CDP-choline Significantly Restores Phosphatidylcholine Levels by Differentially Affecting Phospholipase A2 and CTP: Phosphocholine Cytidylyltransferase after Stroke*

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTα protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 ± 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTα after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.

Focal cerebral ischemia or stroke is characterized by an obstruction of blood flow to the brain, resulting in disruption of the glucose and oxygen that supply the brain's energy needs. Energy failure results in rapid loss of ATP and uncontrolled leakage of ions across the cell membrane, causing membrane depolarization and release of neurotransmitters such as glutamate and dopamine (1,2). Excess glutamate release and stimulation of its receptors result in activation of phospholipases (3)(4)(5) and phospholipid hydrolysis and arachidonic acid release (6). Ultimately, these processes lead to apoptotic or necrotic cell death (7).
Phosphatidylcholine (PtdCho) 2 (8,9) is the major membrane phospholipid and constitutes ϳ50% of the total phospholipid content of mammalian cells. In addition to being an essential structural component of cell membranes, PtdCho is the biosynthetic precursor for other phospholipids such as sphingomyelin and phosphatidylserine; serves as a reservoir for several lipid messengers; and is the source of bioactive lipids such as phosphatidates, 1,2-diacylglycerol (DAG), and arachidonic acid, among others (8). PtdCho homeostasis is regulated by a balance between the opposing actions of hydrolysis and synthesis (10). PtdCho can be hydrolyzed by phospholipase A 2 (PLA 2 ), PtdCho-specific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PLA 2 isozymes occur in multiple forms (11)(12)(13) in the mammalian cell and are classified as the calcium-independent form (iPLA 2 ; 84 kDa) and the calcium-dependent cytosolic (cPLA 2 ; 85-110 kDa) and secretory (sPLA 2 ; 14 -18 kDa) forms. cPLA 2 preferentially hydrolyzes arachidonic acid in the sn-2 position of phospholipids, whereas sPLA 2 and iPLA 2 generally lack specificity for the fatty acid in the sn-2 position (14 -17). CTP:phosphocholine cytidylyltransferase (CCT) synthesizes CDP-choline and is the rate-limiting enzyme (18) in PtdCho synthesis. The final step of PtdCho synthesis is catalyzed by CDP-choline:DAG cholinephosphotransferase (19). Another route of PtdCho synthesis is phosphatidylethanolamine N-methyltransferase; however, this pathway is believed to be of significance only in the liver (20,21). PtdCho synthesis in extrahepatic tissues, including the brain, is primarily through the CDP-choline pathway (22). As a PtdCho precursor, CDP-choline (citicoline or Somazina) is virtually without any side effects and has undergone clinical trials for stroke treatment in the United States (2,23,24). CDP-choline has shown benefit in transient cerebral ischemia (25)(26)(27)(28)(29) and is in clinical use for stroke treatment in 70 countries, including Europe and Japan.
Inhibition of PtdCho synthesis through inactivation of CCT is sufficient to induce cell death (8). In transient global cerebral ischemia (a situation relevant to cardiac arrest), loss of membrane PtdCho resulting from activation of PLA 2 , PtdCho-PLC, and PLD and loss of CCT activity might have contributed significantly to cerebral ischemic injury (2,30,31). Here, we report that transient focal cerebral ischemia (a model for clinical stroke condition) differentially up-regulated sPLA 2 -IIA, Ptd-Cho-PLC, and PLD and down-regulated CCT, resulting in net loss of * This work was supported by NINDS Grant NS42008 from the National Institutes of PtdCho. Treatment with CDP-choline attenuated sPLA 2 -IIA mRNA and protein expression, PtdCho-PLC activity, and loss of CCT activity and protein levels; significantly restored PtdCho levels; and reduced the infarction volume after stroke.

EXPERIMENTAL PROCEDURES
Materials-Unless stated otherwise, all chemicals and reagents were purchased from Sigma. CDP-choline was obtained from BIOMOL (Plymouth Meeting, PA). The following antibodies were obtained from the indicated suppliers: rabbit polyclonal anti-sPLA 2 (Upstate, Charlottesville, VA); rabbit polyclonal anti-PLD, goat polyclonal anti-CCT␣, rabbit polyclonal anti-cPLA 2 , rabbit polyclonal anti-iPLA 2 , and horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Detection of Western blots was performed using SuperSignal from Pierce.
Focal Cerebral Ischemia-All surgical procedures were conducted according to the animal welfare guidelines set forth in the "Guide for the Care and Use of Laboratory Animals" (National Academy Press Washington, D. C. 1996) and were approved by the Animal Care and Use Committee of the University of Wisconsin-Madison. In these studies, we used transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rats; we (32) and others (33) have shown that spontaneously hypertensive rats provide a consistent infarction volume with a low variability. The coefficient of variation in the injury volumes is much less in spontaneously hypertensive rats compared with Sprague-Dawley rats (32). Male spontaneously hypertensive rats (250 -300 g) were purchased from Charles River Laboratories International, Inc. (Wilmington, MA) and subjected to 1 h of tMCAO as described (25,32,34). Under halothane anesthesia (1-2%) in a 50:50 O 2 /N 2 O mixture, a 3-0 monofilament nylon suture was introduced through the left internal carotid to occlude the middle cerebral artery. Reduction in blood flow was confirmed using a laser Doppler blood perfusion monitor (Vasamedics LLC, St. Paul, MN). After 1 h of occlusion, the suture was withdrawn to restore the blood flow, which was confirmed by laser Doppler flowmetry. Mean arterial blood pressure and blood gases PaO 2 and PaCO 2 were monitored via a catheter inserted into the left femoral artery. Body temperature was maintained at 37-38°C using a thermostatically controlled water blanket.
Drug Treatment-CDP-choline (500 mg/kg administered intraperitoneally) was dissolved in saline and administered at the onset of reperfusion and at 3 h and thereafter once daily until 1 day before euthanasia. This dose and schedule for CDP-choline provided maximum neuroprotection in previous studies (23,25). Controls received a corresponding volume of saline. Treatment with CDP-choline did not affect the physiological variables and did not cause any hypothermia (35).
Brain Tissue Collection-For PCR and Western blot analyses, rats were terminated by decapitation under deep anesthesia. Brain tissue was rapidly dissected at 0°C and placed in RNAlater solution (Qiagen Inc.) for PCR analyses or flash-frozen in liquid N 2 for Western blot analyses. For enzyme assays and lipid analyses, brains of anesthetized rats were frozen in situ, dissected at 0°C, and stored at Ϫ80°C until analyzed.
Real-time PCR-Expression of sPLA 2 -IIA mRNA was quantified by real-time PCR as described (36). RNA was extracted from cerebral cortices using a mini total RNA purification kit (Qiagen Inc.). Total RNA (1 g) was reverse-transcribed using oligo(dT) random hexamer primers (Promega Corp.) and Moloney murine leukemia virus reverse transcriptase (Promega) in a final volume of 20 l. 10 ng of cDNA and gene-specific primers were added to SYBR Green PCR Master Mixture (Applied Biosystems, Foster City, CA) and subjected to amplification in the ABI PRISM 7000 sequence detection system (Applied Biosystems). The amplified transcripts were quantified with the comparative C T method using 18 S rRNA as the internal control. The primers were designed based on rat GenBank TM accession number NM_031598 for rat sPLA 2 -IIA using Primer Express software (Applied Biosystems) and purchased from Integrated DNA Technologies, Inc. (Skokie, IL). The primer sequences were 5Ј-ACAAGAAGCCATACCACCATCCCA-3Ј (forward) and 5Ј-ACAGTCATGAGTCACACAGCACCA-3Ј (reverse), which amplified a 252-bp fragment of rat sPLA 2 -IIA. The primer sequences for 18 S rRNA were 5Ј-CGCCGCTAGAGGTGAAATTC-T-3Ј (forward) and 5Ј-CGAACCTCCGACTTTCGTTCT-3Ј (reverse), which amplified a 101-bp fragment of rat 18 S rRNA.
PtdCho-PLC activity was measured using the Amplex Red PtdChospecific PLC assay kit (38) from Molecular Probes (Eugene, OR). The principle of the assay is that PtdCho-PLC hydrolyzes PtdCho to DAG and phosphocholine. Phosphocholine is hydrolyzed to choline by alkaline phosphatase. Choline is oxidized by choline oxidase to betaine and H 2 O 2 . Amplex Red reagent is oxidized stoichiometrically by H 2 O 2 in the presence of horseradish peroxidase to generate the fluorescent product resorufin. The reaction mixture contained 200 M Amplex Red, 1 unit/ml horseradish peroxidase, 4 unit/ml alkaline phosphatase, 0.1 unit/ml choline oxidase, and 0.5 mM PtdCho in 50 mM Tris-HCl (pH 7.4), 140 mM NaCl, 10 mM dimethyl glutarate, 2 mM CaCl 2 , and an aliquot of tissue homogenate. Samples were incubated at 37°C, and fluorescence was measured (Ex 530 nm and Em 590 nm ). Activity was calculated using purified bacterial PtdCho-PLC as a reference.
CCT Assay-Brain tissue was homogenized in 50 mM Tris (pH 7.4) containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 0.025% sodium azide, and protease inhibitor mixture (39). Homogenates were centrifuged at 400 ϫ g for 10 min, and the supernatant was centrifuged at 140,000 ϫ g for 35 min. The resulting supernatant was taken as the cytosol. CCT activity was determined by measuring the formation of radioactive CDP-choline from [methyl-14 C]phosphocholine (Amersham Biosciences) as described previously (31).
Western Blot Analyses-Brain tissue was processed as per the respective enzyme assays. 150 g of protein was loaded onto polyacrylamide gels. SDS-PAGE was performed using the Criterion system (Bio-Rad) at a constant voltage of 200 V. Proteins were subsequently transferred to nitrocellulose at a constant voltage of 100 V for 1 h. Nonspecific binding sites were blocked with 5% nonfat milk powder in 1ϫ Tris-buffered saline with 0.05% Tween 20 (1ϫ TBST) at room temperature for 1 h. Blots were incubated overnight with primary antibodies (diluted in 3% bovine serum albumin in 1ϫ TBST with 0.02% sodium azide) at 4°C, washed with 1ϫ TBST, and then incubated with the appropriate secondary antibodies for 1 h at room temperature. After washing, protein bands were visualized with SuperSignal for 5 min at room temperature and exposure to x-ray film. Relative changes in protein expression were estimated from the mean pixel density using the Scion Image program, normalized to ␤-actin, and calculated as ipsilateral/contralateral cortex ratios.
Lipid Analysis-All solvents and extracts were purged with N 2 during the extraction, TLC, and methylation of lipids. Total lipids were extracted from brain tissue into CHCl 3 /MeOH (1:2 by volume) contain-ing 0.01% butylated hydroxytoluene, separated by TLC, converted to methyl esters, and quantitated using a Hewlett-Packard 6890 gas chromatograph as described (31,40).
Ischemic Injury (Infarction) Volume-Infarction volumes were measured using 2,3,5-triphenyltetrazolium chloride staining as described previously (25,32). Brains were cut in 2-mm coronal slices, incubated with 2% 2,3,5-triphenyltetrazolium chloride for 30 min at 37°C, rinsed with saline, and fixed in 4% paraformaldehyde. Stained sections were scanned, and the ischemic injury volumes were computed by the numeric integration of data from individual slices using the Scion Image program. To compensate for edema in the ischemic hemisphere, corrected infarction volumes were calculated as follows: corrected infarc-tion volume ϭ right hemisphere volume Ϫ (left hemisphere volume Ϫ measured infarction volume) (41).
Statistical Analyses-Data are presented as means Ϯ S.D. (n ϭ 3-5/ group) and were analyzed by analysis of variance, followed by Bonferroni's multigroup comparisons post-test using Prism software (Graph-Pad Software, Inc., San Diego, CA). A value of p Ͻ 0.05 was considered significant.

CDP-choline Attenuates the Increase in sPLA 2 -IIA mRNA and Protein
Expression and PLA 2 Activity after tMCAO-Expression of sPLA 2 -IIA mRNA was measured by real-time-PCR following 1 h of tMCAO and ). There were no significant differences in sPLA 2 -IIA mRNA expression in the contralateral cortex compared with the cortices of sham-operated animals that underwent similar surgical procedures except that the artery was not occluded. **, p Ͻ 0.01 compared with the contralateral cortex; #, p Ͻ 0.05 compared with the respective ipsilateral cortex (1-and 3-day saline-treated groups; n ϭ 4/group). B, Western blot using rabbit polyclonal anti-sPLA 2 antibody and showing the time course of sPLA 2 -IIA protein expression in the 18,000 ϫ g supernatant of ipsilateral (Ipsi-cortex) and contralateral (Contra-) cortical homogenates at various reperfusion times after 1 h of tMCAO. An extract of rat platelets was used as a reference source for sPLA 2 -IIA (42). The bar graph represents the relative increases in sPLA 2 -IIA protein expression estimated from the mean pixel density (Scion Image program), normalized to ␤-actin, and calculated as ipsilateral/contralateral cortex ratios. In sham-operated animals, there was no difference between the left and right cortices (ratio of 1). * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with sham-operated animals (n ϭ 5/group). C, effect of CDP-choline (500 mg/kg administered intraperitoneally at 0 and 3 h of reperfusion for 1 day and at 0 h, 3 h, 1 day, and 2 days of reperfusion for 3 days) on sPLA 2 -IIA protein levels after 1 h of tMCAO. A representative Western blot is shown for 1 day of reperfusion. The bar graph represents the relative sPLA 2 -IIA protein expression calculated as ipsilateral/contralateral cortex ratios. CDP-choline significantly attenuated sPLA 2 -IIA expression in the ipsilateral cortex to contralateral cortex levels at 1 and 3 days of reperfusion. **, p Ͻ 0.01 compared with sham-operated animals; ##, p Ͻ 0.01 compared with saline-treated groups (n ϭ 4/group). There was no statistically significant difference in sPLA 2 expression in the contralateral cortex between saline and CDP-choline treatment. D, effect of CDP-choline on PLA 2 activity in 18,000 ϫ g supernatants of ipsilateral (Ipsi.) and contralateral (Contra.) cortical homogenates after 1 h of tMCAO and reperfusion for the indicated times. PLA 2 activity was determined by the release of labeled arachidonic acid from 1-palmitoyl-2-[1-14 C]arachidonyl-sn-glycero-3-phosphocholine as described under "Experimental Procedures" (30,37). * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with the contralateral cortex; # and ##, p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with the ipsilateral cortex upon saline treatment (n ϭ 4/group). CDP-choline attenuated sPLA 2 activity to near contralateral levels (no statistically significant differences between the ipsilateral cortex/CDP-choline group versus the contralateral cortex/saline group). CDP-choline did not affect PLA 2 activity in the contralateral cortex. PtdCho present in the tissue samples had a minimum effect (Յ1%) on the results of the PLA 2 assay, as elaborated under "Results." reperfusion for 1 and 3 days using the primers given under "Experimental Procedures." sPLA 2 -IIA mRNA increased by 4.7-and 6.2-fold in the ipsilateral (ischemic) cortex compared with the contralateral (non-ischemic) cortex at 1 and 3 days of reperfusion (Fig. 1A), respectively. Treatment with CDP-choline (500 mg/kg administered intraperitoneally at 0 and 3 h of reperfusion; thereafter, on days 1 and 2 for mRNA at 3 days) resulted in 39 and 37% reductions (to 2.87-and 3.9-fold increases compared with the contralateral cortex) in sPLA 2 -IIA mRNA expression in the ipsilateral cortex at 1 and 3 days of reperfusion, respectively (p Ͻ 0.05 compared with the ipsilateral cortex upon saline treatment). sPLA 2 -IIA protein expression following 1 h of tMCAO was determined by Western blotting using rabbit polyclonal anti-sPLA 2 antibody. An extract from rat platelets was used as an electrophoresis standard for sPLA 2 -IIA (42). sPLA 2 -IIA increased in the ipsilateral cortex beginning at 3 h of reperfusion and remained elevated for up to 7 days (Fig. 1B). No significant changes in sPLA 2 -IIA protein levels were observed in the contralateral cortex over 7 days of reperfusion. Treatment with CDPcholine attenuated sPLA 2 -IIA protein levels in the ipsilateral cortex to nearly contralateral cortex levels at 1 and 3 days of reperfusion (Fig. 1C). There was no statistically significant difference in sPLA 2 expression in the contralateral cortex between saline and CDP-choline treatment. Western blotting was also conducted using rabbit polyclonal anti-cPLA 2 and anti-iPLA 2 antibodies. No differences were observed in the cPLA 2 or iPLA 2 protein levels between ipsilateral and contralateral cortices over 7 days of reperfusion after tMCAO (data not shown).
PLA 2 activity was measured by the release of labeled arachidonic acid from 1-palmitoyl-2-[1-14 C]arachidonyl-sn-glycero-3-phosphocholine as described under "Experimental Procedures" (37). Most of the PLA 2 activity required 5 mM Ca 2ϩ , characteristic of sPLA 2 (data not shown). 1 h of tMCAO resulted in 71, 100, and 130% increases in PLA 2 activity in the ipsilateral cortex compared with the contralateral cortex at 3 h, 6 h, and 1 day of reperfusion, respectively (Fig. 1D). CDP-choline treatment attenuated PLA 2 activities in the ipsilateral cortex by 31% (3 h), 29% (6 h), and 46% (1 day) compared with the ipsilateral cortex upon saline treatment. These differences in PLA 2 activity could not be attributed to dilution of the specific activity of labeled PtdCho in the PLA 2 assay by PtdCho present in the tissue samples. PLA 2 activity (Fig. 1D) was determined in the 18,000 ϫ g supernatants of tissue homogenates, in which most of the PtdCho, which was localized to the membrane fraction, was removed. The PLA 2 assay contained 50 nmol of PtdCho in 0.5 ml (37); the 18,000 ϫ g supernatant (0.2 mg of protein/assay) contributed ϳ3.7 nmol of PtdCho, diluting the specific activity by ϳ7%. Although PtdCho levels in brain tissue declined following tMCAO (see "CDP-choline Significantly Restores PtdCho Levels after Stroke" below), these changes probably resulted in Յ1% variability in PtdCho-specific activity.
CDP-choline Attenuates Loss of CCT Protein and Activity after tMCAO-Western blots for CCT␣ protein expression (goat polyclonal antibody) showed that CCT␣ was decreased over 1-7 days of reperfusion in the ipsilateral cortex ( Fig. 2A). There were no significant changes in CCT␣ levels in the contralateral cortex over 7 days of reperfusion and no significant differences between ipsilateral/contralateral cortex ratios at 1, 3, and 7 days (Fig. 2A). Treatment with CDP-choline significantly increased CCT␣ protein expression in the ipsilateral cortex at 1, 3, and 7 days of reperfusion (Fig. 2B). The decrease in CCT␣ protein levels was reflected in loss of CCT activity in the ipsilateral cortex. 1 h of tMCAO and 1 day of reperfusion resulted in 41 and 57% loss of CCT activity in the ipsilateral cortical total homogenate and cytosol, respectively, compared with the contralateral cortex (Fig. 2C). CDP-choline treatment significantly increased CCT activity in the ipsilateral cortical total homogenate and cytosol by 63 and 54%, respectively, compared with the The bar graph represents the relative levels of CCT␣ protein expression (estimated from the mean pixel density (Scion Image program) and normalized to ␤-actin) given as ipsilateral/contralateral cortex ratios. There was significant loss of CCT protein in the ipsilateral cortex over 1-7 days (d) of reperfusion. * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with sham-operated animals (n ϭ 4/group). B, effect of CDPcholine on CCT␣ protein expression after 1 h of tMCAO. A representative Western blot is shown for 1 day of reperfusion. The bar graph represents the relative levels of CCT␣ protein expression given as ipsilateral (IC)/contralateral cortex (CC) ratios. CDP-choline (500 mg/kg administered intraperitoneally at 0 and 3 h of reperfusion and thereafter once daily until 1 day before euthanasia) significantly restored CCT␣ protein levels after 1 h of tMCAO and 1, 3, and 7 days of reperfusion. * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively, compared with sham-operated animals; #, p Ͻ 0.05 compared with saline treatment (n ϭ 3-4/group). C, effect of CDP-choline on CCT activity in total (Tot.) homogenates and cytosolic fractions from ipsilateral (Ipsi.) and contralateral (Contra.) cortices after 1 h of tMCAO and 1 day of reperfusion. CCT activity was determined by measuring the formation of radioactive CDP-choline from [methyl-14 C]phosphocholine as described under "Experimental Procedures" (31). **, p Ͻ 0.01 compared with the contralateral cortex; #, p Ͻ 0.05 compared with the ipsilateral cortex upon saline treatment (n ϭ 4/group). Phosphocholine present in the brain tissue homogenate did not significantly affect the results of the CCT enzyme assay as elaborated under "Results." corresponding fractions from the saline-treated ipsilateral cortex (Fig.  2C). CDP-choline treatment did not significantly alter CCT activity in the contralateral cortex. In transient forebrain ischemia, CDP-choline treatment also significantly restores CCT activity (31). It should be noted that the CCT assay measures formation of labeled CDP-choline from [methyl-14 C]phosphocholine and that assay results might be affected by alterations in brain levels of phosphocholine. Phosphocholine levels in normal rat brain are ϳ228 nmol/g of tissue (43). Based on the amount of protein used in the CCT assay (60 g/assay), the labeled phosphocholine in the assay (1.6 mM) was estimated to be at least 400fold greater than the phosphocholine added by the aliquot of the tissue homogenate.
PLD2 Protein Expression Is Unaffected by CDP-choline after Stroke-Expression of PLD2 protein by Western blotting using rabbit polyclonal antibodies from Santa Cruz Biotechnology, Inc. demonstrated that PLD2 increased beginning at 3 h of reperfusion and remained elevated up to 7 days (Fig. 3A). Caki-1 cell lysate (Santa Cruz Biotechnology, Inc.) was used as an electrophoresis reference for PLD (designated as a control). Treatment with CDP-choline did not alter PLD2 protein expression in either the contralateral cortex (Fig. 3B, lanes a and b) or the ipsilateral cortex (lanes c and d) at 1 day of reperfusion compared with saline treatment. Expression of PLD1 (ϳ120 kDa) was much lower than that of PLD2 and showed a slight increase at 1 day of reperfusion, which was unaffected by CDP-choline treatment (data not shown).
CDP-choline Attenuates the Increase in PtdCho-PLC Activity after tMCAO-PtdCho-PLC activity was measured using the Amplex Red assay kit from Molecular Probes; activity in brain tissue homogenates was calculated using purified bacterial PtdCho-PLC as a reference (provided with the kit). PtdCho-PLC activity significantly increased in the 18,000 ϫ g supernatant of the ipsilateral cortex (400 Ϯ 40 milliunits/mg of protein) at 1 day of reperfusion following 1 h of tMCAO compared with the contralateral cortex (100 Ϯ 15 milliunits/mg of protein (p Ͻ 0.01), n ϭ 4/group). Activity in the assay was abolished by inclusion of the specific PtdCho-PLC inhibitor D609 (tricyclodecan-9-yl potassium xanthate) (44), thus confirming the activity as PtdCho-PLC. CDP-choline treatment significantly attenuated the PtdCho-PLC activity in the ipsilateral cortex to 150 Ϯ 23 (p Ͻ 0.05) compared with the ipsilateral cortex upon saline treatment (n ϭ 4/group). Due to lack of commercially available antibodies for PtdCho-PLC, protein expression by Western blotting could not be pursued.
CDP-choline Significantly Restores PtdCho Levels after Stroke-Total lipids were extracted from cortical tissue using CHCl 3 /MeOH (1:2, by volume). PtdCho was separated by TLC, converted to fatty acid methyl esters, and quantitated by gas chromatography. PtdCho levels significantly decreased by 22 and 17% in the ipsilateral cortex at 1 and 3 days of reperfusion, respectively, after 1 h of tMCAO (p Ͻ 0.05 compared with the contralateral cortex) (Fig. 4). Treatment with CDP-choline resulted in 17 and 13% increases in PtdCho levels in the ipsilateral cortex at 1 and 3 days of reperfusion, respectively (p Ͻ 0.05 compared with the ipsilateral cortex upon saline treatment). PtdCho levels in ipsilateral cortices following CDP-choline treatment tended to be less than those in contralateral cortices, but these differences were not statistically significant. The effect of CDP-choline is not a time shift, as CDP-choline treatment significantly attenuated loss of PtdCho in the ipsilateral cortex compared with saline at 3 days of reperfusion. There was no significant difference in PtdCho levels in the ipsilateral cortex upon saline treat-  Total lipids were extracted from cortices into CHCl 3 /MeOH, separated by TLC, converted to methyl esters, and quantitated using a Hewlett-Packard 6890 gas chromatograph as described (31,40). PtdCho levels were calculated as one-half the sum of fatty acids derived from PtdCho because each PtdCho molecule contains two fatty acid residues. These values are therefore approximately one-half those reported by Narita et al. (46) because PtdCho levels in that report were given as the sum of fatty acids. PtdCho levels significantly decreased in the ipsilateral cortex (Ipsi.) following 1 h of tMCAO and 1 and 3 days (d) of reperfusion. *, p Ͻ 0.05 compared with the contralateral (Contra.) cortex (n ϭ 4/group). CDP-choline (500 mg/kg administered intraperitoneally at 0 and 3 h of reperfusion for 1 day and at 0 h, 3 h, 1 day, and 2 days of reperfusion for 3 days) significantly restored PtdCho levels in the ipsilateral cortices. #, p Ͻ 0.05 compared with the ipsilateral cortex upon saline treatment (n ϭ 4/group). PtdCho levels in ipsilateral cortices following CDP-choline treatment tended to be less than those in contralateral cortices, but these differences were not statistically significant. There was no significant difference between the saline-treated ipsilateral cortices at 1 versus 3 days of reperfusion. ment at 1 versus 3 days of reperfusion. Similar losses were observed for total phospholipids (as the sum of PtdCho, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, and sphingomyelin) at 1 and 3 days of reperfusion, which were partially restored by CDP-choline treatment (data not shown).
CDP-choline Decreases the Infarction Volume after Stroke-Cerebral infarction volumes were measured using 2,3,5-triphenyltetrazolium chloride staining. Brain sections were scanned, and the ischemic injury volumes were computed by the numeric integration of data from individual slices using the Scion Image program and corrected for edema. CDP-choline treatment significantly (p Ͻ 0.01) reduced the infarction volume by 55 Ϯ 5% compared with saline-treated controls (Fig. 5).

DISCUSSION
In this study, we have reported that transient focal cerebral ischemia (a model closely paralleling the clinical stroke condition) up-regulates sPLA 2 -IIA, PtdCho-PLC, and PLD2 and down-regulates CCT, collectively resulting in loss of PtdCho. CDP-choline (an intermediate in Ptd-Cho synthesis) counteracts some of these changes and significantly preserves membrane PtdCho in the ischemic cortex; similar results were observed in the ischemic striatum (data not shown). To the best of our knowledge, this is the first report showing simultaneous up-regulation of sPLA 2 -IIA and down-regulation of CCT after tMCAO. This study also suggests that part of the neuroprotective actions of CDP-choline in stroke may be due to preserving membrane PtdCho by differentially affecting sPLA 2 -IIA, PtdCho-PLC, and CCT protein expression. The effect of CDP-choline was determined over 1-7 days of reperfusion because PLA 2 , PtdCho-PLC, and PLD, CCT and PtdCho levels all showed significant changes during this period.
The release of free fatty acids as indirect evidence for phospholipase activation has been shown in both global (45) and focal (46) cerebral ischemia models (47). Most of the previous studies on activation of PLA 2 in cerebral ischemia focused on the cytosolic form (cPLA 2 ) (33,48,49). Transgenic mice lacking cPLA 2 were generated by targeted disruption of its gene. Following transient focal cerebral ischemia, cPLA 2deficient mice have smaller infarction volumes and fewer neurological deficits compared with wild-type mice (48,50), demonstrating a role for cPLA 2 in ischemic injury. It should be noted that mouse strains C57BL/6J and 129/SV used for transgenic studies have a naturally occurring mutation in the gene for sPLA 2 -IIA (51, 52); and thus, the cPLA 2 knockout mice are deficient in both cPLA 2 and sPLA 2 -IIA. Transgenic mice expressing the human sPLA 2 -IIA gene have been developed (53), but this mouse strain apparently has not yet been used in stroke research to assess the role of sPLA 2 -IIA. sPLA 2 -IIA, also known as inflammatory PLA 2 , is believed to play an important role in inflammation and injury (54, 55), and its expression in rat brain has been shown both in global (56) and focal (54) cerebral ischemia models. Our results showing increased sPLA 2 -IIA mRNA following tMCAO and 1 and 3 days of reperfusion (Fig. 1A) are consistent with a previous report demonstrating similar increases in male Long-Evans rats (54). In our studies, increased sPLA 2 -IIA protein expression by Western blotting was observed as early as 3 h of reperfusion and persisted for up to 7 days (Fig. 1B). The studies by Lin et al. (54) also reported increased sPLA 2 -IIA expression by immunohistochemistry at 1 and 3 days; however, immunoreactivity before 1 day following tMCAO could not be detected. The increase in sPLA 2 -IIA protein expression over 1 day of reperfusion in our studies was reflected by increased PLA 2 activity (Fig. 1D). Treatment with CDP-choline significantly decreased the sPLA 2 -IIA mRNA (Fig. 1A) and protein (Fig. 1C) levels and PLA 2 enzyme activity (Fig. 1D). We have previously demonstrated that CDP-choline attenuates the increase in PLA 2 activity in transient forebrain (global) ischemia (30).
Tumor necrosis factor-␣ and interleukin-1 are up-regulated in the brain after cerebral ischemia (57)(58)(59)(60). They are potent inducers of the transcription factor NF-B, and many of their effects are mediated through this transcription factor (61,62). sPLA 2 -II mRNA and protein expression is induced by tumor necrosis factor-␣ and interleukin-1, and inhibition of NF-B activation suppresses this activation (63), indicating that NF-B mediates cytokine-induced gene activation of sPLA 2 -IIA. NF-B exists normally in an inactive form associated with IB inhibitory proteins. Activation of NF-B involves phosphorylation of IB by IB kinases, followed by ubiquitination and degradation to release active NF-B (64). Several upstream kinases have been implicated in activation of IB kinase, including the MAPK family of enzymes. A recent study showed that CDP-choline attenuates MAPK signaling (65), suggesting that CDP-choline may decrease IB kinase phosphorylation, NF-B activation, and subsequent transcriptional activation of sPLA 2 -IIA.
Exogenous CDP-choline is rapidly hydrolyzed to cytidine and choline (66), and it is these metabolites that reach the brain. Following a single bolus injection in rats, serum CDP-choline levels peak at 1 min, but decline to undetectable levels within 5 min (67). Plasma cytidine levels increase markedly at 1 min and then decline rapidly, but remain elevated for at least 1 h (67). Brain levels of choline show a transient increase beginning 10 min after CDP-choline dosing and return to near basal levels by 1 h (68). However, a significant increase in brain levels of CDP-choline cannot be detected following CDP-choline treatment (69), even though increased incorporation of labeled fatty acids into brain lipids has been demonstrated, indicating increased lipid biosynthesis after CDP-choline dosing (70). Because CDP-choline is the rate-limiting intermediate in PtdCho synthesis, this is probably due to rapid utilization of CDP-choline, thus maintaining brain levels.
Due to rapid hydrolysis of exogenous CDP-choline, CDP-choline has to be resynthesized in the brain from phosphocholine and CTP by CCT (18,71). The status of CCT following cerebral ischemia would therefore be important in CDP-choline administration (25). CCT␣ protein expression ( Fig. 2A) significantly decreased following tMCAO, which was reflected in loss of CCT activity (Fig. 2C). CCT activity decreased in the total cortical homogenate, indicating net loss of cellular CCT. CDPcholine treatment significantly attenuated loss of CCT␣ protein (Fig.  2B) and CCT activity (Fig. 2C). During apoptosis, CCT␣ has been shown to undergo caspase-mediated proteolytic cleavage that coincides with poly(ADP-ribose) polymerase cleavage (72). It has been shown that CDP-choline reduces the expression of procaspase-1, -2, -3, -6, and -8 as well as the expression of cleaved caspase-3 and caspase-cleaved products of poly(ADP-ribose) polymerase following tMCAO (73). Thus, CDP-choline may increase CCT in the ischemic cortex by attenuating caspase activation and proteolytic cleavage of CCT.
CDP-choline significantly reduced the infarction volume following tMCAO (Fig. 5). It has been generally believed that CDP-choline, as a precursor for PtdCho, provides neuroprotection by increasing PtdCho synthesis and membrane integrity. This study indicates that CDP-choline may affect PtdCho levels through additional mechanisms (2), particularly by attenuating PtdCho hydrolysis. In addition to preserving membrane PtdCho, attenuating PLA 2 and PtdCho-PLC activation provides neuroprotection by reducing the release of free fatty acids, including arachidonic acid, and oxidative damage resulting from arachidonic acid metabolism (11,37). PtdCho-PLC releases phosphocholine and DAG; DAG subsequently can be hydrolyzed to free fatty acids.
CDP-choline also provides choline for synthesis of the neurotransmitter acetylcholine (2). Cholinergic neurons are unique in the utilization of choline in two metabolic pathways: synthesis of PtdCho and acetylcholine (77,78). These two pathways compete for the available choline, with acetylation favored when neurons are physiologically active (79). If choline becomes depleted (for example, by excessive neuronal stimulation due to release of excitatory amino acids in cerebral ischemia), choline phospholipids, especially PtdCho, are hydrolyzed to provide a source of choline. Total PLD activity in rat brain has been estimated to release ϳ13-17 nmol of choline/min from PtdCho, approximately the rate of acetylcholine turnover (74). Acetylcholine synthesis is thus favored when the availability of choline is limited; neurotransmission is maintained, but at the expense of phospholipids, a process referred to as "autocannibalism," which ultimately causes neu-ronal death (78,79). It has been shown in vitro that choline deficiency results in loss of membrane PtdCho and sphingomyelin and induction of apoptosis (80). CDP-choline can thus prevent PtdCho hydrolysis and death in cholinergic neurons.
Activation of nicotinic acetylcholine receptors provides neuroprotection in focal cerebral ischemia (81), suggesting that neuroprotection by CDP-choline could involve stimulation of acetylcholine receptors. However, brain levels of acetylcholine after transient cerebral ischemia are not affected by CDP-choline treatment (82), and thus, it seems unlikely that this pathway is significant in CDP-choline neuroprotection.
Recent studies have shown that the actions of CDP-choline may extend beyond its effects on PtdCho levels. CDP-choline reduces the expression of procaspases and cleaved caspase-3 following tMCAO (73), which may impact on PtdCho synthesis as discussed previously. As effectors of the apoptotic cascade, attenuation of caspase activation should also provide neuroprotection independent of effects on PtdCho synthesis. CDP-choline also reduces the phosphorylation of MAPK family members ERK1/2 (extracellular signal-regulated kinase-1/2) and MEK1/2 (MAPK/extracellular signal-regulated kinase kinase-1/2), which may provide anti-inflammatory effects (65). To this extent, a hypothetical scheme (Scheme 1) is proposed integrating results from this study and from recent studies in the literature (65,73).