Detection of elevated protein modification in human cerebellum in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known for some time to occur in the liver, the main site of ethanol metabolism. In more recent studies of laboratory animals, similar modifications have been detected in organs with lesser ability to metabolize ethanol, such as skeletal and cardiac muscle and brain. Such modification may alter protein function or form a neoantigen, making it a target for immune attack. We now report an analysis of protein modification derived from ethanol metabolites in human brain tissue by ELISA using adduct-specific antibodies. We obtained autopsy cerebellum samples from 10 alcoholic cerebellar degeneration cases and 10 matched controls under informed written consent from the next of kin and clearance from the UQ Human Ethics Committee. Elevated levels of protein modifications derived from acetaldehyde (unreduced-acetaldehyde and acetaldehyde-advanced glycation end-product adducts), from malondialdehyde (malondialdehyde adducts) and from combined adducts (malondialdehydeacetaldehyde (MAA) adducts) were detected in alcoholic cerebellar degeneration samples when compared to controls. Other adduct types found in liver samples, such as reduced-acetaldehyde and those derived from hydroxyethyl radicals, were not detected in brain samples. This may reflect the different routes of ethanol metabolism in the two tissues. This is the first report of elevated protein modification in alcoholic cerebellar degeneration, and suggests that such modification may play a role in the pathogenesis of brain injury. Supported by NIAAA under grant NIH AA12404 and the NHMRC (Australia) under grant #981723.

Can restricting calories help you to live longer?

Excess calorie consumption is associated with metabolic disorders and increased incidence of morbidity. Restricting calorie content, either by daily calorie restriction or intermittent fasting periods, has multiple benefits including weight loss and improved body composition. Previous research has shown that restricting calories in this way can increase longevity and slow the ageing process in laboratory animals, although only sparse data exist in human populations. This review critically evaluates the benefits of these dietary interventions on age-related decline and longevity.

The use of erythrocytic and animal models in the study of protein phosphorylation

Phosphorylation processes are common post-transductional mechanisms, by which it is possible to modulate a number of metabolic pathways. Proteins are highly sensitive to phosphorylation, which governs many protein-protein interactions. The enzymatic activity of some protein tyrosine-kinases is under tyrosine-phosphorylation control, as well as several transmembrane anion-fluxes and cation exchanges. In addition, phosphorylation reactions are involved in intra and extra-cellular 'cross-talk' processes. Early studies adopted laboratory animals to study these little known phosphorylation processes. The main difficulty encountered with these animal techniques was obtaining sufficient kinase or phosphatase activity suitable for studying the enzymatic process. Large amounts of biological material from organs, such as the liver and spleen were necessary to conduct such work with protein kinases. Subsequent studies revealed the ubiquity and complexity of phosphorylation processes and techniques evolved from early rat studies to the adaptation of more rewarding in vitro models. These involved human erythrocytes, which are a convenient source both for the enzymes, we investigated and for their substrates. This preliminary work facilitated the development of more advanced phosphorylative models that are based on cell lines. © 2005 Elsevier B.V. All rights reserved.

Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity:an animal-free CNS cell culture model

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

Cage-induced stereotypies, perseveration and the effects of environmental enrichment in laboratory mice.

When kept in barren and restrictive cages, animals frequently develop stereotypic behaviour patterns that are characterized by high repetition rates, conspicuous invariance and an apparent lack of function. Although millions of animals are affected, the underlying causes and mechanisms are still unclear. Growing evidence suggests that cage-induced stereotypies may reflect pathological dysfunction within basal ganglia circuitry expressed by perseverative behaviour. In order to assess whether variation in stereotypy performance and variation in perseverative behaviour may have a common cause in ICR CD-1 mice, we assessed the effects of environmental enrichment on both phenomena. We raised 48 female ICR CD-1 mice in standard or enriched cages from three weeks to either 6 or 11 months of age and measured stereotypy level in the home cage and perseveration on an extinction task. We further examined whether enriched rearing conditions (early enrichment) protect mice from the developing stereotypies later in life and whether stereotypies developed in barren cages would persist in an enriched environment (late enrichment) by transferring standard mice to enriched cages and vice versa for 14 weeks after completion of the extinction task. We found no evidence for a causal relation between stereotypy and perseveration in mice. However, transfer to enriched cages reduced stereotypy levels significantly both at 6 and 11 months of age indicating that stereotypies had not become established yet. Finally, we found that removing enrichments at both ages did not induce higher stereotypy levels, thereby confirming earlier reports of a neuroprotective effect of early enrichment.

Effects of stereotypic behaviour and chronic mild stress on judgement bias in laboratory mice

Cognitive processes are influenced by underlying affective states, and tests of cognitive bias have recently been developed to assess the valence of affective states in animals. These tests are based on the fact that individuals in a negative affective state interpret ambiguous stimuli more pessimistically than individuals in a more positive state. Using two strains of mice we explored whether unpredictable chronic mild stress (UCMS) can induce a negative judgement bias and whether variation in the expression of stereotypic behaviour is associated with variation in judgement bias. Sixteen female CD-1 and 16 female C57BL/6 mice were trained on a tactile conditional discrimination test with grade of sandpaper as a cue for differential food rewards. Once they had learned the discrimination, half of the mice were subjected to UCMS for three weeks to induce a negative affective state. Although UCMS induced a reduced preference for the higher value reward in the judgement bias test, it did not affect saccharine preference or hypothalamic–pituitary–adrenal (HPA) activity. However, UCMS affected responses to ambiguous (intermediate) cues in the judgement bias test. While control mice showed a graded response to ambiguous cues, UCMS mice of both strains did not discriminate between ambiguous cues and tended to show shorter latencies to the ambiguous cues and the negative reference cue. UCMS also increased bar-mouthing in CD-1, but not in C57BL/6 mice. Furthermore, mice with higher levels of stereotypic behaviour made more optimistic choices in the judgement bias test. However, no such relationship was found for stereotypic bar-mouthing, highlighting the importance of investigating different types of stereotypic behaviour separately.

Curcumin-Arteether Combination Therapy of Plasmodium berghei-Infected Mice Prevents Recrudescence Through Immunomodulation

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha, beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2-3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INF gamma and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INF gamma levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2(-/-) and IL-10(-/-) animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR22/2 mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naive animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria.

Comparative seasonal acclimatization of food and energy consumption in adjacent populations of common spiny mice (Acomys cahirinus)

Animals inhabiting environments with low productivity and food availability commonly have reduced energy demands and increased digestive efficiencies. The dry matter intake (DMI), apparent digestible dry matter (ADDM), digestible efficiency (DE) and digestible energy intake (DEI) of two populations of common spiny mouse Acomys cahirinus were compared during both winter and summer under conditions of simulated water stress. Mice were captured from the north- and south-facing slopes (NFS and SFS) of the same canyon that represent mesic and xeric habitats, respectively. Measured variables were also compared between F-1 mice that had been born to either NFS or SFS mice, and raised in the laboratory. SFS mice were able to assimilate energy more efficiently than NFS mice during the summer. By comparison, NFS mice were able to assimilate more energy during the winter. During winter, NFS mice assimilated more energy at low levels of water stress, whereas SFS mice assimilated more energy at higher levels. Differences were also apparent in F-1 mice. It is therefore suggested that local climatic conditions can impose physiological adaptations that are retained in succeeding generations, creating unique meta-populations.

Transport of animals between rooms: A little-noted aspect of laboratory procedure that may interfere with memory

This study investigated the effects of transporting animals from the experimental room to the animal facility in between experimental sessions, a procedure routinely employed in experimental research, on long-term social recognition memory. By using the intruder-resident paradigm, independent groups of Wistar rats exposed to a 2-h encounter with an adult intruder were transported from the experimental room to the animal facility either 0.5 or 6h after the encounter. The following day, residents were exposed to a second encounter with either the same or a different (unfamiliar) intruder. Resident`s social and non-social behaviors were carefully scored and subjected to Principal Component Analysis, thus allowing to parcel out variance and relatedness among these behaviors. Resident rats transported 6h after the first encounter exhibited reduced amount of social investigation towards familiar intruders, but an increase of social investigation when exposed to a different intruder as compared to the first encounter. These effects revealed a consistent long-lasting (24h) social recognition memory in rats. In contrast, resident rats transported 0.5 h after the first encounter did not exhibit social recognition memory. These results indicate that this common, little-noted, laboratory procedure disturbs long-term social recognition memory. (C) 2011 Elsevier B.V. All rights reserved.

Effect of the bacillus of Calmette-Guerin, Propionibacterium acnes and avridine as immunomodulators in antirabies vaccination of mice using the Fuenzalida-Palacios mouse brain vaccine

Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Plopionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). The IPI test was not effective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-gamma were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results. (C) 1999 Elsevier B.V. Ltd. All rights reserved.

Orchidopexy restores morphometric-stereologic changes in the caput epididymis and daily sperm production in cryptorchidic mice, although sperm transit time and fertility parameters remain impaired

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of canine brain decomposition on laboratory diagnosis of rabies.

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29 degrees C for up to 168 h. At 24 h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72 h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48 h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.

Poisoning of mice by Baccharis coridifolia: An experimental model

The poisonous plant Baccharis coridifotia causes necrosis in lymphoid tissues and the gastrointestinal tract of cattle, horses, sheep and rabbits. An experimental poisoning of mice was undertaken to establish an experimental model in a laboratory animal specie. A single 5 to 8-g/kg dose of a suspension of the plant was administered by gavage to II mice. To 3 other control mice, the same volume of water was administered. Plant-dosed mice manifested clinical effects after 12 h: tachipnea, trembles, dehydration and prostration. Most of the dosed mice died 14 to 33 h after plant administration-3 survived for 12 d. Six mice had remarkable necrosis of the germinative center of secondary follicles in lymph nodes and spleen; 3 mice had necrosis of lymphoid tissues in intestine and thymus. Mice reproduce most of the lesions observed in naturally poisoned cattle and the use of this specie as an experimental model is valid.

Refining the intestinal motility test in mice to reduce animal stress

A new protocol using 3-h fast animal for intestinal motility test was developed in our laboratory aiming the 3R's concept to reduce the stress of animals. Our results may aid in formulating recommendations that can be included in revised guidelines with regard to fasting time of mice.