Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

Human elastin-like polypeptides as a versatile platform for exploitation of ultrasensitive bilirubin detection by UnaG.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

Thoracic cage plasticity in prepubertal New Zealand white rabbits submitted to T1-T12 dorsal arthrodesis: computed tomography evaluation, echocardiographic assessment and cardio-pulmonary measurements.

PURPOSE: We aimed to describe the morphological changes in the thoracic cage and spinal column induced in New Zealand White (NZW) prepubertal rabbits subjected to dorsal arthrodesis and observed at skeletal maturity by computed tomography (CT) scans. This was done to evaluate the plasticity of the thoracic cage of rabbits with non-deformed spine, by highlighting its modifications after spinal arthrodesis. Emogas data analysis, echocardiographic assessment and cardio-pulmonary measurements completed the evaluation. METHODS: Surgery was performed in 16 female rabbits, 6 weeks old. Nine were subjected to T1-T12 dorsal arthrodesis, while seven were sham-operated. Surgery involved the implant of two C-shaped stainless steel bars and heterologous bone graft. CT scans were performed before surgery, 2, 6 and 12 months after surgery. One week after the last CT scan, echocardiographic and emogas evaluations were performed. RESULTS: Chest depth (8%), thoracic kyphosis (ThK) (23%), dorsal and ventral length of the thoracic spine (11%) and sternal length (7%) were significantly reduced in operated compared to sham-operated rabbits. Mean values ± standard deviation (SD) of PaCO2, PaO2 and sO2 were not significantly different. Mean values ± SD of echocardiographic measurements were not significantly different between the two groups of rabbits, except for thickness of the interventricular septum in systole, contractile capacity of the left ventricle and ejection fraction. CONCLUSIONS: T1-T12 dorsal arthrodesis in prepubertal NZW rabbits with non-deformed spine induced changes of the thoracic cage morphology. However, those changes are source of cardio-pulmonary complications not severe enough to reproduce a clinical picture comparable to thoracic insufficiency syndrome in humans.

In vivo visualization of gold-loaded cells in mice using x-ray computed tomography.

The ability to perform cell tracking using x-ray computed tomography combined with gold nanoparticles has been demonstrated recently on ex vivo samples using different malignant and nonmalignant cell lines. Here we proved the concept of the method for in vivo assessment in a small-animal model of malignant brain tumors. The limitations of the method due to radiation dose constraints were investigated using Monte Carlo simulations. Taking into consideration different x-ray entrance doses and the spatial resolution, the visibility of the cell clusters was evaluated. The results of the experiments conducted on mice implanted with F98 tumor cells confirmed the prediction of the Monte Carlo calculations. Small clusters of cells exogenously loaded with gold nanoparticles could be visualized using our in vivo method. FROM THE CLINICAL EDITOR: This article discusses the use of CT-based detection of gold nanoparticle loaded cells of interest in small-animal models of malignant brain tumors, where small clusters of cells loaded with gold nanoparticles could be visualized.

Investigation on the change of spermatozoa flagellar beating forces before and after capacitation.

The swimming forces exerted by mammalian spermatozoa during the pathway to the ovary and during the interaction with the oocyte are thought to play a fundamental role in the fertilization of the egg. In particular, a process named capacitation is of key relevance for its success. Capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit different motility called hyperactivation with a change in the sperm cell tail motion from symmetric to a more asymmetric beating, characterized by wider flagellar bending at lower frequencies. Despite several studies about the mechanism that underlies capacitation, no quantitative information is available about the forces associated with sperm motility. Sperm cell motility has been widely studied with digital imaging tools and video microscopy, but these methodologies cannot provide information about the forces exerted by spermatozoa during the motion and the contribution of every single frequency of flagellar beating to the sperm cell movement. For this purpose, fluidic force microscopy was used to trap single swimming spermatozoa allowing to evaluate these parameters. We observe significant differences between capacitated and non-capacitated spermatozoa in terms of force exerted and beating frequencies. The description of the dynamics of this process is of great interest in the field of reproductive medicine. Such information could be useful to clarify unknown causes of male infertility or for the development of novel methods to assess the quality of semen samples.

Cyanidin 3-glucoside targets a hepatic bilirubin transporter in rats.

One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

Atomic force spectroscopy-based essay to evaluate oocyte postovulatory aging.

Postovulatory aging is a process occurring in the mature (MII) oocyte leading the unfertilized ones to apoptosis. The optimal time window of fertility for different mammalian species after oocytes maturation depends on its timeliness: the higher the time elapsed from the accomplishment of the MII stage, the lower are the chances of fertilization and of development of a viable embryo. In the in vitro fertilization, the selection of competent oocytes for intracytoplasmic sperm injection (ICSI) is mostly made by the visual inspection of the MII oocyte morphology, which does not allow to determine the oocyte postovulatory age. On the other hand, more specific tests usually involve some kind of staining, thus compromising the viability of the oocyte for reproductive purposes. Hence, the need of a noninvasive analysis of oocyte aging to improve the success rate of in vitro fertilization procedures. Here, we exploit atomic force microscopy to examine the evolution of the mechanical properties of mouse oocytes during in vitro postovulatory aging. Three hours before the occurrence of any visual morphological feature related to degradation, we observe a sudden change of the mechanical parameters: the elastic modulus doubles its initial value, while the viscosity decreases significantly. These mechanical variations are temporally correlated with the release of the cortical granules, investigated by fluorescence microscopy. Interestingly, the oocyte mechanics correlates as well with the yield of embryo formation, evaluated up to the blastocyst formation stage. These results demonstrate that minimally invasive mechanical measurements are very sensitive to the aging of the oocyte and can be used as a label-free method to detect the age of the postovulatory oocytes.

Anti-idiotypic response in mice expressing human autoantibodies.

Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.

Gadolinium tissue deposition in the periodontal ligament of mice with reduced renal function exposed to Gd-based contrast agents.

Gadolinium deposition in tissue is linked to nephrogenic systemic fibrosis (NSF): a rare disorder occurring in patients with severe chronic kidney disease and associated with administration of Gd-based contrast agents (GBCAs) for Magnetic Resonance Imaging (MRI). It is suggested that the GBCAs prolonged permanence in blood in these patients may result in a Gd precipitation in peripheral or central organs, where it initiates a fibrotic process. In this study we investigated new sites of retention/precipitation of Gd in a mouse model of renal disease (5/6 nephrectomy) receiving two doses (closely after each other) of a linear GBCA. Two commercial GBCAs (Omniscan® and Magnevist®) were administered at doses slightly higher than those used in clinical practice (0.7 mmol/kg body weight, each). The animals were sacrificed one month after the last administration and the explanted organs (kidney, liver, femur, dorsal skin, teeth) were analysed by X-ray fluorescence (XRF) at two synchrotron facilities. The XRF analysis with a millimetre-sized beam at the SYRMEP beamline (Elettra, Italy) produced no detectable levels of Gd in the examined tissues, with the notable exception of the incisors of the nephrectomised mice. The XRF analyses at sub-micron resolution performed at ID21 (ESRF, France) allowed to clearly localize Gd in the periodontal ligaments of teeth both from Omniscan® and Magnevist® treated nephrectomised mice. The latter results were further confirmed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The study prompts that prolonged permanence of GBCAs in blood may result in Gd retention in this particular muscular tissue, opening possibilities for diagnostic applications at this level when investigating Gd-related toxicities.

TRAIL inhibits osteoclastic differentiation by counteracting RANKL-dependent p27Kip1 accumulation in pre-osteoclast precursors.

Experimental evidences indicate that the TNF family member TNF-related apoptosis inducing ligand (TRAIL) might be involved in modulating osteoclastic differentiation. The ability of recombinant soluble TRAIL to affect bone density in vivo was evaluated by using 4-week-old mice subcutaneously (s.c.) injected with TRAIL for 8 days. TRAIL injection induced a significant increase of tibia trabecular thickness and total bone mass in 4-week-old mice, accompanied by a significant decrease of TRAP serum levels, without modulation of osteocalcin and osteoprotegerin (OPG). Parallel experiments performed in vitro showed that inhibition of osteoclastic differentiation, induced by treatment of human peripheral blood osteoclast precursors with TRAIL, was associated to inhibition of receptor activator of nuclear factor kappa B ligand (RANKL)-induced accumulation of p27(Kip1). The potential role of p27(Kip1) pathway in mediating the anti-osteoclastic activity of TRAIL was further suggested by in vitro gene knock-down experiments performed in osteoclast precursor cultures. Taken together, our data strongly suggest that recombinant TRAIL inhibits osteoclastogenesis by inducing the ubiquitin-mediated degradation of p27(Kip1).

Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models.

BACKGROUND: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated. RESULTS: We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host. CONCLUSION: In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.

Dose and time-dependent apoptotic effects by angiotensin II infusion on left ventricular cardiomyocytes.

OBJECTIVE: To gain insight into the regulation of cardiac apoptosis we studied the dose-response and time-course effects of angiotensin II (Ang II) infusion on ventricular cardiomyocyte apoptosis and on the expression of Bax and Bcl-2 genes and proteins. STUDY DESIGN AND METHODS: In the dose-response study, Ang II was infused subcutaneously at doses of 100, 200, 400, 800 and 1200 ng/kg per min for 14 days. In the time-course study, rats infused with Ang II at doses of 200 and 400 ng/kg per min were followed for 7 and 14 days. The cardiomyocyte apoptotic density was assessed by DNA end labelling (terminal deoxynucleotide nick-end labelling; TUNEL). Gene and protein expression of Bcl-2 and Bax were evaluated by reverse transcriptase-polymerase chain reaction and by Western blots. RESULTS: Systolic blood pressure and left ventricular mass were increased in a dose-dependent manner in Ang II-infused rats. A statistically significant increase in the rate of cardiac apoptosis and pro-apoptotic changes of Bcl-2 and Bax gene and protein expression was observed when high doses of Ang II (800-1200 ng/kg per min) were infused. A positive correlation of apoptotic density with Bax and a negative correlation with Bcl-2 and Bcl-2/Bax ratio were found. Cardiac apoptosis was greatly influenced by the timing of Ang II infusion. Losartan-treated Ang II-infused rats exhibited normalized systolic blood pressure, left ventricular weight, apoptosis, and Bax and Bcl-2 levels. CONCLUSIONS: Our results are consistent with the pathophysiological role of Ang II in induction of cardiac apoptosis, and explain the cardioprotective effect of Ang II receptor antagonists.

Gluten ataxia: passive transfer in a mouse model.

Gluten sensitivity is an autoimmune disease that usually causes intestinal atrophy resulting in a malabsorption syndrome known as celiac disease. However, gluten sensitivity may involve several organs and is often associated with extraintestinal manifestations. Typically, patients with celiac disease have circulating anti-tissue transglutaminase and anti-gliadin antibodies. When patients with gluten sensitivity are affected by other autoimmune diseases, other autoantibodies may arise like anti-epidermal transglutaminase in dermatitis herpetiformis, anti-thyroid peroxidase antibodies in thyroiditis, and anti-islet cells antibodies in type 1 diabetes. The most common neurological manifestation of gluten sensitivity is ataxia, the so-called gluten ataxia (GA). In patients with GA we have demonstrated that anti-gliadin and anti-tissue transglutaminase antibodies cross-react with neurons but that additional anti-neural antibodies are present. The aim of the present article is to review the knowledge on animal models of gluten sensitivity, as well as reviewing the role of anti-neural antibodies in GA.

Relocation and Hair Cortisol Concentrations in New Zealand White Rabbits.

To investigate how long relocation modified hair cortisol concentrations in New Zealand white rabbits, 19 rabbits were subjected to a change in their breeding facility at the beginning of the trial and then were kept under stable environmental conditions. Hair samples were collected at the time of arrival to the nonhuman animal facility and at 40-day intervals from the same skin area for up to 440 days after the animals' arrival to the facility. A period effect on the hair cortisol concentration was found (p < .01). The transfer of the rabbits to the new facility might have induced an increase in the hypothalamic-pituitary-adrenal axis activity (p < .01). A second increase in hair cortisol concentration (p < .01) occurred at 320 days, after a change of personnel at the facility that occurred at 280 days, which was the only environmental change. The relocation of rabbits to the facility resulted in a stress response leading to elevated cortisol levels. The effect of relocation on mean cortisol concentrations was exhausted within 120 days when all environmental factors were kept stable.

Moderate caloric restriction, but not physiological hyperleptinemia per se, enhances mitochondrial oxidative capacity in rat liver and skeletal muscle--tissue-specific impact on tissue triglyceride content and AKT activation.

The study aimed at determining, in lean tissues from nonobese rats, whether physiological hyperleptinemia with leptin-induced reduced caloric intake and/or calorie restriction (CR) per se: 1) enhance mitochondrial-energy metabolism gene transcript levels and oxidative capacity; and 2) reduce triglyceride content. Liver and skeletal muscles were collected from 6-month-old Fischer 344 rats after 1-wk leptin sc infusion (0.4 mg/kg . d: leptin + approximately 3-fold leptinemia vs. ad libitum-fed control) or moderate CR (-26% of those fed ad libitum) in pair-fed animals (CR). After 1 wk: 1) leptin and CR comparably enhanced transcriptional expression of mixed muscle mitochondrial genes (P < 0.05 vs. control); 2) CR independently increased (P < 0.05 vs. leptin-control) hepatic mitochondrial-lipooxidative gene expression and oxidative capacity; 3) hepatic but not muscle mitochondrial effects of CR were associated (P < 0.01) with increased activated insulin signaling at AKT level (P < 0.05 vs. leptin-control); 4) liver and muscle triglyceride content were comparable in all groups. In additional experiments, assessing time course of posttranscriptional CR effects, 3-wk superimposable CR (P < 0.05): 1) increased both liver and muscle mitochondrial oxidative capacity; and 2) selectively reduced muscle triglyceride content. Thus, in nonobese adult rat: 1) moderate CR induces early increments of mitochondrial-lipooxidative gene expression and time-dependent increments of oxidative capacity in liver and mixed muscle; 2) sustained moderate CR alters tissue lipid distribution reducing muscle but not liver triglycerides; 3) mitochondrial-lipid metabolism changes are tissue-specifically associated with hepatic AKT activation; 4) short-term physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression. Increased lean tissue oxidative capacity could favor substrate oxidation over storage during reduced nutrient availability.

Dysregulation of the endothelial nitric oxide synthase-soluble guanylate cyclase pathway is normalized by insulin in the aorta of diabetic rat.

Antiatherogenic effects of nitric oxide (NO) are mediated by activation of soluble guanylate cyclase (sGC) and are impaired by diabetes in animals and humans. We investigated whether uncontrolled diabetes and insulin therapy effect expression and function of the main enzymes of the endothelial nitric oxide (eNOS)-sGC signaling pathway in vivo. Expression and function of eNOS, sGC and protein kinase G (PKG) were studied by Western blot analysis and vasorelaxation to NO-donor in thoracic aortas from control (CON) and streptozotocin (SZT)-induced diabetic rats during uncontrolled diabetes (DM) and insulin treatment (INS) for 8 weeks. Protein level of eNOS was increased (+300%, P < 0.05), while sGC (-50%) and PKG (-65%) proteins were reduced (P < 0.03) in aortas of DM. Insulin treatment normalized these defects resulting in eNOS, sGC and PKG aortic protein content comparable to control. In aortic rings, diethylamine NONOate (DEA-NONOate)-induced vasorelaxation was attenuated (P< or =0.05) in DM compared to control and returned to normal in INS. Thus, experimental diabetes decreases sGC and PKG expression and their NO-dependent activation in aorta despite overexpression of eNOS. These abnormalities are normalized by insulin treatment and improved metabolic control.

Dual sugar gut-permeability testing on blood drop in animal models.

BACKGROUND: Intestinal permeability is determined by measuring nonmetabolized sugars. In animals, intestinal permeability is determined in urine, using cumbersome and expensive metabolic cages. We developed an HPLC method for determining concentrations of lactulose (L) and L-rhamnose (R) in blood-drop of rabbits and mice, and we compared these results with the procedure based on sugars excreted in urine. We measured the intestinal permeability induced by a fragment (DeltaG) of the zonula occludens toxin which opens the paracellular pathway. METHODS: The animals received sugar solution and later received the same solution+DeltaG. Five-hour urine collection and timed blood tests were performed after ingestion of sugars. Sugars were measured with HPLC, and the percentage of recovered sugars was expressed as L/R ratio. RESULTS: At 60 min after administration of sugars, the mean L/R ratio for rabbits and mice was 0.026 and 0.052, respectively. At 60 min after administration of sugars+DeltaG, the mean L/R ratio for rabbits and mice was 0.22 and 0.53. The mean L/R ratio in the urine was 0.023 at basal condition and 0.25 after DeltaG ingestion. CONCLUSIONS: Testing small serum samples for sugar permeability is effective for monitoring changes in permeability of the gut in animals. This cheap simple method allows us to measure in vivo the biological activity of other molecules which modulate the paracellular pathway.

Lack of direct effect of moderate hyperleptinemia to improve endothelial function in lean rat aorta: role of calorie restriction.

Leptin stimulates vascular sympathetic activity and NO release. The resulting effect of hyperleptinemia on endothelial function is unknown. Reduction of food intake associated with hyperleptinemia may mediate some of the vascular effects of leptin. This study was designed to assess the relative effects of short-term moderate caloric restriction and hyperleptinemia on vascular function in lean rat. Endothelium-dependent and -independent vasorelaxation, nitric oxide synthase (eNOS) expression and nitrite production were measured in lean rats receiving leptin subcutaneous infusion (-26% caloric intake, -4% body weight versus control) or moderate caloric restriction (pair-feeding) for one week. Plasma leptin was increased (P < = 0.05) by approximately 300% in the leptin-infused group, while it decreased (P < or = 0.05) by approximately 40% in calorie-restricted animals. Both leptin infusion and calorie restriction resulted in improved (P < 0.05) endothelium-dependent vasorelaxation to acetylcholine (EC50: -6.1 +/- 0.2 and -6.2 +/- 0.2 versus -5.4 +/- 0.2 in control) and unaltered endothelium-independent vasodilation to DEA-NONOate. Furthermore, in aortas from leptin-infused and calorie-restricted rats, expression of eNOS and nitrite production were increased (P < = 0.05) to similar extent compared to control animals. These findings suggest that moderate short-term calorie restriction with adaptive hypoleptinemia has independent beneficial effects on endothelial function in lean animals by enhancing eNOS expression and function. In addition, physiological hyperleptinemia does not independently contribute to improved vascular function above and beyond the effect of calorie restriction.

Myostatin expression is not altered by insulin deficiency and replacement in streptozotocin-diabetic rat skeletal muscles.

BACKGROUND AND AIMS: Insulin is a major post-prandial muscle-anabolic hormone. A substantial loss of skeletal muscle mass occurs in insulin-deprived diabetes and is reversed by insulin treatment. Myostatin is a negative regulator of muscle mass upregulated in several chronic catabolic conditions. Whether myostatin expression is altered in insulin-deprived diabetes is unknown. In spite of opposite effects on muscle mass the potential role of basal circulating insulin in the regulation of myostatin expression is also undetermined. METHODS: We measured (Northern Blot) myostatin transcript levels in muscle groups with different fiber composition in streptozotocin-diabetic male rats receiving one of the following treatments for eight weeks: (1) control (C); (2) diabetes without treatment (DM); (3) diabetes with once-daily slow-acting insulin treatment (INS). RESULTS: INS normalized plasma insulin and prevented weight reduction observed in DM. In fast-twitch gastrocnemius muscle myostatin transcript levels were unchanged (P>0.4) in both DM and INS compared to C. Myostatin transcripts were not measurable in any group in slow-twitch soleus muscle. CONCLUSIONS: Muscle-specific myostatin expression is not increased under catabolic conditions in insulin-deprived diabetes. Insulin treatment also does not change myostatin transcript levels. The data provide the first assessment of potential interplay between insulin and myostatin and they do not support a major role of circulating insulin in the in vivo regulation of myostatin gene expression. A role of myostatin in muscle catabolism in chronic insulin-deprived diabetes is also not indicated by the current results.

Ganstigmine and donepezil improve neurodegeneration in AD11 antinerve growth factor transgenic mice.

Ganstigmine (CHF2819) is an acetylcholinesterase inhibitor that increases acetylcholine in rat hippocampus and ameliorates scopolamine-induced amnesia. In this article, we examined whether and how ganstigmine might prevent or rescue the neurodegenerative phenotype in AD11 antinerve growth factor (anti-NGF) mice, a transgenic model for Alzheimer's disease. The effects of ganstigmine were compared with those obtained after administration of donepezil. Results demonstrate that intraperitoneal and oral administration of ganstigmine and donepezil can reverse the cholinergic and behavioral deficit in AD11 mice but not the amyloid and phosphotau accumulation, uncovering different mechanisms leading to neurodegeneration in AD11 mice.