Soluble Epoxide Inhibition Is Protective Against Cerebral Ischemia via Vascular and Neural Protection
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080544
ISSN1525-2191
AutoresAlexis N. Simpkins, R. Daniel Rudic, Derek A. Schreihofer, Sid Roy, Marlina Manhiani, Hsing-Ju Tsai, Bruce D. Hammock, John D. Imig,
Tópico(s)Alcohol Consumption and Health Effects
ResumoInhibition of soluble epoxide hydrolase (SEH), the enzyme responsible for degradation of vasoactive epoxides, protects against cerebral ischemia in rats. However, the molecular and biological mechanisms that confer protection in normotension and hypertension remain unclear. Here we show that 6 weeks of SEH inhibition via 2 mg/day of 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) in spontaneously hypertensive stroke-prone (SHRSP) rats protects against cerebral ischemia induced by middle cerebral artery occlusion, reducing percent hemispheric infarct and neurodeficit score without decreasing blood pressure. This level of cerebral protection was similar to that of the angiotensin-converting enzyme inhibitor, enalapril, which significantly lowered blood pressure. SEH inhibition is also protective in normotensive Wistar-Kyoto (WKY) rats, reducing both hemispheric infarct and neurodeficit score. In SHRSP rats, SEH inhibition reduced wall-to-lumen ratio and collagen deposition and increased cerebral microvessel density, although AUDA did not alter middle cerebral artery structure or microvessel density in WKY rats. An apoptosis mRNA expression microarray of brain tissues from AUDA-treated rats revealed that AUDA modulates gene expression of mediators involved in the regulation of apoptosis in neural tissues of both WKY and SHRSP rats. Hence, we conclude that chronic SEH inhibition protects against cerebral ischemia via vascular protection in SHRSP rats and neural protection in both the SHRSP and WKY rats, indicating that SEH inhibition has broad pharmacological potential for treating ischemic stroke. Inhibition of soluble epoxide hydrolase (SEH), the enzyme responsible for degradation of vasoactive epoxides, protects against cerebral ischemia in rats. However, the molecular and biological mechanisms that confer protection in normotension and hypertension remain unclear. Here we show that 6 weeks of SEH inhibition via 2 mg/day of 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) in spontaneously hypertensive stroke-prone (SHRSP) rats protects against cerebral ischemia induced by middle cerebral artery occlusion, reducing percent hemispheric infarct and neurodeficit score without decreasing blood pressure. This level of cerebral protection was similar to that of the angiotensin-converting enzyme inhibitor, enalapril, which significantly lowered blood pressure. SEH inhibition is also protective in normotensive Wistar-Kyoto (WKY) rats, reducing both hemispheric infarct and neurodeficit score. In SHRSP rats, SEH inhibition reduced wall-to-lumen ratio and collagen deposition and increased cerebral microvessel density, although AUDA did not alter middle cerebral artery structure or microvessel density in WKY rats. An apoptosis mRNA expression microarray of brain tissues from AUDA-treated rats revealed that AUDA modulates gene expression of mediators involved in the regulation of apoptosis in neural tissues of both WKY and SHRSP rats. Hence, we conclude that chronic SEH inhibition protects against cerebral ischemia via vascular protection in SHRSP rats and neural protection in both the SHRSP and WKY rats, indicating that SEH inhibition has broad pharmacological potential for treating ischemic stroke. Epoxyeicosatrienoic acids (EETs), lipid metabolites produced from arachidonic acid by CYP450 enzymes, are novel mediators that antagonize the sequela of hypertension,1Imig JD Cardiovascular therapeutic aspects of soluble epoxide hydrolase inhibitors.Cardiovasc Drug Rev. 2006; 24: 169-188Crossref PubMed Scopus (76) Google Scholar match cerebral blood flow to increased neural activity and metabolic demand, promotes angiogenesis,2Fleming I Vascular cytochrome p450 enzymes: physiology and pathophysiology.Trends Cardiovasc Med. 2008; 18: 20-25Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar and protect against ischemia.3Liu M Alkayed NJ Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.J Cereb Blood Flow Metab. 2005; 25: 939-948Crossref PubMed Scopus (82) Google Scholar, 4Seubert JM Sinal CJ Graves J DeGraff LM Bradbury JA Lee CR Goralski K Carey MA Luria A Newman JW Hammock BD Falck JR Roberts H Rockman HA Murphy E Zeldin DC Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function.Circ Res. 2006; 99: 442-450Crossref PubMed Scopus (161) Google Scholar Because ischemic stroke occurs with loss of cerebral blood flow and is strongly associated with hypertension, modulation of epoxide degradation has potential in managing ischemic stroke. Unfortunately, pharmacological utility of exogenous EETs is impractical because the epoxides are rapidly degraded by the soluble epoxide hydrolase (SEH) into their less active diol, dihydroxyeicosatrienoic acids.5Spector AA Fang X Snyder GD Weintraub NL Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function.Prog Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (481) Google Scholar In fact, human SEH polymorphisms are linked to the incidence of ischemic stroke,6Fornage M Lee CR Doris PA Bray MS Heiss G Zeldin DC Boerwinkle E The soluble epoxide hydrolase gene harbors sequence variation associated with susceptibility to and protection from incident ischemic stroke.Hum Mol Genet. 2005; 14: 2829-2837Crossref PubMed Scopus (80) Google Scholar and this association could be related to modifications in SEH activity, and thus epoxide catabolism.7Przybyla-Zawislak BD Srivastava PK Vazquez-Matias J Mohrenweiser HW Maxwell JE Hammock BD Bradbury JA Enayetallah AE Zeldin DC Grant DF Polymorphisms in human soluble epoxide hydrolase.Mol Pharmacol. 2003; 64: 482-490Crossref PubMed Scopus (132) Google Scholar An alternative strategy that has been used to increase EETs systemically is SEH inhibition.1Imig JD Cardiovascular therapeutic aspects of soluble epoxide hydrolase inhibitors.Cardiovasc Drug Rev. 2006; 24: 169-188Crossref PubMed Scopus (76) Google ScholarWe previously showed that the SEH inhibitor 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) protects against cerebral ischemia in spontaneously hypertensive stroke-prone (SHRSP) rats, an animal model of essential hypertension.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar Interestingly, chronic AUDA treatment in SHRSP rats effectively decreased infarct size induced by middle cerebral artery occlusion (MCAO) without decreasing blood pressure.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar Although blood pressure is an important variable in controlling infarct size in the SHRSP, the cerebrovasculature is also a key determinant of increased sensitivity to cerebral ischemia and thus infarct size.9Coyle P Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY).Anat Rec. 1987; 218: 40-44Crossref PubMed Scopus (42) Google Scholar, 10Coyle P Jokelainen PT Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats.Stroke. 1983; 14: 605-611Crossref PubMed Scopus (110) Google Scholar Our study provided preliminary evidence that AUDA protection in the SHRSP involved changes in vascular structure.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar More recently, a report suggested that administration of AUDA-butyl ester protects against cerebral ischemic reperfusion injury in normotensive mice by mechanisms that may involve neural protection rather than vascular protection.11Zhang W Koerner IP Noppens R Grafe M Tsai HJ Morisseau C Luria A Hammock BD Falck JR Alkayed NJ Soluble epoxide hydrolase: a novel therapeutic target in stroke.J Cereb Blood Flow Metab. 2007; 27: 1931-1940Crossref PubMed Scopus (169) Google Scholar As a result, the contribution of vascular and neural protection to the cerebral protective effects of SEH inhibition during states of hypertension and normotension are unclear and require further investigation. The present study tested the hypothesis that SEH inhibition provides cerebral protection via different mechanisms in normotensive WKY and hypertensive SHRSP rats.Materials and MethodsAnimalsAll male animals were housed and fed a normal rat chow (Tekads 8604; Harlan, Indianapolis, IN) in the animal care facility at the Medical College of Georgia approved by the American Association for the Accreditation of Laboratory Animal Care and all protocols were approved by the institutional animal care and use committee at this institution. All drug treatments were administered via drinking water. Six- to seven-week-old SHRSP rats (Charles River, Wilmington, MA) were divided into four treatment groups as follows: 6 weeks of vehicle (500 mg/L of cyclodextrin and 0.075% of ethanol), 6 weeks of AUDA (2 mg/day via drinking water, 50 mg/L, dissolved using vehicle to aid in solubilization), 6 weeks of enalapril (2.5 mg/day via drinking water dissolved in vehicle), and 5 weeks of AUDA (2 mg/day via drinking water dissolved via vehicle followed by a 7- to 12-day washout period). In addition, a group of 12- to 13-week-old SHRSPs was treated with trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), an SEH inhibitor,12Hwang SH Tsai HJ Liu JY Morisseau C Hammock BD Orally bioavailable potent soluble epoxide hydrolase inhibitors.J Med Chem. 2007; 50: 3825-3840Crossref PubMed Scopus (205) Google Scholar, 13Chiamvimonvat N Ho CM Tsai HJ Hammock BD The soluble epoxide hydrolase as a pharmaceutical target for hypertension.J Cardiovasc Pharmacol. 2007; 50: 225-237Crossref PubMed Scopus (141) Google Scholar 2 mg/day for 7 to 12 days via drinking water (50 mg/L) prepared using vehicle to aid in solubulization. Six- to seven-week-old WKY (Harlan) were treated with either 6 weeks of vehicle or 6 weeks of AUDA, 2 mg/day. Blood pressure was monitored by tail plethysmography (IITC Life Science, Woodland Hills, CA) during the experimental period in acclimatized conscious rats. At the end of the treatment, all animals were anesthetized with pentobarbital (50 mg/kg) for middle cerebral artery occlusion (MCAO) experiments and tissue collection.Plasma and Brain AUDA and Metabolite MeasurementsAUDA levels were measured in homogenized brain, and AUDA metabolites were assessed in plasma samples by reverse phase high performance liquid chromatography followed by negative mode electron spray ionization and tandem mass spectroscopy as previously described.11Zhang W Koerner IP Noppens R Grafe M Tsai HJ Morisseau C Luria A Hammock BD Falck JR Alkayed NJ Soluble epoxide hydrolase: a novel therapeutic target in stroke.J Cereb Blood Flow Metab. 2007; 27: 1931-1940Crossref PubMed Scopus (169) Google ScholarMCAOMCAO was conducted as previously described by Longa and colleagues.14Longa EZ Weinstein PR Carlson S Cummins R Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke. 1989; 20: 84-91Crossref PubMed Scopus (6586) Google Scholar In brief, anesthetized rats with body temperature maintained at 37°C had a 3–0 dermalon monofilament with a rounded tip inserted cranially until a drop in the Laser Doppler (Perimed, North Royalton, OH) attached to the skull 2 mm down and 5 mm lateral to bregma confirmed MCA occlusion.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar Physiological parameters (PO2, PCO2, hematocrit, and blood glucose) were measured via blood sampling with a femoral arterial line using a blood gas analyzer (GemPremier 3000; Instrumentation Laboratory, Lexington, MA). There were no differences in physiological parameters measured during the MCAO at baseline between vehicle and AUDA treatment (see Supplemental Table S1 at http://ajp.amjpathol.org).Six hours after MCAO, neurodeficit was assessed using a combination of a modified form of the Bederson and colleagues15Bederson JB Pitts LH Tsuji M Nishimura MC Davis RL Bartkowski H Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.Stroke. 1986; 17: 472-476Crossref PubMed Scopus (2398) Google Scholar score and an abbreviated adaptation of the modified Neurological Severity Score described by Chen and colleagues.16Chen J Sanberg PR Li Y Wang L Lu M Willing AE Sanchez-Ramos J Chopp M Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats.Stroke. 2001; 32: 2682-2688Crossref PubMed Scopus (1079) Google Scholar The neurodeficit score for WKY animals consisted of a tail suspension test (score range, 1 to 4) to assess postural reflexes and a pad walk to assess motility (range, 1 to 3) that were combined for an overall neurodeficit score. SHRSP neurodeficit score also included an additional spontaneous activity score (1 to 4) to assess the more dramatic deficits displayed in the SHRSP rats, assessing general condition (calm to aggressive) and abnormal movements (curvilinear walk to seizure-like activity).Next, brains were collected and sliced into 2-mm sections coronally from the frontal pole. Noninfarcted hemisphere, infarcted hemisphere, and infarct were measured using the National Institutes of Health (Bethesda, MD) Image software on digitized images of the slices stained with 2% triphenyltetrazolium chloride (TTC). Percent hemisphere infarct was calculated using the Swanson equation to account for swelling.17Swanson RA Morton MT Tsao-Wu G Savalos RA Davidson C Sharp FR A semiautomated method for measuring brain infarct volume.J Cereb Blood Flow Metab. 1990; 10: 290-293Crossref PubMed Scopus (1433) Google ScholarHistology of MCAAnimals were perfused with a vasodilator cocktail (papaverine 0.3 mmol/L/L, adenosine 0.2 mmol/L, diltiazem 0.2 mmol/L/L) prepared in phosphate-buffered saline. A section of the MCA, 4 mm away from the Circle of Willis, was embedded. Serial sections of 5 μm in thickness were taken equidistantly along 650 μm, totaling 16 measurements per vessel. Outer and inner perimeters were measured via by a blinded reviewer using Axiovision 4.0 software (Axio Vision Rel.4.6.3; Carl Zeiss, Thornwood, NY). These measurements were used to calculate wall thickness and wall to lumen (W:L) ratio using the equation of a circle. The MCA was also stained for collagen with the Masson's trichrome stain and picrosirius red stain. The MCA was scored on a scale of 1 to 10 by two blinded observers.Microvessel DensityBecause the reduction in infarct size evident in SEH knockout mice was associated with increased perfusion in the cortex and striatum during MCA occlusion and early reperfusion18Zhang W Kooper D Alkayed NJ Soluble epoxide hydrolase gene deletion is associated with increased cbf and reduced stroke damage.Stroke. 2006; 37: 683Crossref Scopus (93) Google Scholar and we previously found that AUDA treatment reduced infarcted tissue in cortex and striatum,8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar we measured microvessel density in the cortex and the striatum of treated and control rats. To measure the microvessel density, the brains were collected and sliced into five 2-mm slices in the same manner used for TTC staining and infarct quantification. The slices are flash-frozen in a methylbutane dry-ice bath. Serial sections, 5 μm thick, were cut of each slice. After fixation with 10% formalin and blocking in 10% normal goat serum, frozen 5-μm serial sections of brain were incubated overnight with von Willebrand factor antibody (1:250, F3520; Sigma, St. Louis, MO) at 4°C, totaling five sections per animal. After washing with phosphate-buffered saline and incubating with secondary antibody, Cy3 goat anti-rabbit IgG conjugate (1:200; Zymed, South San Francisco, CA), slides were mounted with a coverslip using Prolong Antifade Reagent mounting media (Molecular Probes, Carlsbad, CA). Fluorescence was visualized with a Zeiss LSM 510 Meta confocal laser-scanning microscope (Thornwood, NY 543 nm wavelength). Twenty-five pictures of the cortex and striatum per animal at ×200 magnification were analyzed using Axiovision 5.2 (Carl Zeiss, Thornwood, NY) by two blinded reviewers. Data were reported as area (mm2) of fluorescence per area of field (mm2).Real-Time Polymerase Chain Reaction (PCR) Apoptosis Gene Expression ArrayAs previously described,19Knight SF Quigley JE Yuan J Roy SS Elmarakby A Imig JD Endothelial dysfunction and the development of renal injury in spontaneously hypertensive rats fed a high-fat diet.Hypertension. 2008; 51: 352-359Crossref PubMed Scopus (94) Google Scholar total RNA was extracted from 50 mg of whole brain from male rats using the RNeasy lipid mini kit and DNase digestion (Qiagen, Valencia, CA) according to the manufacturer's protocol and RNA concentrations were determined using absorbance at 260 nm. Using RT2 PCR array first strand kit (SuperArray Bioscience, Frederick, MD), 1 μg of RNA was converted to cDNA. The cDNA was then incubated with RT2 real-time SYBR Green PCR mastermix (SuperArray Bioscience) into a 96-well PCR array plate, one sample per plate (three samples per experimental group). Thermal cycling and real-time detection via a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA) was conducted following the instructions of apoptosis array (Superarray Bioscience). The Superarray RT2 PCR arrays are equipped with controls for genomic DNA contamination, reverse transcription, and PCR. Threshold cycle (Ct) values were normalized to β-actin. The significance analysis of microarrays (SAM) software (Stanford University, Stanford, CA) was used to determine the significance of changes in gene expression in the apoptosis microarray20Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9705) Google Scholar with a median false discovery rate of 17% and q-value (comparable with P value) of 10%.StatisticsAll data are expressed as mean ± SEM. Differences were assessed using analysis of variance and Student's t-tests with P values <0.05 being statistically significant.ResultsBrain AUDA and Plasma AUDA Metabolite LevelsAUDA is metabolized by β oxidation into an inactive metabolite 12-(3-adamantyl-ureido)-butyl acid (AUBA). AUBA levels are used as an indication of AUDA exposure. Because AUDA reversibly and competitively inhibits the SEH enzyme, measuring SEH activity in the tissue to determine the degree of SEH inhibition is not feasible.13Chiamvimonvat N Ho CM Tsai HJ Hammock BD The soluble epoxide hydrolase as a pharmaceutical target for hypertension.J Cardiovasc Pharmacol. 2007; 50: 225-237Crossref PubMed Scopus (141) Google Scholar, 21Newman JW Denton DL Morisseau C Koger CS Wheelock CE Hinton DE Hammock BD Evaluation of fish models of soluble epoxide hydrolase inhibition.Environ Health Perspect. 2001; 109: 61-66Crossref PubMed Scopus (34) Google Scholar, 22Wolf NM Morisseau C Jones PD Hock B Hammock BD Development of a high-throughput screen for soluble epoxide hydrolase inhibition.Anal Biochem. 2006; 355: 71-80Crossref PubMed Scopus (65) Google Scholar Plasma levels of AUBA reached 4 ng/ml in the SHRSP rats and 13 ng/ml in the WKY rats after 6 weeks of SEH inhibition. In the brain, AUDA levels reached 2 μmol/g in the SHRSP rats and 3 μmol/g in the WKY rats. These levels suggest that treatment was sufficient to inhibit SEH enzymatic activity.AUDA Decreases Infarct Size Without Preventing HypertensionAUDA treatment in the SHRSP rats did not prevent the development of hypertension in the 12-week-old SHRSP rats (Figure 1) consistent with our previous findings.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar To determine whether the protective effects of AUDA were independent of blood pressure lowering, additional SHRSP rats were treated with an angiotensin-converting enzyme inhibitor and served as a blood pressure control group for the SHRSP AUDA-treated rats. As expected, administration of the angiotensin-converting enzyme inhibitor enalapril (2.5 mg/day) for 6 weeks was effective at preventing the development of hypertension in the SHRSP rats. Despite the absence of an effect on blood pressure, the reduction in infarct size and neurodeficit achieved with AUDA was similar to that achieved by 6 weeks of enalapril treatment (Figure 1, A–D).Previous studies have suggested that high concentrations of AUDA activate the orphan nuclear receptor PPAR-α.23Fang X Hu S Watanabe T Weintraub NL Snyder GD Yao J Liu Y Shyy JY Hammock BD Spector AA Activation of peroxisome proliferator-activated receptor alpha by substituted urea-derived soluble epoxide hydrolase inhibitors.J Pharmacol Exp Ther. 2005; 314: 260-270Crossref PubMed Scopus (60) Google Scholar, 24Ng VY Morisseau C Falck JR Hammock BD Kroetz DL Inhibition of smooth muscle proliferation by urea-based alkanoic acids via peroxisome proliferator-activated receptor alpha-dependent repression of cyclin D1.Arterioscler Thromb Vasc Biol. 2006; 26: 2462-2468Crossref PubMed Scopus (24) Google Scholar Thus, we treated a separate set of SHRSP rats with tAUCB, a potent SEH inhibitor12Hwang SH Tsai HJ Liu JY Morisseau C Hammock BD Orally bioavailable potent soluble epoxide hydrolase inhibitors.J Med Chem. 2007; 50: 3825-3840Crossref PubMed Scopus (205) Google Scholar, 13Chiamvimonvat N Ho CM Tsai HJ Hammock BD The soluble epoxide hydrolase as a pharmaceutical target for hypertension.J Cardiovasc Pharmacol. 2007; 50: 225-237Crossref PubMed Scopus (141) Google Scholar that lacks potential for PPAR agonistic activity (Figure 1). As expected, tAUCB protected against cerebral ischemia in the SHRSPs, reducing infarct size to 43 ± 3%, P < 0.05, hemispheric infarct size and neurodeficit score to 6.3 ± 0.7, n = 9, P < 0.05. These results indicate that the protective properties of AUDA are not attributable to PPAR agonistic activity but are the result of SEH inhibition.Like the SHRSPs, AUDA treatment in the WKY did not alter blood pressure in the 12-week-old WKY rats (143 ± 1 mmHg versus 141 ± 3 mmHg). However, we still found a protective effect against cerebral ischemia in the WKY animals treated with AUDA. SEH inhibition significantly reduced percent infarct size 40% and neurodeficit 13% (Figure 2, A–C).Figure 2AUDA treatment decreases infarct size and neurodeficit in WKY rats. A: AUDA decreases infarct size after MCAO in WKY rats as demonstrated by the representative TTC-stained coronal slices arranged caudally starting at the frontal pole from each treatment group (black line delineates infarct from viable tissue). B: Quantification of percent hemispheric infarct size in WKY groups. *P < 0.05 relative to WKY. C: Quantification of neurodeficit score in WKY groups. *P < 0.05 relative to WKY. WKY, n = 11; and WKY AUDA, n = 12.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AUDA Provides Vascular Protection in the SHRSP RatsPrevious studies have suggested that AUDA may mediate cerebral protection by increasing MCA compliance.8Dorrance AM Rupp N Pollock DM Newman JW Hammock BD Imig JD An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats.J Cardiovasc Pharmacol. 2005; 46: 842-848Crossref PubMed Scopus (109) Google Scholar However, direct evidence implicating AUDA in vascular structure and remodeling is lacking. Thus, we analyzed blood vessel structure in the MCA in the SHRSP and WKY rats. The wall thickness and W:L ratio, two measures of vascular remodeling, and collagen deposition around the MCA were increased in the adult SHRSP in comparison with the adult WKY animals. Treatment with AUDA attenuated remodeling of MCA in SHRSP rats, manifest as a reduction in wall thickness (26%) and the W:L ratio (27%) (Figure 3, A and B). In addition, there was an appreciable reduction in collagen deposition around the MCA of SHRSP AUDA-treated animals as determined by the Masson's trichrome stain and picrosirius red staining (Figure 3, C–J). Semiquantitative scoring by blinded reviewers determined that untreated SHR-SP had a collagen score of 7.8 ± 0.3 and that this significantly decreased to 4.6 ± 1.3 in AUDA-treated SHRSP. In contrast, we found that MCA wall thickness and W:L ratio of the WKY rats treated with AUDA was not altered (Figure 3, A and B). There was also no noticeable reduction in collagen deposition around the MCAs of AUDA-treated WKYs (Figure 3, C–J).Figure 3Middle cerebral artery (MCA) remodeling and collagen deposition is attenuated by AUDA treatment in SHRSP. A: AUDA attenuates the increase in wall to lumen ratio in the SHRSP. ##P < 0.05 relative to WKY, *P < 0.05 relative to SHRSP. B: AUDA attenuates the increase in wall thickness in the SHRSP. ##P < 0.05 relative to WKY, *P < 0.05 relative to SHRSP. C–F: Staining the MCAs for collagen (pointed to by the black arrowhead) via Masson's trichrome stain. G–J: Staining the MCAs for collagen via picrosirius red stain (collagen red). Stainings demonstrate that AUDA attenuates the increase in collagen deposition around the MCA as depicted by representative images taken at ×200 magnification. WKY (C and G), AUDA-treated WKY (D and H), SHRSP (E and I), AUDA-treated SHRSP (F and J). WKY, n = 4; WKY AUDA, n = 5; SHRSP, n = 6; and SHRSP AUDA, n = 6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further investigate the impact of AUDA on the vasculature, we assessed cerebral microvessel density [area (mm2) of fluorescence per area of field (mm2)]. Assessment of immunofluorescently labeled von Willebrand factor in the brain parenchyma of SHRSP and WKY rats at the end of the treatment period revealed that the area of microvessel density in the adult SHRSP was 33% lower than the adult WKY rats (Figure 4, A, B, and D). AUDA was again protective by increasing the microvessel density by 20% in the SHRSP rats (Figure 4, A, D, and E). This supports the notion that SEH inhibition could be providing cerebral protection by reducing the area at risk to ischemia in the SHRSP rats. WKY rats did not exhibit any change in cerebral microvessel density in the WKY animals treated with AUDA (Figure 4, A–C).Figure 4Cerebral microvascular density is increased by AUDA treatment in SHRSP. A: The area of fluorescently labeled von Willebrand factor (mm2) per area of the field (mm2) in the WKY (n = 4), WKY AUDA (n = 4), SHRSP (n = 4), and SHRSP AUDA (n = 5). ##P < 0.05 relative to WKY, *P < 0.05 relative to AUDA. B–E: Representative von Willebrand factor-labeled immunofluorescent images (red). Original magnifications, ×200.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the protection achieved by chronic AUDA treatment could be sustained after discontinuation of treatment, a separate group of SHRSP rats were treated with AUDA, 2 mg/day, for 5 weeks followed by 7 to 12 days of AUDA withdrawal. The area under the curve (AUC) of orally administered AUDA is 0.4 × 104nmol/L.minute,12Hwang SH Tsai HJ Liu JY Morisseau C Hammock BD Orally bioavailable potent soluble epoxide hydrolase inhibitors.J Med Chem. 2007; 50: 3825-3840Crossref PubMed Scopus (205) Google Scholar and the half-life for AUDA after oral gavage is 7.3 hours.29Watanabe T Schulz D Morisseau C Hammock BD High-throughput pharmacokinetic method: cassette dosing in mice associated with minuscule serial bleedings and LC/MS/MS analysis.Anal Chim Acta. 2006; 559: 37-44Crossref PubMed Scopus (48) Google Scholar As a result, the withdrawal period should be sufficient to exclude the effects of the plasma presence of the drug AUDA. As anticipated, a trend for protection was still evident after the withdrawal period (46 ± 9% hemispheric infarct, n = 5, P < 0.26 and 7.6 ± 0.8 neurodeficit, n = 5, P < 0.20) further supporting the notion that chronic AUDA treatment protection from ischemic damage was in part attributable to structural changes
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