Rat Carotid Endarterectomy Research Articles

Homocysteine Upregulates Expression of NR2D-containing N-methyl-D-aspartate Receptors in a Rat Carotid Endarterectomy Model

Homocysteine Upregulates Expression of NR2D-containing N-methyl-D-aspartate Receptors in a Rat Carotid Endarterectomy Model

Estrogen Replacement Attenuates Exaggerated Neointimal Hyperplasia Following Carotid Endarterectomy in Rats

To investigate whether estrogen may attenuate neointima formation in hyperhomocysteinemic rat carotid endarterectomy. 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. 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. 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

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. 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. 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. Glucose control by acarbose in rat carotid endarterectomy model of diet-induced insulin resistance resulted in attenuation of intimal hyperplasia.

Combined Superoxide Dismutase Mimetic and Peroxynitrite Scavenger Protects Against Neointima Formation After Endarterectomy in Association with Decreased Proliferation and Nitro-oxidative Stress

Objective Reactive oxygen and nitrogen species (e.g., peroxynitrite) may trigger neointima formation leading to restenosis. In a rat carotid endarterectomy (CEA) model, we investigated the effects of the manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase (SOD) mimetic and peroxynitrite scavenger on neointima formation. Methods CEA was performed in male Sprague–Dawley rats. Animals received either vehicle (control group; n = 15) or 15 mg kg −1 day −1 MnTBAP intraperitoneally for 3 weeks (treatment group; n = 13). Four groups of carotids were analysed: the left, uninjured carotids (sham) and the right, injured carotids (control CEA) from the control group, the right, injured carotids from the treatment group (CEA + MnTBAP) and an additional group of carotids that were harvested 1 h following endarterectomy. The analysis of carotid arteries was performed by histology, immunohistochemistry and real-time polymerase chain reaction (PCR). Plasma malondialdehyde (MDA) levels were measured by lipid hydroperoxidase assay. Results Stenosis rate (10.5 ± 8.1% vs. 45.4 ± 28.3%), the percentage of proliferating cell nuclear antigen-positive cells (13.4 ± 7.1% vs. 23.3 ± 11.0%) and nitrotyrosine immunoreactivity (5.8 ± 1.9 vs. 8.0 ± 2.0) were significantly reduced in the vascular wall of the CEA + MnTBAP group compared with control CEA group. Ratio of Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL)-positive nuclei was significantly lower after antioxidant therapy (41.7 ± 26.7% vs. 64.9 ± 18.5%). Plasma MDA levels increased after endarterectomy (11.7 ± 4.8 vs. 4.1 ± 2.0 μmol l −1) and reduced in the treatment group (3.2 ± 2.1 μmol l −1). No significant gene regulation after MnTBAP treatment could be noted. Conclusions MnTBAP decreased neointima formation, which was associated with reduced vascular smooth muscle cell proliferation and attenuated local and systemic nitro-oxidative stress.

Combined superoxide dismutase mimetic and peroxynitrite scavenger protects against neointima formation after endarterectomy in association with decreased proliferation and nitro-oxidative stress

Abstract Objective Reactive oxygen and nitrogen species (e.g., peroxynitrite) may trigger neointima formation leading to restenosis. In a rat carotid endarterectomy (CEA) model, we investigated the effects of the manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase (SOD) mimetic and peroxynitrite scavenger on neointima formation. Methods CEA was performed in male Sprague–Dawley rats. Animals received either vehicle (control group; n = 15) or 15 mg kg−1 day−1 MnTBAP intraperitoneally for 3 weeks (treatment group; n = 13). Four groups of carotids were analysed: the left, uninjured carotids (sham) and the right, injured carotids (control CEA) from the control group, the right, injured carotids from the treatment group (CEA + MnTBAP) and an additional group of carotids that were harvested 1 h following endarterectomy. The analysis of carotid arteries was performed by histology, immunohistochemistry and real-time polymerase chain reaction (PCR). Plasma malondialdehyde (MDA) levels were measured by lipid hydroperoxidase assay. Results Stenosis rate (10.5 ± 8.1% vs. 45.4 ± 28.3%), the percentage of proliferating cell nuclear antigen-positive cells (13.4 ± 7.1% vs. 23.3 ± 11.0%) and nitrotyrosine immunoreactivity (5.8 ± 1.9 vs. 8.0 ± 2.0) were significantly reduced in the vascular wall of the CEA + MnTBAP group compared with control CEA group. Ratio of Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL)-positive nuclei was significantly lower after antioxidant therapy (41.7 ± 26.7% vs. 64.9 ± 18.5%). Plasma MDA levels increased after endarterectomy (11.7 ± 4.8 vs. 4.1 ± 2.0 μmol l−1) and reduced in the treatment group (3.2 ± 2.1 μmol l−1). No significant gene regulation after MnTBAP treatment could be noted. Conclusions MnTBAP decreased neointima formation, which was associated with reduced vascular smooth muscle cell proliferation and attenuated local and systemic nitro-oxidative stress.

Effects of VCAM-1 siRNA on restenosis following carotid endarterectomy: experiment with rats

To evaluate the inhibitory effects of VCAM-1 siRNA on VCAM-1 protein expression and restenosis following carotid endarterectomy in rats. Lentivirus-based VCAM-1 siRNA was constructed and its efficacy of blocking VCAM-1 protein expression in endothelial cells and carotids was identified by Western blot. Doppler ultrasonography and morphometric analysis were performed to measure the degree of restenosis. VCAM-1 siRNA decreased the protein expression of VCAM-1 in cultured endothelial cells and carotids. Treatment of VCAM-1 siRNA showed a significant reduction in the restenosis and manifested as an increased blood velocity and linear diameter as compared with control siRNA (P < 0.05). Morphometric analysis showed that the ratio of intima to media area (I/M) increased significantly in CEA group (3.99 +/- 0.65) versus sham-operated group (0.35 +/- 0.13) (P < 0.05). Furthermore, VCAM-1 siRNA resulted in an evident decrease in the neointimal area (1.79 +/- 0.43) as compared with that of the control siRNA (4.33 +/- 0.59) (P < 0.05). VCAM-1 plays an important role in pathogenesis of restenosis after carotid endarterectomy. VCAM-1 siRNA blocks VCAM-1 protein expression and alleviates the restenosis following carotid endarterectomy in rats.

High resolution duplex visualization details development of normal and exaggerated luminal stenosis following endarterectomy

Reparative surgical procedures in the carotid are limited by intimal hyperplasia. To understand initial patterns of hyperplasia development, we studied circumferential measurements of luminal narrowing, arterial peak systolic velocity (PSV) and end diastolic velocity (EDV) measurements in a rat carotid endarterectomy (CEA).Three male Sprague Dawley rats (450 g BW) served as a normal hyperplasia development group (CON) and N=4 rats had dietary 4.5 g/kg DL‐homocystine (HCYS) added for exaggerated proliferation. Carotids were visualized in real time and duplexed pre‐operatively and 4, 9 and 14 days post‐CEA (Pre‐OP or D4, D9 and D14). PSV/EDV measurements and lesion development was noted longitudinally and circumferential measurements were taken across the suture line. Volume calculations measured lumenal narrowing and were compared to Pre‐Op.Post‐Op PSV/EDV measurements were increased above Pre‐Op levels at D4 through D14 at p&lt;0.05. In HCYS both PSV/EDV were higher at D14 vs. CON at p=0.051 and p=0.018, respectively. Along the anastomosis lumenal narrowing was indicated for both CON and HCYS in Pre‐OP vs. Post‐Op beginning at D4 (p=0.0241 and p=0.0450, respectively). At D14, there was more luminal narrowing in HCYS rats vs. CON at p=0.0001.We observed by duplex that lumenal stenosis is dynamic and changes occur as early as D4 with concomitant influences on arterial pressure. Funded by US Gov, VA Merit Review.

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.

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.

Saratin (an inhibitor of platelet-collagen interaction) decreases platelet aggregation and homocysteine-mediated postcarotid endarterectomy intimal hyperplasia in a dose-dependent manner

Background This study investigated Saratin’s (Merck KGaA, Darmstadt, Germany) prevention of platelet adhesion and intimal hyperplasia at different doses and in the hyperhomocystinemia rat carotid endarterectomy (CEA) model. Methods Rats were divided into two groups: (1) platelet adhesion or (2) luminal stenosis because of intimal hyperplasia. At CEA, rats received 0, 0.5, 5.0, 10.0, or 20.0 μg Saratin on the artery. Post-CEA platelet aggregation was evaluated by standard error of the mean. Intimal hyperplasia group received either (1) control or (2) 4.5 g/kg DL-homocystine diets for two weeks followed by CEA and treated with diluent or 5.0 μg Saratin. Endpoints included platelet adhesion, intimal hyperplasia, plasma homocysteine (HCys), and its metabolic enzymes cystathionine β-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR). Results Platelet adhesion: post-CEA, platelet adhesion was reduced by 63%, 67%, and 67% in Saratin doses ≥5.0 μg. Intimal hyperplasia: 5.0 μg Saratin in the HCys group decreased intimal hyperplasia by 45% compared with the non-Saratin-treated HCys group. Plasma HCys levels were not altered with Saratin treatment in the HCys groups nor were CBS or MTHFR. Conclusions Saratin significantly inhibited platelet adhesion at ≥5.0 μg, and Saratin at 5.0 μg attenuated luminal stenosis in a hyperhomocysteinemic rat CEA model.

Plasma homocysteine measurements after carotid artery manipulation and clamping in a rat CEA model

The effect of a rat carotid endarterectomy (CEA) on homocysteine and the metabolic enzymes methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS) was studied. Rats were placed into 7 groups: (1) no anesthesia (NA), (2) anesthesia only (AO), (3) skin opened and closed (O/C), (4) skin opened with exposure of the carotid artery and closed (O/E/C), (5) carotid isolated and clamped (CO), (6) open CEA, and (7) open femoral endarterectomy (FEA). End points included homocysteine, hepatic MTHFR, and CBS activity. Homocysteine in the NA, AO, O/C, O/E/C, and FEA were low and not different. CEA produced a 6-fold increase in homocysteine when compared with non-CEA groups. Specifically, CEA produced an increase in homocysteine versus the AO group at 2 weeks (11.3 +/- 0.7 vs 2.1 +/- 0.9 micromol/L;P < .001), 4 weeks (8.9 +/- 0.7 vs 3.5 +/- 0.9 micromol/L; P = .004) and 6 weeks (7.7 +/- 0.9 vs 3.1 +/- 1.5 micromol/L; P = .03). The CO group had increased homocysteine versus the O/C, O/E/C, and FEA, but was lower than the CEA group. CEA produced an increase in MTHFR and CBS versus the AO group. CEA resulted in elevated levels of homocysteine; however, when broken down into its component parts, no elevation was observed except for a small increase with the CO procedure. Manipulation of the femoral artery did not raise homocysteine levels. The increase in homocysteine is possibly due to the combination of vessel wall damage and changes in cerebral blood flow dynamics.

Cigarette smoke increases intimal hyperplasia and homocysteine in a rat carotid endarterectomy

Homocysteine and smoking are independent risks for CVD; however their importance in post-CEA intimal hyperplasia is unclear. We performed a CEA in rats exposed to cigarette smoke with the hypothesis that smoking would increase intimal hyperplasia that may be associated with an elevated serum homocysteine. Folic acid (FA) and the homocysteine metabolic enzymes MTHFR and CBS were used to test for the significance of homocysteine elevation. Rats underwent an open CEA. N = 13 rats received smoke exposure 2 weeks prior, and 2 weeks post-CEA and N = 12 received no smoke. Each group was divided into either control or an FA-added diet resulting in four groups. Rats were sacrificed at 2 weeks post-CEA; liver, urine, blood, and carotid arteries samples were obtained. Smoked rats had increased urinary peak and trough cotinine levels versus non-smoke rats, which decreased with FA. Smoke exposure increased intimal hyperplasia versus non-smoke controls by nearly 120% (57.8 +/- 6.2 versus 26.8 +/- 5.4% luminal stenosis, P = 0.005). Smoke-exposed rats had an increased serum homocysteine versus non-smoke controls (8.3 +/- 0.8 versus 5.7 +/- 0.8 microm, P = 0.014). Smoked rats given FA had decreased serum homocysteine compared to the smoke group. Along with reductions in homocysteine, FA eliminated the increase in intimal hyperplasia seen with smoke exposure (33.5 +/- 6.1 versus 57.8 +/- 6.2% luminal stenosis, P = 0.03). CBS activity decreased in smoked rats by nearly 20% versus non-smoke rats. FA supplementation in smoked rats both (1) increased CBS activity and (2) decreased MTHFR compared to control non-smoke-exposure levels. Smoking increases plasma homocysteine and post-CEA intimal hyperplasia. This suggests homocysteine has an etiological role in the intimal hyperplasia increase observed with smoking, since both were negated with FA.

Identification of a novel smooth muscle associated protein, smap2, upregulated during neointima formation in a rat carotid endarterectomy model

Proliferation of aortic smooth muscle cells is an important event in vascular lesion formation. To identify new genes that are involved in neointima formation, we constructed an aortic 3′-directed cDNA library. The novel cDNA of a gene designated smooth muscle associated protein 2 (smap2) was isolated. The full-length cDNA of smap2 is 2914 base pairs long and contains an open reading frame of 1338 base pairs. Dot blot analysis revealed that smap2 was expressed particularly in aorta. The deduced amino acid sequence of smap2 contains two thyroglobulin type-1 domains, two EF-hand calcium-binding domains and putative signal peptide. Furthermore, we demonstrated that smap2 mRNA was upregulated during neointima formation in a rat carotid endarterectomy model. These findings suggest that smap2 might be involved in the progression of atherosclerosis in aorta.

Saratin, an inhibitor of von Willebrand factor–dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model

Purpose: Post–carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis , which is thought to act by binding to collagen, and inhibits von Willebrand factor–collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. Methods: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. Results: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 ± 17 vs 155 ± 33 platelets per grid, P =.05), and 24 hours after carotid endarterectomy (35 ± 11 vs 149 ± 37 platelets per grid, P =.0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% ± 1.8% vs 29.8% ± 6.8%, P =.0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P =.0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. Conclusion: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation. (J Vasc Surg 2001;34:724-9.)

Folate supplementation inhibits intimal hyperplasia induced by a high-homocysteine diet in a rat carotid endarterectomy model

Objective: Hyperhomocysteinemia has been implicated as a causative factor in intimal hyperplasia development. The addition of dietary folate in a hyperhomocysteinemia, carotid endarterectomy rat model is postulated to decrease plasma homocysteine levels and, in turn, reduce post–carotid endarterectomy intimal hyperplasia. Methods: Each rat was fed one of six diets: (1) lab chow with no folate (n = 7), (2) lab chow with 10 mg/kg folate added (n = 3), (3) lab chow with 25 mg/kg folate added (n = 3), (4) a homocysteine diet with no folate (n = 7), (5) a homocysteine diet with 10 mg/kg folate added (n = 5), or (6) homocysteine diet with 25 mg/kg folate added (n = 5). Each rat then underwent an open carotid endarterectomy. In 2 weeks, intimal hyperplasia in the carotid artery was measured. Plasma homocysteine and folate levels were measured. Results: Plasma folate levels rose with folate administration. Plasma homocysteine in the lab chow group was 5.4 ± 0.5 μmol/L and did not change with the addition of folate. In the homocysteine diet group, plasma homocysteine rose 10-fold over the lab chow group (51.9 ± 6.5 vs 5.4 ± 0.5, μmol/L, P <.0001). In the group fed a homocysteine diet with 10 mg/kg folate added, a significant decrease in plasma homocysteine was observed (17.5 ± 8.5 vs 51.9 ± 6.5, μmol/L, P =.0003). In the group fed a homocysteine diet with 25 mg/kg folate added, plasma homocysteine levels were further reduced to levels seen in the lab chow group (12.6 ± 2.6 vs 5.4 ± 0.5, μmol/L, P = not significant). The relationship between plasma folate and homocysteine was inverse (R = 0.39, P =.0036). Luminal stenosis due to intimal hyperplasia was minimal in lab chow groups and unaffected by folate. The homocysteine diet group demonstrated post–carotid endarterectomy luminal stenosis due to intimal hyperplasia (60.9% ± 9.2%). In the group fed a homocysteine diet with 10 mg/kg folate added, intimal hyperplasia was reduced, compared with the homocysteine diet group (32.6% ± 7.4% vs 60.9% ± 9.2%, P =.009). In the group fed a homocysteine diet with 25 mg/kg folate added, intimal hyperplasia was reduced to lab chow group levels (10.8% ± 0.8% vs 4.8% ± 1.0%, P = not significant) and was reduced, compared with the group fed a homocysteine diet with 10 mg/kg folate added. Conclusion: The use of folate in this hyperhomocysteinemia carotid endarterectomy model and the resultant attenuation of plasma homocysteine elevation and intimal hyperplasia development lend strong support to homocysteine being an independent etiologic factor in post–carotid endarterectomy intimal hyperplasia. (J Vasc Surg 2001;34:474-81.)

Abstracts of literature

Abstracts of literature

Hyperhomocysteinemia increases intimal hyperplasia in a rat carotid endarterectomy model

Purpose: This preliminary study investigated the ability to elevate the serum homocysteine (H[e]) levels and investigated the increases in postoperative neointimal hyperplasia (IH) in an environment with hyperhomocysteinemia and the resultant restenosis in a rat carotid endarterectomy (CEA) model. Method: The 9 rats for the control group were fed rat chow, and the 8 rats for the H(e) group were fed H(e)-supplemented rat chow for 2 weeks before and after CEA. The animals underwent anesthesia, and a left common CEA was performed. After 14 days, the serum H(e) levels were measured and the left carotid artery was harvested and elastin stained. Morphometric measurements were used to calculate the area of stenosis of the lumen. The mean and the standard deviation of the mean were determined. The 2 groups were compared with the Mann-Whitney test and a linear regression model. Three additional rats per group were studied, with carotid artery sectioning with double immunohistochemical staining for 5-bromodeoxyuridine (BrdU) and α–smooth muscle (α-SM) actin. Results: The serum H(e) level in the H(e) group was 36.32 μmol/L ± 15.28, and in the control group the level was 5.53 μmol/L ± 2.06 ( P = .0007). IH presented as percent lumen stenosis was 21.89% ± 4.82% in the H(e) group and 4.82% ± 1.64% in the control group ( P = .0007). The linear regression model of the serum H(e) levels and the percent stenosis showed a linear relationship ( r 2 = .72). The α-SM actin staining revealed that nearly all of the cells in the IH area were of smooth muscle or myofibroblast origin and that 10.1% ± 2.6% of the cells were stained for BrdU in the control group versus 23% ± 7.1% in the H(e) group. Also, 9.3% ± 2.6% of the cells in the IH area were stained for BrdU and for α-SM actin versus 19.1% ± 5.6% stained for both BrdU and α-SM actin in the H(e) group. Conclusion: This is the first study to examine IH after CEA and hyperhomocysteinemia in rats. The study shows that the elevation of serum H(e) levels can be obtained by feeding rats modified diets with added H(e). The consistent elevation of serum H(e) levels was associated with more than 4 times the amount of IH after a CEA in a rat model. (J Vasc Surg 1998;28:909-18.)

Enhancement of Platelet Deposition by Cross-Linked Hemoglobin in a Rat Carotid Endarterectomy Model

Purified human cross-linked hemoglobin, which is now being used in clinical trials, increases mean arterial pressure through binding of nitric oxide (NO). We postulated that binding of NO by cross-linked hemoglobin (alpha alpha Hb) could also increase platelet deposition at sites of subintimal injury. Male Sprague-Dawley rats were infused with alpha alpha Hb (0.88 g/kg, n = 8) or with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 30 mg/kg, n = 7) before undergoing microsurgical carotid endarterectomy. 111In-labeled platelets were infused after endarterectomy, and platelet deposition was measured 20 minutes later. In control endarterectomized rats (n = 8), mean platelet deposition was 7.7 +/- 0.7 x 10(6)/mm2. Platelet deposition was significantly increased above controls in rats that received alpha alpha Hb (13.2 +/- 0.9 x 10(6)/mm2, P = .0004) and in rats infused with L-NMMA (13.9 +/- 1.0 x 10(6)/mm2, P = .0002). The increase was prevented by infusion of L-arginine (150 mg/kg) immediately after alpha alpha Hb or L-NMMA. To determine whether aspirin (ASA) blocked the increased deposition induced by alpha alpha Hb, rats received oral ASA (10 mg/kg) 18 hours before endarterectomy. Platelet deposition in animals receiving ASA alone was 6.4 +/- 0.9 x 10(6)/mm2 (n = 8). This was significantly increased to 10.8 +/- 0.8 x 10(6)/mm2 (P = .002) for the ASA-treated group that received alpha alpha Hb at the time of endarterectomy (n = 8). The prolonged bleeding times induced by ASA were unaffected by the infusion of alpha alpha Hb. These data suggest that in a rat endarterectomy model, alpha alpha Hb increases platelet deposition at sites of subintimal injury by binding NO. Increased deposition induced by alpha alpha Hb can be prevented by administration of L-arginine but not by pretreatment with aspirin.

Cell-Free Hemoglobin as an Oxygen Carrier Removes Nitric Oxide, Resulting in Defective Thromboregulation

Development of a clinically safe and effective substitute for erythrocytes that is capable of efficient oxygen delivery in vivo has progressed to the stage where cross-linked hemoglobin preparations are now undergoing clinical trials.1 Such preparations withstand storage for prolonged periods of time, can be administered without the need for cross-matching, and are free of contamination by infectious agents. Utilization of cell-free hemoglobin as an erythrocyte substitute was initially hampered by nephrotoxicity and an affinity for oxygen that prevented efficient oxygen delivery to tissues.2 These disadvantages were overcome when Bunn and Jandl3 chemically cross-linked the hemoglobin molecule to produce stable hemoglobin oligomers that do not pass through the glomerular filtrate. In addition, Benesch and Benesch4 developed reagents that modified the 2,3-diphosphoglycerate binding site of hemoglobin, thereby reducing its oxygen affinity. Administration of cell-free hemoglobin solutions results in systemic vasoconstriction in research animals.5 This is thought to be a consequence of the high avidity of hemoglobin for nitric oxide (NO, endothelium-derived relaxing factor [EDRF]), which it binds and inactivates. The NO-hemoglobin interaction results in rapid formation of nitrite/nitrate and methemoglobin. This blocks vasodilation induced by …

Antithrombotic Effects of Hirulog in a Rat Carotid Endarterectomy Model

Although hirulog, a specific, direct inhibitor of thrombin, can prevent thrombosis in unstable angina and angioplasty without inducing excessive bleeding, it has not been used in a surgical setting. In the present study, the antithrombotic activity of hirulog was assessed in rats undergoing carotid endarterectomy. Three groups of anesthetized male Sprague–Dawley rats received either intravenous heparin (10 U/kg bolus followed by 90 U/kg/hr,n= 4), high-dose hirulog (0.8 mg/kg bolus followed by 2.2 mg/kg/hr,n= 7), or saline (n= 6) before endarterectomy and until termination of the protocol 30 min later. Platelet deposition, as measured by scanning electron microscopy, in rats receiving this high dose of hirulog was reduced by 63% (±14%, SE) compared to controls (P= 0.004) and by 36% (±16%) in heparinized rats (P= 0.07). Both groups had prolonged postsurgical bleeding. Infusion of hirulog at a lower dose (0.4 mg/kg bolus followed by 1.0 mg/kg/hr,n= 8) was not associated with prolonged bleeding; however, platelet deposition was reduced by only 16% (±27%,P= 0.30), although125I-fibrin deposition was reduced by 64% (±11%,P= 0.004). In the high-dose hirulog group, plasma hirulog levels, as determined with a quantitative thrombin time, were three times higher (95% CI: 1.5–4.5 times) than in the group receiving the lower hirulog dose [11.6 ± 2.3 (SE) μg/ml vs 3.9 ± 0.6 μg/ml,P= 0.0022]. However, the mean activated partial thromboplastin time with the higher dose was similar to that of the lower dose (110 ± 4 vs 90 ± 13 sec,P= 0.09). The antithrombotic activity of hirulog can be maximized by titrating the dose, monitoring plasma drug levels, and possibly administering the drug after surgery to avoid prolonged bleeding.