Function In Laboratory Animals Research Articles

Complex Assessment of the Functional State of the Urinary System in Preclinical Studies. Part 1. Instrumental and Laboratory Assessment Methods (Review)

INTRODUCTION. Functional examination of the urinary system, and particularly the kidneys, is an important challenge in preclinical studies. Currently, there is no generally recognised and detailed approach to drug-induced nephrotoxicity detection in vivo, nor are there clear criteria for its assessment.AIM. This study aimed to analyse and systematise instrumental and laboratory methods for the assessment of urinary system function in laboratory animals and to identify the basic principles for studying drug-induced nephrotoxic effects.DISCUSSION. The study analysed the advantages and limitations of the methods used to study the nephrotoxicity of medicinal products, with considerations for the use of these methods in small and large laboratory animals. The effects of a test substance on the urinary system should first be evaluated using minimally invasive methods. One of these methods is urinalysis. For urinalysis, important considerations include the sampling technique, the volume of the biomaterial, and the turnaround time between urine collection and analysis. Ultrasonography is the most accessible instrumental method in preclinical studies. Ultrasonography can assess organ position, size, structure, and echogenicity and detect abnormalities and changes in real time. Dif ferent method settings are preferred for each species of laboratory animal. Further analysis can include macroscopic examination of organs, measurement of their masses, and microscopic analysis of tissues. Visual assessment should cover the size, colour, and consistency of the ureters, bladder, and kidneys. Nephrotoxicity may manifest as increased apoptosis, vacuolation of renal tubular epithelial cells, epithelial degeneration or dystrophy, oedema, diapedesis-associated haemorrhages, acute tubular and papillary necrosis, necrosis of the Bowman–Schumlansky capsule, casts and crystals in the tubular lumen, glomerulopathy with the corresponding changes, and inflammatory and vascular reactions.CONCLUSIONS. The study analysed and systematised instrumental and laboratory methods for assessing the functional state of the urinary system in preclinical studies. The authors outlined the basic principles for a structured and comprehensive study of the potential nephrotoxicity of novel medicines. The assessment of nephrotoxicity should start with simple and minimally invasive laboratory and instrumental methods, which include general urinalysis and microscopic examination of urine sediment. These methods can detect organ dysfunction that has not yet presented with an associated anatomical lesion. A more in-depth analysis should involve histological and immunohistochemical methods to examine the urinary tissues of laboratory animals.

Preclinical Studies on the Safety and Toxicity of Photoditazine in the Antibacterial Photodynamic Therapy of Uropathogenic Bacteria.

The 'dusting' technique of lithotripsy for the removal of infected urinary calculi and the wide use of drainage after endoscopic surgery may stimulate spreading of multidrug-resistant bacterial strains. Antibacterial photodynamic therapy (PDT) is one promising method for the elimination these strains. The purpose of our study was to evaluate alterations of renal pelvis morphology and renal function in laboratory animals after bactericidal regimens of PDT. Renal pelvises of pigs were filled with Photoditazine and then assessed either by examining the accumulation of Photoditazine in the urothelium or by illumination with a laser at a wavelength of 662 nm. A renal test and a complete blood count was performed to assess a negative effect of the treatment on health. Structural alterations of the kidney tissues were analyzed by histological examination. No photosensitizer fluorescence was detected in the urothelium of the pelvis. Histological study showed that PDT caused minor changes to the urothelium of the renal pelvis but did not affect the underlying connective tissue. No renal function abnormalities were found after PDT. Thus, the study indicates that antibacterial PDT is a safety technique that can complement common antibiotic therapy in the surgical treatment of urolithiasis.

Sex differences in immune gene expression in the brain of a small shorebird

Males and females often exhibit differences in behaviour, life histories, and ecology, many of which are typically reflected in their brains. Neuronal protection and maintenance include complex processes led by the microglia, which also interacts with metabolites such as hormones or immune components. Despite increasing interest in sex-specific brain function in laboratory animals, the significance of sex-specific immune activation in the brain of wild animals along with the variables that could affect it is widely lacking. Here, we use the Kentish plover (Charadrius alexandrinus) to study sex differences in expression of immune genes in the brain of adult males and females, in two wild populations breeding in contrasting habitats: a coastal sea-level population and a high-altitude inland population in China. Our analysis yielded 379 genes associated with immune function. We show a significant male-biased immune gene upregulation. Immune gene expression in the brain did not differ in upregulation between the coastal and inland populations. We discuss the role of dosage compensation in our findings and their evolutionary significance mediated by sex-specific survival and neuronal deterioration. Similar expression profiles in the coastal and inland populations suggest comparable genetic control by the microglia and possible similarities in pathogen pressures between habitats. We call for further studies on gene expression of males and females in wild population to understand the implications of immune function for life-histories and demography in natural systems.

Optical coherence tomography angiography in preclinical neuroimaging.

Preclinical neuroimaging allows for the assessment of brain anatomy, connectivity, and function in laboratory animals, such as mice and this imaging field has been a rapidly growing aimed at bridging the translation gap between animal and human research. The progress in the animal research could be accelerated by high-resolution in vivo optical imaging technologies. Optical coherence tomography-based angiography (OCTA) estimates the scattering from moving red blood cells, providing the visualization of functional micro-vessel networks within tissue beds in vivo without a need for exogenous contrast agents. Recent advancement of OCTA methods have expanded its application to neuroimaging of small animal models of brain disorders. In this paper, we overview the recent development of OCTA techniques for blood flow imaging and its preclinical applications in neuroimaging. In specific, a summary of preclinical OCTA studies for traumatic brain injury, cerebral stroke, and aging brain on mice is reviewed.

Effects of Stroke on Motor Function in Laboratory Animals

Effects of Stroke on Motor Function in Laboratory Animals

Supplementation with macular carotenoids improves visual performance of transgenic mice

Carotenoid supplementation can improve human visual performance, but there is still no validated rodent model to test their effects on visual function in laboratory animals. We recently showed that mice deficient in β-carotene oxygenase 2 (BCO2) and/or β-carotene oxygenase 1 (BCO1) enzymes can accumulate carotenoids in their retinas, allowing us to investigate the effects of carotenoids on the visual performance of mice. Using OptoMotry, a device to measure visual function in rodents, we examined the effect of zeaxanthin, lutein, and β-carotene on visual performance of various BCO knockout mice. We then transgenically expressed the human zeaxanthin-binding protein GSTP1 (hGSTP1) in the rods of bco2−/− mice to examine if delivering more zeaxanthin to retina will improve their visual function further. The visual performance of bco2−/− mice fed with zeaxanthin or lutein was significantly improved relative to control mice fed with placebo beadlets. β-Carotene had no significant effect in bco2−/− mice but modestly improved cone visual function of bco1−/− mice. Expression of hGSTP1 in the rods of bco2−/−mice resulted in a 40% increase of retinal zeaxanthin and further improvement of visual performance. This work demonstrates that these “macular pigment mice” may serve as animal models to study carotenoid function in the retina.

Comparison of the Response of Male BALB/c and C57BL/6 Mice in Behavioral Tasks to Evaluate Cognitive Function

To evaluate several cognitive parameters during the execution of behavioral tasks assessing cognitive function in laboratory animals, the parameters are reported within a range. This situation entails that each laboratory must establish the conditions under which the behavioral task to evaluate the cognitive function can be carried out. C57BL/6 and BALB/c inbred strains are used more often in behavioral studies relating to anxiety, stress, fear and cognitive function. The aim of this work was to compare the behavioral response of mice of the strains BALB/c and C57BL/6 to evaluate memory and learning as cognitive functions. Young male mice, 7–8 weeks of age, from each strain were used. Y maze, object recognition and passive avoidance tasks were performed. Both strains of mice showed differences in the response to the passive avoidance and Y maze task. This study advances knowledge about the baseline behavior of laboratory mice strains and their response during the experimental procedures, which are due to the treatment, genetic influence, procedural differences, genetic background variance, or any combination of these elements.

A non-invasive restraining system for awake mouse imaging

BackgroundPreclinical neuroimaging allows for the assessment of brain anatomy, connectivity and function in laboratory animals, such as mice and rats. Most of these studies are performed under anesthesia to avoid movement during the scanning sessions. MethodDue to the limitations associated with anesthetized imaging, recent efforts have been made to conduct rodent imaging studies in awake animals, habituated to the restraint systems used in these instances. As of now, only one such system is commercially available for mouse scanning (Animal Imaging Research, Boston, MA, USA) integrating the radiofrequency coil electronics with the restraining element, an approach which, although effective in reducing head motion during awake imaging, has some limitations. In the current report, we present a novel mouse restraining system that addresses some of these limitations. Results/Comparison to other methodsThe effectiveness of the restraining system was evaluated in terms of three-dimensional linear head movement across two consecutive functional MRI scans (total 20min) in 33 awake mice. Head movement was minimal, recorded in roughly 12% of the time-series. Respiration rate during the acclimation procedure dropped while the bolus count remained unchanged. Body movement during functional acquisitions did not have a significant effect on magnetic field (B0) homogeneity. Conclusion/noveltyCompared to the commercially available system, the benefit of the current design is two-fold: 1) it is compatible with a range of commercially-available coils, and 2) it allows for the pairing of neuroimaging with other established techniques involving intracranial cannulation (i.e. microinfusion and optogenetics).

Alteration of Energy Metabolism and Antioxidative Processing in the Hippocampus of Rats Reared in Long-Term Environmental Enrichment

Environmental enrichment (EE) is a typical experimental method that promotes levels of novelty and complexity that enhance experience-dependent neuroplasticity and cognitive behavior function in laboratory animals. Early EE is associated with resilience in the face of later-life challenges. Since increased synaptic activity enhances endogenous neuronal antioxidant defenses, we hypothesized that long-term EE beginning at an early stage may alter the levels of oxidative stress. We investigated global protein expression and oxidative stress in hippocampal proteins from rats nurtured for a 6-month EE beginning in the prenatal period. The analysis of protein expression was carried out using 2-dimensional gel electrophoresis with matrix-associated laser desorption ionization time-of-flight mass spectrometry. Proteins with altered expression were involved in energy metabolism (phosphoglycerate mutase 1, α-enolase isoform 1, adenylate kinase 1, and triose phosphate isomerase) and antioxidant enzymes (superoxide dismutase 1, glutathione S-transferase ω type 1, peroxiredoxin 5, DJ-1, and glial maturation factor β). Using Western blot assays, some of the proteins with altered expression and NADPH oxidase 2 were confirmed to be decreased. Further confirmation was demonstrated with attenuated expression of 7,8-dihydro-8-oxo-deoxyguanine, a DNA oxidative stress marker, in the hippocampus of EE group rats. Our data demonstrate that a long-term EE program beginning in the prenatal and early postnatal phase of development modulates energy metabolism and reduced oxidant stress possibly through enhanced synaptic activity. We provide evidence that EE can be developed as a tool to protect the brain from oxidative stress-induced injury.

Reliability of Transcutaneous Measurement of Renal Function in Various Strains of Conscious Mice

Measuring renal function in laboratory animals using blood and/or urine sampling is not only labor-intensive but puts also a strain on the animal. Several approaches for fluorescence based transcutaneous measurement of the glomerular filtration rate (GFR) in laboratory animals have been developed. They allow the measurement of GFR based on the elimination kinetics of fluorescent exogenous markers. None of the studies dealt with the reproducibility of the measurements in the same animals. Therefore, the reproducibility of a transcutaneous GFR assessment method was investigated using the fluorescent renal marker FITC-Sinistrin in conscious mice in the present study. We performed two transcutaneous GFR measurements within three days in five groups of mice (Balb/c, C57BL/6, SV129, NMRI at 3–4 months of age, and a group of 24 months old C57BL/6). Data were evaluated regarding day-to-day reproducibility as well as intra- and inter-strain variability of GFR and the impact of age on these parameters. No significant differences between the two subsequent GFR measurements were detected. Fastest elimination for FITC-Sinistrin was detected in Balb/c with significant differences to C57BL/6 and SV129 mice. GFR decreased significantly with age in C57BL/6 mice. Evaluation of GFR in cohorts of young and old C57BL/6 mice from the same supplier showed high consistency of GFR values between groups. Our study shows that the investigated technique is a highly reproducible and reliable method for repeated GFR measurements in conscious mice. This gentle method is easily used even in old mice and can be used to monitor the age-related decline in GFR.

Feeding the immune system

A well-functioning immune system is key to providing good defence against pathogenic organisms and to providing tolerance to non-threatening organisms, to food components and to self. The immune system works by providing an exclusion barrier, by identifying and eliminating pathogens and by identifying and tolerating non-threatening sources of antigens, and by maintaining a memory of immunological encounters. The immune system is complex involving many different cell types distributed throughout the body and many different chemical mediators some of which are involved directly in defence while others have a regulatory role. Babies are born with an immature immune system that fully develops in the first few years of life. Immune competence can decline with ageing. The sub-optimal immune competence that occurs early and late in life increases susceptibility to infection. Undernutrition decreases immune defences, making an individual more susceptible to infection. However, the immune response to an infection can itself impair nutritional status and alter body composition. Practically all forms of immunity are affected by protein-energy malnutrition, but non-specific defences and cell-mediated immunity are most severely affected. Micronutrient deficiencies impair immune function. Here, vitamins A, D and E, and Zn, Fe and Se are discussed. The gut-associated lymphoid tissue is especially important in health and well-being because of its close proximity to a large and diverse population of organisms in the gastrointestinal tract and its exposure to food constituents. Certain probiotic bacteria which modify the gut microbiota enhance immune function in laboratory animals and may do so in human subjects.

Pharmacological Enhancement of Memory and Executive Functioning in Laboratory Animals

Investigating how different pharmacological compounds may enhance learning, memory, and higher-order cognitive functions in laboratory animals is the first critical step toward the development of cognitive enhancers that may be used to ameliorate impairments in these functions in patients suffering from neuropsychiatric disorders. Rather than focus on one aspect of cognition, or class of drug, in this review we provide a broad overview of how distinct classes of pharmacological compounds may enhance different types of memory and executive functioning, particularly those mediated by the prefrontal cortex. These include recognition memory, attention, working memory, and different components of behavioral flexibility. A key emphasis is placed on comparing and contrasting the effects of certain drugs on different cognitive and mnemonic functions, highlighting methodological issues associated with this type of research, tasks used to investigate these functions, and avenues for future research. Viewed collectively, studies of the neuropharmacological basis of cognition in rodents and non-human primates have identified targets that will hopefully open new avenues for the treatment of cognitive disabilities in persons affected by mental disorders.

PHYSIOLOGICAL AND HISTOLOGICAL EFFECT OF LEAD ACETATE IN KIDNEY OF MALE MICEMus musculus.

: Human exposure to lead continues to be a serious public health problem, because lead can cause renal disease and the kidney has the highest concentrations among the soft tissues. Exposure to lead is associated with adverse effects on renal function in laboratory animals and man. An experiment was conducted to study the effect of oral feeding of lead acetate on kidney by estimation the levels of uric acid, urea and creatinine in the serum of mice and the histological parameters of kidney in three different durations. Mice were treated with 0.4 mg/100 ml lead acetate (LA) for 10 days (group A) and for 20 days (group B) and for 30 days (group C).levels of urea in the serum of group A, B, and C increased significantly comparing with the control group, also there was significant differences between group A and group B and between group A and group C, but there was no significant differences between Group B and group C. Level of serum uric acid in groups A, B, C increased significantly comparing with the control group but there was no significant differences among treated group.Serum creatinine levels in group A increased none significantly comparing with the control group. While increased significantly in both of group B and group C, there was significant differences between group A and group C, but there was no significant differences between group A and group B.Kidney sections in group A characterized by foci of congestion and heamorrhage.Glomerular swelling revealed in the kidneys of group B, but the kidney of group C revealed vasculitis, heamorrhage and early hyalinization, normal gromeruli appear in all treated groups.

Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers Alter Striatal Dopamine Neurochemistry in Synaptosomes from Developing Rats in an Additive Manner

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with changes in behavior and neurochemical function in laboratory animals and behavioral deficits in children. PCBs and PBDEs are found in food, especially in seafood and dairy products, and coexposure to these contaminants is likely. We examined the effects of an environmentally relevant mixture of PCBs (Fox River Mix [FRM]) and a PBDE mixture (DE-71) alone and in combination on synaptosomal and medium dopamine (DA) levels and the levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in striatal synaptosomes derived from postnatal days (PND) 7, PND14, or PND21 rats. FRM elevated medium DA and reduced synaptosomal DA concentrations with greater potency than equimolar concentrations of DE-71. The effects of FRM, but not DE-71, were dependent on the age of the animals from which the synaptosomes were derived, with greater effects observed in synaptosomes from the youngest animals. We used Bliss' model of independence to assess the possible interaction(s) of a 1:1 mixture of FRM and DE-71 on synaptosomal DA function and found that the effects of the FRM/DE-71 mixture were additive. Furthermore, as for FRM alone, the effects of the FRM/DE71 mixture were greater in synaptosomes prepared from PND7 rats than in synaptosomes from PND14 and PND21 rats. Because the effects of these contaminants are additive, it is necessary to take into account the cumulative exposure to organohalogen contaminants such as PCBs and PBDEs during risk assessment.

Perfluorooctanoic Acid (PFOA) and Pubertal Maturation in Young Girls

ISEE-0763 Background: Polyfluoroalkyl compounds (PFCs) and their salts, such as perfluorooctanoic acid (PFOA), have been reported to change mammary gland structure and function in laboratory animals. We explored the relationship between serum PFOA concentration and timing of pubertal maturation in young girls. Methods: Within the NIH Breast Cancer and the Environment Research Centers (BCERC), we conducted a study of multiple environmental biomarkers, including PFOA and other PFCs in serum of young girls (age 6–7 years at entry) from two sites (N = 689 girls). Pubertal staging (breast (B) and pubic hair (PH)) has been conducted by clinicians or trained research staff, every year or more frequently, for as long as four years. After calculating adjusted geometric means for all PFCs, we examined the relationship between PFOA serum concentration at the beginning of the study with body mass index (BMI) and pubertal Stage 2 at baseline and one year follow-up. Results: Detectable serum levels of five PFCs, including PFOA, were found in >95% of the girls. The PFOA median was 6.4 ng/ml (range < LOD 0.1 to 55.9 ng/ml), with 24.9% having values above the 95th percentile for children 12–19 years (NHANES 2003–2004 population (8.6 ng/ml)). At the follow-up visit, 28.3% of girls had reached Tanner stage B2+, 19.2% were PH2+ and 30.3% had a BMI percentile for age >85. In analyses where serum PFOA was modeled as a continuous variable, we found a direct relationship with pubertal breast status and an inverse relationship with BMI percentile at the follow-up visit, with adjustment for age, race, site and caregiver education. Conclusions: It appears that PFOA acts as an endocrine disruptor although perhaps not by the usual mechanism. Although the relationship with BMI was inverse, there was a direct relationship with breast maturation. We continue to explore these complex relationships in models including other covariates.

Spatial deficits in a virtual water maze in amnesic participants with hippocampal damage

The Morris water maze is a standard paradigm for the testing of hippocampal function in laboratory animals. Virtual versions of the Morris water maze are now available and can be used to assess spatial learning and memory ability in both healthy and brain injured participants. To evaluate the importance of the hippocampus in spatial learning and memory, we tested five amnesic participants with selective hippocampal damage using a virtual water maze called the Arena Maze. The amnesic participants with hippocampal damage were impaired on the invisible platform (place) task that required them to use distal cues, but were able to navigate almost as well as comparison participants when the invisible platform was marked by a single proximal cue. These results not only confirm that the hippocampus plays a necessary role in human navigation in large-scale environments but also provides a new link between the mnemonic and navigational roles of the hippocampus.

Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug toxicology and safety pharmacology

Background: Echocardiography (EC) is a method used for the investigation of cardiac morphology and function. Two-dimensional EC gives a visualisation of the morphology of the heart. M-mode EC allows heart function to be monitored. Pulsed Doppler EC is the method of choice to measure blood flows. Objective: To describe the information EC can provide for cardiovascular investigation in laboratory animals, with a special focus on the potential helpfulness of EC in preclinical toxicology and safety pharmacology. Methods: This review includes publications describing the methodology of EC and its application to several animal species used in biological experimentation. Results/conclusion: EC has been established in dogs, monkeys, rodents, rabbits and pigs. As demonstrated by experiments in different species, EC can be particularly helpful in toxicology and safety pharmacology, based on the amount of information it can give on the causes and consequences of drug adverse effects on the cardiovascular system. Furthermore, EC does not require any surgery and is therefore a key refinement compared to invasive methods generally used for investigating the cardiovascular function in laboratory animals. Despite some limitations of the method (the need for trained people, time required for an accurate EC recording, lack of current validation), EC should be further developed in preclinical toxicology and safety pharmacology.

Irradiation of Varying Volumes of Rat Lung to Same Mean Lung Dose: a Little to a Lot or a Lot to a Little?

To investigate whether irradiating small lung volumes with a large dose or irradiating large lung volumes with a small dose, given the same mean lung dose (MLD), has a different effect on pulmonary function in laboratory animals. WAG/Rij/MCW male rats were exposed to single fractions of 300 kVp X-rays. Four treatments, in decreasing order of irradiated lung volume, were administered: (1) whole lung irradiation, (2) right lung irradiation, (3) left lung irradiation, and (4) irradiation of a small lung volume with four narrow beams. The irradiation times were chosen to accumulate the same MLD of 10, 12.5, or 15 Gy with each irradiated lung volume. The development of radiation-induced lung injury for < or =20 weeks was evaluated as increased breathing frequency, mortality, and histopathologic changes in the irradiated and control rats. A significant elevation of respiratory rate, which correlated with the lung volume exposed to single small doses (> or =5 Gy), but not with the MLD, was observed. The survival of the rats in the whole-lung-irradiated group was MLD dependent, with all events occurring between 4.5 and 9 weeks after irradiation. No mortality was observed in the partial-volume irradiated rats. The lung volume irradiated to small doses might be the dominant factor influencing the loss of pulmonary function in the rat model of radiation-induced lung injury. Caution should be used when new radiotherapy techniques that result in irradiation of large volumes of normal tissue are used for the treatment of lung cancer and other tumors in the thorax.

Virtual environment navigation tasks and the assessment of cognitive deficits in individuals with brain injury

Navigation in real environments is often impaired by traumatic brain injury (TBI). These deficits in wayfinding appear to be due to disruption of cognitive processes underlying navigation and may in turn be due to damage to the hippocampus and frontal lobes. These wayfinding problems after TBI were investigated using a virtual simulation of a Morris Water Maze (MWM), a standard test of hippocampal function in laboratory animals. The virtual environment consisted of a large virtual arena in a very large virtual room whose walls provided views of a naturalistic landscape. Eleven community-dwelling TBI survivors and 12 comparison participants, matched for gender, age and education were tested to see if they could find a location in the arena marked by one of the following: (a) a visible platform, (b) a single proximal object, (c) a single proximal object among seven other distracter objects, or (d) distal features inside and outside the room. The proximal objects allowed participants to use egocentric (body-centered) navigational strategies that rely on relatively simple stimulus-response associations. The absence of proximal cues forced the participants to rely on distal features of the environment (room walls, landscape elements) and tested their ability to use allocentric (world-based) navigational strategies requiring cognitive mapping. Results indicated that the navigation of TBI survivors was not impaired when the proximal cues were present but was impaired when proximal cues were absent. These results provide more evidence that the navigational deficit after TBI is due to an inability to form, remember or use cognitive maps.

Effect of Na-,Fe-,Co-,Cu-polygalacturonate on hemopoietic function in laboratory animals

The effects of water-soluble Na-,Fe-,Co-,Cu-polygalacturonate on hemopoiesis were studied in laboratory animals of various species, age, and functional groups. The compound had a strong positive effect on hemopoiesis, which manifested in an increase in hemoglobin concentration and erythrocyte count and rapid recovery of blood parameters after acute blood loss.