Normal New Zealand White Rabbits Research Articles

Morphine-induced nitric oxide production in isolated, iris-ciliary bodies

Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an integral role in opioid receptor-mediated responses in the cardiovascular and immune systems. Previous studies in our laboratory and others have shown that nitric oxide (NO) plays a role in morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) in the New Zealand white (NZW) rabbit. The present study is designed to determine the effect of morphine on NO production in the isolated, iris-ciliary body (ICB), site of aqueous humor production, as this effect could be associated with morphine-stimulated changes in aqueous humor dynamics and iris function. ICBs obtained from normal NZW rabbits were utilized in these experiments. In some experiments, ICB samples were treated with morphine (1, 10 and 100 μM) for 1 h and later examined for changes in NO levels using a NO detection kit. In other experiments, tissue samples were pretreated with naloxone (non-selective opioid receptor antagonist), L-NAME (non-selective NO-synthase inhibitor) or GSH (sulfhydryl reagent) for 30 min, followed by treatment with morphine (10 μM). Morphine caused a concentration-dependent increase in the release of NO from ICBs. Levels of NO detected in the incubation medium of ICB samples increased from 1.49 ± 0.19 (control) to 8.81 ± 2.20 μM/mg protein (morphine-treated; 100 μM). Morphine-stimulated release of NO was significantly inhibited in tissues pretreated with 10 μM naloxone, L-NAME, or GSH. Results obtained from this study suggest that morphine stimulates NO release from the ICB through a mechanism that involves activation of NO-releasing opioid receptors. These results support the in vivo effects of morphine demonstrated in previous studies.

Radiolabeled MDA2, an oxidation-specific, monoclonal antibody, identifies native atherosclerotic lesions in vivo

Background. Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal arteries and plays a crucial role in the pathogenesis and adverse consequences of atherosclerotic lesions. We previously generated a series of monoclonal antibodies (MoAb) against oxidation-specific neo-epitopes formed during the oxidative modification of LDL. MDA2, a prototype MoAb, recognizes malondialdehyde-lysine epitopes (eg, in malondialdehyde-modified LDL) within atherosclerotic lesions. We describe the in vivo characteristics of MDA2 and initial noninvasive imaging studies of atherosclerosis in rabbits. Methods. To assess the in vivo specificity of MDA2 for atherosclerotic lesions, iodine 125-MDA2 was intravenously injected into 7 LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) and 2 normal New Zealand white (NZW) rabbits, and the aortic plaque uptake was evaluated 24 hours later. 125I-Halb, an isotype-matched irrelevant MoAb that binds to human albumin, was injected into 5 WHHL and 2 NZW rabbits as a control. Aortic autoradiography was performed, and the mean uptake of MoAbs was measured as the percent injected dose per gram aortic tissue. Gamma camera imaging was then carried out in 7 WHHL rabbits and 2 NZW rabbits with 99mTc-MDA2. Imaging was carried out at 10 minutes and at 12 or 24 hours. Malondialdehyde-LDL was then injected to clear the blood pool signal, and final images were obtained 2 hours later. Results. Mean uptake of 125I-MDA2 in the entire aorta was 17.4-fold higher in WHHL than in NZW aortas ( P < .001), and 2.8-fold higher than 125I-Halb in WHHL aortas. 125I-MDA2 also had higher specificity for lesioned areas than 125I-Halb (plaque/normal ratio 6.3 vs 2.9, P < .001). Autoradiograph of aortas of 125I-MDA2-injected WHHL rabbits revealed uptake in lipid-stained lesions with absence of signal in adjacent normal arterial tissue. Immunostaining of WHHL lesions, which accumulated MDA2 as noted on autoradiography, revealed that uptake was highest in areas with abundant foam cells and in lipid-rich necrotic core areas. Autoradiograph of aortas from NZW rabbits injected with 125I-MDA2 did not yield any visible signal. Planar gamma camera in vivo scintigraphy revealed a visible signal in 4 7 WHHL rabbits, which was confirmed by aortic Sudan staining. Conclusion. Radiolabeled MDA2 shows excellent in vivo uptake and specificity for atherosclerotic lesions containing abundant oxidation-specific epitopes. The in vivo imaging studies suggest that noninvasive imaging of oxidation-rich atherosclerotic lesions with radiolabeled MDA2 may be feasible in human beings with optimization of the imaging methods.

Adenovirus-mediated gene transfer is augmented in basilar and carotid arteries of heritable hyperlipidemic rabbits.

There are major differences in susceptibility of intracranial and extracranial arteries to atherosclerosis. The goal of this study was to examine adenovirus-mediated gene transfer to basilar and carotid arteries of Watanabe heritable hyperlipidemic (WHHL) rabbits, which have spontaneous hypercholesterolemia and atherosclerosis, and normal New Zealand White (NZW) rabbits. We used 2 different adenoviral vectors, driven by either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. Basilar and carotid arteries were removed from WHHL and NZW rabbits and cut into rings. The arteries were incubated with an adenoviral vector that expresses beta-galactosidase and is driven by either a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter (AdCMVbetagal or AdRSVbetagal). Arteries were incubated with virus for 2 hours, and then incubated in medium for 24 hours to allow expression of transgene. Transgene expression was assessed by enzyme activity (Galacto-Light assay) and by a histochemical method after X-Gal staining. After gene transfer, beta-galactosidase was expressed in endothelium and adventitia but not media. There were moderately severe atherosclerotic lesions in carotid arteries and early lesions in basilar arteries. Enzyme activity after gene transfer with AdCMVbetagal (3x10(11) particles/mL) was greater in the basilar artery of WHHL than NZW (137+/-40 versus 25+/-10 mU/mg protein, P<0.05) (mean+/-SE) and in the carotid artery (133+/-27 versus 34+/-11 mU/mg protein, P<0.05). After gene transfer with AdRSVbetagal, transgene expression was similar in arteries from WHHL and normal NZW rabbits. This is the first study to examine gene transfer to intracranial and extracranial arteries from atherosclerotic animals. The findings suggest that an adenoviral vector with a CMV, but not RSV, promoter provides greater transgene expression in the basilar and carotid arteries from spontaneously atherosclerotic rabbits than from normal rabbits.

Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme results in a reduction of plasma LDL levels in normal and watanabe heritable hyperlipidemic rabbits

Apolipoprotein (apo) B exists in two forms, the full length protein apoB-100 and the carboxyterminal-truncated apoB-48 that is synthesized in the intestine due to editing of the apoB mRNA which generates a premature stop codon. To determine whether gene transfer of the catalytic subunit of the apoB mRNA editing enzyme APOBEC-1 (apoB mRNA editing enzyme catalytic polypeptide 1) into the liver of rabbits reconstitutes hepatic apoB mRNA editing and how this affects the plasma levels of apoB-containing lipoproteins, we constructed an APOBEC-1 recombinant adenovirus (Ad APOBEC-1). After injection of Ad APOBEC-1 into normal New Zealand White (NZW) or Watanabe heritable hyperlipidemic (WHHL) rabbits, up to 50% of the hepatic apoB mRNA was edited and freshly isolated hepatocytes secreted predominantly apoB-48-containing lipoproteins. VLDL isolated from Ad APOBEC-1-treated NZW and WHHL rabbits contained both apoB-100 and apoB-48, whereas that from control rabbits infected with a beta-galactosidase recombinant adenovirus (Ad LacZ) contained exclusively apoB-100. VLDL from WHHL rabbits treated with Ad APOBEC-1 had the same particle size, lipid composition, and content of apolipoprotein E as VLDL from Ad LacZ-infected control animals. An increase of VLDL was observed in NZW and WHHL rabbits after infection with Ad APOBEC-1 as well as Ad LacZ. After injection of Ad APOBEC-1, LDL became undetectable in the plasma of NZW rabbits and was reduced by an average of 65% in the plasma of WHHL rabbits compared to Ad LacZ-infected controls. LDL from Ad APOBEC-1-infected WHHL rabbits contained only apoB-100. VLDL isolated from Ad APOBEC-1-infected WHHL rabbits were rapidly cleared from the circulation after injection into NZW rabbits. These results provide further evidence that the switch in the hepatic synthesis from exclusively apoB-100 to partly apoB-48 can result in a reduction of LDL formation that requires the full-length apoB-100.

Effects of alloxan-induced diabetes on hemorheology in rabbits.

Plasma viscosity (PV), apparent whole blood viscosity (WBV), relative blood viscosity (RV) and erythrocyte deformability (filterability) (EDF) were determined in 13 New Zealand White (NZW) rabbits with alloxan induced-diabetes (AID) and 8 normal NZW rabbits, matched for age, sex and weight. AID rabbits were divided into two groups depending on the duration of hyperglycemia (long-term, greater than 6.0 months (n = 7), and short-term, less than or equal to 3.0 months of hyperglycemia, n = 6). Comparing long-term AID rabbits to normal animals, we found significant increases in WBV (P less than 0.001, 0.005 for high and low rates of shear, respectively), and a marked reduction in EDF (P less than 0.001). There was no significant difference in PV between long-term AID and normal rabbits. Conversely, PV was significantly increased in rabbits with short-term diabetes (P less than 0.01) while there was a concurrent significant increase in WBV measured at high and low rates of shear (P less than 0.001, 0.001, respectively). No difference was detected in EDF between normal and short-term AID rabbits. Furthermore, in long-term AID rabbits there was a strongly positive correlation between RV and reduced erythrocyte deformability (r = 0.94, P = 0.006) while WBV strongly correlated with PV (r = 0.92, P = 0.004) in the short-term AID subgroup. We conclude from these data: (1) elevated blood viscosity in long term AID rabbits is associated with reduced erythrocyte filterability; and (2) elevated WBV in short-term AID rabbits is associated with increased PV.

Augmented vasoconstrictor responses to serotonin precede development of atherosclerosis in aorta of WHHL rabbit.

Watanabe heritable hyperlipidemic (WHHL) rabbits have elevated concentrations of plasma cholesterol and develop progressive atherosclerosis. The present investigation was undertaken to evaluate the vascular responses to vasoactive compounds of aorta from WHHL rabbits and normal New Zealand White (NZW) rabbits at 1 and 6 months of age. Rings of distal thoracic aorta were suspended under isometric tension in oxygenated Krebs buffer. Developed tension was measured in response to graded concentrations of agonists. Maximal responses to KCl (40 mM) were the same in aortas from the 1-month-old and 6-month-old WHHL and NZW rabbits. Aortas from 1-month-old animals were more sensitive to serotonin than aortas from 6-month-old animals. Aortas from WHHL rabbits exhibited an increased maximal response to serotonin when compared with NZW controls. In contrast, the constrictor responses to norepinephrine were reduced in WHHL rabbits compared with NZW rabbits at both age groups. Methacholine decreased tension development in serotonin-contracted vessels. This relaxation was greatest in aortas from NZW rabbits. In 1-month-old NZW rabbits fed a high cholesterol diet, the constrictor responses to serotonin and the relaxation responses to methacholine did not differ from NZW rabbits ingesting a normal diet. However, the responses to norepinephrine were markedly attenuated in the hypercholesterolemic NZW rabbits. Microscopic evaluation of the aortas revealed occasional adherent leukocytes and irregularities in the vascular endothelium in 1-month-old WHHL animals. These changes were greater in aortas from 6-month-old WHHL animals, with more adherent leukocytes, adherent platelets, and severe irregularities in the endothelial surface.(ABSTRACT TRUNCATED AT 250 WORDS)