18.

Inokuchi

Mizutani

Jimbo

Usuki

Yamagishi

Mochizuki

Muramoto

Kobayashi

Kuroda

Iwasaki

Ohgami

Fujiwara

(1998) A synthetic ceramide analog (L-PDMP) up-regulates neuronal function. Ann N Y Acad Sci 845:219–224

19.

Irie

Hirabayashi

(1998) Application of exogenous ceramide to cultured rat spinal motoneurons promotes survival or death by regulation of apoptosis depending on its concentrations. J Neurosci Res 54:475–485

20.

Ito

Horigome

(1995) Ceramide prevents neuronal programmed cell death induced by nerve growth factor deprivation. J Neurochem 65:463–466

21.

Jarvis

Kolesnick

Fornari

Traylor

Gewirtz

Grant

(1994) Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway. Proc Natl Acad Sci U S A 91:73–77

22.

Kato

Chen

Liu

Nakata

Kogure

(1993) Immunohistochemical localization of ubiquitin in gerbil hippocampus with induced tolerance to ischemia. Brain Res 619:339–343

23.

Kato

Kogure

Araki

Liu

Kato

Itoyama

(1995) Immunohistochemical localization of superoxide dismutase in the hippocampus following ischemia in a gerbil model of ischemic tolerance. J Cereb Blood Flow Metab 15:60–70

24.

Kirino

Tsujita

Tamura

(1991) Induced tolerance to ischemia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 11:299–307

25.

Kitagawa

Matsumoto

Tagaya

Hata

Ueda

Niinobe

Handa

(1990) ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res 528:21–24

26.

Kitagawa

Matsumoto

Kuwabara

Tagaya

Ohtsuki

Hata

Ueda

Handa

Kimura

Kamada

(1991) ‘Ischemic tolerance’ phenomenon detected in various brain regions. Brain Res 561:203–211

27.

Kolesnick

Golde

(1994) The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77:325–328

28.

Leach

Swan

Eisenthal

Dopson

Nobbs

(1993) BW619C89, a glutamate release inhibitor, protects against focal cerebral ischemic damage. Stroke 24:1063–1067

29.

Liu

Ginis

Spatz

Hallenbeck

(2000) Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide. Am J Physiol 278:C144–153

30.

Matsushima

Hakim

(1995) Transient forebrain ischemia protects against subsequent focal cerebral ischemia without changing cerebral perfusion. Stroke 26:1047–1052

31.

Michel

Feron

(1997) Nitric oxide synthases: which, where, how, and why? J Clin Invest 100:2146–2152

32.

Morikawa

Moskowitz

Huang

Tazuka

Irikura

Dalkara

(1994) L-arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat. Stroke 25:429–435

33.

Murohara

Buerke

Margiotta

Ruan

Igarashi

Hakomori

Lefer

(1995) Myocardial and endothelial protection by TMS in ischemia-reperfusion injury. Am J Physiol 269:H504–514

34.

Nawashiro

Tasaki

Ruetzler

Hallenbeck

(1997) TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice. J Cereb Blood Flow Metab 17:483–490

35.

Ping

Barrett

(1998) Ceramide can induce cell death in sensory neurons, whereas ceramide analogues and sphingosine promote survival. J Neurosci Res 54:206–213

36.

Puisieux

Deplanque

Souil

Bastide

Bordet

(2000) Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance. Eur J Pharmacol 389:71–78

37.

Santana

Pena

Haimovitz-Friedman

Martin

Green

McLoughlin

Cordon-Cardo

Schuchman

Fuks

Kolesnick

(1996) Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 86:189–199

38.

Shamloo

Rytter

Wieloch

(1999) Activation of the extracellular-regulated protein kinase cascade in the hippocampal CA1 region in a rat model of global cerebral ischemia preconditioning. Neuroscience 93:81–88

39.

Shimazaki

Ishida

Kawai

(1994) Increase in bcl-2 oncoprotein and the tolerance to ischemia-induced neuronal death in the gerbil hippocampus. Neurosci Res 20:95–99

40.

Shimuzu

Graham

Simon

(1998) The role of bcl-2 family genes in the induction of tolerance to focal ischemia. In: Pharmacology of cerebral ischemia ( Krieglstein

, ed), Stuttgart, Germany: Wissenschaftliche Verlagsgesellschaft mbH, pp 389–394

41.

Tamura

Graham

McCulloch

Teasdale

(1981) Focal cerebral ischaemia in the rat. 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1:53–60

42.

Tasaki

Ruetzler

Ohtsuki

Martin

Nawashiro

Hallenbeck

(1997) Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats. Brain Res 748:267–270

43.

Tomasevic

Shamloo

Israeli

Wieloch

(1999) Activation of p53 and its target genes p21 ^WAF1/Cip1 and PAG608/Wig-1 in ischemic preconditioning. Mol Brain Res 70:304–313

44.

Wiegmann

Schutze

Machleidt

Witte

Kronke

(1994) Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005–1015

45.

Zheng

Wang

Altura

(1999) C2–ceramide attenuates phenylephrine-induced vasoconstriction and elevation in [Ca²⁺]_i in rat aortic smooth muscle. Lipids 34:689–695

Cell Permeable Exogenous Ceramide Reduces Infarct Size in Spontaneously Hypertensive Rats Supporting In Vitro Studies That Have Implicated Ceramide in Induction of Tolerance to Ischemia

Abstract

Keywords

MATERIALS AND METHODS

Reagents

Animals

Experimental group

Experiment 1: intracisternal administration

Experiment 2: intravenous administration

Physiologic variables

Assessment of infarct size

Statistics

RESULTS

Physiologic variables

Infarct volumes in experiment 1 (intracisternal administration)

Time and treatment effect on infarct volumes in experiment 2 (intravenous administration)

DISCUSSION

Footnotes

Abbreviations used

Acknowledgments

References