Animal models in neuroscience with alternative approaches: Evolutionary, biomedical, and ethical perspectives

Abstract

Animal models have been a crucial tool in neuroscience research for decades, providing insights into the biomedical and evolutionary mechanisms of the nervous system, disease, and behavior. However, their use has raised concerns on several ethical, clinical, and scientific considerations. The welfare of animals and the 3R principles (replacement, reduction, refinement) are the focus of the ethical concerns, targeting the importance of reducing the stress and suffering of these models. Several laws and guidelines are applied and developed to protect animal rights during experimenting. Concurrently, in the clinic and biomedical fields, discussions on the relevance of animal model findings on human organisms have increased. Latest data suggest that in a considerable amount of time the animal model results are not translatable in humans, costing time and money. Alternative methods, such as in vitro (cell culture, microscopy, organoids, and micro physiological systems) techniques and in silico (computational) modeling, have emerged as potential replacements for animal models, providing more accurate data in a minimized cost. By adopting alternative methods and promoting ethical considerations in research practices, we can achieve the 3R goals while upholding our responsibility to both humans and other animals. Our goal is to present a thorough review of animal models used in neuroscience from the biomedical, evolutionary, and ethical perspectives. The novelty of this research lies in integrating diverse points of views to provide an understanding of the advantages and disadvantages of animal models in neuroscience and in discussing potential alternative methods.

Animal models have played a key role in neuroscience research for years. Nevertheless, their usage brings up ethical and transability issues concerning humans. In the recent years, alternative approaches have been developed, containing a potential for replacement and reduction of animal experimenting, associated with more relatable results. This review covers the role the animal models have played so far, the ethical issues, and new replacing technologies.

1. INTRODUCTION

Neuroscience is a field that studies the genetic, molecular, cellular, and physiological processes of the nervous system, including the brain, spinal cord, and peripheral neural system (PNS). ¹ Animal models have been a fundamental tool in neuroscience research for decades. ² The use of animal models in neuroscience dates to the ancient Egyptians, Greeks, and Romans. ³ The first records of animal models in neuroscience included chickens, dogs, fish, pigeons, pigs, and mice, and then, frogs became widely used. ^{4 , 5} In the late 1800s, several invertebrate organisms started being used as model animals in research, such as nematodes, insects, planarians, mollusks, and crustaceans. ⁶ In the 21st century, the most used animal models include rodents, nematodes, fruit flies, zebrafish, guinea pig, ferret, hamsters, chicken, nonhuman primates, and large domesticated animals. ^{2 , 7 , 8} Animal models provide scientists with an approach to understand the mechanism and evolution of various neurological conditions, as well as to test different therapies. ⁹ Some of the widely used animal models in neuroscience include primates (chimpanzees and macaques), rodents (mice and rats), zebrafish, salamanders, and Aplysia. ^{10 , 11 , 12} These models have provided important insight into the nervous system, cognitive processes, neurodegenerative diseases, evolution, and drug development. ^{9 , 10 , 11 , 12} Even though no animal model can entirely recapitulate the human brain in a complete way because of the unique evolutionary history of all species, ¹³ they provide a significant tool for making new discoveries and developing new therapies. ¹⁴ Each model has its own advantages and disadvantages based on the research type. ¹⁵ Moreover, the comparative method and One Health perspective connecting humans with other animals are increasing day by day to understand neurological basis and to solve common neurological diseases in terms of evolution and biomedicine ^{16 , 17} (Figure 1; Table 1).

FIGURE 1.

Animal models in neuroscience within evolutionary, biomedical, and ethical perspectives. The figure was created on BioRender (https://www.biorender.com).

TABLE 1.

Animal models in neuroscience based on evolutionary and biomedical perspectives.

Animal model	Evolutionary perspective	Biomedical perspective	Reference
Chimpanzee (Pan troglodytes)	Human‐specific neurological changes	Nonhuman primate neurodegenerative disease model	18, 19, 20
Bonobo (Pan paniscus)	Human‐specific neurological changes	Nonhuman primate neurodegenerative disease model	18, 19, 21
Rhesus macaque (Macaca mulatta)	Mirror neurons, evolution of behavior and cognition	Neotenic brain development, PD, HD	22, 23, 24, 25, 26, 27
Rat (Rattus norvegicus)	Neuronal architecture, brain evolution, and evolution of neural tube closure patterns	Depression, anxiety disorders, and antidepressant drugs	28, 29, 30, 31, 32
Mouse (Mus musculus)	Neuronal architecture, brain evolution, and evolution of neural tube closure patterns	Humanized chimeric nervous system, HD, PD, Down syndrome, congenital hypomyelination treatment	32, 33, 34, 35, 36, 37, 38, 39, 40
European rabbit (Oryctolagus cuniculus)	Evolution of neural tube closure patterns	MS treatment, AD, neurological effects of vaccines and antiviral drugs in pregnant rabbits, anencephaly, and spina bifida	40, 41, 42, 43
Pig (Sus scrofa)	Evolution of neural tube closure patterns	HD, Neurofibromatosis type 1	40, 44, 45
Dog (Canis lupus familiaris)	Evolution of age‐related changes and brain atrophy	Multiple brain tumors (glioma and leptomeningeal sarcoma), epilepsy, spinal cord injury, stroke, and AD	17, 46, 47
Cat (Felis catus)	Evolution of age‐related changes and brain atrophy	Brain tumors, epilepsy, spinal cord injury, stroke, and AD	17, 46
Zebrafish (Danio rerio)	Vertebrate brain evolution, motor function, cognition	Epilepsy, schizophrenia, intellectual disability, autism spectrum disorders, depression, anxiety	11, 48, 49, 50, 51
Amphioxus (Branchiostoma lanceolatum)	Evolutionary origins of vertebrate brains, frontal brain‐like region	Neural tube and notochord regeneration	52, 53, 54, 55
Fruit fly (Drosophila melanogaster)	Conserved molecular neural pathways	AD, PD, neural tumors, CNS damage, and epilepsy	56, 57
Nematode (Caenorhabditis elegans)	Evolution of neural processes like circuit function, synaptic transmission, and minimal nervous system	Neurodegeneration, autism, and diseases such as PD and AD	58, 59, 60, 61, 62, 63, 64, 65
California sea slug (Aplysia californica)	Learning, short‐term and long‐term memory	AD	66, 67, 68

Abbreviations: AD, Alzheimer's disease; CNS, central nervous system; HD, Huntington's disease; MS, multiple sclerosis; PD, Parkinson's disease.

Despite their contributions and importance in research, the usage of animal models comes together with ethical concerns and specific limitations. Moral standards and laws related to animal testing are topics of ongoing discussion and discord. ⁶⁹ During experiments, model animals experience a poor quality of life, facing physical, mental, and physiological pain and distress. ⁷⁰ Ethically, the scientists are trying to reduce, refine, and replace the animal models (3Rs). Furthermore, the results obtained from animal experimenting are not always translatable in humans. In drug development, Food and Drug Administration (FDA) acknowledges that animal models don't predict the drug safety in humans. ⁷¹ Almost 90% of the drug developments fail, one of the reasons being the failure of getting the same results in humans as in animal models. ^{72 , 73} In behavioral neuroscience, the results from animal experimenting fail to predict the outcomes in humans in more than 90% of the cases. ⁷⁴ In the case of neurodegenerative diseases, even though animal models have helped gain insights in several diseases, their usage in preclinical trials resulted in disappointment. ^{75 , 76} Moreover, the cost of animal research is high and delays the drug development process. ⁷³ These challenges have increased the interest in alternative approaches to animal models.

An increasing number of studies are focusing on alternative approaches contrary to animal model usage, including in vitro (cell culture and microscopy) techniques and in silico (computational) modeling in neuroscience studies ^{21 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99} (Figure 2; Table 2). Advancements in technology have led to the development of innovative strategies such as organoids, microphysiological systems (organs on chips [OoCs]), induced pluripotent stem cells (iPSCs), advanced imaging methods, molecular docking, physiologically based pharmacokinetic (PBPK) models, and quantitative structure–activity relationship (QSAR) models. ^{100 , 101 , 102 , 103 , 104}

FIGURE 2.

Alternative approaches to animal models in neuroscience. The figure was created on BioRender (https://www.biorender.com).

TABLE 2.

Alternative approaches (in vitro and in silico) to animal models in neuroscience.

Approach	Disease model	Cell type	Reference
In vitro (3D cell culture and microscopy) technique In silico (computational) modeling	AD	Microglia, astrocyte Neural cells	81, 91
In vitro (cell culture and microscopy) technique	FTD	Peripheral blood mononuclear cells (PBMCs) Fibroblast cells	86, 87
In vitro (3D, organoid cell culture, and microscopy) technique In silico (computational) modeling	PD	SH‐SY5Y neuroblastoma cell line LUHMES	93, 96, 98
In vitro (cell culture and microscopy) technique In silico (computational) modeling	HD	Specific mutation‐carrying human embryonic stem cells	85, 99
In vitro (cell culture and functional neuroimaging) technique	Schizophrenia	Microglia	79, 95
In vitro (3D, organotypic ex vivo‐based cell culture and microscopy) technique	Peripheral nerve degeneration	Schwann cells Neuronal cells	88
In vitro (organotypic cell culture and microscopy) technique	MS	Human oligodendrocytes or oligodendrocyte precursor cells (OPCs) IPSC‐derived OPCs	21, 83, 89
In vitro (organ‐like 3D coculture system and microscopy) technique	ALS	IPSC‐derived cell lines Immortalized cell line	84, 90, 92
In silico (computational) modeling	Motor control and stroke	Brain and spinal cord neurons	97
In vitro (microfluidic cell culture and microscopy) technique	Axonal response to injury Synaptic formation and function Myelination Neuronal response to chemical inducements	CNS neurons and glial cells	77, 78, 80, 82

Abbreviations: AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; CNS, central nervous system; FTD, frontotemporal dementia; HD, Huntington's disease; MS, multiple sclerosis; PD, Parkinson's disease.

The aim of this study is to summarize the use of animal models in neuroscience, highlighting their importance in evolutionary and biomedical research while also discussing the limitations and ethical concerns associated with these models (Figure 1; Table 1). Furthermore, alternative approaches, including in vitro techniques and in silico methods and the way these innovations are shaping neuroscience research, are also discussed (Figure 2; Table 2).

2. EVOLUTIONARY AND BIOMEDICAL PERSPECTIVES

Animal models have been used over the years in evolutionary studies. ¹⁰⁵ Vertebrates have evolved in more specialized tissues and complex neurological structure, including several nerve branches and a vascular system, which is highly branched, making them suitable models for human neurological conditions. ¹⁰⁶ Also, from a biomedical point of view, model animals have contributed greatly to the explanation and understanding of many mechanisms and development of new therapies (Figure 1; Table 1).

2.1. Mammals

The inability to directly study the origin of the complex brain and the central nervous system (CNS) is one of the biggest issues it faces. For instance, although there is much to be learned about evolutionary patterns and the genetic basis of mechanisms of different phenotypes in a variety of different animals, it is much more challenging and likely impossible to directly study the evolution of complex circuits in mammalian brains. Fortunately, two methods, the comparative method and the developmental method, can be used to determine the kinds of alterations that have taken place in the neocortex over the history of mammalian evolution and how those alterations were accomplished. ²⁸

The comparative approach examines similar features that all brains share, as well as derivations or specializations that have arisen in different mammalian brains because of adaptation to a unique lifestyle and environment, comparing distinct features of the neocortex of select mammals that ideally represent several phylogenetic branches of evolution, rather than just a few species such as monkeys, cats, and mice. ²⁸ The developmental approach studies the developmental mechanisms and their modifications that result in different adult phenotypes. The creation of the body and the brain is influenced by inherent genetic and activity‐dependent mechanisms, and changes to one or both cause species differences, according to studies of development. ²⁸

2.1.1. Nonhuman primates

Because of their close phylogenetic relations with human and nonhuman primates, studies yield the most promising results that can be translated to humans. Similarities in the brain organization, social complexity, and involved cognitive abilities make this animal model very valuable in neuroscience research. ²⁹ The nonhuman primate model in brain research was apparent in 1981 Nobel Prize in Physiology or Medicine, when Wiesel and Hubel were awarded for their findings in the information processing by the visual system. ^{30 , 107} It was the macaque parkinsonian model that led to the discovery that brain stimulation of subthalamic brain structure was an effective therapy for the disease, improving the lives of 250 000 Parkinson's patients all over the world. ²² Rhesus macaque (Macaca mulatta) was also used in neuroscience studies in terms of evolution of human brain, behavior, and cognition in addition to mirror neurons discovered for the first time in this animal model species. ^{23 , 24 , 25} Among the nonhuman primate animal models, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) are the closest great ape relatives of humans, and they are the most important animal models to understand human‐specific neurological/genetic changes (cortex expansion, behavior, cognition, and complex language) and human neurodegenerative diseases (Alzheimer's disease [AD], Parkinson's disease [PD], Huntington's disease [HD], and dementia) ^{18 , 19} (Table 1).

Latest advances that come from monkey research include new therapies for stroke patients, improved understanding of the healthy brain cognitive functions and symptoms associated with psychiatric disorders, and the practice of brain–machine interfaces for reestablishing movement in paralyzed patients. ¹⁰⁸ Comparative genomic data resulted in human lineage‐specific (HLS) sequence identification. ³¹ Because apes are humans' closest evolutionary relatives, the transgenic introduction of HLS in them contains a great potential to produce human phenotypes. For example, the transgenic rhesus monkeys that carry the MCPH1 human gene variant showed human‐like neotenic brain development, leading to short reaction time and improved short‐term memory compared to the wild type. ²⁶ Another example is the transgenic rhesus macaque that developed HD, opening a way to better understanding of the disease and the development of new therapies. ²⁷ Such studies demonstrate the potential of transgenic nonhuman primates to provide important understandings in human neurodegenerative, cognitive, and social behavior disorders.

2.1.2. Rodents

In recent years, 18%–20% of mammalian nervous system research has relied on rat (Rattus norvegicus) and mouse (Mus musculus) animal models, respectively, and rats and mice have been popular mammalian experimental models in various neuroscience research studies. ³³ They have been used to understand neuronal architecture and brain evolution in neuroscience studies. ³⁴ Nowadays, more than 200 000 mutant mouse strains can be generated among over 24 000 existing strains and over 209 000 altered mouse embryonic stem cells. ³⁵ Furthermore, rat strains are increasing, making rodents a remarkable genetic research model in biomedical research. ³⁵ Other advantages include their short life cycle, size, and maintenance simplicity. A difference has also been the generation of mouse–human chimaeras, which defines a tissue or organism that originated from at least two genetically diverse populations derived from different zygotes. ¹⁰⁹ An example is the creation of a humanized mouse chimeric nervous system that allows the study of human neural growth and the pathogenesis of several diseases. ³² Chimeric mouse models have also been used to study HD, congenital hypomyelination treatment, AD, and Down syndrome ^{36 , 37 , 38 , 39} (Table 1).

Several aspects of PD, such as sleep disorders, behavior, and motor disability, can be modeled in rodents. ^{110 , 111} Depression, anxiety disorders, and antidepressant drugs have been studied in rats. ¹¹² Nonrodent animal models, such as SOD1 mutations and aged nonhuman primates, have been studied for AD, but have not been effective, making rodents an important model. ⁷⁶ Nevertheless, preclinical research should be more rigorous, with acceptable replication and group sizes, an assessment of sex effects, and, if necessary, testing in several disease‐relevant models. ⁷⁶

2.1.3. Other mammals

Rabbits (Oryctolagus cuniculus) can also be used in preclinical studies to identify fetal diseases in several parts of the brain after maternal exposure to different viruses and to identify active fetal viral infections after exposure during pregnancy. ⁴¹ This model can be used for testing chemical substances for multiple sclerosis (MS) treatment, vaccines and antiviral drugs in pregnant rabbits and their probable effects on the neurological condition of embryos or fetuses, and the valuation of the effects of maternal anesthesia or surgery during pregnancy. ⁴¹ In addition, rabbits are viewed as an ideal model for the development of maternal vaccines due to the embryological similarities with humans in the development of the CNS. ¹¹³ Rabbits, having the amyloid amino acid sequence same as humans, present a nontransgenic model for AD study. ^{42 , 43} Furthermore, in human and rabbit embryos, the axial curvature increases throughout development, whereas the rate of neural tube closure reduces, and the parallels between these events could be used to examine illnesses like anencephaly and spina bifida that result from the failure of embryonic neural tube closure. ^{40 , 114} Moreover, from a comparative approach, evolution of neural tube closure patterns in rabbits was investigated in addition to various mammalian species, including rat, mouse, and pig ⁴⁰ (Table 1).

Pigs (Sus scrofa) are another mammalian animal models with both evolutionary (neural tube closure patterns) and biomedical (HD, neurofibromatosis type 1) perspectives in neuroscience studies with anatomical, physiological, and developmental similarities to humans. ^{40 , 44 , 45} Cats (Felis catus) and dogs (Canis lupus familiaris) are remarkable companion animal models in neuroscience studies because of their bridge position between rodents and humans, and several human neurological diseases have also been reported in these companion animals, including brain tumors, epilepsy, spinal cord injury, stroke, and AD. ^{17 , 46} Moreover, there are evolutionary studies in neuroscience, including age‐related changes and brain atrophy in cats and dogs ¹⁷ (Table 1).

2.2. Nonmammalian vertebrates

Zebrafish (Danio rerio) has become a widely used animal model in neuroscience because its transparent embryos and larvae allow for real‐time visualization, making it easy to breed and cost‐efficient. It is mostly used in brain function and disorder studies, behavior, drug‐induced conditions, and drug responses. ¹¹ Zebrafish are used as a model organism for several neurobehavioral disorders, such as depression, ⁴⁸ anxiety, ⁴⁹ and neurodevelopmental disorders, such as epilepsy, schizophrenia, intellectual disability, and autism spectrum disorders. ⁵⁰ Furthermore, behavior, motor functions during development, and cognition can be studied using zebrafish as an experimental model. ⁵⁰ Most importantly, zebrafish share with mammals the three basic divisions of a vertebrate brain: the forebrain, midbrain, and hindbrain. ⁵¹ Zebrafish models in translational neuroscience have also gained a lot of importance because of their translational relevance to humans ¹¹⁵ (Table 1).

2.3. Invertebrates

Comparisons among the vertebrate CNS and their invertebrate chordate relatives, such as tunicates and cephalochordates (amphioxus), give an understanding of the evolution of the vertebrate CNS. Nonetheless, there is still no consensus on the evolution of the chordate CNS. ^{116 , 117} The cephalochordate amphioxus remains to be a focus for the argumentative evolutionary origins of vertebrates after two centuries of discussion, with its phylogenetic position that makes amphioxus privileged to shed light on some of the key transitions of animal evolution, such as the origin of deuterostomes, the origin of chordates, or the origin of vertebrates. ⁵² Gene expression patterns demonstrate that the frontal brain‐like region of the amphioxus nerve cord is locally subdivided in a similar way as the vertebrate brain. ^{53 , 54} Additionally, European amphioxus (Branchiostoma lanceolatum) has been used in regeneration studies, as its adults can regenerate structures like the neural tube and notochord. ⁵⁵ Comprehension of the evolution of the regenerative capacity in Metazoa depends on labeling the developmental and molecular foundations of organ regeneration in basal chordates, including amphioxus ⁵⁵ (Table 1).

Neuronal evolution research has depended on several animal models, and the first theory on neuron evolution was studied using Hydra as a model organism. ¹¹⁸ Other studies and theories followed, using models such as medusas, ¹¹⁹ sponge, and coelenterates (cnidarians and ctenophores). ¹²⁰ Gastropod mollusks, Lymnae, Helix, Aplysia, Clione, and Tritonia, include the most used animals during the 20th century. ¹²¹ Molecular mechanisms and gene identification studies used mostly Drosophila as a model. ¹²² Caenorhabditis elegans, Aplysia, and Drosophila have been used in discovering the molecular and cellular aspects of learning and memory. ¹²³ Significantly, fundamental memory attainment mechanisms of invertebrates seem to be organized in analogous principles as those of vertebrates. ¹²⁴

The fruit fly (Drosophila melanogaster) model is used for the study of molecular and cellular mechanisms related to different human disorders, mostly AD and PD, neural tumors, CNS damage, and epilepsy. ⁵⁶ Anatomically the difference between fruit flies and humans is evident, but the essential molecular neural pathways are still conserved. ⁵⁷ Nevertheless, conditions such as brain hemorrhage and brain infarcts cannot be studied in Drosophila because of the lack of vessels and the mostly primitive hemocytes made up of blood. ⁵⁶ However, using targeted gene expression, human proteins can be expressed in Drosophila animal models ¹²⁵ (Table 1).

The nematode (C. elegans) is used for neural processes like circuit function, synaptic transmission, autism, and diseases such as PD and AD. ^{58 , 59 , 60 , 61 , 62 , 63} C. elegans offers advantages such as short lifespan, sequenced genome, and a minimal nervous system, making it an ideal organism to study aging, deoxyribonucleic acid (DNA) repair, and neurodegeneration, as well as to study potential interventions to prevent or delay it. ^{64 , 65} It has a nervous system of around 300 neurons and a well‐defined synaptic connections network ^{126 , 127} (Table 1).

The California sea slug (Aplysia californica) is the popular experimental animal in neuroscience studied by Eric Kandel, who was awarded the Nobel Prize for Physiology or Medicine in 2000 for discovering the link between the brain and the environment in terms of learning short‐term and long‐term memory formations at the molecular level. ^{66 , 67} Aplysia offers a good system to study the molecular, physiological, and behavioral aspects of AD, with 898 potential protein orthologs to humans. ⁶⁸ Furthermore, Aplysia provides a neural model for the behavior and learning research in the neural circuit, molecular, cellular, and organism level. ^{128 , 129 , 130} The aging effects in the sensory neurons' transcriptome can also be studied at a molecular level in this model animal ^{131 , 132} (Table 1).

3. ETHICAL PERSPECTIVE

Animals have been used for centuries in scientific research. The main aim of animal experiments was to improve animal and human health by understanding the mechanisms and sources of several diseases and disorders, developing and testing new therapies, treatments, surgical interventions, and medical appliances. ⁴ Together with their big contribution to science, nevertheless, the use of animals in research raises concerns when it comes to the ethical implications in terms of animal welfare ¹³³ .

Concerns about human and animal use in research were raised during the late 1800s and the start of the 20th century when medical research greatly expanded. ^{134 , 135} With the expose of numerous unethical research endeavors, such as the Tuskegee syphilis study and several medical tests performed on numerous prisoners during World War II, distrusts concerning the use of humans in research increased, and it was due to these violations, the Nuremberg Code, National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research (1974), and the ensuing Belmont Report were founded. ¹³⁶ Today, these rules serve as a groundwork for the safety of human research subjects.

Ethical issues on animal experiments were initiated in 1959, with the 3Rs' principle of animal use. ¹⁶ Government legislation, moral stands, and public opinions for a long time accompanied animal model usage. Government regulations constrain the researchers from causing pain, injury, or suffering during their animal model experiments. ¹³⁷ Each country has its own laws and guidelines for animal experimenting. In the United States of America, the Laboratory Animal Welfare Act was the first federal regulation related to animal research. ¹³⁸ The Guide for the Care and Use of Laboratory Animals must be followed by the scientists using animal models, and each research center must include an Institutional Animal Care and Use Committee (IACUC) and report its The Association for Assessment and Acreditation of Laboratory Animal Care (AAALAC) International accreditation. ¹³⁸ Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy) is another guideline related to animal research. ¹³⁸ In the United Kingdom, the most significant legislation for animal experimenting is Animals (Scientific Procedures) Act 1986 (ASPA). The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) is an organization that promotes the 3Rs. ^{138 , 139} The European Union legislation for animal welfare includes the Directive 2010/63/EU of the European Parliament and the Council. ¹⁴⁰ Other institutions and guidelines related to animal welfare and 3Rs over the world include Canadian Council on Animal Care (CCAC), the Australian Code for the Care and Use of Animals for Scientific Purposes, the Chinese National Guidelines (GB/T 35892‐20181) for the Ethical Review of Laboratory Animal Welfare, the Japanese Law for the Humane Treatment and Management of Animals. ^{141 , 142 , 143 , 144}

Studies have confirmed that animals experience pain and distress ¹⁴⁵ ; thus even in gentle handling, they show hormonal stress markers and physiological changes. ¹⁴⁶ Furthermore, animals exhibit emotional states and pain responses like those of humans. ¹⁴⁷ Although the use of animal models played a significant role in numerous research studies, several data show that the results from animal experimentation did not relate or predict the human outcomes in clinical research. ^{148 , 149} This raised questions whether human diseases can be sufficiently mimicked in animals. Promising nonanimal methods are being developed, including the field of neurology. ¹⁵⁰ A significant turning point in the toxicology field occurred in 2007, when the U.S. National Research Council highlighted the necessity of in vitro and in silico (computational) methods to obtain more accurate data in the prediction of toxic effects in humans. ¹⁵¹

Legislative, scientific, and ethical priorities are driving the replacement of animal testing by practices such as in vitro cell and tissue research, volunteer studies, physicochemical approaches, and computer modeling. Nonanimal methods are presently regarded as cutting‐edge ways that can address many of the problems of animal experiments. In vitro assays have avoided the use of a vast number of animals in testing drugs and chemicals. Research on neurological, reproductive, and dental issues has replaced certain animal models. ¹⁵⁰ To address significant areas of medical research and development, many researchers have started to exclusively use cell and tissue experiments, as well as human data. For example, to predict the immune response to a certain medicine or vaccination, an in vitro human immune system has been created in the field of vaccine testing and development. ^{152 , 153}

There is constant discussion and debate about the moral principles and laws governing animal experimentation. ⁶⁹ Alternative methods that replace animal models can make it necessary to reexamine the rules and laws governing research, notwithstanding their great impact on science. The relevance of the research question and the viability of replacing animals with current methods from molecular and cell biology, genetics, biochemistry, and computational biology must be evaluated by researchers and oversight boards. Although none of these tools can reproduce an entire organism on their own, they do propose a mechanistic understanding of molecular activities. It is critical for researchers and reviewers to consider the anatomical, physiological, and genetic variations that may affect the applicability of findings, as well as changes in the clinical presentation and expression of diseases between species. ¹⁵⁴

4. ALTERNATIVE APPROACHES TO ANIMAL MODELS

Alternative approaches, such as in vitro and in silico techniques, have potential applications in neuroscience studies today and in the future, and they may eventually replace animal models ^{21 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99} (Figure 2; Table 2). In vitro techniques include two‐dimensional (2D) and 3D cell culture models, which can be used for cell‐based assays and high‐throughput screening (HTS), organoids (3D structure with different cell types), microphysiological systems (organs on chips [OoCs]), and advanced imaging techniques. ⁷¹ Up to date, the 2D cell culturing methods have been the most used because of their low cost and simplicity; however, they do not accurately reflect the fundamental physiology of real tissues. ¹⁵⁵ Controversially, 3D models can be adjusted to mimic the in vivo cell environment, leading to more accurate data on several diseases, drug discovery, stem cells research, metabolic profiling, and cell‐to‐cell interactions. ¹⁵⁶ Furthermore, the 3D models provide the possibility to study the organ dynamics using organoids, closing the gap among 2D cell culture techniques and animal models. ¹⁵⁶ For example, brain organoids recapitulate the tissue construction, the cell variety, and maturation, reducing the gap with in vivo features. ¹⁵⁷ OoCs is a new and innovative method in biomedical research. Brain‐on‐chip models can reproduce the essential functions of the organ in both the normal and pathophysiological settings. ¹⁵⁸ Other OoCs used in neuroscience include the neurovascular unit, blood–brain barrier, and nerve signal transduction chips. ¹⁵⁹

These methods have several advantages compared to animal models. In behavioral neuroscience, the results from animal experimenting fail to predict the outcomes in humans more than 90% of the cases. ⁷⁴ Animal models have weakened immune systems and lack the stroma–tumor interactions like humans. ¹⁶⁰ Finding concordance among animal models and clinical cancer trials is still difficult with the average percentage of concordant results being <8%. ^{161 , 162} Moreover, the cost of animal research is high and delays the drug development process. ⁷³ 3D models eliminate these problems making possible direct human testing. ¹⁵⁶ In the case of neurodegenerative diseases, even though animal models have helped gain insights in several diseases, their usage in preclinical trials resulted in failure. ^{75 , 76} Similarly, for HD, researchers used cell culture techniques in specific mutation‐carrying human embryonic stem cells and in silico (computational) modeling approaches. ^{85 , 99} In Schizophrenia, cell culture techniques targeting microglial cells and microscopy (functional neuroimaging) were applied to reveal the disease pathology. ^{79 , 95} Moreover, cell culture (3D and organotypic/organ‐like coculture) and microscopy techniques were also used for neurological diseases, such as peripheral nerve degeneration, MS, and amyotrophic lateral sclerosis (ALS). ^{21 , 83 , 84 , 88 , 89 , 90 , 92} In vitro cell culture (microfluidic) and microscopy techniques were also applied for CNS neurons and glial cells to understand several situations, including axonal response to injury, synaptic formation and function, myelination, and neuronal response to chemical inducements. ^{77 , 78 , 80 , 82} Neural organoids have been generated from human pluripotent stem cells to mimic the developing brain, and when combined with machine learning they can be used to predict neural toxicity, promising potential use in drug and chemical safety testing. ¹⁶³ Furthermore, organoids have successfully used for creating models such as lissencephaly, Miller–Dieker syndrome, ¹⁶⁴ microcephaly, and cerebral cortex. ¹⁶⁵ Human induced pluripotent stem cells (hiPSC) and embryonic stem cells (ESC) have been used to construct the human blood–brain barrier. ¹⁶⁶ hiPSC has also been used to study genetic diseases such as AD ¹⁶⁷ and Rett syndrome. ¹⁶⁸

In silico (computational) methods include molecular docking, QSAR models, and physiologically based pharmacokinetic (PBPK) models. ^{102 , 169 , 170} In silico methods have also been used in several evolutionary studies such as phylogenetic and structural analysis ^{171 , 172} and positive selection studies. ¹⁷³ In neuroscience, in silico methods have been used to study the evolution of neural networks linked to language. ¹⁷⁴ In a study, only in silico (computational) modeling approach was used to model brain and spinal cord neurons in motor control and stroke. ⁹⁷ The development of advanced statistical techniques and integrated experimentation design has reduced the use of animal models; for example, live embryos can now be replaced by in vitro embryonic stem cell testing. ¹⁷⁵

Nevertheless, the usage and applications of these alternative methods are associated with their own challenges. Organoid technology is associated with challenges such as expanding the cellular heterogeneity, simulating the micro‐ and matrix‐environment that cells encounter, creating reliable protocols that the in vitro organoid maturation remains fetal‐like. ¹⁷⁶ Furthermore, challenges associated to them include the absence of vascular system; for example as the organoid grows to a particular size, it becomes difficult for the cells in the center to get nutrition or excrete the metabolic waste. ¹⁷⁷ Other issues include the hydrogel biocompatibility and cost and creating the immune microenvironment of the human organism. ¹⁷⁷ Organoid use is associated with several ethical concerns, such as the stem cell source, cell donors' consent and privacy, moral and legal organoid status, the potential of attaining the human characteristics, gene editing usage, chimera creation, transplantation, patentability, commercialization, and storage. ¹⁷⁸ Specific guidelines and regulation may be needed to monitor the development in this field. ¹⁷⁸ In silico methods are associated with challenges such as an abundance of data, particularly important in the field of personalized medicine, and the need for developing biological systems' computational models (executable biology) to replicate the biological phenomena. ^{179 , 180} Furthermore, the deficiencies in standardized evaluation metrics, development of biomedical condition‐specific extraction methods, feature selection, interpretability, and the combination of several computational methods can impact performance. ¹⁸¹

Alternative methods have many benefits, such as lower cost, less time requirements, and less complex experimental procedure. ¹⁸² Adapting alternative methods to replace the animal models is associated with its own scientific and ethical challenges. Several researchers believe that development of alternative methods is still in its initial phase, and therefore they cannot utterly replace the animal models in preclinical research yet. ¹⁸³ At the moment, a limited number of these methods have been approved by the federal authorities, and extra funding will be required for the development and testing of these new methods. ¹⁸³

5. CONCLUSION

The advantages and disadvantages of animal models in neuroscience were discussed in terms of evolutionary and biomedical studies, and it was revealed that animal welfare and ethics should also be considered. In addition, in vitro (such as cell culture and microscopy) and in silico (computational) approaches have emerged as potential alternatives to animal models. By adopting alternative approaches and promoting ethical considerations in research practice, we can achieve these goals while maintaining our responsibility toward both humans and other animals. Furthermore, when designing an animal model study, we propose that evaluating in vitro and in silico analyses, and the using these alternative models, should be prerequisites for obtaining ethical approval in the future.

6. FUTURE DIRECTIONS

The intention is to increase the application of alternative methods in neuroscience research and improve animal welfare. The relevance of in vitro models for studying intricate cellular interactions and disease mechanisms can increase with further breakthroughs in this field, such as the creation of 3D cultures, organoids, and organ‐on‐a‐chip systems. Genetic engineering methods and patient‐derived cells could increase the usefulness of these models for medication screening and personalized therapy. Improved computational modeling methods, like machine learning algorithms and network‐based studies, will make it possible to forecast disease progression, treatment outcomes, and biomarker discovery with more accuracy. To leverage big data and convert computational discoveries into therapeutic applications, collaborations between neuroscientists, physicians, and computational biologists will be essential.

AUTHOR CONTRIBUTIONS

Sabina Neziri: Conceptualization; formal analysis; investigation; methodology; writing – original draft. Ahmet Efe Köseoğlu: Conceptualization; formal analysis; investigation; methodology; supervision; writing – original draft; writing – review and editing. Gülsüm Deniz Köseoğlu: Formal analysis; investigation; writing – original draft. Buminhan Özgültekin: Formal analysis; investigation; writing – original draft. Nehir Özdemir Özgentürk: Supervision; writing – review and editing.

FUNDING INFORMATION

This study was not funded.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

No animal models were used in this study.

Neziri S, Köseoğlu AE, Deniz Köseoğlu G, Özgültekin B, Özgentürk NÖ. Animal models in neuroscience with alternative approaches: Evolutionary, biomedical, and ethical perspectives. Anim Models Exp Med. 2024;7:868‐880. doi: 10.1002/ame2.12487