The wobbler mouse, an ALS animal model

This review article is focused on the research progress made utilizing the wobbler mouse as animal model for human motor neuron diseases, especially the amyotrophic lateral sclerosis (ALS). The wobbler mouse develops progressive degeneration of upper and lower motor neurons and shows striking similarities to ALS. The cellular effects of the wobbler mutation, cellular transport defects, neurofilament aggregation, neuronal hyperexcitability and neuroinflammation closely resemble human ALS. Now, 57 years after the first report on the wobbler mouse we summarize the progress made in understanding the disease mechanism and testing various therapeutic approaches and discuss the relevance of these advances for human ALS. The identification of the causative mutation linking the wobbler mutation to a vesicle transport factor and the research focussed on the cellular basis and the therapeutic treatment of the wobbler motor neuron degeneration has shed new light on the molecular pathology of the disease and might contribute to the understanding the complexity of ALS.     

Myofibrillar responsiveness to cAMP,PKA, and caffeine in an animal model of heart failure

We investigated whether an alteration of myofilament calcium responsiveness and contractile activation may in part contribute to heart failure. A control group of Broad Breasted White turkey poults was given regular feed without additive, whereas the experimental group was given the control ration with 700 ppm of furazolidone at 1 week of age for 3 weeks (DCM). At 4 weeks of age, left ventricular trabeculae carneae were isolated from hearts and calcium-force relationships studied. No differences in calcium-activation between fibers from control or failing hearts were noted under standard experimental conditions. Also failing hearts demonstrated no significant shift in the population of troponin T isoforms but we did observe a significant 4-fold decrease in TnT content in failing hearts compared to non-failing hearts. Addition of caffeine, however, resulted in a greater leftward shift on the calcium axis in fibers from failing hearts. At pCa 6, caffeine increased force by 26+/-2.1% in control fibers and 44.5+/-8.7% in myopathic fibers. Cyclic AMP resulted in a greater rightward shift on the calcium axis in failing myocardium. In control muscles, the frequency of minimum stiffness (f(min)) was higher than in muscles from failing hearts. cAMP and caffeine both shifted f(min) to higher frequencies in control fibers whereas in fibers from failing hearts both caused a greater shift. These results lead us to conclude that heart failure exerts differential effects on cAMP and caffeine responsiveness. Our data suggest that changes at the level of the thin myofilaments may alter myofilament calcium responsiveness and contribute to the contractile dysfunction seen in heart failure.     

Rat ear reattachment as an animal model

The external ear of the rat is an excellent model for practicing microsurgical dissection and for the refinement of microvascular anastomoses, techniques that are crucial for microvascular en bloc tissue transfer and replantation. Preparation of the rat ear for replantation requires familiarity with the vascular anatomy and gentle tissue handling with atraumatic dissection of arterial and venous pedicles, steps similarly crucial in raising free flaps for microvascular transfer. The strategy of performing accurate reduction and stabilization of the tubal cartilage prior to vessel repairs, anastomosing the more deeply seated external carotid artery prior to the more superficial posterior facial vein, is as critical to rat ear replantation as for digital reattachment. In addition, the rat ear as compared to other animal models such as the rabbit ear or canine hindlimbs is much less expensive. Compared to the rat hindlimb model, rat ears are much easier to observe, which is a distinct advantage when used as a model for long-term study of replantation, revascularization, or transplantation.     

Analysing gametic variation with an animal model

Summary A method is presented to treat gametes as homozygous diploid individuals allowing their inclusion into the relationship matrix between animals. In this way standard techniques developed for the analysis of individual genetic variation may be used to analyze gametic variation. An example is given for maternal gametic imprinting and equivalence is shown to a gametic model. The method may also be adopted for the analysis of species (like the honey bee) with one haploid sex.AGBU is a joint initiative of UNE and the NSW Department of Agriculture     

Abnormal lipid metabolism in cystathionine beta-synthase-deficient mice, an animal model for hyperhomocysteinemia

Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine beta-synthase (CBS). Although HHCY is an important and independent risk factor for cardiovascular diseases that are commonly associated with hepatic steatosis, the mechanism by which homocysteine promotes the development of fatty liver is poorly understood. CBS-deficient (CBS(-/-)) mice were previously generated by targeted deletion of the Cbs gene and exhibit pathological features similar to HHCY patients, including endothelial dysfunction and hepatic steatosis. Here we show abnormal lipid metabolism in CBS(-/-) mice. Triglyceride and nonesterified fatty acid levels were markedly elevated in CBS(-/-) mouse liver and serum. The activity of thiolase, a key enzyme in beta-oxidation of fatty acids, was significantly impaired in CBS(-/-) mouse liver. Hepatic apolipoprotein B100 levels were decreased, whereas serum apolipoprotein B100 and very low density lipoprotein levels were elevated in CBS(-/-) mice. Serum levels of cholesterol/phospholipid in high density lipoprotein fractions but not of total cholesterol/phospholipid were decreased, and the activity of lecithin-cholesterol acyltransferase was severely impaired in CBS(-/-) mice. Abnormal high density lipoprotein particles with higher mobility in polyacrylamide gel electrophoresis were observed in serum obtained from CBS(-/-) mice. Moreover, serum cholesterol/triglyceride distribution in lipoprotein fractions was altered in CBS(-/-) mice. These results suggest that hepatic steatosis in CBS(-/-) mice is caused by or associated with abnormal lipid metabolism.     

Ultrastructural observations in the carbonyl iron-fed rat, an animal model for hemochromatosis

Rats fed a carbonyl iron-supplemented diet for 4-15 months were studied for iron content and morphologic changes in the liver, spleen, intestinal mucosa, pancreas and heart. All organs had an increased iron content measured by atomic absorption, with the highest concentrations in the liver and spleen. The periportal distribution of stored iron in the liver was similar to that in human hemochromatosis. In animals treated beyond 6 months Kupffer cells and sinusoidal lining cells also showed cytosiderosis. Electron microscopy provided information on ferritin and hemosiderin content and distribution within parenchymal and sinusoidal cells of the liver but no excessive fibrosis was found. Except for the spleen, the other organs showed less iron deposition. Iron-filled lysosomes (siderosomes) were found in macrophages in the intestinal lamina propria and pancreas, as well as in enterocytes, pancreatic acinar cells and heart muscle cells. Heavily iron-laden siderosomes had increased membrane instability which was demonstrated both morphologically and by measurements of latent lysosomal enzyme activities. Even though cirrhosis was not found, the distribution pattern of accumulated storage iron and lysosomal lability indicated that the carbonyl iron-fed rat is a suitable experimental model for human hemochromatosis.     

Neuropathy in experimental diabetes: an animal model.

A morphometric study of the common peroneal nerve in early experimental diabetes in rats showed that fibre size was diminished. The reduction in the size of the axon was twice that of the myelin sheath. This may contribute to the understanding of the impaired motor conduction velocity found in diabetics shortly after the onset of their disease.     

Narcolepsy: cholinergic receptor changes in an animal model

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.     

Myocardial dysfunction in an animal model of cancer cachexia

AimsFatigue is a common occurrence in cancer patients regardless of tumor type or anti-tumor therapies and is an especially problematic symptom in persons with incurable tumor disease. In rodents, tumor-induced fatigue is associated with a progressive loss of skeletal muscle mass and increased expression of biomarkers of muscle protein degradation. The purpose of the present study was to determine if muscle wasting and expression of biomarkers of muscle protein degradation occur in the hearts of tumor-bearing mice, and if these effects of tumor growth are associated with changes in cardiac function.Main methodsThe colon26 adenocarcinoma cell line was implanted into female CD2F1 mice and skeletal muscle wasting, in vivo heart function, in vitro cardiomyocyte function, and biomarkers of muscle protein degradation were determined.Key findingsExpression of biomarkers of protein degradation were increased in both the gastrocnemius and heart muscle of tumor-bearing mice and caused systolic dysfunction in vivo. Cardiomyocyte function was significantly depressed during both cellular contraction and relaxation.SignificanceThese results suggest that heart muscle is directly affected by tumor growth, with myocardial function more severely compromised at the cellular level than what is observed using echocardiography.     

Relative nephrotoxicity of cephalosporin antibiotics in an animal model

An animal model is described in which mild transitory renal impairment is induced with glycerol and the nephrotoxic effects of cephalosporin antibiotics and furosemide studied. Cephaloridine and cephalothin were found to produce extensive acute tubular necrosis in rats when given in subnephrotoxic doses in combination with furosemide; this damage occurred at serum antibiotic levels not much higher than those obtained in clinical practice. No significant renal damage was found with cephalexin or Cephapirin given in equivalent dosage. It is suggested that the cephalosporin antibiotics should be used with caution in the presence of even minor transient renal impairment and particularly if furosemide is being given concurrently.     

Nitrosative stress in an animal model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of newborns characterized by gut barrier failure. We reasoned that upregulation of inducible nitric oxide synthase (iNOS) may result in nitrosative stress and accumulation of nitroso species in the intestine. Newborn rats were either breast-fed (BF), or formula-fed and additionally subjected to hypoxia (FFH). At Day 4 after birth, the distal ilea were harvested and processed for Western blot analysis and measurement of NO-related metabolites. While BF neonates showed normal morphology, FFH neonates developed signs of NEC by Day 4. These pathological changes correlated with upregulation of iNOS and increases in tissue nitrite, nitrosothiol, and nitrosamine concentrations. Enhanced nitroso levels were most prominent in the mucosal layers of the ileum and iNOS inhibition resulted in a significant decrease in both nitroso species and incidence of NEC. In contrast, increased nitrite levels were distributed evenly throughout the ileum and remained unchanged following iNOS inhibition. Similarly, specimens from NEC patients had higher intestinal levels of NO-related metabolites compared to non-NEC controls. This is the first report of tissue levels of nitroso species in the gut of an animal model of NEC and of human specimens. The results suggest that local nitrosative stress contributes to the pathology associated with NEC. Unexpectedly, the NO breakdown product nitrite, previously considered biologically inert, was found to be present throughout the ileal wall, suggesting that cellular NO metabolism is altered significantly in NEC. Whether nitrite plays a protective or deleterious role remains to be investigated.     

Proteomic profiling in an animal model of acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disease of the pancreas, which evolves in approximately 20% of the patients to a severe illness associated with a high mortality rate. In this study, we performed a comparative proteomic analysis of pancreatic tissue extracts from rats with AP and healthy rodent controls in order to identify changes in protein expression related to the pathobiological processes of this disease. Pancreatic extracts from diseased and controls rats were analyzed by 2-DE and MS/MS. A total of 125 proteins were identified from both samples. Comparative analysis allowed the detection of 42 proteins or protein fragments differentially expressed between diseased and control pancreas, some of them being newly described in AP. Interestingly, these changes were representative of the main pathobiological pathways involved in this disease. We observed activation of digestive proteases and increased expression of various inflammatory markers, including several members of the alpha-macroglobulin family. We also detected changes related to oxidative and cell stress responses. Finally, we highlighted modifications of 14-3-3 proteins that could be related to apoptosis regulation. These results showed the interest of proteomic analysis to identify changes characterizing pancreatic tissue damage and, therefore, to highlight new potential biomarkers of AP.     

Element concentrations and cataract: an experimental animal model

The determination of inorganic ions in cataractous human lenses has been the subject of several investigations; nevertheless, few studies have been concerned with trace element contents in lenses, and data are sometimes contradictory. An animal experimental model of induced cataract is here proposed with the aim of evaluating the changes of Ca, Na, K, Cu and Zn concentrations. The cataract was produced by an Nd: YAG Laser treatment of the right eye of sexteen male rabbits. The determination of the elements was performed by atomic absorption spectrometry (both flame and flameless methods) after an acid digestion of samples. Compared with the results obtained in left lenses used as a control (Ca 14.4±5.7 mg/kg d.w.; Na 1.3±0.5 g/kg d.w.; K 9.9±1.1 g/kg d.w.; Cu 0.24±0.09 mg/kg d.w.; Zn 24.8±2.3 mg/kg d.w.), the mean concentration values of opaque lenses showed some significant changes for Ca, Na, and Cu (Ca 123.7±106.6 mg/kg d.w.; Na 4.5±4.3 g/kg d.w; Cu 0.43±0.21 mg/kg d.w.). Potassium showed a tendency to decrease, and zinc to increase. Positive correlations were found between calcium and sodium both in controls (r=0.73, p<0.001) and in treated lenses (r=0.87, p< 0.0001). An inverse correlation between Ca and K confirmed the tendency of potassium to decrease.     

Interactions between Zn and Cu in LEC rats, an animal model of Wilson's disease

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.     

Phenobarbital inducibility and differences in protein expression of an animal model

Aldehyde dehydrogenases (ALDHs) are a group of enzymes which catalyze the conversion of aldehydes to the corresponding carboxylic acids in a NAD(P)⁺-dependent reaction. In mammals, different ALDHs are constitutively expressed in liver, stomach, eye and skin. In addition, inducible ALDH-isoenzymes are detectable in many tissues; apart from other physico- and immuno-chemical differences, two cytosolic ALDHs (ALDH1A3 and ALDH3A1) are known to be activated in rat liver, by different types of inducers of drug metabolism. Phenobarbital-type inducers increase the ALDH1A3, while polycyclic hydrocarbons (such as BaP and TCDD) increase the expression of the two members of ALDH3A subfamily (3A1 and 3A2). In this study, we used two Wistar rat substrains which have been well-characterized for different inducibility of ALDH1A3 enzyme activity after treatment with phenobarbital. Animals that respond (RR) or do not respond (rr) to treatment have been inbred for almost 25 years, offering a useful experimental model. Apart from the level of ALDH1A3 induced enzyme expression after phenobarbital treatment, no other differences between the two substrains have been noticed, as far as drug metabolizing enzyme activities (like the pentoxy- and ethoxy-O-dealkylation rate) are concerned. According to the present results, the ALDH1A3 expression is still the only difference between the two substrains. Immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or/and CYP1A1/1A2 showed no differences between the two substrains. Additionally, data concerning time- and dose-response induction of ALDH1A3 after phenobarbital and griseofulvin treatment are presented. It is concluded that these two Wistar rat substrains represent a unique animal model for studying what seems to be the only difference between these substrains — the genetic basis of the phenobarbital induction.     

Phenobarbital inducibility and differences in protein expression of an animal model

Aldehyde dehydrogenases (ALDHs) are a group of enzymes which catalyze the conversion of aldehydes to the corresponding carboxylic acids in a NAD(P)(+)-dependent reaction. In mammals, different ALDHs are constitutively expressed in liver, stomach, eye and skin. In addition, inducible ALDH-isoenzymes are detectable in many tissues; apart from other physico- and immuno-chemical differences, two cytosolic ALDHs (ALDH1A3 and ALDH3A1) are known to be activated in rat liver, by different types of inducers of drug metabolism. Phenobarbital-type inducers increase the ALDH1A3, while polycyclic hydrocarbons (such as BaP and TCDD) increase the expression of the two members of ALDH3A subfamily (3A1 and 3A2). In this study, we used two Wistar rat substrains which have been well-characterized for different inducibility of ALDH1A3 enzyme activity after treatment with phenobarbital. Animals that respond (RR) or do not respond (rr) to treatment have been inbred for almost 25 years, offering a useful experimental model. Apart from the level of ALDH1A3 induced enzyme expression after phenobarbital treatment, no other differences between the two substrains have been noticed, as far as drug metabolizing enzyme activities (like the pentoxy- and ethoxy-O-dealkylation rate) are concerned. According to the present results, the ALDH1A3 expression is still the only difference between the two substrains. Immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or/and CYP1A1/1A2 showed no differences between the two substrains. Additionally, data concerning time- and dose-response induction of ALDH1A3 after phenobarbital and griseofulvin treatment are presented. It is concluded that these two Wistar rat substrains represent a unique animal model for studying what seems to be the only difference between these substrains - the genetic basis of the phenobarbital induction.     

Genetic effects on an animal model of anxiety

Genetic effects on behavioural measures thought to model anxiety have been reported on 15 mouse chromosomes. In general the individual effect from each locus is small, contributing to 10% or less of the total variation, but through use of crosses between inbred rodents the power to detect such effects is high: 39 loci have been reported at stringent levels of significance. Novel multivariate analyses of these data go some way to characterizing the genetic architecture of anxiety and also to validating the tests that are used for its measurement. However, we are still some way from finding the molecular variants that explain the heritability of the trait.