Heterozygous Rabbits Research Articles

Abstract 14182: A Missense Mutation in Ryanodine Receptor 2 Leads to Structural Remodeling of the Heart and Cardiac Arrhythmia in a Rabbit Model

Ryanodine receptor 2 (RyR2) is the Ca 2+ release channel of sarcoplasmic reticulum that provides the majority of Ca 2+ necessary for contractions of the heart. RyR2 mutations are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disorder with normal heart structure. There are reports suggesting that RyR2 mutations can also lead to structural heart disease, notably arrhythmogenic right ventricular cardiomyopathy (ARVC); however, additional research is required to support this association. We generated a rabbit model carrying the missense mutation RyR2-V2475F, linked to CPVT in humans. Heterozygous (V2475F +/- ) rabbits show a CPVT-like syndrome and display arrhythmia under pharmacological stress. Homozygotes (V2475F +/+ ) show a stronger phenotype, with 37% of the animals (13/35) experiencing sudden death at 22.5±3.8 weeks old during regular housing. Remarkably, gross examination of V2475F +/+ hearts showed significant structural remodeling, characterized by fibrous infiltrations and thinning of the right ventricle with or without left ventricle involvement. This phenotype is reminiscent of ARVC. Lead II ECG obtained in conscious rabbits by telemetry showed that V2475F +/+ animals have significant bradycardia (144.6±11.1 bpm) with prolonged QT interval (QTc: 180.4±8.2 ms, n=6) compared to WT (HR: 224.9±4.2 bpm, p=0.04; QTc: 157.4±5.3 ms, p=0.02, n=5). After subcutaneous injection of 0.5 mg/kg of isoproterenol, WT rabbits showed the expected positive chronotropic response (HR, 367±10 bpm, n=3) while V2475F +/+ animals developed bouts of ventricular tachycardia. Mild emotional stress applied by transporting the animal around the laboratory, also induced ventricular arrhythmia in 100% (5/5) V2475F +/+ rabbits, while WT littermates were unaffected. RyR2-V2475F expressed in a rabbit model triggers remarkably different phenotypes based on the zygosity of the animals, ranging from a purely arrhythmogenic syndrome (CPVT) in V2475F +/- animals to severe structural disease compatible with ARVC in V2475F +/+ rabbits. The RyR2-V2475F rabbit model is therefore an excellent model to dissect the intersection of arrhythmic and structural phenotypes due to RyR2 dysfunction.

Truncated C-terminus of fibrillin-1 induces Marfanoid-progeroid-lipodystrophy (MPL) syndrome in rabbit.

ABSTRACTVarious clinical differences have been observed between patients with the FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system. FBN1 heterozygous (FBN1 Het) rabbits faithfully recapitulated the phenotypes of MFS, including muscle wasting and impaired connective tissue, ocular syndrome and aortic dilation. Moreover, skin symptoms, lipodystrophy, growth retardation and dysglycemia were also seen in these FBN1 Het rabbits, and have not been reported in other animal models. In conclusion, this novel rabbit model mimics the histopathological changes and functional defects of MPL syndrome, and could become a valuable model for studies of pathogenesis and drug screening for MPL syndrome.

6 CRISPR-Generated ZP4 Knockout Rabbits Exhibit Impaired In Vivo Pre-Implantation Development

Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P < 0.05). These morphological differences suggest that rabbit ZP without ZP4 may fail to protect the embryo during development or may physically impede blastocyst expansion. This study is supported by the projects AGL2014-58739-R and RYC-2012-10193 (to PBA), AGL2015-70159-P (to MA) and AGL2015-65572-C2-1-R (to PGR and PL). ILT and NFB are supported by FPI grants.

Effects of dietary hypercholesterolemia on plasma lipids levels male and female Japanese white and heterozygous Kurosawa-Kusanagi hypercholesterolemic rabbits

High plasma cholesterol levels constitute a major risk factor for coronary heart diseases. In this study, we compared the response to dietary hypercholesterolemia with respect to plasma lipids levels in male and female Kurosawa-Kusanagi hypercholesterolemic (KHC) rabbits, which have Low density lipoprotein (LDL) receptor deficiency. Male and female Japanese white (JW) and heterozygous KHC rabbits were given a standard milled rabbit diet or diets with different concentrations of cholesterol. All the male and female JW and KHC rabbits given a standard milled rabbit diet did not show increased plasma cholesterol levels over the 24-week experiments. The plasma cholesterol levels in male JW rabbits fed a 0.1 % cholesterol-containing diet did not increase, but the levels in male heterozygous KHC rabbits fed the same diet transiently increased for 4-8 weeks (to 300 mg/dL) and then gradually decreased till the initial level. In contrast, in female JW rabbits fed the same diet, the plasma cholesterol  levels increased for 4-24 weeks (to 300 mg/dL), and in heterozygous KHC rabbits, the levels gradually increased (to 675 mg/dL after 24 weeks) after consumption of the 0.1 % cholesterol-containing diet. In ovariectomized JW and KHC rabbits fed the 0.1 % cholesterol-containing diet, the plasma cholesterol levels were half value the levels in non-ovariectomized rabbits  after 24 weeks. Gene expression of the LDL receptor in the liver significantly increased in male JW and KHC rabbits, but significantly decreased in female KHC rabbits. These results indicate that female rabbits had a greater response to the cholesterol diet than male rabbits and that the responsiveness to dietary hypercholesterolemia was predominantly genetic.

A critical reassessment of murine and rabbit models of atherosclerosis: focus on lesion progression and remodelling

Background Experimental animal atherosclerosis models are frequently regarded as an adequate surrogate for human vascular disease. The external validity of these models should be approached critically.Objectives The current study provides a direct comparison of atherosclerosis progression in four different animal models: C57BL/6 apolipoprotein (apo) E-/- mice, C57BL/6 low density lipoprotein receptor deficient mice (LDLr/- mice), heterozygous LDL receptor deficient rabbits (LDLr+/- rabbits), and homozygous LDL receptor deficient rabbits (LDLr-/- rabbits). The main objective was to perform a longitudinal analysis of arterial remodelling and of the evolution of the medial area during atherosclerosis progression. Secondary objectives were to analyse sex differences in atherosclerosis progression and to determine intersite correlations.Results Progression of atherosclerosis in all models was accompanied by expansive (overcompensatory) remodelling leading not only to the absence of luminal narrowing but also to an increase of the absolute lumen size. Atherosclerosis progression in mice and rabbits is often accompanied by an increase of the medial area. Female mice are more susceptible or equally susceptible to atherosclerosis development compared to male mice notwithstanding lower plasma cholesterol levels. However, this sex difference was not reiterated in both rabbit models. Whereas cholesterol-fed LDLr/- mice show a moderate or strong correlation between the extent of advanced atherosclerosis in the aortic root and the brachiocephalic artery, no such correlation was observed in apo E-/- mice.Conclusion The extensive morphometric data in the current study provide a framework to critically reassess the potential and limitations of animal models of atherosclerosis.

Effects of NO-1886, a Lipoprotein Lipase Promoting Agent, on Homozygous and Heterozygous Watanabe Heritable Hyperlipidaemic Rabbits

The novel compound NO-1886 ([4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester, CAS 133208-93-2) is a lipoprotein lipase (LPL) activator, and long term administration of NO-1886 protects against the development of experimental atherosclerosis in rats and rabbits. In the present experiments, the effects of this compound were examined in Watanabe heritable hyperlipidaemic (WHHL) rabbits, an animal model for familial hypercholesterolemia lacking low density lipoprotein (LDL) receptors. NO-1886 increased postheparin plasma LPL activity, resulting in a reduction of plasma triglycerides with concomitant elevation of HDL cholesterol in heterozygous WHHL rabbits. However, the compound did not cause any changes in plasma lipids and postheparin plasma LPL activity in homozygous WHHL rabbits. The different responses suggest that the effects of NO-1886 may be either mediated by LDL receptors, or that persistent exposure to extreme hypercholesterolemia might affect the cellular response to this particular compound in homozygous WHHL rabbits.

Co-existence of somatic hypermutation and gene conversion in hypervariable regions of single Igkappa clones.

In the rabbit, recent investigations have provided evidence that gene conversion leads to the generation of diversity of heavy chain rearranged VH-DH-JH genes. No data have been published on a similar mechanism for rabbit light chains. In our laboratory, we initially infected rabbits with Trypanosoma brucei, which stimulates B-cell hyperplasia and hypergammaglobulinaemia. The heterozygous rabbits exhibited the Ckappa1 b4 and b9 kappa light chain allotypes. After reverse transcription of mRNA, and cloning and sequencing of cDNA, the Vkappa-Jkappa-Ckappa genes provided evidence for both somatic hypermutation and gene conversion. We saw that in each of the b4 and b9 kappa light chain cDNA, CDR1 and CDR3 carried both point mutation and provisional gene conversion traits. In the CDR2 region, point mutation and gene conversion inserts were observed in the b4 genes, with only gene conversion in two b9 genes. In the CDR regions, although some genes exhibited only somatic hypermutation or gene conversion, others showed linkage of both somatic hypermutation and gene conversion in the same sequence. This also marks the first time that somatic hypermutation and gene conversion in the same cloned CDR region has been observed in Vkappa1 genes; however, it has been seen earlier in rabbit heavy chain VH sequences. Furthermore, the addition of several codons to the CDR3 segment by gene conversion may have provided a mechanism for length variation. In addition, we demonstrated that Jkappa and framework region segments contained examples of somatic hypermutation. Confirmation of gene conversion necessitates that donor sequences be identified as providing the templated inserts. Thus after cloning two pseudogenes we found putative CDR3 donor segments for two CDR3 rearranged genes. The results offer additional mechanisms for the generation of diversity among rearranged rabbit kappa light chain genes. Whether there is a relationship or influence of gene conversion upon somatic hypermutation or vice versa is not discernable at present.

Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion

The effect of cholesterol feeding (3 g/day) on bile acid synthesis was examined in 10 New Zealand white rabbits (NZW), 8 Watanabe heterozygous and 10 homozygous rabbits with partial and complete deficiencies of LDL receptors. After 10 days of cholesterol feeding, bile fistulas were constructed and bile acid pool sizes were measured. Cholesterol feeding increased plasma and hepatic cholesterol levels in all rabbit groups. Baseline bile acid pool sizes were smaller (P < 0.01) in heterozygotes (139 ± 3 mg) and homozygotes (124 ± 30 mg) than NZW rabbits (254 ± 44 mg). After feeding cholesterol, bile acid pool sizes doubled with increased cholic acid synthesis in NZW and, to a lesser extent, in Watanabe heterozygous rabbits but not in homozygotes. Baseline cholesterol 7α-hydroxylase activity in NZW and heterozygotes declined 69% and 53% (P < 0.001), respectively, after cholesterol feeding. Sterol 27-hydroxylase activity reflecting alternative bile acid synthesis increased 66% (P < 0.01) in NZW and 37% in Watanabe heterozygotes but not in homozygotes after feeding cholesterol. Bile fistula drainage stimulated cholesterol 7α-hydroxylase activity but not sterol 27-hydroxylase activity in all thr ee rabbit groups.▪ These results demonstrated that dietary cholesterol increased hepatic sterol 27-hydroxylase activity and alternative bile acid synthesis to expand the bile acid pool and inhibited cholesterol 7α-hydroxylase in NZW and in Watanabe heterozygous rabbits but not in homozygotes with absent hepatic LDL receptor function. Thus, in rabbits, sterol 27-hydroxylase is up-regulated by the increased hepatic cholesterol that enters the liver via LDL receptors whereas cholesterol 7α-hydroxylase is controlled by the circulating hepatic bile acid flux.—Xu, G., G. Salen, S. Shefer, G. S. Tint, L. B. Nguyen, T. T. Parker, T. S. Chen, J. Roberts, X. Kong, and D. Greenblatt. Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion.

Survival and cardiovascular pathology of heterozygous Watanabe heritable hyperlipidaemic rabbits treated with pravastatin and probucol on a low-cholesterol (0.03%)-enriched diet.

This study was aimed at determining the effects of a combined pravastatin and probucol regimen on survival and vascular pathology of heterozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits fed a low-cholesterol (0.03%)-enriched diet. Pravastatin monotherapy preceded the combined treatment. In animals receiving pravastatin and the enriched diet (verum group; n = 6), mean total serum cholesterol levels were consistently lowered at a dosage of 5 mg/kg pravastatin and with the combined treatment. Survival was increased (median 45 vs 25 months), while coronary atherosclerosis was less obstructive and altered to a more fibrous type than in controls (n = 8). The extent of aortic lesions, as determined by the relative plaque volume, was not related to survival in either group. However, aortic plaque types in verum group animals revealed less severe stages with a different composition and architecture, with a lower relative content of macrophage-derived foam cells and necrosis and a higher relative content of extracellular matrix. There was also a thicker fibrous cap than in control animals of similar age. Our data reveal a beneficial effect on survival of heterozygous WHHL rabbits when lipid-lowering and antioxidative treatment are combined. This appears to be due both to reduced coronary atherosclerosis and to a different, more stable type of atherosclerotic disease in this animal model.

1.P.93 Effects on plasma lipids levels in cholesterol feeding male/female heterozygous KHC rabbits

1.P.93 Effects on plasma lipids levels in cholesterol feeding male/female heterozygous KHC rabbits

Measurement of expired carbon dioxide to assess the metabolism of remnant lipoproteins.

The cholesteryl esters transported from the intestine in chylomicrons are delivered to the liver. Hepatocytes take up chylomicron remnants by receptor-mediated endocytosis and the cholesteryl esters are subsequently degraded. In this study we measured the appearance in breath of labeled carbon dioxide after injection of chylomicron-like emulsions labeled with radioactive cholesteryl [1-14C]oleate. Measurements by the breath test provide an integrated assessment of capacity for clearance and subsequent metabolism of the remnants of the triglyceride-rich lipoproteins. In normal rats, mice, and rabbits injected with the radioactive emulsions, label appeared in the breath after a delay of approximately 30 min, appreciably slower than the appearance of label after injection of emulsions labeled with [14C]triolein or of [14C]oleic acid complexed with albumin. To test for the ability of the procedure to detect defects in remnant clearance, labeled emulsions were injected into diabetic rats, apoE-deficient mice, low density lipoprotein receptor (LDLr)-deficient mice, and Watanabe heritable hyperlipidemic (WHHL) rabbits with defective low density lipoprotein receptors. In rats made diabetic by treatment with streptozotocin the appearance of 14CO2 in breath was slower than in normal control rats. This finding was consistent with previous evidence from our laboratory that remnant clearance is defective in diabetic rats. In LDLr-deficient mice the appearance of 14CO2 was slower when compared with control mice and in apoE-deficient mice the appearance of 14CO2 was extremely small. In homozygous WHHL rabbits, the appearance of 14CO2 in breath was much slower than in normal control rabbits, while in heterozygous WHHL rabbits an intermediate level of appearance was found, consistent with our previous findings of defective remnant clearance in WHHL rabbits. Emulsions with cholesterol omitted, previously found to be cleared from plasma much slower than chylomicron-like emulsions, had much slower appearance of label in breath. The breath test as described is consistent with predicted metabolism of chylomicron remnants and therefore provides a useful means of assessment of remnant catabolism in the intact animal.

Development and progression of atherosclerosis in aorta from heterozygous and homozygous WHHL rabbits. Effects of simvastatin treatment.

This study was conducted to define progression of atherosclerosis in both homozygous and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits and to investigate the ability of the HMG CoA reductase inhibitor simvastatin to attenuate progression of the disease. We examined contractile responses to phenylephrine and endothelium-dependent relaxation in response to carbachol in thoracic aorta at 3, 6, 9, and 12 months in control New Zealand White (NZW) rabbits, homozygous WHHL rabbits, and heterozygous WHHL rabbits. Homozygous and heterozygous rabbits were treated with simvastatin (10 mg/kg per day) from 3 to 6 months and from 9 to 12 months of age. Simvastatin significantly reduced serum cholesterol levels in young heterozygotes, with a nonsignificant trend toward a reduction in older heterozygotes. In homozygotes, no significant fall was observed. Contractile function declined progressively with age in all groups--most in homozygotes and least in NZW rabbits. Relaxation was unaffected by age in NZW rabbits; relaxation declined in the heterozygotes and declined to a greater extent in homozygotes. Simvastatin retarded the loss of function in the young heterozygotes. Similar trends were observed in young homozygotes and older heterozygotes, with no effect in older homozygotes. Histological studies revealed the progressive development of early atherosclerosis in heterozygotes, and more advanced atherosclerosis was observed in homozygotes. Simvastatin did not inhibit development of atheroma. A correlation was observed between vascular function and structure. However, functional changes preceded the development of atheroma. In addition, we have demonstrated that simvastatin can help to reduce the loss of vascular function associated with the progression of atherosclerosis in the heterozygous WHHL rabbit.

Restriction fragment-length polymorphism analysis of the major histocompatibility complex in New Zealand white rabbits.

As a model system, rabbits are particularly useful in transplantation studies because of their size and accessibility. However, inbred rabbits of known MHC haplotype are not commercially available, and there are few monoclonal antibody reagents available to permit serological typing of experimental animals prior to transplant. Here we present a rapid and reliable method to distinguish rabbits that differ at their MHC class I and class II loci, and present 34 class I and 16 class II haplotypes determined by restriction fragment-length polymorphism analysis of outbred heterozygous rabbits. The applicability of this molecular typing system to transplantation experiments in the New Zealand White rabbit is discussed.

Atherosclerosis in Watanabe heritable hyperlipidaemic rabbits

The spontaneous development of atherosclerotic disease in 38 homozygous and 34 heterozygous Watanabe heritable hyperlipidaemic rabbits was evaluated by qualitative and quantitative light microscopy in aorta, coronary, pulmonary and renal arteries, by naked eye and macroscopic morphometric estimation of aortic atherosclerosis extent and by biochemical analysis of aortic cholesterol content. No noteworthy atherosclerosis was demonstrated within 19 months in heterozygous rabbits. In homozygous rabbits, atherosclerotic lesions were seen from the age of 4 months and progressed with age. All 19-month-old rabbits had severe atherosclerotic disease. As much as 64% of the variation in atherosclerosis extent/severity could be explained by serum cholesterol and age. A highly significant correlation between the various methods for quantitation of atherosclerosis extent and/or severity was demonstrated, suggesting that quantitative microscopy, macroscopic morphometry and determination of aortic cholesterol content may be equally valid as a measure of atherosclerosis in WHHL rabbits and are therefore interchangeable.

Immunoglobulin (Ig) allotypic markers in Kawasaki disease (KD): Studies in racially diverse populations

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b4b5 heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b4b5 heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.

Relationship of vascular 15-lipoxygenase induction to atherosclerotic plaque formation in rabbits

Aortas from atherosclerotic rabbits have increased levels of 15-lipoxygenase, but the relationship between induction of this enzyme and the atherosclerotic process has not been defined. We found that dietary administration of cortisone acetate significantly suppressed atherosclerotic plaque formation in both Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed WHHL NZW heterozygous rabbits. There was, however, no corresponding decrease in the elevated 15-lipoxygenase activity. In addition, the elevated 15-lipoxygenase activity in atherosclerotic rabbit aortas was uniformly distributed throughout the aorta, and was not preferentially localized in the lesions. These results indicate that induction of the 15-lipoxygenase is not necessarily causally related to plaque development, and that plaques are not the major source of the increased enzyme activity. However, the results confirm that hypercholesterolemia is a necessary condition for both atherosclerosis and 15-lipoxygenase induction, suggesting that perhaps the 15-lipoxygenase may represent a protective response to the hyperlipidemic stress. This possibility is supported by the finding that the induced 15-lipoxygenase converts linoleic acid, which is the predominant essential fatty acid in aorta, to 13-hydroxyoctadecadienoic acid (13-HODE). This compound is a chemorepellant factor for platelets, inhibits platelet thromboxane synthesis, and stimulates prostacyclin synthesis by endothelial cells.

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination.

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.

Cholesterol-fed heterozygous Watanabe heritable hyperlipidemic rabbits: a new model for atherosclerosis

Homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits are used widely to study atherosclerosis, but the WHHL heterozygous rabbit has received little attention. To study their potential as a model for atherosclerosis, heterozygous WHHL and New Zealand white (NZW) rabbits were fed diets containing 0%, 0.5% and 1.0% cholesterol. Plasma lipids were analyzed at 0, 4, 8, 12, 16 and 24 weeks, and animals were killed at 12 and 24 weeks. Plasma cholesterol levels were significantly higher in cholesterol-fed WHHL heterozygotes at 8 weeks compared with NZW rabbits, but no differences were apparent at other times. Atherosclerotic plaques in the aortas of cholesterol-fed WHHL heterozygous rabbits differed from those in NZW rabbits, in that the WHHL had complicated lesions with necrosis, cholesterol clefts, fibrous caps and calcification, similar to that found in humans and homozygous WHHL rabbits. In contrast, NZW rabbits had predominantly foam cell lesions. Heterozygous WHHL rabbits also had less extensive extravascular foam cell deposits. Our results suggest that the cholesterol-fed heterozygous WHHL rabbit may provide a promising model for studying the pathogenesis of atherosclerosis.

ヘテロ接合体WHHL家兎に対するLDL吸着型モジュールを用いた反復的血漿交換の効果

ヘテロ接合体WHHL家兎に対するLDL吸着型モジュールを用いた反復的血漿交換の効果

Sequential double immunocytochemical staining for in situ identification of an auto-anti-allotype immune response in allotype-suppressed rabbits.

Immunocytochemical staining has been used to detect putative autoimmune B-cells in rabbits undergoing chronic allotype suppression. This condition is seen in heterozygous rabbits exposed perinatally to antibody against the paternal immunoglobulin allotype. Such animals develop lifelong suppression for this allotype and have been used as models for study of antibody-induced disturbance of immune regulation. Normal rabbits deliberately immunized against a heterologous allotype were used to establish the feasibility of identifying cells forming anti-allotypic antibodies in cryostat sections of rabbit lymphoid tissues. Incubation and staining of tissue sections from suppressed rabbits then revealed the presence of autoimmune B-cells, with antibody specificity for the suppressed allotype, in all chronically suppressed adult rabbits tested. Sequential incubation and staining with allotype- and anti-allotype-enzyme conjugates established that such cells were of non-suppressed origin. Auto-anti-allotype antibody-forming cells were not found in normal heterozygotes or in chimeric rabbits. The immunocytochemical techniques described here permitted simultaneous detection of specificity (i.e., anti-allotype) and origin (allotype) of antibody-forming cells involved in an autoimmune response, as well as their anatomical correlation with other B-cells of suppressed or non-suppressed origin. Since the method described can be adapted to detection of alternate cell markers, we believe it to have potential application to the study of other autoimmune phenomena.