Animal Handling Research Articles

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.

Enhancing sustainability through resource efficiency in beef production systems using a sliding time window-based approach and frame scores

The food needs of the increasing global population, inefficiencies in supply chains, customer expectations and environmental concerns are the challenges to meeting resource-intensive protein needs sustainably. Collectively, this increases the need to enhance sustainability in the beef sector. This study proposes a sliding time-window-based multi-period livestock production model using mixed-integer linear programming (MILP) to simultaneously balance economic and environmental losses. It identifies the optimal finishing time using frame score (FS) and feed conversion ratio (FCR), targeting flexibility by allowing variable growth periods to reduce food/nutritional losses while meeting the variability in demands with minimum inventory levels. Furthermore, sequencing and assigning animals to facilities with optimum separation time is applied to avoid bad handling of animals and ensure quality meat with hygienic standards for longer shelf life. The system boundary of the proposed model includes beef farms and processing facilities. Compared to the recently proposed batch processing models over seven months with a herd size of 1980 animals, the findings reduce the average forage needed by ∼126.90 kips and methane emissions by ∼2560 kg, with a significant benefit in terms of the live animals' weight gain by ∼10,276 lbs.

Immobilization of Raccoons (Procyon lotor) with Nalbuphine, Medetomidine, and Azaperone.

Chemical immobilization is widely used by wildlife and veterinary professionals for the safe handling of animals. A combination of nalbuphine (40 mg/mL), azaperone (10 mg/mL), and medetomidine (10 mg/mL), known as NAM, is a low-volume combination with field immobilization practicality and fewer regulations restricting its use in the US than some other drug combinations. We evaluated the safety and effectiveness of NAM as an immobilizing agent for raccoons (Procyon lotor). From May 2021 to February 2022, 16 adult raccoons were captured in cage traps and immobilized with 0.3 mL NAM intramuscularly (12 mg nalbuphine, 3 mg medetomidine, and 3 mg azaperone, regardless of body weight). After administration, time to sedation was measured; body temperature, heart rate, respiratory rate, and oxygen saturation were monitored and recorded every 5 min for 20 min. Each raccoon was weighed; the dose administered was calculated (range 2.2-4.1 mg/kg, mean 3 mg/kg). Mean induction time was 6 min (4-17 min); time to recovery following administration of 15 mg atipamezole, 7.5 mg naltrexone for reversal, was 10 min (6-18 min). Heart rate, oxygen saturation, and respiration rate remained steady during immobilization. Rectal temperature steadily declined. Overall, NAM appeared to be a practical option for raccoon immobilization, providing rapid induction and reversal as well as adequate sedation for short-term handling and minimally invasive sampling.

Generation of the NeoThy mouse model for human immune system studies.

Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function.

Evidence of the Atlantic nurse shark (Ginglymostoma cirratum) population shrink at Rocas Atoll, Southwestern Atlantic

Abstract The Atlantic nurse shark, Ginglymostoma cirratum, was recently listed as Vulnerable by IUCN Red List, however, as highlighted, little is known about its populations in the Southwestern Atlantic. Therefore, population studies are essential to propose status evaluations and new conservation measures. Natural marks present on the animals' bodies are used as an alternative non‐invasive method rather than the usual tagging system for individual identification, thus avoiding the need for animal handling. This study aimed to evaluate the population size trend of G. cirratum at the Rocas Atoll Marine Biological Reserve, the only atoll located in the South Atlantic. Data were collected through underwater filming, photo‐identification, and visual censuses. Three expeditions were carried out in 2018 and the abundance of sharks was sampled in the inner part of Rocas Atoll Marine Biological Reserve. Sharks were identified individually with the aid of the software Interactive Identification System – Contour (I3S). Based on the re‐sightings of identified animals, two capture–recapture estimators of population size were used (Petersen–Bailey; Jolly–Seber). The estimates were then compared to the only previous estimate made 20 years before, which employed the same number of expeditions and estimators. A total of 139 sharks were identified from 444 sightings in 63 h of diving effort. Population estimates varied from 200 to 205 sharks, which are significantly lower than the previous estimate (339–368 sharks) made 20 years ago, suggesting a demographic decline in Southwestern Atlantic populations. Population studies and movement of G. cirratum are essential to inform the implementation of additional protective measures, including the establishment of protected corridors between marine protected areas and the recovery of G. cirratum populations. It is crucial to consider that the distribution of individuals may extend beyond the borders of the non‐take marine protected area. Therefore, the observed decline in the local population could potential indicate a broader decline across the Southwestern Atlantic.

Reasons for the Sex Bias in Osteoarthritis Research: A Review of Preclinical Studies.

Osteoarthritis (OA) is one of the most common degenerative diseases of articular cartilage. During OA, all the elements that contribute to the joint undergo physiological and structural changes that impair the joint function and cause joint pain and stiffness. OA can arise naturally, with the aging population witnessing an increase in diagnoses of this pathology, but the root causes of OA have yet to be identified, and increasing interest is arising towards investigating biological sex as a risk factor. Clinical studies show increased prevalence and worse clinical outcomes for female patients, yet most clinical and preclinical studies have disproportionately focused on male subjects. This review provides a critical overview of preclinical practices in the context of OA, highlighting the underlying need for taking biological sex as both a risk factor and an important component affecting treatment outcome. A unique insight into the possible reasons for female underrepresentation in preclinical studies is offered, including factors such as lack of specific guidelines requiring the analysis of sex as a biological variable (SABV), research-associated costs and animal handling, and wrongful application of the reduction principle. Additionally, a thorough investigation of sex-related variables is provided, stressing how each of them could add valuable information for the understanding of OA pathophysiology, as well as sex-dependent treatment strategies.

Working animal’s welfare in antiquity. An interdisciplinary comparison of ancient horse breeding and training methods, as well as historical equipment, with modern horse welfare research results

Working animals, the method of their breeding, preparation for work and equipment related to their use, are inextricably linked with production and trade around the world from prehistoric times to the present day. Typically, archaeological research focus on analysing the effects of human-animal cooperation, not the breeding and training process itself. The article presents an interdisciplinary approach to the subject, combining the analysis of ancient texts, obtained through excavations, elements of the horse tack, as well as the effects of the work of reenactors with the approach to animals presented by modern animal psychologists and trainers and their research on the welfare of working horses. The first part focuses on the comparison of ancient texts with current guidelines regarding the handling of farm animals and the principles of natural training methods. The second part of the is devoted to the comparison of the construction of Scythian, Roman and Medieval saddles with modern saddles, as well as the results of a meta-analysis of research on the pressure exerted on the horse's back by various types of saddles. The presented analysis was performed using the ANOVA method. The indicators used in it were the pressure force of the saddle on the back, measured in kPa, by the mat system Novel GmbH, Novel Electronic and Pliance-X System, as well as the presence of sweat stains on the back under the saddle. The obtained values were compared with known pain scales in order to identify the saddle exerting the least harmful pressure on the horse's back.

Horse behavior and facial movements in relation to food rewards.

Food rewards are believed to have a positive valence in horses. The aim of this study was to assess the effect of food rewards on horse behavior before entering a horse chute, and behavior and facial movements while restrained in it. Thirteen female adult horses were brought once daily to an animal handling facility for three weeks. In week 1, baseline period, no reinforcement was applied. In weeks 2 and 3, experimental phase, half of the horses received positive reinforcement treatment after entering and remaining in the chute; the remaining horses were considered as controls (no positive reinforcement applied). There was a cross-over between the groups during the experimental phase. Horses were individually brought to the restraining chute and videos recorded during 60-sec. The duration and number of entries into the area close to the gate leading to the chute were measured before restraining and body posture, neck position, and tail swinging were recorded in the chute. Facial movements were also recorded and scored using EquiFACS methodology. Multilevel linear and logistic models were built to assess behavioral changes from baseline to the treatment phase and between phases (control and positively reinforced). Horses did not change their body posture or tail swings across the different phases (P > 0.1) and were less likely to show lowered neck during the positively reinforced phase (OR: 0.05; CI95%: 0.00-0.56; P = 0.05) compared to baseline. The likelihood of a lowered neck did not differ between the positive reinforcement and control phases (P = 0.11). In the positively reinforced phase, horses seemed to be more attentive (ears forward) and active (less eye closures, more nose movements) than in the control phase. A three-day positive reinforcement phase did not elicit major changes in body behavior in the chute but affected the facial movements of group-housed mares.

The Changing Cost of Cattle Transportation

Diesel is one of the primary inputs in the transportation of goods throughout the United States. Livestock hauling is no exception. Feeder cattle are generally transported long distances in the fall as they move from summer grazing to placement in backgrounding operations or directly into feedlots. Cattle, and other livestock in general, can handle transportation stress fairly well – to a point. Trucking feeder cattle long distances can impact animal health outcomes, and improper animal handling can lead to bruised or injured cattle. These negative impacts can generally be offset by appropriate stocking densities, proper trailer ventilation, correct animal handling techniques during loading and unloading, and truck and trailer sanitation.

Relação entre o peso corporal e a circunferência escrotal em ovinos Pelibuey em crescimento criados em condições tropicais

ABSTRACT The body weight (BW) of an animal is a vital economic trait that might help in decision-making in the handling of animals. The objective of the present study was to develop equations for the prediction of BW in Pelibuey sheep using scrotal circumference (SC). The BW (23.40 ± 6.96 kg) and SC (20.25 ± 6.19 cm) have been recorded in 405 male Pelibuey at the Southeastern Center for Ovine Integration, Mexico. Linear, logarithmic, quadratic, exponential, cubic, and power regression models were used for data analysis. Pearson correlation (R), Coefficient of determination (R2), Adjusted coefficient of determination (Adj.R2) Root mean square error (RMSE), Akaike information criterion (AIC), and Bayesian information criterion (BIC) were used to select the best model. Power regression model showed the highest R (0.93), R2 (0.86), Adj.R2 (0.86) and lowest RMSE (0.02), AIC (-989.44) and BIC (-981.44). The current study suggests that SC might be used as the only predictor for BW of growing Pelibuey sheep raised under tropical conditions.

Towards understanding host–pathogen dynamics of cetacean poxvirus: attainable approach through the application of a repetitive non-invasive skin sampling in bottlenose dolphins (Tursiops truncatus) under human care

Nowadays, zoos and aquariums, along with the constant advancement of sociocultural moral values, are proactively committed to ensuring and safeguarding cetacean health standards. This entails developing new approaches to health assessments by embracing minimally invasive sampling methods and enhanced animal handling and management, among other aspects. Hence, in the present survey, to appraise skin diseases, the implementation of cytology cell samplers as a non-invasive skin sampling device on 18 bottlenose dolphins housed in two facilities in the Canary Islands during the months of April, October, and December 2019 was performed to isolate cetacean poxvirus in tattoo-like lesions through a real-time PCR-based method using the DNA polymerase gene. Samples were repeatedly collected over time from eleven tattoo-like lesions and from apparently healthy skin to serve as a control for all study animals. From a total of 55 skin samples, detection of the poxvirus was attained in 31 (56.36%); specifically, on 20 of 21 samples collected from tattoo-like lesions (95.23%) and on 11 of 34 samples acquired from apparently healthy skin (32.35%). Correspondingly, the current study constitutes the first report of the isolation of cetacean poxvirus in skin samples without macroscopical signs of tattoo lesions in cetaceans. Likewise, ten of the eleven dolphins that showed tattoo lesions housed in Facility 1 were positive for tattoo skin disease, while four dolphins held in Facility 2 were positive for cetacean poxvirus without ever showing clinical evidence of the disease. This raises the question of whether this pathogen can produce latent infections and whether progression of the disease may depend on environmental stimuli, viral load, or the good health/immunological status of individual animals. Accordingly, further scientific research on cetaceans under human care could provide the knowledge, skills, and resources to understand the host–pathogen dynamics of cetacean poxviruses and their effect on cetaceans’ health.

Spiral volumetric optoacoustic tomography for imaging whole-body biodynamics in small animals.

Fast tracking of biological dynamics across multiple murine organs using the currently commercially available whole-body preclinical imaging systems is hindered by their limited contrast, sensitivity and spatial or temporal resolution. Spiral volumetric optoacoustic tomography (SVOT) provides optical contrast, with an unprecedented level of spatial and temporal resolution, by rapidly scanning a mouse using spherical arrays, thus overcoming the current limitations in whole-body imaging. The method enables the visualization of deep-seated structures in living mammalian tissues in the near-infrared spectral window, while further providing unrivalled image quality and rich spectroscopic optical contrast. Here, we describe the detailed procedures for SVOT imaging of mice and provide specific details on how to implement a SVOT system, including component selection, system arrangement and alignment, as well as the image processing methods. The step-by-step guide for the rapid panoramic (360°) head-to-tail whole-body imaging of a mouse includes the rapid visualization of contrast agent perfusion and biodistribution. The isotropic spatial resolution possible with SVOT can reach 90 µm in 3D, while alternative steps enable whole-body scans in less than 2 s, unattainable with other preclinical imaging modalities. The method further allows the real-time (100 frames per second) imaging of biodynamics at the whole-organ level. The multiscale imaging capacity provided by SVOT can be used for visualizing rapid biodynamics, monitoring responses to treatments and stimuli, tracking perfusion, and quantifying total body accumulation and clearance dynamics of molecular agents and drugs. Depending on the imaging procedure, the protocol requires 1-2 h to complete by users trained in animal handling and biomedical imaging.

An 8-cage imaging system for automated analyses of mouse behavior

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4–5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer’s disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer’s disease.

Psychological Determinants of Turkish Farmers’ Health and Safety Behaviors: An Application of the Extended Theory of Planned Behavior

Background: Structural and behavioral factors are among the causes of occupational accidents in agriculture. The SACURIMA Cost network developed a questionnaire to measure the determinants of farmers’ safety behavior based on the extended theory of planned behavior (TPB). Extended TPB adds subjective norms, subjective control, physical barriers and safety culture to TPB. Objective: The aim of this study is to test the psychometric properties of the “Farmers Safety Behaviors questionnaire” developed by the SACURIMA Cost network. Methods: A Turkish version of the questionnaire was applied to 305 farmers producing six different agricultural products in Turkey. The tool consists of 64 items measuring the determinants of four risk-related behaviors (fall prevention, machine handling, chemical-pesticide use, and animal handling) in a single extended TPB model. Results: The alpha values for the six dimensions ranged from 0.69 to 0.89. The confirmatory factor analysis results for all dimensions were at acceptable levels (CFI range = 0.93–0.99; RMSEA range = 0.03–0.09). Four path models were used to test the behaviors and their predictors, and the results were found to be predictive. The criterion and known groups’ validity analyses results were sufficient. Conclusion: The “Farmers Safety Behaviors Questionnaire” is a valid and reliable tool to measure the determinants of occupational safety behaviors in Turkish farmers.

Wizard: Unsupervised goats tracking algorithm

Computer vision is an interesting tool for animal behavior monitoring, mainly because it limits animal handling and it can be used to record various traits using only one sensor. From previous studies, this technic has shown to be suitable for various species and behavior. However it remains challenging to collect individual information, i.e. not only to detect animals and behavior on the video frames, but also to identify them. Animal identification is a prerequisite to gather individual information in order to characterize individuals and compare them. A common solution to this problem, known as multiple objects tracking, consists in detecting the animals on each video frame, and then associate detections to a unique animal ID. Association of detections between two consecutive frames are generally made to maintain coherence of the detection locations and appearances. To extract appearance information, a common solution is to use a convolutional neural network (CNN), trained on a large dataset before running the tracking algorithm. For farmed animals, designing such network is challenging as far as large training dataset are still lacking. In this article, we proposed an innovative solution, where the CNN used to extract appearance information is parameterized using offline unsupervised training. The algorithm, named Wizard, was evaluated for the purpose of goats monitoring in outdoor conditions. 17 annotated videos were used, for a total of 4H30, with various number of animals on the video (from 3 to 8) and different level of color differences between animals. First, the ability of the algorithm to track the detected animals was evaluated. When animals were detected, the algorithm found the correct animal ID in 94.82% of the frames. When tracking and detection were evaluated together, we found that Wizard found the correct animal ID in 86.18% of the video length. In situations where the animal detection rate could be high, Wizard seems to be a suitable solution for individual behavior analysis experiments based on computer vision.

Effects of blood meal source on blood consumption and reproductive success of cat fleas, Ctenocephalides felis.

Cat fleas, small blood-feeding ectoparasites that feed on humans and animals, cause discomfort through their bites, and can transmit numerous diseases to animals and humans. Traditionally, fleas have been reared for research on live animals, but this process requires animal handling permits, inflicts discomfort on animals, and requires money and time to maintain the host animals. Although artificial membrane-based feeding systems have been implemented, these methods are not sustainable in the long term because they result in lower blood consumption and egg production than those with rearing on live hosts. To maximize these parameters, we tested blood from four hosts to determine the most suitable blood, on the basis of blood consumption and egg production. We also tested the effects of adding the phagostimulant adenosine-5´-triphosphate to the blood to maximize blood consumption. In 48 hours, fleas fed dog blood consumed the most blood, averaging 9.5 μL per flea, whereas fleas fed on cow, cat, or human blood consumed 8.3 μL, 5.7 μL, or 5.2 μL, respectively. Addition of 0.01 M and 0.1 M adenosine-5´-triphosphate to dog and cow blood did not enhance blood consumption. In a 1-week feeding period, the total egg production was also greatest in fleas fed dog blood, with females producing 129.5 eggs, whereas females on cat, human, and cow blood produced 97.2, 83.0, and 70.7 eggs, respectively. The observed results in dog blood indicate an improvement over previously reported results in cat fleas fed with an artificial feeding system. Improving the sustainability of rearing cat flea colonies without feeding on live animals will enable more humane and convenient production of this pest for scientific research.

Pendampingan dan Evaluasi Pelaksanaan Kurban di Desa Kedung Umpal, Kecamatan Cibinong, Kabupaten Bogor pada Masa Pandemi Covid-19

The purpose of this community service is to provide assistance in the slaughtering and handling of sacrificial animals during the Implementation of Emergency Restrictions on Community Activities (PPKM) against Ministry of Religion regulation No 17 of 2021. This community service uses a qualitative descriptive method obtained from observations, questionnaires and interviews with qurban officers in Kedung Umpal Village, Cibinong Sub District, Bogor Districts, West Java Province The results showed that the slaughter and handling of sacrificial animals in the village of Kedung Umpal Cibinong did not follow the rules issued by the ministry of religion No. 17 of 2021 regarding the procedures for slaughtering sacrificial animals during the emergency PPKM period, including the application of social distancing, health protocols and cleanliness of sacrificial officers and hygiene. tool. Based on these results, it can be concluded that the awareness of the officers handling and slaughtering sacrificial animals during the emergency PPKM period was still not aware of the rules set by the government.

Establishing the Most Important Clinical Skills for New Graduate Veterinarians by Comparing Published Lists with Regional Stakeholder Expectations: A Bangladesh Experience.

Veterinary clinical skills training is evolving rapidly around the world and there is increasing interest in Bangladesh in opening clinical skills laboratories and using models in teaching. The first clinical skills laboratory was opened at Chattogram Veterinary and Animal Sciences University in 2019. The current study aimed to identify the most important clinical skills for veterinarians in Bangladesh to inform the further development of clinical skills laboratories and ensure resources are deployed effectively and efficiently. Lists of clinical skills were collated from the literature, national and international accreditation standards, and regional syllabi. The list was refined through local consultation, focused on farm and pet animals, and was disseminated via an online survey to veterinarians and final-year students who were asked to rate the level of importance of each skill for a new graduate. The survey was completed by 215 veterinarians and 115 students. A ranked list was generated with injection techniques, animal handling, clinical examination, and basic surgical skills among the most important. Some techniques requiring specific equipment and some advanced surgical procedures were considered less important. As a result of the study, the most important clinical skills for a new graduate in Bangladesh have been identified for the first time. The results will inform the development of models, the use of clinical skills laboratories, and the design of clinical skills courses for veterinary training. Our approach of drawing upon existing lists followed by local stakeholders consultation is recommended to others to ensure clinical skills teaching is regionally relevant.

SHIFT OF THE HUMAN LEPTOSPIROSIS RISK FACTORS IN SOUTH ANDAMAN ISLAND, INDIA

Introduction: Leptospirosis is a zoonosis, endemic in south Andaman with 12.9% prevalence rate for rural population. Objective: identify the changes in environmental and behavioral risk factors associated with leptospirosis, which will help in the prevention and control strategies design. Cross-sectional study design with Methods: a universal sampling of laboratory-positive cases of 385 and 360 controls. Multilogistic regression analysis with 21 signicant variabl Results: es revealed, that leptospirosis positivity is signicantly associated with the presence of cats (OR 3.774, P < 0.05, CI 316, 6.151), rodents (OR 2.041, P < 0.05, CI 1.270, 3.282) and cattle's (OR 6.350 P < 0.05, CI 3.789, 10.640) and animal handling, bathing in the pond and working in the eld and open defecation (OR 4.128, P < 0.05, CI 2.270, 7.508). Our results emphasize the importance of creating awareness about leptospi Conclusion: rosis, many identied risk factors can be easily controlled by proper sanitation, proper drainage system and easy access to personal protective equipment

A flow cytometry-based protocol for syngenic isolation of neurovascular unit cells from mouse and human tissues.

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.