Animal Preparation Research Articles

Breathing stimulation mediated by 5-HT1A and 5-HT3 receptors within the preBötzinger complex of the adult rabbit

Serotonin (5-HT) has been reported to play excitatory effects on respiration by acting on preBötzinger complex (preBötC) neurons in neonatal or juvenile rodents. However, whether its action is circumscribed to the preBötC and present in other animal species, particularly in adult preparations, is unknown. We investigated the respiratory role of 5-HT within the preBötC and neighbouring respiration-related regions. Experiments were performed on α-chloralose-urethane anesthetized, vagotomized, paralyzed and artificially ventilated rabbits making use of bilateral microinjections (30–50 nl). 5-HT caused excitatory effects on respiratory activity only when applied to the preBötC. These effects were mediated by 5-HT1A and 5-HT3 receptors as shown by microinjections of specific agonists of the different types of 5-HT receptors. Unexpectedly, the blockade of 5-HT1A receptors by methysergide or the specific antagonist (S)-WAY 100135 induced excitatory respiratory effects. Microinjections of the 5-HT3 receptor antagonist ondansetron did not influence respiration, but prevented (S)-WAY 100135-induced responses. The blockade of GABAA receptors by bicuculline within the preBötC prevented the effects of the 5-HT1A receptor agonist 8-OH-DPAT. The involvement of GABAergic inhibition and 5-HT1A receptor-mediated disinhibition is also corroborated by immunohistochemical data. The results show for the first time in an adult animal preparation that 5-HT plays a pivotal role in the modulation of the preBötC activity probably via both presynaptic and postsynaptic mechanisms and highlight the importance of disinhibition phenomena. Present findings may be relevant to some respiratory disorders in which an impairment of central 5-HT mechanisms has been reported, such as sleep apnoea and sudden infant death syndrome.

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in β-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

Tracking Cell Recruitment and Behavior within the Tumor Microenvironment Using Advanced Intravital Imaging Approaches.

Recent advances in imaging technology have made it possible to track cellular recruitment and behavior within the vasculature of living animals in real-time. Using approaches such as resonant scanning confocal and multiphoton intravital microscopy (IVM), we are now able to observe cells within the intact tumor microenvironment of a mouse. We are able to follow these cells for extended periods of time (hours) and can characterize how specific cell types (T cells, neutrophils, monocytes) interact with the tumor vasculature and cancer cells. This approach provides greater insight into specific cellular behaviors and cell–cell interactions than conventional techniques such as histology and flow cytometry. In this report, we describe the surgical preparation of animals to expose the tumor and both resonant scanning confocal and multiphoton imaging approaches used to track leukocyte recruitment, adhesion, and behavior within the tumor microenvironment. We present techniques for the measurement and quantification of leukocyte behavior within the bloodstream and tumor interstitium. The use of IVM to study leukocyte behavior within the tumor microenvironment provides key information not attainable with other approaches, that will help shape the development of better, more effective anticancer drugs and therapeutic approaches.

Visualizing Viral Infection In Vivo by Multi-Photon Intravital Microscopy.

Viral pathogens have adapted to the host organism to exploit the cellular machinery for virus replication and to modulate the host cells for efficient systemic dissemination and immune evasion. Much of our knowledge of the effects that virus infections have on cells originates from in vitro imaging studies using experimental culture systems consisting of cell lines and primary cells. Recently, intravital microscopy using multi-photon excitation of fluorophores has been applied to observe virus dissemination and pathogenesis in real-time under physiological conditions in living organisms. Critical steps during viral infection and pathogenesis could be studied by direct visualization of fluorescent virus particles, virus-infected cells, and the immune response to viral infection. In this review, I summarize the latest research on in vivo studies of viral infections using multi-photon intravital microscopy (MP-IVM). Initially, the underlying principle of multi-photon microscopy is introduced and experimental challenges during microsurgical animal preparation and fluorescent labeling strategies for intravital imaging are discussed. I will further highlight recent studies that combine MP-IVM with optogenetic tools and transcriptional analysis as a powerful approach to extend the significance of in vivo imaging studies of viral pathogens.

Robust and accurate decoding of motoneuron behaviour and prediction of the resulting force output.

The spinal alpha motoneuron is the only cell in the human CNS whose discharge can be routinely recorded in humans. We have reengineered motor unit collection and decomposition approaches, originally developed in humans, to measure the neural drive to muscle and estimate muscle force generation in the in vivo cat model. Experimental, computational, and predictive approaches are used to demonstrate the validity of this approach across a wide range of modes to activate the motor pool. The utility of this approach is shown through the ability to track individual motor units across trials, allowing for better predictions of muscle force than the electromyography signal, and providing insights in to the stereotypical discharge characteristics in response to synaptic activation of the motor pool. This approach now allows for a direct link between the intracellular data of single motoneurons, the discharge properties of motoneuron populations, and muscle force generation in the same preparation. The discharge of a spinal alpha motoneuron and the resulting contraction of its muscle fibres represents the functional quantum of the motor system. Recent advances in the recording and decomposition of the electromyographic signal allow for the identification of several tens of concurrently active motor units. These detailed population data provide the potential to achieve deep insights into the synaptic organization of motor commands. Yet most of our understanding of the synaptic input to motoneurons is derived from intracellular recordings in animal preparations. Thus, it is necessary to extend the new electrode and decomposition methods to recording of motor unit populations in these same preparations. To achieve this goal, we use high-density electrode arrays and decomposition techniques, analogous to those developed for humans, to record and decompose the activity of tens of concurrently active motor units in a hindlimb muscle in the in vivo cat. Our results showed that the decomposition method in this animal preparation was highly accurate, with conventional two-source validation providing rates of agreement equal to or superior to those found in humans. Multidimensional reconstruction of the motor unit action potential provides the ability to accurately track the same motor unit across multiple contractions. Additionally, correlational analyses demonstrate that the composite spike train provides better estimates of whole muscle force than conventional estimates obtained from the electromyographic signal. Lastly, stark differences are observed between the modes of activation, in particular tendon vibration produced quantal interspike intervals at integer multiples of the vibration period.

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

In Utero MRI of Mouse Embryos.

Genetically engineered mouse models are used extensively as models of human development and developmental diseases. Conventional histological approaches are static and two-dimensional, and do not provide a full understanding of the dynamic, spatiotemporal changes in developing mouse embryos. Magnetic resonance imaging (MRI) offers a noninvasive and longitudinal approach for three-dimensional in utero imaging of normal and mutant mouse embryos. In this chapter, we describe MRI approaches that have been developed for imaging the living embryonic mouse brain and vasculature. Details are provided on the animal preparation and setup, MRI equipment, acquisition and reconstruction methods that have been found to be most useful for in utero MRI, including examples of applications to fetal mouse neuroimaging.

Anesthesia and Monitoring of Animals During MRI Studies.

The use of imaging represents a major impact on the refinement and the reduction of in vivo studies in animal models, in particular for allowing longitudinal monitoring of the onset and the progression of disease within the same animal, and studying the biological effects of drug candidate and their therapeutic effectiveness. But the use of imaging procedures can affect animal physiology, and the need to anesthetize the animals for imaging entails potential health risks. During anesthesia, there is an inevitable autonomic nervous system depression which induces cardiovascular depression, respiratory depression, and hypothermia. Also other procedures associated with imaging such as animal preparation (e.g., fasting, premedication), blood sampling, and dosage/contrast agent injections can also affect physiology and animal welfare. All these factors are likely to have confounding effect on the outcome of the imaging studies and pose important concerns regarding the animal's well-being, particularly when imaging immune deprived animals or diseased animals. We will discuss these challenges and considerations during imaging to maximize efficacious data while promoting animal welfare.

In Vivo Two-Photon Imaging of the Olfactory System in Insects.

This chapter describes how to apply two-photon neuroimaging to study the insect olfactory system in vivo. It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological information from the living brain. We discuss the most important choices to make when buying or building a two-photon laser-scanning microscope. We illustrate different possibilities of animal preparation and brain tissue labeling for in vivo imaging. Finally, we give an overview of the main methods of image data processing and analysis, followed by a short description of pioneering applications of this imaging modality.

Effect of xylazine, medetomidine and dexmedetomidine on cardiac conduction in pigs

The majority of anaesthetics used in studies regarding heart arrhythmias may affect the cardiac conduction system, thus influencing the results. In veterinary medicine, xylazine, medetomidine and dexmedetomidine are commonly used for premedication in laboratory and companion animals. To date, there have been no studies assessing the effect of these substances on the cardiac conduction system. The aim of this study was to assess the effect of xylazine, medetomidine and dexmedetomidine on the parameters of the cardiac conduction system in pigs. The study was carried out on 18 Great White Polish male pigs weighing from 21 to 40 kg. The animals were divided into three equal groups. The animals from the first group received xylazine at a dose of 2 mg/kg i.v.; those from the second group received medetomidine at 40 mcg/kg i.v.; and those from the third group received dexmedetomidine at 10 mcg/kg i.v. The electrophysiological activity of the heart was analysed using an invasive electrophysiological study (EPS). During the EPS, a decrease in the heart rate after substance administration was observed in all animals, but there were no statistically significant differences in the cardiac conduction parameters. A pro-arrhythmic effect of xylazine was observed, but no statistically significant changes in the EPS parameters were noted. Our results indicate that medetomidine and dexmedetomidine may be used as standard premedication drugs in electrophysiological studies in pigs. Their use may facilitate animal preparation procedures without affecting study results..

The influence of transport on pig meat quality

During the transport of slaughter pigs several typical forms of stress have a strong influence on the animals. Preparation of animals (early handling, indirect transfer systems, fasting period), transport (driving and loading of pigs to the vehicle, vehicle design, weather condition, loading density, driving style, transport time and distance, unloading of pigs), driving and lairage time of the pigs at the slaughterhouse are critical points within the pre-slaughter period. Although halothane status is the most important factor influencing pork quality, optimum-handling procedures pre-slaughter may make significant improvements to pork quality in those pigs carrying the halothane gene. Stress-reduced measures during the pre-slaughter handling of the animals are discussed. The elimination of the Hal gene, the duration of the fasting period (12-18 h), the careful driving and loading of animals in small groups, the vehicle design, the optimal loading density (200-235 Kgr pig/m2or 0,425-0,5m2/l00 Kgr pig), the considerate driving style, the fast unloading of the animals, and the adequate resting period before slaughter (2-4 h) are measures of outmost importance. The occurrence of the déviances in meat quality (PSE-, DFD-meat) in our country calls for the implementation of the abovementioned measures. All these measures require a lot of material, staff and time and can therefore increase transport costs. Good transport management on the other hand can avoid total losses and minimizes losses in carcass weight, damages of the carcass as well as the déviances in meat quality. It is therefore clear that animal welfare and operational efficacy are not necessarily irreconcilable.

Public Awareness in ensuring Animal Originated Food Safety : A Review on “One Health” Approach in Veterinary Medicine

Human and animals live in the same ecosystem bound one to another interdependently and build an harmony in an ecosystem. Since the time of Hippocrates, the One Health concept has existed, which is now we can find in AVMA’s web page, the words “on Airs, Waters, and Places”. One of the One Health’s founders is an epidemilogist, Dr. Calvin W. Schwabe, captured the word “One Medicine” in his book, Veterinary Medicine and Human Health. Now we live in this century can feel the existence of various health problems involving human health and animal health affected one to another.Antimicrobial resistance issues is nowadays one of the challenge facing the medicine. Veterinary Services has a plenty history of applying veterinary public health principles to protect human health through animals and animal products saftey. The Veterinary role in the AMR problems is not a question. The use of drugs, includes antimicrobial, in veterinary practices can not be hindered, that is all to ensure safety not just for animals but also for humanbeing. Microbes contamination in the preparation of animal originated food, from farm to fork, is the main problem in connection to the animal originated food safety. Public services in this field is the entry point of microbes contamination which causing the decrease of food quality. To change the people’s behaviour in processing of animal originated food chain is not easy. Continuing public awareness in preparing safety food is one of athe best way to ensure the intelectuality of a nation Keywords: Global health problem; Animal originated food ; Food safety in Indonesia; Human-animal health interactions; From farm to fork; Public awareness

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging.

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

Composição miofibrilar do glúteo médio de equinos de concurso completo de equitação na fase inicial do treinamento

The studies of myofibrillar composition in athletic horses make it possible to best direct them according to their specific fitness and certainly saves time and resources in the preparation of these athletic animals. Through the technique of percutaneous muscle biopsy and different enzyme-histochemical techniques have been possible to classify these fibers in three different types, type I, IIA and IIX. This information can be applied in the selection of those horses and to the possibility the proper conduct of training protocols. In order to evaluate the myofibrillar composition of Gluteus Medius to the selected mode of Eventing by Army Riding School -RJ, thirteen horses, crossbred breeds of Hanoverian, Thoroughbred and Brazilian Equestrian, in the age range 6-12 years, were used. All animals were submitted to a percutaneous muscle biopsy procedure and the fragments subjected to enzyme-histochemical techniques for distinguishing the three different types of muscle fibers by assessing the reactivity of the enzyme adenosine triphosphate miosinica ( mATPase ) and succinate dehydrogenase ( SDH). The animals used had an average relative distribution of different types of muscle fibers apparently quite homogeneous, with 25.9 %, 35.7 % and 38.4 % of fibers of type I, IIA and IIX, respectively, but lying statistically significant difference , with a higher proportion of type II fibers compared to type I , suggesting that it was a direct genetic influence of the training squad races of the Brazilian Army .

Basics of ultrasonographic examination in sheep

Diagnostic ultrasound has become an essential tool in veterinary medicine and is well established in a variety of species. Since its introduction into sheep clinical practice, it has been mainly used in pregnancy diagnosis and identification of litter size, as well as in experimental settings in reproductive medicine. Furthermore, sheep frequently serve as an experimental model in several disciplines and sub-disciplines of human medicine. However, despite of its great potential, ultrasonography does not yet play a major role in general clinical practice. The review focuses on ultrasonography in sheep and provides information in basics of ultrasound imaging. These include physical principles, instrumentation, handling and preparation of animals, imaging modes (amplitude-depth mode, brightness mode, time-motion mode, 3- and 4-dimensional ultrasound, Doppler ultrasonography), and interpretation of ultrasound images and artefacts. Examples of clinical and experimental applications are included with regard to the different imaging modes, to demonstrate how ultrasonography can be used in sheep clinical practice and research. Additionally, animal welfare as well as safety and hygiene aspects are considered.

Compensation of pulsation artifacts during optical imaging with and without cranial chamber.

Functional Optical Imaging (OI) through the opened skull forms a group of Neuroimaging techniques characterized by a high temporal and spatial resolution on a meso-to macroscopic scale. State of the art OI experiments are generally difficult to execute, with a very timely surgical preparation preceding the experiment, that requires a skilled surgeon to mount a sealed imaging chamber onto the skull. The chamber reduces brain pulsation artifacts and swelling of the brain through movement restriction. In this work, we present preliminary results of a novel approach that does not rely on the usage of an imaging chamber with the goal to facilitate heavily the surgical animal preparation and to allow straightforward joint Electroencephalography - Optical Imaging recordings in the future. We carried out experiments to compare the movement restricting properties of the imaging chamber with the movement in a recording of an unconstrained and periodically irrigated brain. We used high-level image processing techniques to reduce brain pulsation artifacts and did a quantitative movement analysis of the recordings. Our results suggest that while recordings with imaging chamber show less sagittal movement, both with and without imaging chamber comprise the same lateral movements.

Spatial organization of acute myocardial ischemia

IntroductionMyocardial ischemia is a pathological condition initiated by supply and demand imbalance of the blood to the heart. Previous studies suggest that ischemia originates in the subendocardium, i.e., that nontransmural ischemia is limited to the subendocardium. By contrast, we hypothesized that acute myocardial ischemia is not limited to the subendocardium and sought to document its spatial distribution in an animal preparation. The goal of these experiments was to investigate the spatial organization of ischemia and its relationship to the resulting shifts in ST segment potentials during short episodes of acute ischemia. MethodsWe conducted acute ischemia studies in open-chest canines (N=19) and swines (N=10), which entailed creating carefully controlled ischemia using demand, supply or complete occlusion ischemia protocols and recording intramyocardial and epicardial potentials. Elevation of the potentials at 40% of the ST segment between the J-point and the peak of the T-wave (ST40%) provided the metric for local ischemia. The threshold for ischemic ST segment elevations was defined as two standard deviations away from the baseline values. ResultsThe relative frequency of occurrence of acute ischemia was higher in the subendocardium (78% for canines and 94% for swines) and the mid-wall (87% for canines and 97% for swines) in comparison with the subepicardium (30% for canines and 22% for swines). In addition, acute ischemia was seen arising throughout the myocardium (distributed pattern) in 87% of the canine and 94% of the swine episodes. Alternately, acute ischemia was seen originating only in the subendocardium (subendocardial pattern) in 13% of the canine episodes and 6% of the swine episodes (p<0.05). ConclusionsOur findings suggest that the spatial distribution of acute ischemia is a complex phenomenon arising throughout the myocardial wall and is not limited to the subendocardium.

Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe.

This protocol is an extension to:Nat. Protoc. 1, 1552-1558 (2006); doi:10.1038/nprot.2006.276; published online 9 November 2006This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50-90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.