Ethical Use Of Animals Research Articles

Glial Response and Neuronal Modulation Induced by Epidural Electrode Implant in the Pilocarpine Mouse Model of Epilepsy.

In animal models of epilepsy, cranial surgery is often required to implant electrodes for electroencephalography (EEG) recording. However, electrode implants can lead to the activation of glial cells and interfere with physiological neuronal activity. In this study, we evaluated the impact of epidural electrode implants in the pilocarpine mouse model of temporal lobe epilepsy. Brain neuroinflammation was assessed 1 and 3 weeks after surgery by cytokines quantification, immunohistochemistry, and western blotting. Moreover, we investigated the effect of pilocarpine, administered two weeks after surgery, on mice mortality rate. The reported results indicate that implanted mice suffer from neuroinflammation, characterized by an early release of pro-inflammatory cytokines, microglia activation, and subsequent astrogliosis, which persists after three weeks. Notably, mice subjected to electrode implants displayed a higher mortality rate following pilocarpine injection 2 weeks after the surgery. Moreover, the analysis of EEGs recorded from implanted mice revealed a high number of single spikes, indicating a possible increased susceptibility to seizures. In conclusion, epidural electrode implant in mice promotes neuroinflammation that could lower the seizure thresholds to pilocarpine and increase the death rate. An improved protocol considering the persistent neuroinflammation induced by electrode implants will address refinement and reduction, two of the 3Rs principles for the ethical use of animals in scientific research.

Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

A technique for repeated blood and cerebrospinal fluid sampling from individual rats over time without the need for repeated anesthesia

Ethical animal use follows the 3R’s: Replacement, Reduction and Refinement. Here, we present the use of simultaneous jugular vein and cisterna magna catheterization via a port system in rats for repeated fluid sampling for 14 consecutive days without loss of catheter patency. This technique allows repeated intra-animal sampling without anesthesia and, if used with pooling samples from a cohort of animals, replaces the need for terminal collections for sufficient sample volumes.

Software for animal randomization: A tool for increasing the reproducibility of science.

Poorly designed preclinical studies may compromise human health due to erroneous conclusions regarding treatment effects in addition to contributing to experimental irreproducibility and wasted resources. Randomization is one of the crucial steps to enhance scientific rigor and is a commonly recognized bias-reducing instrument that increases the reliability and reproduction of studies involving animals (even with syngeneic animals). This procedure should be considered when planning a study and reported during data publication. In this context, this work aimed to highlight the importance of adopting quality measures in preclinical trials, with an emphasis on animal randomization. The 'Mouse Randomization' app was developed to help researchers estimate an adequate sample size to obtain significant statistical power, ensuring the ethical use of animals. This app is freely available on the internet to carry out animal randomization and calculate sample sizes for in vivo experiments. We believe that this brief discussion about animal randomization could raise awareness among researchers on how to improve the quality of preclinical research, increasing reproducibility and avoiding animal misuse.

Introducing Graduate Students in Biomedical Sciences to an Institutional Animal Research Program.

At the University of Oklahoma Health Sciences Center, graduate students enrolled in biomedical science programs comprise a significant portion of personnel working with research animals. Although the University requires that all personnel receive appropriate training before working with animals, veterinarians and research mentors agreed students would benefit from additional training. As a result, a course entitled "Laboratory Animal Use and Concepts" was added to the curriculum of the University's largest graduate program in biomedical sciences starting in 2017. The course introduces students to a number of topics relevant to the use of animals in biomedical research, with an emphasis on mice. Here we present a summary of the course and an assessment of its impact during the initial 5 y, from 2017-2021. Enrollment, student outcomes, and student evaluation surveys were included in this assessment. The course was offered to 6 classes totaling more than 120 students during this period. After completing the course, nearly 80% of students used animals as part of their graduate training. Among those, at least 21% sought additional training through formal workshops that offered supplemental opportunities to practice animal handling techniques. Student feedback suggested strong satisfaction with the course content and an appreciation for wet lab sessions. Providing enhanced training for incoming graduate students through this structured course appears to help students improve knowledge, skills, and attitudes that facilitate the responsible and ethical use of animals in biomedical research.

First report of Eimeria myoxi in the garden dormouse (Eliomys quercinus Linnaeus, 1766) from Doñana Natural Area (Andalusia, SW Spain).

This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2±1.2μm and width 17.4±1.1μm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.

Partition of variation for predicting experimental power with a broiler chicken example

A 1932 editorial in Poultry Science stated that sampling theory, or experimental power, could be useful for "the investigator to know how many … birds to put into each experimental pen." Nevertheless, in the past 90 yr, appropriate experimental power estimates have rarely been applied to research with poultry. To estimate the overall variation and appropriate use of resources with animals in pens, a nested analysis should be conducted. Bird-to-bird and separate pen-to-pen variances were separated for 2 datasets, one from Australia and one from North America. The implications of using variances for birds per pen and pens per treatments are detailed. With 5 pens per treatment, increasing birds per pen from 2 to 4 decreased the SD from 183 to 154, but increasing birds/pen from 100 to 200 only decreased the SD from 70 to 60. With 15 birds per treatment, increasing pens/treatment from 2 to 3 decreased SD from 140 to 126, but increasing pens/treatment from 11 to 12 only decreased the SD from 91 to 89. Choosing the number of birds to include in any study should be based on expectations from historical data and the amount of risk investigators are prepared to accept. Too little replication will not allow relatively small differences to be detected. On the other hand, too much replication is wasteful in terms of birds and resources, and violates the fundamental principles of the ethical use of animals in research. Two general conclusions can be made from this analysis. First, it is very difficult to detect 1% to 3% differences in broiler chicken body weight with only one experiment consistently because of inherent genetic variability. Second, increasing either birds per pen or pens per treatment decreased the SD in a diminishing returns fashion. The example presented here is body weight, of primary importance to production agriculture, but it is applicable whenever a nested design is used (multiple samples from the same bird or tissue, etc.).

Simple and Efficient 3D-Printed Superfusion Chamber for Electrophysiological and Neuroimaging Recordings In Vivo.

In vitro and in vivo experimentation in the central nervous system are effective approaches to study its functioning. Manipulations in vitro are characterized by easy experimental control and stable experimental conditions. However, transferring these advantages to in vivo research remains technically and ethically challenging, preventing many research teams from acquiring critical recordings in their animal models. In order to transfer the benefits of in vitro experimentation to in vivo experimentation, we developed a suite of 3D-printed tools (a superfusion chamber with an independent brain presser and animal stand). Using the immature rat barrel cortex as a model, we show that our set of tools (further "superfusion preparation") provides stable conditions for electrophysiological and neuroimaging recordings in the neonatal rat neocortex in vivo Highly correlated intracellular and extracellular activity was recorded during spontaneous and evoked cortical activity, supporting the possibility of simultaneous long-lasting electrophysiological recordings from a single cortical column in vivo The optical intrinsic signal of evoked cortical responses was also recorded from the skull-free neocortex, suggesting the effective combination of the superfusion preparation with neuroimaging approaches. Modulation of immature activity by epicortical application of pharmacological agents via superfusion equally supports the use of the superfused cortex preparation in pharmacological screening. In addition to high efficiency (in affordability, reliability, and ease of use in vivo), the 3D-printed set of tools developed should reduce animal use, supporting the 3Rs principle (Replacement, Reduction, and Refinement) of ethical use of animals.

Micromechanical characterisation of 3D bioprinted neural cell models using Brillouin microspectroscopy

Biofabrication of three-dimensional (3D) in vitro neural cell models that closely mimic the central nervous system (CNS) is an emerging field of research with applications from fundamental biology to regenerative medicine, and far reaching benefits for the economy, healthcare and the ethical use of animals. The micromechanical properties of such models are an important factor dictating the success of modelling outcomes in relation to accurate reproduction of the processes in native tissues. Characterising the micromechanical properties of such models non-destructively and over a prolonged span of time, however, are key challenges. Brillouin microspectroscopy (BM) could provide a solution to this problem since this technology is non-invasive, label-free and is capable of micro-scale 3D imaging. In this work, the micromechanical properties of 3D bioprinted neural cell models consisting of NG 108-15 neuronal cells and Gelatin methacryloyl (GelMA) hydrogels of various concentrations were investigated using BM. We demonstrate changes in the volume-averaged (VA) and local micro-scale mechanical properties of these models over a 7 day period, in which the hydrogel component of the model are found to soften as the cells grow, multiply and form stiffer spheroid-type structures. These findings signify the necessity to resolve in microscopic detail the mechanics of in vitro 3D tissue models and suggest Brillouin microspectroscopy to be a suitable technology to bridge this gap.

Computer-based virtual laboratory simulations: LabHEART cardiac physiology practical.

Practical demonstration of cardiomyocyte function requires substantial preparation, a source of freshly isolated animal hearts, and specialized equipment. Even where such resources are available, it is not conducive for demonstration to any more than a few students at a time. These approaches are also not consistent with the 3R principle (replacement, reduction, and refinement) of ethical use of animals. We present an implementation of the LabHEART software, developed by Donald Bers and Jose Puglisi, for medical students. Prior to the activity, students had lectures covering the physiological and pharmacological aspects of cardiac excitation-contraction (EC) coupling. We used this problem-based activity to help students consolidate their knowledge and to allow a hands-on approach to explore the key features of EC coupling. Students simulate and measure action potentials, intracellular calcium changes, and cardiomyocyte contraction. They also apply drugs that target ion channels (e.g., nifedipine or tetrodotoxin) or sympathetic input (using isoproterenol) and explore changes to EC coupling. Furthermore, by modifying the biophysical parameters of key ion channels involved in the electrical activity of the heart, students also explore the effect of channelopathies such as long QT syndromes. We describe approaches to implement this activity in a flipped classroom format, with recorded lecture materials provided ahead of the practical to facilitate active learning. We also describe our experiences implementing this activity online. The content and difficulty of the activity can be altered to suit individual courses and is also amenable to promote peer-driven learning.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

Public attitudes toward dairy farm practices and technology related to milk production.

Dairy farm systems have intensified to meet growing demands for animal products, but public opposition to this intensification has also grown due, in part, to concerns about animal welfare. One approach to addressing challenges in agricultural systems has been through the addition of new technologies, including genetic modification. Previous studies have reported some public resistance towards the use of these technologies in agriculture, but this research has assessed public attitudes toward individual practices and technologies and few studies have examined a range of practices on dairy farms. In the present study, we presented participants with four scenarios describing dairy practices (cow-calf separation, the fate of excess dairy calves, pasture access and disbudding). Citizens from Canada and the United States (n = 650) indicated their support (on a 7-point scale) toward five approaches (maintaining standard farm practice, using a naturalistic approach, using a technological approach, or switching to plant-based or yeast-based milk production) aimed at addressing the welfare issues associated with the four dairy practices. Respondents also provided a text-based rationale for their responses and answered a series of demographic questions including age, gender, and diet. Participant diet affected attitudes toward milk alternatives, with vegetarians and vegans showing more support for the plant-based and yeast-based milk production. Regardless of diet, most participants opposed genetic modification technologies and supported more naturalistic practices. Qualitative responses provided insight into participants’ values and concerns, and illustrated a variety of perceived benefits and concerns related to the options presented. Common themes included animal welfare, ethics of animal use, and opposition toward technology. We conclude that Canadian and US citizens consider multiple aspects of farm systems when contemplating animal welfare concerns, and tend to favor naturalistic approaches over technological solutions, especially when the latter are based on genetic modification.

From Mice to Monkeys? Beyond Orthodox Approaches to the Ethics of Animal Model Choice.

Simple SummaryNew tools, allowing scientists to make precise changes to mammal genomes, have made possible future increased use of larger mammals in biomedical research, such as primates, pigs, and dogs. This paper addresses ethical issues that are raised by using larger mammals instead of smaller ones in this research. Because scientists who use animals in research follow strict guidelines, we first examine what those guidelines suggest for using larger mammals. We then consider what philosophers, who write about the ethics of animal use, consider as the important questions in evaluating which (if any) animals are acceptable to use in research. We find that philosophical perspectives have typically been interested in the question of when or if animal use is justified, while biomedical research guidance has assumed that animal use is justified but defined specific limits to that use. To address directly the ethical questions that arise in the practice of biomedical research in selecting which animals to use, we consider an approach to ethics that is focused on character and living well (or flourishing). This paper is valuable to society in drawing attention to the ethical questions, rather than merely the scientific issues, that are important in selecting which animals to use in biomedical research.Recent developments in genome editing tools, along with limits in the translational potential of rodent models of human disease, have spurred renewed biomedical research interest in large mammals like nonhuman primates, pigs, and dogs. Such scientific developments raise ethical issues about the use of these animals in comparison with smaller mammals, such as mice and rats. To examine these ethical questions, we first consider standard (or “orthodox”) approaches, including ethics oversight within biomedical research communities, and critical theoretical reflections on animal research, including rights-based and utilitarian approaches. We argue that oversight of biomedical research offers guidance on the profession’s permitted uses of animals within a research setting and orthodox approaches to animal ethics questions when and whether animals should be used in biomedicine; however, neither approach sufficiently investigates the nuances of ethical practices within the research setting. To fill this lacuna, we consider a virtue ethical approach to the use of specific animal models in biomedicine. From this perspective, we argued that limitations on flourishing for large mammals in a research setting, as well as potential human-animal bonds, are two sources of likely ethical tensions in animal care and use in the context of larger mammals.

Responsible Science and Research Animal Use.

This issue of the ILAR Journal focuses on the topic of responsible science as it relates to animal research. We start with the concept of the scientist as a responsible citizen and then move through multiple phases of research including careful experimental planning, reporting, and incorporation of laboratory animal science. The work of the Institutional Animal Care and Use Committee (IACUC) or animal ethical/oversight body in reviewing both animal use and contributing to scientific excellence is explored. Additional topics include protection of animal handlers from multiple experimental hazards, use of agricultural animals and wildlife studies, regulatory ambiguities, and harmonization of animal research. Rounding out the issue is a discussion of how animal care and use programs can enhance animal welfare while mitigating regulatory burden, and our responsibility to clearly communicate the ethical use of animals in advancing biomedical research. A deeper understanding of these topics can assist scientists in simultaneously advancing their research and animal welfare.

A Series Circuit from CA1 of Dorsal Hippocampus to Basolateral Amygdala via Postrhinal Cortex Participates in Conditioned Context-Induced Retrieval of Morphine Withdrawal Memory

Background: Conditioned context-induced retrieval of drug withdrawal memory contributes to drug relapse. The basolateral amygdala (BLA) plays an important role in conditioned context-induced retrieval of morphine withdrawal memory. However, the upstream pathways of the activation of the BLA by conditioned context remains to be studied. Methods: Using the conditioned place aversion model, chemical-genetic method, trans-neuronal virus tracing technique and immunohistochemical method, we studied the upstream neural circuits of the activation of the BLA during conditioned context-induced retrieval of morphine withdrawal memory. Findings: The present results show that the dorsal CA1 of the hippocampus is an upstream brain region of the activation of the BLA during conditioned context-induced withdrawal memory retrieval; the indirect connection from dorsal CA1 of the hippocampus to the BLA is enhanced in mice with conditioned place aversion; the postrhinal cortex (POR) is a brain region that connects the dorsal CA1 of the hippocampus and the activation of the BLA in conditioned context-induced retrieval of morphine withdrawal memory. Interpretation: These results suggest that a series circuit from CA1 of dorsal hippocampus to basolateral amygdala via postrhinal cortex participates in conditioned context-induced retrieval of morphine withdrawal memory. Funding Statement: This study was supported by the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2009CB52201 and 2013CB835100) and Project of Foundation of National Natural Science of China (31121061, 91332204, 81371466 and 31070932). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: All experimental procedures conformed to Fudan University as well as international guidelines on the ethical use of animals. All efforts were made to minimize animal suffering and reduce the number of animals used.

Abstract B134: Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin

Abstract Background: A variety of methods have been shown to induce immunogenic cell death (ICD) and overcome the immunosuppressive tumor microenvironment, thus generating an effective immunotherapy. In our previous work, we have shown that non-replicating adenovirus mediated gene transfer of p19Arf and interferon-β (IFNβ) forms a strategic alliance between gene function and the vector, resulting in a multi-modal mechanism of cell death consistent with necroptosis and with the liberation of ICD molecules HMGB1, ATP and calreticulin in B16F10 mouse melanoma cells. We have performed vaccine and immunotherapy assays as well as in situ vaccination in order to show that our gene transfer approach contributes to an antitumor immune response that involves neutrophils, NK, CD4+ and CD8+ T-cells and cytokine production that is consistent with a Th1 profile. While promising so far, we strive to boost tumor cell killing while preserving the well-being of the study subject. Objective: To use our gene transfer approach in association with doxorubicin (Dox), a well-known inducer of ICD, in order to promote cell killing while permitting the use of reduced drug dosages, thus ameliorating collateral effects, including cardiopathy, and possibly benefitting the antitumor immune response. Materials and Methods: MCA205 and B16F10 cells were used in this study. The AdRGDPG vectors are non-replicating recombinant Ad5 viruses containing the RGD tripeptide modification of the virus fiber protein and which utilize a p53-responsive promoter (termed PG) to control transgene expression. The AdRGDPG-GFP, AdRGDPG-p19, and AdRGDPG-IFNβ vectors encode enhanced green fluorescent protein (GFP), the mouse cDNAs for p19Arf (a functional partner of p53) and IFNβ, respectively. Using flow cytometry (Attune, Thermo Fisher), accumulation of hypodiploid cells was revealed upon fixation and propidium iodide staining while caspase 3/7 activity was visualized using Cell Event Caspase 3/7 (Thermo Fisher). Alternatively, BLI (IVIS Spectrum, Perkin Elmer) was used to detect a DEVD-dependent luciferase reporter construct in vivo. Cardiac function was monitored using echocardiography (VisualSonics Vevo 2100 Imaging System) and data analyzed using VevoCQ LV analysis software (VisualSonics). All animal assays were performed according to and with the approval of the Committee for the Ethical Use of Animals, FM-USP. Results: Association of p19Arf/IFNβ gene transfer with Dox treatment in vitro improved cell killing and central composite rotational design analysis revealed that indeed, the association permits the application of less drug and/or virus while preserving high levels of death in either cell line. In vitro, caspase 3/7 activity was additive upon exposure of MCA205 cells to the associated treatments, yet Dox was sufficient in B16F10. In vivo, Dox (20 mg/kg) alone or in combination with in situ gene therapy was correlated with inhibition of MCA205 tumor progression, increased caspase 3/7 activity and prolonged survival. However, 20 mg/kg Dox provoked reduced cardiac function (left ventricular ejection and systolic fractions, stroke and systolic volumes). Strikingly, the sub-therapeutic dose of 10 mg/kg Dox was not correlated with cardiopathy, yet only when used in combination with in situ gene transfer did it effectively block tumor progression. Finally, immunotherapy of previously established MCA205 tumors using a therapeutic vaccine consisting of dying cells treated ex vivo with both gene transfer and Dox resulted in effective challenge tumor inhibition. Conclusions: Here we have clearly shown that the association of gene transfer with Dox enables the use of lower drug doses in order to both preserve cardiac function and inhibit tumor progression. Early evidence also suggests that this combination increases the immunogenicity of treated cells, inviting the development of potential immunotherapeutic strategies. Funding: Sao Paulo Research Foundation (FAPESP). Citation Format: Bryan E. Strauss, Ruan F.V. Medrano, Rodrigo Tamura, Samir A. Mendonça, Valker A. Feitosa, Rafael Dariolli, Thiago A. Salles, Aline Hunger, João P.P. Catani, Elaine G. Rodrigues. Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B134.

The Animal Is Present: The Ethics of Animal Use in Contemporary Art

The Animal Is Present: The Ethics of Animal Use in Contemporary Art

Conducting and reporting animal experimentation: Quo vadis?

Most scientific journals ask authors to include a statement in their articles that animal studies have been carried out in agreement with international regulations on the use and care of laboratory animals. This statement implies that all the experiments conducted on animals have been evaluated and accepted by an Ethical Committee and, that animal welfare has been put as a priority throughout the experimental protocol. Nevertheless, discrepancies are commonly found between the described procedures and the guidelines that are claimed to have been followed; this reveals a double dilemma. First, animal welfare is not always considered, implicating discomfort or even worse, suffering to animals involved. Secondly, revisions of manuscripts are sometimes done without taking into account ethical and regulatory aspects concerning the use of animals. Underestimation of pain or suffering, disregard for physiological parameters, and other examples recently reported in scientific journals by neuroscientists from all over the world are discussed in this article. In a period of great debate about the ethical use of animals, with society being involved and engaged in the discussion, this Neuro-Opinion intends to call the attention of researchers, ethical committee members, and journal editors about the need of strictly endorsing international regulations and placing animal welfare as the top priority.

Produção de lágrima após exérese parcial da glândula lacrimal principal em cães

Background : : : : The partial loss of the lacrimal gland by obstruction, injury, removal of tumor tissue or other pathological changes may lead to loss of tear flow, bringing harmful consequences to the body. The aim was to evaluate the production of tears after removal of approximately 50% of the main lacrimal gland. Materials, Methods & Results : For this, lacrimal glands from the left eyes of seven dogs, male, adult, with weight ranging from 6 to 13kg were partially removed. In each animal, an incision of approximately 3.0 cm of skin, orbicularis and cutaneous muscle of the eye between the eyeball and the zygomatic process of frontal bone of the lateral left side was performed. After incision of the conjunctiva, close to the zygomatic process, the orbital ligament was located and partially sectioned to expose the main lacrimal gland. With a stainless steel wire graph, the gland was measured and retired approximately 50% of the parenchyma. The fragment was placed in a bottle containing 10% formalin for histological evaluation. The tears production was measured with lacrimal Schirmer test at 28 oC and 70% relative humidity 24 hours before surgery and on the 15th, 30th and 60th days after surgery. At the 60th day, samples of gland lacrimal of the same animals were colleted for histopathologic examination of number, mean diameter of the acini and their nuclei, nucleus: cytoplasm ratio and cell area in a sample of 30 acini per animal and the density of collagen fibers. In the evaluations of the tears production with the Schirmer Test, between the pre-operative (PE), 15, 30 and 60 days post-operatively (PO) were noticed significant differences (p=0.0033) at times between PE and 15 days of PO and between 15 and 60 days of PO. By comparing histological evaluation of lacrimal gland samples in the PE and after 60 days, there were significant differences in the lacrimal gland of nuclear area (p > 0), nuclear cytoplasm proportion (p=0.0006) and cell area (p=0.0083). The collagen fibers formation did not differ significantly between PE and 60 days after partial removal of the gland (p=0.7104). All the ethical principles of ethical use of animals in experiments recommended by the Brazilian College of Animal Experimentation and Ethics Committee of the use of animals at the Federal University of Uberlandia. Discussion : The partial removal allowed the lacrimal gland remaining to offset the production of aqueous tear film: the values found in tear production were above normal throughout the trial period. Induction of cells secreting tears to hyperactivity occurred to offset the demand of production after removing part of the gland. Thus, it was stimulated increase nuclear - giant and multiple nuclei in constant transcription-protein synthesis – and small cytoplasm with little storage tear. This fact explains the increase in cell at 60 days, the decrease in tear production after 24 hours and its return to normal parameters at 60 days postoperatively. In this experiment, the density of collagen fibers found throughout the parenchyma tear between the PE and after 60 days postoperatively was not significant, possibly due to the deposition of fibers are directed to the incision site and the inflammatory reaction, along with the deposition collagen fibers was terminated with 60 days postoperatively. Therefore, the partial removal of the main tear gland in dogs does not interfere significantly in the fall of tear production on day 60 of evaluation in which the tears production came back within the normal patterns.

A novel microsurgical anastomosis training model using gradually thawed cryopreserved microvessels of rat cadavers

In consideration of the 3-R-rule (Refine-Replace-Reduce) as a guideline for promoting ethical use of animals for surgical training, we present a novel training model for microvessel anastomosis. In a rat cadaveric study, we evaluated the surgical anatomy of the common carotid artery (CCA), external jugular vein (EJV) and femoral vessels (FV) which were then used as templates for the present investigation. Anatomical dissection of 30 rat cadavers was performed. Two residents without prior microsurgical experience were included in the study and performed 5 CCA, 5 femoral artery, 5 EJV and 5 femoral vein anastomoses. Patency and leakage served as qualitative variables and operation time as a quantitative variable for efficiency control. The average time improved for arterial and venous anastomoses (45 min–22 and 60 to 32 min, respectively) for both surgeons. While both surgeons experienced patency failure or leakage within the first half of performed arterial and venous anastomoses, they could improve to a 100% patency rate without the occurrence of leakage for the last half of trials. The rat head & neck anatomy presents various characteristics related to the harvest of the vessels of interest. We provide anatomical knowledge about the topography related to the harvest of the CCA, EJV, and FV. Our model is an easily accessible, low-cost microsurgical simulation model, allowing a realistic and instructive performance of anastomoses. Since cadaveric vessels are used, an approval of the local ethics committee is not needed.