Wild-type Rabbits Research Articles

Metabolic treatment of an electrical disease? Beneficial APD/QT prolongation by L-Carnitine in transgenic SQT1 rabbit models

Abstract Background Short-QT syndrome 1 (SQT1) is a genetic cardiac channelopathy caused by gain-of-function mutations (KCNH2-N588K) in HERG/IKr, that leads to shortened QT-interval, increased risk for arrhythmias and sudden cardiac death (SCD). An acquired form of SQTS has been described in patients with primary (genetic) carnitine-deficiency, indicating that carnitine might affect cardiac repolarization. Purpose We aimed to investigate potential beneficial (APD/QT-prolonging) effect of L-Carnitine in (genetic) SQTS using transgenic SQT1 rabbits that mimic the human disease phenotype. Methods Effects of L-carnitine on cardiac repolarisation were assessed in adult wildtype (WT) and transgenic SQT1 rabbits (KCNH2-N588K) using in vivo ECG and ex vivo Langendorff-perfused whole-heart or isolated ventricular cardiomyocyte action potential (AP) recordings. Effects on ion currents were assessed by whole-cell patch-clamping. Results In vivo, the heart-rate corrected QT index (QTi) was prolonged significantly by L-carnitine both in WT (QTi, baseline 102.7%±4.9 vs. L-carnitine 106.9%±6.2, p<0.05, n=12) and SQT1 (QTi, baseline 94.8%±7.4 vs. L-carnitine 99.5%±8.2, p<0.05, n=13), leading to normalisation of QTi in SQT1. Ex vivo, whole-heart monophasic and cellular APs were also significantly prolonged by L-carnitine in WT and SQT1 (change in monophasic APD75, ms, WT +13.9±4.4, SQT1 +9.9±7.0; change in cellular APD90, %, WT +10.4%, SQT1 +10.4%, all p<0.05). As underlying mechanisms, we identified acute effects on the main repolarizing ion currents IKr and IKs: IKr-steady, which is significantly increased in SQT1 contributing to accelerated repolarization, was reduced by L-carnitine (WT: −23%, SQT1: −16%). Moreover, L-carnitine accelerated the deactivation kinetics of IKr - while no change was observed in IKr-tail. In addition, IKs-steady was decreased by L-carnitine in SQT1 and WT cardiomyocytes. Conclusion L-carnitine prolongs/normalizes QT and APD in transgenic SQT1 rabbits by decreasing the pathologically increased IKr-steady and also IKs-steady and may therefore serve as potential future anti-arrhythmic therapy in SQTS. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Differences in extent of mechano-induced QT-changes in SQTS, WT and LQTS rabbit models

Abstract Background Electro-mechanical (EMC) and mechano-electrical coupling (MEC) are essential for normal cardiac function. Alterations in these can result in increased arrhythmia formation. In “electrical” cardiac diseases, long-QT and short-QT syndrome, regional mechanical function is altered via EMC. Purpose In this study, we aimed to investigate how acute changes in mechanics may impact on electrical function (MEC) in these diseases. Methods To determine how acute changes in preload impact on QT duration, adult rabbits of both sexes were given a 6ml/kg BW bolus of 0.9% NaCl IV and 12-lead-ECGs were assessed first in wildtype (WT) and acquired drug-induced (E4031 to block IKr) LQT2 (“aLQT2”) rabbits, and in a second step in transgenic short-QT type 1 (“SQT1”, KCNH2-N588K) and WT littermate control rabbits (“WT-LMC”). Results At baseline, aLQT2 rabbits demonstrated a markedly prolonged heart-rate corrected QTc duration compared to WT (p<0.0001; n=13), with increased QT-dispersion (QTMax-Min [ms], WT 21.4±5.7 vs. aLQT2 25.8±5.8; p=0.003; n=13) and increased short-term variability of QT (STVQT [ms], WT 3.5±1.0 vs. aLQT2 5.3±1.7; p=0.02; n=13), markers for regional and temporal heterogeneity of repolarization, respectively. SQT1 rabbits (n=8) demonstrated a shorter QTc duration compared to WT-LMC (n=10; p=0.04), with no differences in QT-dispersion and STVQT between the two groups. Increased preload acutely prolonged QT and heart-rate corrected QTc in all groups (despite a slight increase in heart-rate by an average of 25 beats/min): in WT [ms] 171.6±11.6 to 213.3±20.3 (p<0.0001) vs. aLQT2 208.9±19.6 to 271.0±37.5 (p<0.0001; n=13 each), and in WT-LMC 171.3±4.8 to 199.2±5.4 (p<0.0001; n=10) vs. SQT1 156.0±4.7 to 177.3±3.5 (p=0.0004; n=8). Importantly, the extent of mechano-induced electrical changes differed among genotypes, with less pronounced QTc prolongation in SQT1 compared to WT-LMC (delta QTc [ms], SQT1 21.2±3.4 (n=8) vs. WT-LMC 27.9±2.8 (n=10; p=0.15)), and a more pronounced QTc prolongation in aLQT2 compared to WT (delta QTc [ms], WT 41.6±14.9 vs. aLQT2 62.1±32.1; p=0.006; n=13 each). Moreover, QT-dispersion was increased significantly upon global mechanical change only in aLQTS (QTMax-Min [ms], 25.8±5.5 to 32.7±12.3; p=0.03; n=13). Conclusion Acute changes in mechanical function result in electrical changes via MEC in SQT1, WT and aLQT2 rabbits. The extent of these changes, however, depends on the underlying QTc duration, with the least pronounced QTc prolongation in SQT1 rabbits, with the shortest QTc, and the most pronounced QTc prolongation in aLQT2 rabbits, with the longest QTc. The most pronounced MEC effects on global QT duration as well as on regional QT dispersion in aLQT2 indicate that acute MEC effects may play an additional role in LQTS-related arrhythmogenesis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Research Foundation (DFG) andSwiss National Science Foundation (SNF)

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and β-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or β-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

A loss-of-function mutation in RORB disrupts saltatorial locomotion in rabbits.

Saltatorial locomotion is a type of hopping gait that in mammals can be found in rabbits, hares, kangaroos, and some species of rodents. The molecular mechanisms that control and fine-tune the formation of this type of gait are unknown. Here, we take advantage of one strain of domesticated rabbits, the sauteur d'Alfort, that exhibits an abnormal locomotion behavior defined by the loss of the typical jumping that characterizes wild-type rabbits. Strikingly, individuals from this strain frequently adopt a bipedal gait using their front legs. Using a combination of experimental crosses and whole genome sequencing, we show that a single locus containing the RAR related orphan receptor B gene (RORB) explains the atypical gait of these rabbits. We found that a splice-site mutation in an evolutionary conserved site of RORB results in several aberrant transcript isoforms incorporating intronic sequence. This mutation leads to a drastic reduction of RORB-positive neurons in the spinal cord, as well as defects in differentiation of populations of spinal cord interneurons. Our results show that RORB function is required for the performance of saltatorial locomotion in rabbits.

Intestinal Dysbiosis in Young Cystic Fibrosis Rabbits.

Individuals with cystic fibrosis (CF) often experience gastrointestinal (GI) abnormalities. In recent years, the intestinal microbiome has been postulated as a contributor to the development of CF-associated GI complications, hence representing a potential therapeutic target for treatment. We recently developed a rabbit model of CF, which is shown to manifest many human patient-like pathological changes, including intestinal obstruction. Here, we investigated the feces microbiome in young CF rabbits in the absence of antibiotics treatment. Stool samples were collected from seven- to nine-week-old CF rabbits (n = 7) and age-matched wild-type (WT) rabbits (n = 6). Microbiomes were investigated by iTag sequencing of 16S rRNA genes, and functional profiles were predicted using PICRUSt. Consistent with reports of those in pediatric CF patients, the fecal microbiomes of CF rabbits are of lower richness and diversity than that of WT rabbits, with a marked taxonomic and inferred functional dysbiosis. Our work identified a new CF animal model with the manifestation of intestinal dysbiosis phenotype. This model system may facilitate the research and development of novel treatments for CF-associated gastrointestinal diseases.

Genetically Modified Rabbits for Cardiovascular Research.

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

Rabbit models of intracranial atherosclerotic disease for pathological validation of vessel wall MRI.

Vessel wall magnetic resonance imaging can improve the evaluation of intracranial atherosclerotic disease. However, pathological validation is needed to improve vessel wall magnetic resonance imaging techniques. Human pathology samples are not practical for such analysis, so an animal model is therefore needed. Watanabe heritable hyperlipidemic rabbits and apolipoprotein E knockout rabbits were evaluated against New Zealand white wild-type rabbits. Evaluation of intracranial arteries was performed with vessel wall magnetic resonance imaging and pathological analysis, rating the presence and severity of disease in each segment. Two-tailed t-tests were performed to compare disease occurrence and severity prevalence among rabbit subtypes. Sensitivity and specificity were calculated to assess the diagnostic accuracy of vessel wall magnetic resonance imaging. Seventeen rabbits (five Watanabe heritable hyperlipidemic, four apolipoprotein E knockout and eight New Zealand white) were analysed for a total of 51 artery segments. Eleven segments (five Watanabe heritable hyperlipidemic and six apolipoprotein E knockout) demonstrated intracranial atherosclerotic disease on pathology. Disease model animals had lesions more frequently than New Zealand white animals (P<0.001). The sensitivity and specificity of vessel wall magnetic resonance imaging for the detection of intracranial atherosclerotic disease were 68.8% and 95.2%, respectively. When excluding mild cases to assess vessel wall magnetic resonance imaging accuracy for detecting moderate to severe intracranial atherosclerotic disease lesions, sensitivity improved to 100% with unchanged specificity. Intracranial atherosclerotic disease can be reliably produced and detected using 3T vessel wall magnetic resonance imaging-compatible Watanabe heritable hyperlipidemic and ApoE rabbit models. Further analysis is needed to characterize better the development and progression of the disease to correlate tissue-validated animal findings with those in human vessel wall magnetic resonance imaging studies.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of β-VLDLs-the major atherogenic lipoproteins. β-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than β-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Disease pharmacokinetic-pharmacodynamic modelling in acute intermittent porphyria to support the development of mRNA-based therapies.

Acute intermittent porphyria (AIP) results from haplo-insufficiency of the porphobilinogen deaminase (PBGD) gene encoding the third enzyme in the haem biosynthesis pathway. As liver is the main organ of pathology for AIP, emerging therapies that restore enzyme hepatic levels are appealing. The objective of this work was to develop a mechanistic-based computational framework to describe the effects of novel PBGD mRNA therapy on the accumulation of neurotoxic haem precursors in small and large animal models. Liver PBGD activity data and/or 24-hr urinary haem precursors were obtained from genetic AIP mice and wild-type mice, rats, rabbits, and macaques. To mimic acute attacks, porphyrogenic drugs were administered over one or multiple challenges, and animals were used as controls or treated with different PBGD mRNA products. Available experimental data were sequentially used to build and validate a semi-mechanistic mathematical model using non-linear mixed-effects approach. The developed framework accounts for the different biological processes involved (i.e., mRNA sequence, release from lipid nanoparticle and degradation, mRNA translation, increased PBGD activity in liver, and haem precursor metabolism) in a simplified mechanistic fashion. The model, validated using external data, shows robustness in the extrapolation of PBGD activity data in rat, rabbit, and non-human primate species. This quantitative framework provides a valuable tool to compare PBGD mRNA drug products during early preclinical stages, optimize the amount of experimental data required, and project results to humans, thus supporting drug development and clinical dose and dosing regimen selection.

Hypercholesterolemia negatively influences morphology and molecular markers of epithelial cells within the choroid plexus in rabbits

BackgroundChoroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood–cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known.MethodsWe used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization.ResultsCompared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type.ConclusionCholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits.

APOE-knockout in rabbits causes loss of cells in nucleus pulposus and enhances the levels of inflammatory catabolic cytokines damaging the intervertebral disc matrix.

Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders.

Genetic deletion of a short fragment of glucokinase in rabbit by CRISPR/Cas9 leading to hyperglycemia and other typical features seen in MODY-2.

Glucokinase (GCK) is a key enzyme in glucose sensing and glycemic regulation. In humans, mutations in the GCK gene cause maturity-onset diabetes of the young 2 (MODY-2), a disease that is characterized by an early-onset and persistent hyperglycemia. It is known that Gck knockout (KO) is lethal in mice with Gck KO mice dying within 2weeks after birth. Therefore, Gck KO mice are not suitable for preclinical study and have limited suitability to study the pathophysiological role of glucokinase in vivo. Here, we report the generation of a novel rabbit with a non-frameshift mutation of GCK gene (GCK-NFS) by cytoplasm microinjection of Cas9 mRNA and gRNA. These GCK-NFS rabbits showed typical features of MODY-2 including hyperglycemia and glucose intolerance with similar survival rate and weight compared to wild-type (WT) rabbits. The diabetic phenotype including pancreatic and renal dysfunction was also found in the F1-generation rabbits, indicating that the genetic modification is germline transmissible. Treatment of GCK-NFS rabbit with glimepiride successfully reduced the fasting blood glucose drastically and improved its islet function. In conclusion, this novel GCK mutant rabbit generated with the CRISPR/Cas9 system mimics most, if not all, histopathological and functional defects seen in MODY-2 patients such as hyperglycemia and will be a valuable rabbit model for preclinical studies and drug screening for diabetes as well as for studying the pathophysiological role of glucokinase.

Urotensin II and urantide exert opposite effects on the cellular components of atherosclerotic plaque in hypercholesterolemic rabbits.

Increasing levels of plasma urotensin II (UII) are positively associated with atherosclerosis. In this study we investigated the role of macrophage-secreted UII in atherosclerosis progression, and evaluated the therapeutic value of urantide, a potent competitive UII receptor antagonist, in atherosclerosis treatment. Macrophage-specific human UII-transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet for 16 weeks to induce atherosclerosis. Immunohistochemical staining of the cellular components (macrophages and smooth muscle cells) of aortic atherosclerotic lesions revealed a significant increase (52%) in the macrophage-positive area in only male transgenic rabbits compared with that in the nontransgenic littermates. However, both male and female transgenic rabbits showed a significant decrease (45% in males and 31% in females) in the smooth muscle cell-positive area compared with that of their control littermates. The effects of macrophage-secreted UII on the plaque cellular components were independent of plasma lipid level. Meanwhile the wild-type rabbits were continuously subcutaneously infused with urantide (5.4 µg· kg−1· h−1) using osmotic mini-pumps. Infusion of urantide exerted effects opposite to those caused by UII, as it significantly decreased the macrophage-positive area in male wild-type rabbits compared with that of control rabbits. In cultured human umbilical vein endothelial cells, treatment with UII dose-dependently increased the expression of the adhesion molecules VCAM-1 and ICAM-1, and this effect was partially reversed by urantide. The current study provides direct evidence that macrophage-secreted UII plays a key role in atherogenesis. Targeting UII with urantide may promote plaque stability by decreasing macrophage-derived foam cell formation, which is an indicator of unstable plaque.

Retinoid-Related Orphan Nuclear Receptor RORB is Required for Saltatorial Locomotion in Rabbits

Saltatorial locomotion is a type of gait based on hopping that in mammals can be found in rabbits, hares, kangaroos, and some species of rodents. The molecular mechanisms that control and fine tune the formation of this type of gait are unknown. Here, we take advantage of one breed of domesticated rabbits, the sauteur d’Alfort, that exhibits an abnormal locomotion behavior characterized by the loss of the typical jumping that characterizes wild-type rabbits. Strikingly, individuals from this breed frequently adopt a bipedal gait using their front legs. Using a combination of experimental crosses and whole genome sequencing, we show that a single locus containing the RAR related orphan receptor B gene (RORB) explains the atypical gait of these rabbits. We found that a splice-site mutation in an evolutionary conserved site of RORB results in several aberrant transcript isoforms incorporating intronic sequence. This mutation leads to a drastic reduction of RORB-positive neurons in the spinal cord, as well as defects in differentiation of the DMRT3-expressing population of interneurons. Our results demonstrate that RORB plays an important role in neuronal differentiation of the rabbit spinal cord that is required for normal saltatorial locomotion. They further suggest that the DMRT3 gene appears to have a general role in regulating gait across species and different types of locomotion.

Electrically Evoked Potentials Are Reduced Compared to Axon Numbers in Rhodopsin P347L Transgenic Rabbits With Severe Photoreceptor Degeneration.

To determine the relationship between the amplitudes of the electrically evoked potentials (EEPs) and the number of optic nerve axons at a late stage of retinal degeneration in rhodopsin P347L transgenic (Tg) rabbits, a model of retinitis pigmentosa. Six eyes of six wild-type (WT) (43.8 ± 7.5 months of age) and six eyes of six Tg (40.3 ± 2.6 months of age) rabbits were studied. The EEPs were elicited by 1 to 5 mA of transcorneal electrical stimulation. The first positive wave, the P1 component, was analyzed. After euthanasia, the number of axons in the optic nerve was counted. The threshold current to elicit a P1 was significantly higher in Tg rabbits than WT rabbits. The amplitude of P1 elicited by 5 mA in Tg rabbits was about 24% of that in WT rabbits (P < 0.01). The number of axons in the optic nerve of Tg rabbits was reduced to about 59% of that of WT rabbits (P < 0.01). The correlation between the axon number and the amplitude of the P1 in Tg and WT rabbits was not significant. The mean ratio of the P1 amplitude/axon in Tg rabbits was decreased to 53% of that in WT rabbits (P < 0.05). The degree of reduction in the EEP in Tg rabbits is more severe than the reduction in the number of optic nerve axons. The use of transcorneal electrical stimulation to determine the suitable candidates for prosthesis at the end-stage of retinitis pigmentosa may underestimate the condition of the optic nerves.

'Double-muscling' and pelvic tilt phenomena in rabbits with the cystine-knot motif deficiency of myostatin on exon 3.

Gene mutations at different gene sites will produce totally different phenotypes or biological functions in gene-edited animals. An allelic series of mutations in the myostatin (MSTN) gene can cause the ‘double-muscling’ phenotype. Although there have been many studies performed on MSTN-mutant animals, there have been few studies that have investigated the cystine-knot motif in exon 3 of MSTN in rabbits. In the current study, CRISPR/Cas9 sgRNA anchored exon 3 of a rabbit’s MSTN was used to disrupt the cystine-knot motif to change the MSTN construction and cause a loss of its function. Eleven MSTN-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational MSTN amino acid sequences of the 11 founder rabbits were modeled to the tertiary structure using the SWISS-MODEL, and the results showed that the structure of the cystine-knot motif of each protein in the founder rabbits differed from the wild-type (WT). The MSTN-KO rabbits displayed an obvious ‘double-muscling’ phenomena, with a 20−30% increase in body weight compared with WT rabbits. In the MSTN-KO rabbits, all of the MSTN−/− rabbits showed teeth dislocation and tongue enlargement, and the percentage of rabbits having pelvic tilt was 0% in MSTN+/+, 0% in MSTN+/−, 77.78% in female MSTN−/− rabbits, and 37.50% in male MSTN−/− rabbits. The biomechanical mechanism of pelvic tilt and teeth dislocation in the MSTN-KO rabbits requires further investigation.These newly generated MSTN-KO rabbits will serve as an important animal model, not only for studying skeletal muscle development, but also for biomedical studies in pelvic tilt correction and craniofacial research.

Developing AAV-based gene therapy for adrenoleukodystrophy (X-ALD)

Background & Aim Adrenoleukodystrophy (X-ALD) is an X-linked disease. ALD is caused by mutations of the gene encoding ATP-Binding cassette subfamily D member 1 (ABCD1), a peroxisomal transmembrane protein. Dysfunctional ABCD1 protein results in excessive accumulation of very long chain fatty acid (VLCFA) in neural system and leads to demyelination and neural death. Currently, no effective treatments are available to cure X-ALD except BMT at very early stage of disease. Gene therapy using virus as a vehicle is an efficient method to transfer a target gene to human cells. Adeno-associated virus (AAV)-based gene therapy is of high interest for current research. The mechanism of AAV gene transfer does not cause any insertions of target gene into the human genome, which highly enhance the safety of the therapy. There are different serotypes of AAV, each targets different cell types. Therefore, AAV would be a promising vector to cure different diseases in the future. Methods, Results & Conclusion Here in this project, AAV vector with functional ABCD1 gene is constructed and its effectiveness is being tested in vitro for treating X-ALD. Meanwhile, a new animal model – ABCD1 gene mutated rabbit is used to mimic condition of X-ALD patients. Compared to the same age wild type rabbits, the diseased model shows a 5-10 folds increase in plasma VLCFA level before the treatment, and magnetic resonance imaging (MRI) scan is performed to determine demyelination inside the CNS. After AAV injection, rabbits' blood VLCFA level is monitored regularly, MRI scan and brain tissue sectioning are examined to determine the effectiveness of the AAV-based gene therapy targeting X-ALD.

Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system

ObjectiveTo generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.MethodsCRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.ResultsSix knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.ConclusionLarge fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.

Effects of laser needle-knife therapy on vertebroarterial morphology and protein expression of PI-3K, AKT and VEGF in the carotid artery in a rabbit model of cervical spondylotic arteriopathy.

PurposeTo explore how the use of a laser needle-knife affects vertebroarterial morphology and protein expression of PI-3K, AKT and VEGF in the carotid artery of a rabbit model of cervical spondylotic arteriopathy (CSA), and to determine its primary treatment mechanism.MethodsThe CSA rabbit model consisted of 30 rabbits with CSA that were randomly divided into a model group (n = 10), an acupuncture group (n = 10) and a laser needle-knife group (n = 10) plus a further 10 wild type rabbits placed into a normal group. Rabbits in the acupuncture group were treated by needling the Fengchi (GB 20) and Jiaji (EX-B2) acupoints, while those in the laser needle-knife group were treated with a laser needle-knife on the Jiaji (EX-B2) near the spinous process of the fifth cervical vertebra, the rabbit in both groups immobilized during treatment. Rabbits in the other two groups received no treatment, but were immobilized for a similar duration. The morphology of the right vertebral artery and the distribution of pore size before and after treatment were compared using BET and SEM, and the protein expression of PI-3K, AKT and VEGF in the carotid artery of the four groups measured using Western blot analysis.ResultsThe pore diameter and specific surface area of the right vertebral artery increased after treatment, as did the number of micropores. Compared with the normal group, the protein expression of PI-3K, AKT and VEGF in the carotid artery of the model group was significantly lower (P < 0.05), while that of the laser needle-knife group was significantly higher (P < 0.05 and P < 0.01). In addition, rabbits from the acupuncture and laser needle-knife groups demonstrated significantly higher levels of protein expression of PI-3K and VEGF in the carotid artery compared with the model group (P < 0.05 and P < 0.01).ConclusionsBy promoting micrangium hyperplasia within the vertebral artery of rabbits with CSA, treatment with a laser needle-knife modified the protein expression of PI-3K, AKT and VEGF, suggesting that laser needle-knife therapy possibly treats CSA though these signaling pathways.

Genomic and Transcriptomic Analysis of Hypercholesterolemic Rabbits: Progress and Perspectives.

Rabbits (Oryctolagus cuniculus) are one of the most widely used animal models for the study of human lipid metabolism and atherosclerosis because they are more sensitive to a cholesterol diet than other experimental animals such as rodents. Currently, two hypercholesterolemic rabbit models are frequently used for atherosclerosis studies. One is a cholesterol-fed wild-type rabbit and the other is the Watanabe heritable hyperlipidemic (WHHL) rabbit, which is genetically deficient in low density lipoprotein (LDL) receptor function. Wild-type rabbits can be easily induced to develop severe hypercholesterolemia with a cholesterol-rich diet due to the marked increase in hepatically and intestinally derived remnant lipoproteins, called β-very low density lipoproteins (VLDL), which are rich in cholesteryl esters. WHHL rabbits are characterized by elevated plasma LDL levels on a standard chow diet, which resembles human familial hypercholesterolemia. Therefore, both rabbit models develop aortic and coronary atherosclerosis, but the elevated plasma cholesterol levels are caused by completely different mechanisms. In addition, cholesterol-fed rabbits but not WHHL rabbits exhibit different degrees of hepatosteatosis. Recently, we along with others have shown that there are many differentially expressed genes in the atherosclerotic lesions and livers of cholesterol-fed rabbits that are either significantly up- or down-regulated, compared with those in normal rabbits, including genes involved in the regulation of inflammation and lipid metabolism. Therefore, dietary cholesterol plays an important role not only in hypercholesterolemia and atherosclerosis but also in hepatosteatosis. In this review, we make an overview of the recent progress in genomic and transcriptomic analyses of hypercholesterolemic rabbits. These transcriptomic profiling data should provide novel insight into the relationship between hypercholesterolemia and atherosclerosis or hepatic dysfunction caused by dietary cholesterol.