Emergency transport of stroke suspects in a rural state: opportunities for improvement.

INTRODUCTION: Time delay is the key obstacle for receiving successful stroke treatment. Alteplase therapy must start within 4.5 hours from stroke occurrence. Rapid transport to a primary stroke center (PSC) or acute stroke-ready hospital (ASRH) by the emergency medical system (EMS) paramedics is vital. We determined transport time and destination data for EMS-identified and -delivered stroke suspects in Arkansas during 2013. Our objective was to analyze transport time and the hospital qualification for stroke care across the state. METHODS: The state's 75 counties were placed into 8 geographical regions (R1-R8). Transport time and hospital qualification were determined for all EMS-identified strokes. Each hospital's stroke care status was categorized as PSC, ASRH, a nonspecialty or unknown care facility (NSCF), out-of-state, or nonapplicable designation facilities. RESULTS: There were 9588 EMS stroke ground transports with median within-region transport times of 29-40 minutes. Statewide, only 65% of EMS-transported stroke patients were transported to either PSC (12%) or ASRH (53%) facilities. One-third of the patients (30.6%) were delivered to NSCFs, where acute stroke therapy may rarely be performed. Regions with the highest suspected-stroke cases per capita also had the highest percentage of transports to NSCFs. CONCLUSION: With only a few PSCs in Arkansas, EMS agencies should prioritize transporting stroke patients to ASRHs when PSCs are not regionally located.

Elevation of serum lactate dehydrogenase at posterior reversible encephalopathy syndrome onset in chemotherapy-treated cancer patients.

The pathophysiology of posterior reversible encephalopathy syndrome (PRES) is incompletely understood; however, an underlying state of immune dysregulation and endothelial dysfunction has been proposed. We examined alterations of serum lactate dehydrogenase (LDH), a marker of endothelial dysfunction, relative to the development of PRES in patients receiving chemotherapy. A retrospective Institutional Review Board approved database of 88 PRES patients was examined. PRES diagnosis was confirmed by congruent clinical diagnosis and MRI. Clinical features at presentation were recorded. Serum LDH values were collected at three time points: prior to, at the time of, and following PRES diagnosis. Student's t-test was employed. LDH values were available during the course of treatment in 12 patients (nine women; mean age 57.8 years [range 33-75 years]). Chemotherapy-associated PRES patients were more likely to be normotensive (25%) versus the non-chemotherapy group (9%). LDH levels at the time of PRES diagnosis were higher than those before and after (p=0.0263), with a mean difference of 114.8 international units/L. Mean time intervals between LDH measurement prior to and following PRES diagnosis were 44.8 days and 51.4 days, respectively. Mean elapsed time between last chemotherapy administration and PRES onset was 11.1days. In conclusion, serum LDH, a marker of endothelial dysfunction, shows statistically significant elevation at the onset of PRES toxicity in cancer patients receiving chemotherapy. Our findings support a systemic process characterized by endothelial injury/dysfunction as a factor, if not the prime event, in the pathophysiology of PRES.

Three variations in rabbit angiographic stroke models.

PURPOSE: To develop angiographic models of embolic stroke in the rabbit using pre-formed clot or microspheres to model clinical situations ranging from transient ischemic events to severe ischemic stroke. MATERIALS AND METHODS: New Zealand White rabbits (N=151) received angiographic access to the internal carotid artery (ICA) from a femoral approach. Variations of emboli type and quantity of emboli were tested by injection into the ICA. These included fresh clots (1.0-mm length, 3-6h), larger aged clots (4.0-mm length, 3 days), and 2 or 3 insoluble microspheres (700-900 µm). Neurological assessment scores (NAS) were based on motor, sensory, balance, and reflex measures. Rabbits were euthanized at 4, 7, or 24h after embolization, and infarct volume was measured as a percent of total brain volume using 2,3,5-triphenyltetrazolium chloride (TTC). RESULTS: Infarct volume percent at 24 h after stroke was lower for rabbits embolized with fresh clot (0.45±0.14%), compared with aged clot (3.52±1.31%) and insoluble microspheres (3.39±1.04%). Overall NAS (including posterior vessel occlusions) were positively correlated to infarct volume percent measurements in the fresh clot (r=0.50), aged clot (r=0.65) and microsphere (r=0.62) models (p<0.001). CONCLUSION: The three basic angiographic stroke models may be similar to human transient ischemic attacks (TIA) (fresh clot), major strokes that can be thrombolysed (aged clot), or major strokes with insoluble emboli such as atheromata (microspheres). Model selection can be tailored to specific research needs.

Effect of trifluoperazine on toxicity, HIF-1α induction and hepatocyte regeneration in acetaminophen toxicity in mice.

Oxidative stress and mitochondrial permeability transition (MPT) are important mechanisms in acetaminophen (APAP) toxicity. The MPT inhibitor trifluoperazine (TFP) reduced MPT, oxidative stress, and toxicity in freshly isolated hepatocytes treated with APAP. Since hypoxia inducible factor-one alpha (HIF-1α) is induced very early in APAP toxicity, a role for oxidative stress in the induction has been postulated. In the present study, the effect of TFP on toxicity and HIF-1α induction in B6C3F1 male mice treated with APAP was examined. Mice received TFP (10mg/kg, oral gavage) prior to APAP (200mg/kg IP) and at 7 and 36h after APAP. Measures of metabolism (hepatic glutathione and APAP protein adducts) were comparable in the two groups of mice. Toxicity was decreased in the APAP/TFP mice at 2, 4, and 8h, compared to the APAP mice. At 24 and 48h, there were no significant differences in toxicity between the two groups. TFP lowered HIF-1α induction but also reduced the expression of proliferating cell nuclear antigen, a marker of hepatocyte regeneration. TFP can also inhibit phospholipase A(2), and cytosolic and secretory PLA(2) activity levels were reduced in the APAP/TFP mice compared to the APAP mice. TFP also lowered prostaglandin E(2) expression, a known mechanism of cytoprotection. In summary, the MPT inhibitor TFP delayed the onset of toxicity and lowered HIF-1α induction in APAP treated mice. TFP also reduced PGE(2) expression and hepatocyte regeneration, likely through a mechanism involving PLA(2).

Correlation of MRI findings to histology of acetaminophen toxicity in the mouse.

Acetaminophen (APAP) toxicity is responsible for approximately half of all cases of acute liver failure in the United States. The mouse model of APAP toxicity is widely used to examine mechanisms of APAP toxicity. Noninvasive approaches would allow for serial measurements in a single animal to study the effects of experimental interventions on the development and resolution of hepatocellular necrosis. The following study examined the time course of hepatic necrosis using small animal magnetic resonance imaging (MRI) following the administration of 200 mg/kg ip APAP given to B6C3F1 male mice. Mice treated with saline served as controls (CON). Other mice received treatment with the clinical antidote N-acetylcysteine (APAP+NAC). Mouse liver pathology was characterized using T1- and T2-weighted sequences at 2, 4, 8 and 24 h following APAP administration. Standard assays for APAP toxicity [serum alanine aminotransaminase (ALT) levels and hematoxylin and eosin (H&E) staining of liver sections] were examined relative to MRI findings. Overall, T2 sequences had a greater sensitivity for necrosis and hemorrhage than T1 (FLASH) images. Liver injury severity scoring of MR images demonstrated increased scores in the APAP mice at 4, 8 and 24 h compared to the CON mice. APAP+NAC mice had MRI scores similar to the CON mice. Semiquantitative analysis of hepatic hemorrhage strongly correlated with serum ALT. Small animal MRI can be used to monitor the evolution of APAP toxicity over time and to evaluate the response to therapy.

Dodecafluoropentane emulsion decreases infarct volume in a rabbit ischemic stroke model.

PURPOSE: To assess the efficacy of dodecafluoropentane emulsion (DDFPe), a nanodroplet emulsion with significant oxygen transport potential, in decreasing infarct volume in an insoluble-emboli rabbit stroke model. MATERIALS AND METHODS: New Zealand White rabbits (N = 64; weight, 5.1 ± 0.50 kg) underwent angiography and received embolic spheres in occluded internal carotid artery branches. Rabbits were randomly assigned to groups in 4-hour and 7-hour studies. Four-hour groups included control (n = 7, embolized without treatment) and DDFPe treatment 30 minutes before stroke (n = 7), at stroke onset (n = 8), and 30 minutes (n = 5), 1 hour (n = 7), 2 hours (n = 5), or 3 hours after stroke (n = 6). Seven-hour groups included control (n = 6) and DDFPe at 1 hour (n = 8) and 6 hours after stroke (n = 5). DDFPe dose was a 2% weight/volume intravenous injection (0.6 mL/kg) repeated every 90 minutes as time allowed. After euthanasia, infarct volume was determined by vital stains on brain sections. RESULTS: At 4 hours, median infarct volume decreased for all DDFPe treatment times (pretreatment, 0.30% [P = .004]; onset, 0.20% [P = .004]; 30 min, 0.35% [P = .009]; 1 h, 0.30% [P = .01]; 2 h, 0.40% [P = .009]; and 3 h, 0.25% [P = .003]) compared with controls (3.20%). At 7 hours, median infarct volume decreased with treatment at 1 hour (0.25%; P = .007) but not at 6 hours (1.4%; P = .49) compared with controls (2.2%). CONCLUSIONS: Intravenous DDFPe in an animal model decreases infarct volumes and protects brain tissue from ischemia, justifying further investigation.

Successful microbubble sonothrombolysis without tissue-type plasminogen activator in a rabbit model of acute ischemic stroke.

BACKGROUND AND PURPOSE: Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue-type plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with 3 types of MB using a rabbit embolic stroke model. METHODS: New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3-day-old cylindrical clots (0.6 × 4.0 mm). Groups included: (1) control (n=11) embolized without treatment; (2) tPA (n=20); (3) tPA+US (n=10); (4) perflutren lipid MB+US (n=16); (5) albumin 3 µm MB+US (n=8); and (6) tagged albumin 3 µm MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm²) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3 µm MB was 5 × 109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on hematoxylin and eosin sections. RESULTS: Infarct volume percent was lower for rabbits treated with lipid MB+US (1.0%± 0.6%; P=0.013), 3 µm MB+US (0.7% ± 0.9%; P=0.018), and tagged 3 µm MB+US (0.8% ± 0.8%; P=0.019) compared with controls (3.5%± 0.8%). The 3 MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2% ± 0.6% and 1.7%± 0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=nonsignificant). CONCLUSIONS: Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

Variations in the circle of Willis in the New Zealand white rabbit.

PURPOSE: The New Zealand White rabbit (NZWR) serves an important role as an experimental model for vascular research, specifically in the area of stroke. Here the authors document vascular variations in the circle of Willis (COW). MATERIALS AND METHODS: Subselective internal carotid digital subtraction angiography was performed in 100 NZWRs. RESULTS: Important variations include hypoplasia in 36%, duplication of the middle cerebral artery in 29%, asymmetries of the posterior region in 19%, and multiple variations in 28%. The complete classical symmetric COW without significant variation is present in fewer than 30% of animals. CONCLUSIONS: With recognition of the variations, the NZWR becomes an improved research model.

Microbubble-augmented ultrasound sonothrombolysis decreases intracranial hemorrhage in a rabbit model of acute ischemic stroke.

OBJECTIVES: Increasing evidence confirms that microbubble (MB)-augmented ultrasound (US) thrombolysis enhances clot lysis with or without tissue plasminogen activator (tPA). Intracranial hemorrhage (ICH) is a major complication militating against tPA use in acute ischemic stroke. We quantified the incidence of ICH associated with tPA thrombolysis and MB + US therapy and compared infarct volumes in a rabbit model of acute ischemic stroke. MATERIALS AND METHODS: Rabbits (n = 158) received a 1.0-mm clot, angiographically injected into the internal carotid artery causing infarcts. Rabbits were randomized to 6 test groups including (1) control (n = 50), embolized without therapy, (2) US (n = 18), (3) tPA only (n = 27), (4) tPA + US (n = 22), (5) MB + US (n = 27), and (6) tPA + MB + US (n = 14). US groups received pulsed wave US (1 MHz, 0.8 W/cm) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) given over 30 minutes. Rabbits were killed 24 hours later and infarct volume and incidence, location, and severity of ICH were determined by histology and pathologic examination. RESULTS: Percentage of rabbits having ICH outside the infarct area was significantly decreased (P = 0.004) for MB + US (19%) rabbits compared with tPA + US (73%), US only (56%), tPA (48%), tPA + MB + US (36%), and control (36%) rabbits. Incidence and severity of ICH within the infarct did not differ (P > 0.39). Infarct volume was significantly greater (P = 0.002) for rabbits receiving US (0.97% ± 0.17%) than for MB + US (0.20% ± 0.14%), tPA + US (0.15% ± 0.16%), tPA (0.14% ± 0.14%), and tPA + MB + US (0.10% ± 20%) rabbits; these treatments collectively, excluding US only, differed (P = 0.03) from control (0.45% ± 0.10%). CONCLUSIONS: Treatment with MB + US after embolization decreased the incidence of ICH and efficacy was similar to tPA in reducing infarct volume.

Microbubbles improve sonothrombolysis in vitro and decrease hemorrhage in vivo in a rabbit stroke model.

INTRODUCTION: Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH. MATERIALS AND METHODS: Fresh blood clots (3-5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm²), with or without 1.12 × 108/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm²) for 60 minutes and intravenous tPA (0.1-0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later. RESULTS: In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P < 0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19. CONCLUSION: Combining tPA and MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

Estrogen replacement attenuates exaggerated neointimal hyperplasia following carotid endarterectomy in rats.

BACKGROUND: To investigate whether estrogen may attenuate neointima formation in hyperhomocysteinemic rat carotid endarterectomy. METHODS: Rats were divided into 6 groups: ovariectomized estradiol-treated homocysteine or chow; ovariectomized placebo-treated homocysteine or chow; intact placebo-treated homocysteine or chow. Chow served as controls while homocysteine served as exaggerated intimal hyperplasia. Prior to endarterectomy, rats were implanted with estradiol mini-pump or placebo, diets given 2 weeks before and after surgery. Homocysteine, estrogen, and neointimal hyperplasia were determined. RESULTS: Homocysteine was elevated in homocysteine groups versus controls except in estradiol-treated group. Intimal hyperplasia increased in placebo-treated ovariectomized homocysteine versus intact group. Exaggerated intimal hyperplasia in placebo-treated ovariectomized homocysteine was reduced by estrogen and so was homocysteine. Estrogen replacement in ovariectomized homocysteine group reduced intimal hyperplasia to that of intact or ovariectomized controls. CONCLUSION: Estradiol treatment in this ovariectomized hyperhomocysteinemia carotid endarterectomy and resultant attenuation of homocysteine and neointima may have relevance to the beneficial effects of estrogen on hyperplastic response.

The effects of acarbose treatment on intimal hyperplasia in a rat carotid endarterectomy model of diet-induced insulin resistance.

BACKGROUND: Increased carotid restenosis due to revascularization therapy is associated with insulin resistance. We hypothesize that glucose control using acarbose may attenuate intimal hyperplasia in rat carotid endarterectomy model of diet-induced insulin resistance. METHODS: Rats were fed low-fat complex carbohydrate (control) or high-fat sucrose (insulin resistance) for 4 months. Three days preoperatively, some high-fat-sucrose rats were on acarbose, remainder of the rats received placebo. Rat carotids were assessed with duplex pre-and postoperatively. Acarbose and placebo continued for 2 weeks. Glucose, insulin, blood flow velocities and intimal hyperplasia were determined. RESULTS: High-fat sucrose plus acarbose attenuated intimal hyperplasia. Post-drug high-fat sucrose glucose decreased. Blood flow velocities postoperatively elevated above baseline. High-fat sucrose increased blood flow velocities postoperatively, which was attenuated with acarbose. CONCLUSION: Glucose control by acarbose in rat carotid endarterectomy model of diet-induced insulin resistance resulted in attenuation of intimal hyperplasia.

Stroke location and brain function in an embolic rabbit stroke model.

PURPOSE: Current rabbit stroke models often depend on symptoms as endpoints for embolization and produce wide variation in location, size, and severity of strokes. In a further refinement of an angiographic embolic stroke model, localized infarctions were correlated to neurologic deficits with the goal to create a rabbit model for long-term studies of therapies after stroke. MATERIALS AND METHODS: New Zealand White rabbits (4-5 kg; N = 71) had selective internal carotid artery (ICA) angiography and a single clot was injected. At 24 hours, neurologic assessment score (NAS) was measured on an 11-point scale (0, normal; 10, dead). Brains were removed and stained to identify stroke areas. All animals with single strokes (n = 31) were analyzed by specific brain structure involvement, and NAS values were correlated. RESULTS: Stroke incidence differed by location, with cortex, subcortical, and basal ganglia regions highest. The middle cerebral artery (MCA), at 52%, and anterior cerebral artery (ACA), at 29%, were most commonly involved, with the largest stroke volumes in the ACA distribution. Brainstem and cerebellum strokes had disproportionately severe neurologic deficits, scoring 2.25 +/- 1.0 on the NAS, which represented a significant (P < .02) difference versus cortex (0.5 +/- 0.2), subcortical (1.3 +/- 0.4), and basal ganglia (0.5 +/- 0.3), all in the frontal or parietal regions. CONCLUSIONS: MCA and ACA distributions included 81% of strokes. These sites were relatively silent (potentially allowing longer-term survival studies) whereas others in the posterior circulation produced disproportionately severe symptoms. Symptoms were not reliable indicators of stroke occurrence, and other endpoints such as imaging may be required. These are important steps toward refinement of the rabbit stroke model.

The effects of Toradol on postoperative intimal hyperplasia in a rat carotid endarterectomy model: laboratory research.

Carotid endarterectomy (CEA) and more recently carotid artery stenting are the treatments of choice for atherosclerotic disease of the extracranial carotid arteries; however, early restenosis caused by neointimal hyperplasia confounds surgical therapy. Oxidative stress has been implicated in the progression of intimal hyperplasia. The authors hypothesized that ketorolac tromethamine (Toradol), a nonsteroidal antiinflammatory drug that is a potent cyclooxygenase inhibitor, would decrease oxidative stress and thereby reduce intimal hyperplasia in a rat CEA model. Twenty-nine male Sprague-Dawley rats underwent CEA and were divided into 3 treatment groups as follows: (1) control (placebo), (2) 7.5 mg/kg Toradol, and (3) 10 mg/kg Toradol. Toradol treatment began 2 days before CEA and continued for 2 weeks. Two weeks after endarterectomy, carotid arteries were fixed, harvested, and examined for platelet activity (platelet reactive units), oxidative stress (malondialdehyde and glutathione), and intimal hyperplasia (measured as percentage of luminal stenosis). Platelet activity, malondialdehyde and glutathione, and intimal hyperplasia were all significantly lowered in both 7.5- and 10-mg/kg doses of Toradol versus control. Toradol given daily beginning 2 days before CEA and ending 2 weeks after the procedure was effective at significantly reducing platelet activity, oxidative stress, and intimal hyperplasia development in the rat without any increase in bleeding. Although the mechanism of action of this reduction is not completely understood, one possible explanation may be through the inhibition of reactive oxygen species production.

Selective intracranial magnification angiography of the rabbit: basic techniques and anatomy.

Development of new therapies for stroke requires animal models with well-defined intracranial vasculature. The rabbit as a small animal model has many desirable traits; however, a modern atlas of rabbit angiographic anatomy is not readily available. Improved digital subtraction magnification angiography and superselective small-catheter techniques now allow excellent anatomical definition. Angiographic techniques include selection of the internal carotid artery and subselection with microcatheters that can progress to branches of the circle of Willis and provide high-resolution cerebral angiography. The authors present an overview of current techniques and illustrations of the angiography of cerebral vessels.

Pravastatin and clopidogrel combined inhibit intimal hyperplasia in a rat carotid endarterectomy model.

Intimal hyperplasia, resulting from a complex cascade of events involving platelets, leukocytes, and smooth muscle cells, may be inhibited by the HMG-CoA reductase inhibitor pravastatin, which demonstrates inhibition of platelet activity and leukocyte adhesion and may be associated with inhibition of vascular smooth muscle cell proliferation and migration. Clopidogrel, an adenosine diphosphate (ADP) receptor inhibitor, was shown to decrease platelet activity and aggregation but not intimal hyperplasia (IH). We postulated that the combination of both pravastatin and clopidogrel would significantly decrease IH in a rat carotid endarterectomy model. Male Sprague-Dawley rats (n = 18) divided by treatment regimen underwent treatment for 2 weeks both before and after an open carotid endarterectomy. Serum collected at the time of harvest was measured for C-reactive protein (CRP), platelet activity, and total serum cholesterol; carotid arteries were removed and processed for IH determination. Control rats (n = 7) received oral vehicle daily before and following endarterectomy. Pravastatin-alone rats (n = 6) received oral pravastatin (10 mg/kg/day) before and after endarterectomy. Pravastatin plus clopidogrel rats (n = 5) received oral pravastatin (10 mg/kg/day) plus a preendarterectomy bolus of oral clopidogrel (4.3 mg/kg) before endarterectomy and resumed pravastatin (10 mg/kg/day) plus oral clopidogrel (1 mg/kg/day) postendarterectomy. Pravastatin alone and pravastatin plus clopidogrel significantly decreased CRP compared to controls (120.2 +/-11.2 and 134.1 +/- 9.9 vs 191.1 +/- 9.2 microg/mL, respectively p = 0.003 and p =0.0024). CRP levels were not different between pravastatin alone and pravastatin plus clopidogrel (p = 0.35). Platelet activity was significantly decreased by pravastatin alone and pravastatin plus clopidogrel in comparison to controls (7.3 +/- 2.2 and 6.6 +/- 2.8 vs 19.2 +/- 6.1 platelet reactive units (PRU), respectively p = 0.048 and p = 0.045). No significant difference was noted in platelet activity between pravastatin alone and pravastatin plus clopidogrel (p = 0.89). Pravastatin plus clopidogrel significantly reduced serum cholesterol compared to control and pravastatin alone (84.0 +/- 6.6 vs 110.4 +/- 7.4 and 117.0 +/- 8.8 mg/dL, respectively p = 0.03 and p = 0.01). Pravastatin alone did not decrease serum cholesterol compared to controls (p = 0.54). IH was not reduced by pravastatin alone compared to controls (p = 0.61) but was significantly decreased by pravastatin plus clopidogrel in comparison to control and pravastatin alone (3.0 +/- 1.1 vs 46.3 +/- 13.7 and 37.4 +/- 14.6% luminal stenosis, respectively p = 0.01 and p = 0.05). Pravastatin plus clopidogrel significantly decreased CRP, platelet activity, total serum cholesterol, and IH while pravastatin alone decreased only CRP and platelet activity. Intimal hyperplasia reduction may therefore be dependent on other contributors, possibly growth factors, cytokines, and oxidative stress. The combination of pravastatin plus clopidogrel may have synergistic or even additional inhibitory effects on IH. Pravastatin plus clopidogrel was effective in decreasing IH in a rat carotid endarterectomy model and may prove a useful therapy for IH reduction in the clinical setting.

Homocysteine-induced vascular dysregulation is mediated by the NMDA receptor.

Elevated plasma homocysteine accelerates myointimal hyperplasia and luminal narrowing after carotid endarterectomy. N-methyl D aspartate receptors (NMDAr) in rat cerebrovascular cells are involved in homocysteine uptake and receptor-mediated stimulation. In the vasculature, NMDAr subunits (NR1, 2A-2D) have been identified by sequence homology in rat aortic endothelial cells. Exposure of these cells to homocysteine increased expression of receptor subunits, an effect that was attenuated by dizocilpine (MK801), a noncompetitive NMDA inhibitor. The objective of this study was to investigate the existence of an NMDAr in rat vascular smooth muscle (A7r5) cells, and also the effect of homocysteine on vascular dysregulation as mediated by this receptor. Subunits of the NMDAr (NR1, 2A-2D) were detected in the A7r5 cells by using the reverse transcriptase polymerase chain reaction and Western blotting. Homocysteine induced an increase in A7r5 cell proliferation, which was blocked by MK801. Homocysteine, in a dose and time dependent manner, increased expression of matrix metallinoproteinase-9 and interleukin-1beta, which have been implicated in vascular smooth muscle cell migration and/or proliferation. Homocysteine reduced the vascular elaboration of nitric oxide and increased the elaboration of the nitric oxide synthase inhibitor, asymmetric dimethylarginine. All of these homocysteine mediated effects were inhibited by MK801. NMDAr exist in vascular smooth muscle cells and appear to mediate, at least in part, homocysteine-induced dysregulation of vascular smooth muscle cell functions.

Identification of a homocysteine receptor in the peripheral endothelium and its role in proliferation.

BACKGROUND: Homocysteine, a risk factor for atherosclerosis, increases intimal hyperplasia after carotid endarterectomy with associated smooth muscle cell proliferation and modulation of cytokines. The N-methyl-D-aspartate receptor (NMDAr), a glutamate-gated ion channel receptor, is associated with homocysteine-induced cerebrovascular injury; however, the receptor has not been identified in peripheral vascular cells, nor has any interaction with homocysteine been clarified. Our objectives were first, to identify NMDAr in rat carotid artery and rat aorta endothelial cells (RAEC); and second, to determine whether homocysteine activates NMDAr in the endothelium. METHODS: NR1 and NR2A, two NMDAr subunits, were probed in rat carotid arteries by immunohistochemistry. RNA was isolated from RAECs, and expression of all NMDAr subunits (NR1, 2A, 2B, 2C, and 2D) were examined by RT-PCR and sequencing. For receptor protein expression, RAEC were incubated with different homocysteine concentrations and incubation times and also were treated with 50 microM homocysteine and/or preincubated with 50 microM dizocilpine MK-801, an NMDAr inhibitor. RESULTS: Both NR1 and NR2A were expressed in rat carotid arteries. All NMDAr subunits were expressed in the RAECs, and there was 92% to 100% similarity compared with rat NMDAr from the National Center for Biotechnology Information (NCBI) GenBank. Homocysteine upregulated NR1 expression and increased cell proliferation. RAEC pretreatment with MK-801 reduced homocysteine-mediated cell proliferation. CONCLUSION: This study is the first to show that NMDAr exists in the peripheral vasculature, and that homocysteine may act via NMDAr to increase intimal hyperplasia. CLINICAL RELEVANCE: Our objectives included the identification of a homocysteine receptor in the peripheral vasculature. The possible inhibition of a homocysteine receptor to prevent intimal hyperplasia rather than treat established stenosis would make a significant clinical impact. This will open further avenues of study in determining the role of homocysteine in the pathogenesis of intimal hyperplasia.

Effect of clopidogrel on platelet aggregation and intimal hyperplasia following carotid endarterectomy in the rat.

Intimal hyperplasia results in significant morbidity and mortality following vascular intervention. Both platelets and elevated homocysteine have been implicated in the development of intimal hyperplasia. We previously demonstrated that a locally applied antiplatelet agent decreases the development of intimal hyperplasia. We were therefore interested in a systemic antiplatelet agent, clopidogrel. We hypothesized that clopidogrel would decrease platelet aggregation and activity and intimal hyperplasia. Male Sprague-Dawley rats underwent carotid endarterectomy (CEA) and treatment with either placebo or varying regimens of clopidogrel, including chronic, pre-CEA bolus, chronic plus pre-CEA bolus, and chronic plus post-CEA bolus; a homocystine diet was used to elevate both plasma homocysteine and the degree of intimal hyperplasia. Platelet aggregation, platelet activity, and intimal hyperplasia were then assessed. Platelet aggregation was not decreased with chronic clopidogrel; however, it was decreased with pre-CEA bolus clopidogrel. Similarly, platelet activity was not inhibited by chronic clopidogrel but was inhibited by pre-CEA and chronic plus pre-CEA bolus clopidogrel. Neither chronic, pre-CEA bolus, chronic plus pre-CEA bolus, nor chronic plus post-CEA bolus clopidogrel resulted in a decrease in intimal hyperplasia. Although pre-CEA bolus clopidogrel resulted in a decrease in both platelet aggregation and activity, it was unable to decrease the development of intimal hyperplasia at any dose. Additional factors must therefore contribute to the pathologic development of intimal hyperplasia.