There has always been a debate among the researchers about the significance of animal models, as many experiments yield promising results, whereas, others couldn’t produce desired outcomes, so, that model could be translated to humans too. Owing to their close phylogenetic closeness to humans, non-human primates are proved to be the most potential candidate. They have genetic, biochemical, and psychological activities similar to humans. In this context, the necessity of non-human primates continues to grow in several areas of research of human diseases viz. AIDS, Parkinson’s disease, hepatitis, dentistry, orthopaedic surgical techniques, cardiovascular surgeries, psychological disorders, toxicological studies, drug development, toxicological studies as well as vaccine development [4]. The discovery of vaccines and diagnostic modalities with the animal model does not only benefit humans but also enhances the lifespan of animals and prevents many zoonotic diseases, with the production of many vaccines and drugs like rabies, tetanus, parvo virus, feline leukemia, etc (Table 1).
Table 1 Significance and challenges of different animal modelsFull size table
Animal research adheres to a few dimensions like government legislation, public opinion, moral stand, and search for appropriate alternatives for the research. Mahatma Gandhi opined that to judge the greatness and moral progress of a nation, one should judge the way its animals are being treated. Government legislation restricts the researchers and institutes from likely injury, pain, or suffering that may arise during animal research [33]. On the contrary, many modern countries ruled that before human administration, vaccine testing, lethal dose testing should be done on animals [34]. Social acceptance has also an influential role in animal experiments as it utilizes public money [33]. In their moral view, many people think that dog has more moral impact than pig, rat, fishes, mouse, etc.
Ethical issues on animal experimentation started in 1959, where the emphasis has been given on principles of 3Rs, reduction, refinement, and replacement of animal use [35]. According to this principle, minimum necessary numbers of animals are to be used for scientific experiments i.e. reduction. Pain or distress of the animals during experiments has to be minimized, i.e. refinement. Wherever applicable replacements of the animals are to be done with other non-animal alternatives, i.e. replacement. Though these principles are considered as the cornerstone of animal experimentations, but there are questions regarding the implementation of these regulations [36].
The importance of rat and mouse models has proved their outstanding importance in biomedical research. Besides, other mammalian and non-mammalian small domestic animals like the guinea pig, hamster, rabbit, ferrets, birds, amphibians, fishes, flies, worms have equal importance in terms of anatomical and physiological resemblance with humans. Large animal models also proved their uniqueness due to specific anatomical and physiological characteristics pertinent to those specific researches (Table 2).
Table 2 Biomedical significances and limitations of small animal modelsFull size table
The gene rule and role in the biological system of human diseases has improved many folds with the introduction of the transgenic animal model in biomedical research within the last three decades. The early example of most unique biological research started, when structural gene coding for the human growth hormone (GH) was initiated into mice after fusion with the regulatory region of mouse metallothionein-I gene, as a result, transgenic mouse produced and showed excess GH production [157].
Linking of the genotype with disease phenotype has been expedited with the genome editing with the introduction of the CRISPR–Cas9 system by which disease-causing mutations are done in animal models [158]. Moreover, the production of transgenic animals has been radically changed by the introduction of the CRISPR–Cas9 system. Through the successful use of this model accurate human disease models in animals have been produced and possible therapies have been potentiated. Recapitulation of various disease-causing single nucleotide polymorphisms (SNPs) in animal models is achieved by the introduction of gRNA with the combination of Cas9 and donor template DNA [159], viz. mouse model has enormous importance in carrying human genetic traits, developmental similarities as well as disease translation [158, 160,161,162]. Zhang and Sharp labs at MIT/Broad Institute used CRISPR–Cas9 through AAV and lentivirus [163] both in vivo and ex vivo in neurons as well as endothelial cells of mice for the production of lung cancer model in mice where lung causing genes namely Kras, Tp53, and Lkb1 were mutated. On the other hand, an MIT-Harvard team [164] disrupted the tumor suppressor genes Pten and Tp53, and consequently liver cancer was produced in mice.
In recent advancements, animal models are the most practical tools for pre-clinical drug screening before application into clinical trials. Animal models are considered as most important in vivo models in terms of basic pharmacokinetic parameters like drug efficiency, safety, toxicological studies, as these pre-clinical data are required before translating into humans. Toxicological tests are performed on a large number of animals like general toxicity, mutagenicity, carcinogenicity, and teratogenicity and to evaluate whether the drugs are irritant to eyes and skin. In most instances, both in vitro and in vivo models are corroborated before proceeding to medical trials. In vivo models are mostly conducted in mice, rats, and rabbits [2]. Certain stages are involved in pre-clinical trials with animal models: firstly, if the trial drug shows desirable efficacy then only further studies are carried out; secondly, if a drug in pre-clinical trials on animals proved to be safe, then it is administered in small human volunteer groups, at the same time, the animal trial will go on to evaluate the effect of the drug when administered for an extended period [8, 165]. Mostly, rodents are used for these trials as they have similar biological properties to humans and are easy to handle and rear in laboratories. In new regulations, it is mandatory to carry on the trials on non-rodents such as rabbits, dogs, cats, or primates simultaneously with rodents [166].
There are many conditions involving bone pathologies such as osteomyelitis, osteosarcoma, osteoporosis, etc. Being a complex organ, the treatment of bone needs special care and extensive researches that involves specialized techniques as well as specific animal models for the studies of specific diseases. Herein, the animal models emphasize mostly related to fracture healing (critical size defect), osteoporosis, osteomyelitis, and osteosarcoma (Table 3).
Table 3 Different animal models in orthopaedic researchFull size table
Type 2 diabetes and associated foot ulcer have turned into an epidemic worldwide in recent years causing severe socio-economic trouble to the patients as well as the health care system of the nation as a whole [208]. Various researches depicted that chance of developing an ulcer in diabetic patients varies between 15–25% [209, 210] and the chance of recurrence is about 20–58% among the patients within a year after recovery [211]. Hence, many researchers studied different materials or drugs to treat diabetic wounds. Similarly, burn wounds occur due to exposure to flames, hot surfaces, liquids, chemicals, or even cold exposure [212]. Though with the recent modalities like skin grafting prognosis has improved however, the mortality rate is high [213,214,215].
For developing this model, clinically healthy male Wistar rats (150 ~ 250 g body weight) are used. To induce hyperglycemia, injection nicotinamide (NAD)@ 150 mg/kg BW intraperitoneally, after 15 min injection Streptozotocin (STZ) @ 65 mg/kg BW intraperitoneally [216] are to be injected. The same procedure has to be repeated after 24 h. Blood is to be collected from the tail after 72 h to check hyperglycemia. Rats having high blood glucose levels (≥ 10 mmol/L) are considered to be diabetic [217]. For wound creation, rats are to be anesthetized with a combination of xylazine @10 mg/kg (intramuscular injection) and ketamine @90 mg/kg (intramuscular injection) [218]. After marking the dorsal back area with methylene blue, the site is to be prepared aseptically after shaving [219]. Full-thickness wound creation is to be done with a sterile 6 mm biopsy punch measuring 6 mm diameter and 2 mm depth and left open [218] (Fig. 1c).
Fig. 1a. Bone defect model and implantation of implant b. Vascular graft mode c. Diabetic wound model d. Osteomyelitis model development e. Creation of burn wound model f. Cartilage graft model—All in rabbit
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Because of the severity and types of cause, the management of burn injuries poses a significant challenge to plastic surgeons in humans. In general, primary and secondary burn wounds heal by the primary healing process, but, third-degree burn injuries with the destruction of all the skin layers are resistant to the normal healing process and necessitate the added surgical procedures, such as skin grafting, and the relevance of advanced wound dressing [220]. Several researchers used the albino Winstar male rats (Rattus norvegicus) model weighing 250 ± 50 g for the study of burn wounds. Anesthesia was achieved with intramuscular administration of atropine sulfate (0.04 mg/kg BW) and after 10 min a combination of 10% ketamine (90 mg/kg) and 2% xylazine (10 mg/kg) intramuscularly produced adequate anesthesia [221]. After aseptic preparation of the dorsal back area, thermal injury has to be made with a 10 mm aluminium rod previously heated with 100 °C boiling water. The aluminium rod has to be kept in situ for 15 s. Immediately after the procedure analgesic is to be provided and to be continued for at least 3 days [222,223,224]. A hot air blower has been used to produce a 6% third-degree burn injury in a mouse model [225]. In pig, a partial-thickness burn model in the skin was produced by placing a glass bottle having heated water at 92 °C for 14 s [226] In other studies, a homemade heating device was placed over the skin for 35 s to create burn wound [227]. In rabbits, it was demonstrated to use a dry-heated brass rod for 10 and 20 s at 90 °C to create a deep partial-thickness burn wound in the ear [228]. In mice, a full-thickness burn was created under 3–5% isoflurane anesthesia and intraperitoneal caprofen 5 mg/kg as analgesia. Here, a 4 cm2 brass rod attached to a temperature probe was first heated to 260 °C and then cool to 230 °C and finally placed on the dorsum skin for 9 s [229] (Fig. 1e).
Animal models have enormous importance in the study of cartilage repair. Though in vitro models have been reported, it could not replace the necessity of using animal models prior to clinical implementation [230,231,232,233,234,235,236] (Table 4).
Table 4 Different animal models for cartilage rejuvenation or repairFull size table
With the increase of cardiovascular complications, there is a need for surgical intervention using vascular grafts. Vascular grafting and cardiac valve repair have become important issues to the clinicians for the replacement of damaged vessels [249, 250], hence there is an increased demand for tissue-engineered blood vessel substitute [250, 251]. The main prosthetic options are synthetic grafts such as polytetrafluoroethylene, polyethylene terephthalate, and polyurethane [252], and autologous conduits. Although these types of synthetic grafts provide reasonable outcomes in large-diameter vascular applications, long-term patency is questionable as compared to autologous conduits in small-diameter (< 6 mm) applications due to their inclination to various complications [253]. Despite the superior outcome of autologous grafts, it has some disadvantages such as limited availability and prior use. Moreover, the determination of a suitable animal model needs considerations of various factors. The factors for the selection of animal species depend on diameter and length of conduits, period of implantation, anastomotic site, price, accessibility, reaction to anesthesia and surgery, and flow of blood at sites of graft implantation. Animal applications of these tissue-engineered vessels are, therefore, an utmost necessity as pre-clinical studies before use in humans (Fig. 1b, Table 5).
Table 5 In vivo animal studies of different vascular graftsFull size table
Intervertebral disc degeneration (IVDD) and herniation manifested as lower back pain cause a massive socio-economic burden to the patient and society as a whole [264,265,266,267]. But there is a lack of treatment modalities to cure mildly to moderate degeneration as well as complications associated with surgical interventions associated with the advanced stage; hence, researchers are enormously trying to reinforce regenerative strategies and to lower the suffering by controlling the pain with the injection of stem cells, growth factors hydrogels for replacement of the disc [268]. Diverse animal models have been reported as a pre-clinical trial to translate the procedure in humans (Table 6).
Table 6 Different animal models for the study of IVDDFull size table
Another important aspect of using animals is that they helped understand the transmission of the virus to other domestic species and showed that pets could acquire the SARS-CoV-2 virus through contact with an infected human. However, there is no evidence of active pet-to-human transmission [ 54 ]. Studies with dogs, pigs, chickens, and ducks showed they were not susceptible to COVID-19 infection due to low viral replication [ 55 ]. Identifying susceptible species made it possible to choose appropriate models for developing and testing vaccines [ 55 ]. Ferrets, Syrian hamsters, rabbits, transgenic mice [ 47 ], and cats were all found to be susceptible, the latter even vulnerable to airborne transmission with the development of clinical signs such as hair loss and pulmonary alterations similar to those seen in humans [ 56 57 ]. Apart from domestic cats, wild felines (tigers, lions, pumas, snow leopards) [ 58 ] have been reported to show infections by this virus. Kang et al. [ 59 ], who reported the first Delta variant (SARS-CoV-2 Delta) case in three domestic cats with COVID-19-positive owners in China, insist that transmission to pets is a topic of concern due to their possible role as silent intermediate hosts.
The reinfection processes prevalent in human populations were replicated in studies with. Infection in six animals caused signs such as fever (50%), hypercapnia (66%), 2–7-fold increases in C-reactive protein concentrations (100%), and coagulopathy (100%) were recorded. That research proved that anal, oral, and nasal swabs could detect viral loads up to 15 dpi [ 44 ]. These findings are similar to those from other works with, where viral RNA was found in swabs from the nose, pharynx, and anus, with amounts increasing up to 3 dpi (in an approximate range of 4–7 copies/mL) [ 53 ]. These nonhuman primate models undoubtedly contributed significantly to our understanding of the pathogenicity of COVID-19 and the physiological bases for implementing preventive and diagnostic measures and treatment.
One animal model that shares multiple similarities with humans for the physiopathology of the SARS-CoV-2 virus is based on Rhesus macaques, African green monkeys (), and crab-eating macaques () [ 51 ]. The latter has been utilized to replicate the infection conditions in young (males and females of 3–9 years) and old-aged animals (23–29 years-old females). After intranasal and intratracheal viral inoculations, researchers found that nasal swabs (peak viral load of 10copies/µL) had higher viral loads than pharynx and rectal ones (a maximum of 10copies/µL). Additionally, viruses from nasal and pharynx samples were detected for longer periods in elderly monkeys [ 52 ]. This relation between age and disease mortality was also reported in Rhesus monkeys. Comparative studies of three nonhuman primates (three 3–5 years and two 15 years old macaques) infected intratracheally revealed that the viral replication detected by nasopharyngeal and anal swabs was persistently detected from 3 days post-infection (dpi) to 11 dpi in elderly animals. In older macaques, 104–107.5 copies/mL were also detected (while young individuals had approximately 104 copies/mL), often accompanied by the development of diffuse severe interstitial pneumonia [ 53 ].
The transgenic mice can express the human angiotensin-converting enzyme II (hACE2), a functional receptor for the SARS-CoV-2 virus that mimics clinical signs observed in humans [ 47 ]. Sun et al.’s [ 48 ] research with 4.5–30-week-old transgenic mice successfully replicated the virus after intranasal and intragastric inoculation. It led to the discovery of viral loads in the lung, trachea, brain, and feces. Those authors also detected an immune and inflammatory response due to the presence of interleukins (IL). Adult mice showed more lesions in the alveolar epithelial cells, focal pulmonary hemorrhage, and more significant apoptosis of macrophages. Those findings concurred with human reports showing that COVID-19 affected older adults more severely, with the over-65 population representing 80% of all hospitalizations and a 23-fold greater risk of mortality. Reports emphasized clinical signs, such as respiratory distress and cytokine release syndromes [ 49 ]. Studies with Syrian hamsters found that while the virus is lung-tropic and infects the respiratory tract by binding to the ACE2 cell surface in the alveoli, causing pneumonia in 67% of the animals, the gastrointestinal signs reported in humans are due to viral replication and dissemination in enterocytes [ 50 ].
The choice of an animal model that would allow researchers to observe the histopathological, radiological, or immune changes that the virus caused required that the test animals be susceptible to lung tissue damage and capable of developing an inflammatory process [ 45 ]. Potential species included nonhuman primates, ferrets, rats, mice, Syrian hamsters, lagomorphs, minks, cats, camelids, and even zebrafish [ 46 ].
The SARS-CoV-2 virus is the etiologic agent of the coronavirus 2019 disease (COVID-19) [ 41 ]. This disease has claimed the lives of over 6.3 million people worldwide since 2019 [ 42 43 ]. The lack of knowledge of this virus and its rapid propagation at the onset of the pandemic made it essential to determine its physiopathology and identify therapeutic agents and vaccines that could mitigate its threatening consequences. These fundamental issues were solved using in vivo assays that replicated the virus in animals to untangle its pathogenesis, the immune response, and the adverse effects that might result from the vaccines and therapies proposed before testing in humans and their release to the public [ 41 44 ].
The domestic dog has been postulated as a valuable model for studying chronic morbidities brought on by environmental conditions since they share morbidity and mortality factors with humans. In this field, Hoffman et al. [ 75 ] reported that comorbidities behind chronic conditions such as obesity, arthritis, hypothyroidism, and diabetes reported in humans were also present in 73,835 canines and that those dogs showed a positive association between age and the number of morbidities (< 0.001). Other studies have revealed that obesity in dogs (137/198) is closely linked to the alimentary habits of their owners, finding that the 79.8% of dogs from overweight owners (114 persons) were obese (< 0.001) [ 76 ]. Therefore, studies of these animals could provide information on disease interaction.
Another animal species considered a promising model for studying metabolic syndromes is the zebrafish (). This species has genetic homology with humans, so through genetic mutation, chemical induction, and changes in diet, they can be used to study hyperglycemia, obesity, diabetes, and hypertriglyceridemia [ 71 ]. Pigs, meanwhile, share similarities with humans in terms of organ size, lifespan, anatomy, physiology, and metabolic profile [ 40 ]. A study of obesity in Iberian pigs showed the pathogenesis of chronic kidney disease caused by overweight and obesity. Although the administration of high-fat diets did not generate diabetes in those pigs by day 100, analyses revealed hypercholesterolemia (142 ± 27 mg/dl), hypertriglyceridemia (75 ± 43), insulin resistance, and glomerular hyperfiltration [ 72 ]. These effects also occur in humans [ 73 ] and have been studied in obese male mice and ovariectomized females [ 74 ].
Studies of the human genome have identified hundreds of genetic variants associated with obesity and opened the way to examining these genes in species such as, a nematode capable of storing fat in the form of lipid droplets inside hypodermal and intestinal cells.has 14 genes that promote diet-induced obesity and three that prevent it [ 67 ]. Those genes are now recognized as potential targets for anti-obesity treatment. Ke et al. [ 68 ] found that the knockdown of 23 fat-storing not only reduced excessive fat accumulation but also improved the health and lifespan of this species (< 0.05). The inhibitory effect of flavonoids such as butein on lipogenesis insucceeded in reducing triglyceride levels by up to 27% without altering food intake or energy expenditure, an effect due to the downregulation of proteins involved in lipid metabolism [ 69 ]. Likewise, the appetite suppressant effect of administering vegetable extracts from themushroom (300 and 1000 µg/mL) tofunctioned as a natural means of preventing obesity [ 70 ]. Studies of this kind allow researchers to address obesity as a complex pathology affected by diverse factors: diet, physical activity, developmental stage, age, genes, and environmental interaction [ 67 ].
The importance of physical activity in treating these conditions has been demonstrated in experiments with 48 Sprague-Dawley male rats, where aerobic exercise for 12 weeks combined with prebiotic fiber supplementation prevented knee joint damage, dyslipidemia, endotoxemia and normalized the effects of insulin resistance (< 0.001) [ 61 ]. Studies with these supplements as part of a therapeutic protocol in Wistar rats, administered in presentations such as yogurt, have shown that supplementation with 5% of yogurt reduces levels of oxidative stress (significant decreases in NO levels,< 0.05), and had fewer amounts of inflammatory cell infiltration and collagen deposits in the liver (< 0.05) when compared to animals fed high-fat diets. According to these studies, this supplement could be a potential human therapeutic option [ 66 ].
The role of the different types of adipose tissue in humans and animals is a crucial line of research that has developed with the use of rodents. For example, adipogenesis suppression and the browning of white adipose tissue (WAT) [ 63 ] have been suggested as strategies for preventing obesity [ 60 ]. The browning process creates a brown adipose-like tissue (BAT) that can participate in thermogenesis by transforming caloric intake into heat [ 64 ]. Since this is part of a central nervous system response to cold, certain medications and exercise can trigger browning as has been observed in obese and lean rats subjected to high-intensity training. In C57BL/6J mice, the transformation of beige adipocytes into WAT can be promoted with diets complemented with resveratrol for 16 weeks, as this induces a change in the intestinal microbiota in treated animals (< 0.01) (increasing microorganisms of the genera, and, among others) that modulates lipid metabolism and has anti-inflammatory properties and anti-obesity effects [ 65 ].
Obesity is a public health problem affecting over 600 million people worldwide [ 60 ]. Obesity and its associated metabolic syndromes have consequences such as knee osteoarthritis, a disease prevalent in approximately 60% of the overweight population [ 61 ], but this is also associated with cancer, cardiovascular disease, hypertension, coronary artery disease, stroke, sleep apnea, asthma, gallstones, steatohepatitis, and dyslipidemia. Over one-third of the world’s overweight or obese population is at risk of developing type 2 diabetes mellitus [ 23 ]. Using rodent models, researchers have determined that one element that promotes the development of type 2 diabetes mellitus is adipose tissue inflammation due to insulin resistance and excess fat mass [ 62 ]. The increase in the presentation of these comorbidities has led to the use of animal models to test new, improved strategies for reducing the incidence of this disease.
Nanoparticles and their application, together with in vivo imaging, can help to test novel luminescent particles and assess their tissue penetration to improve cancer therapy [ 93 ]. In vivo imaging enables us to understand tumor growth-related processes such as oxidative mitochondrial metabolism in mouse models with cell lung cancer [ 94 ]. Likewise, in a mouse model of brain tumor –glioblastoma– under general anesthesia, modified in vivo optical imaging (Surface enhanced spatially offset Raman scattering) covers the inability of conventional techniques that rely on subcutaneous inoculation of cancerous cells because they cannot read deep tissues [ 95 ]. These techniques are the basis for imaging-guided phototherapies that are a current research field to find agents capable of inducing tumor cell apoptosis, such as photodynamic y and photothermal therapy [ 96 ].
In addition to the support of laboratory techniques such as immunofluorescence, non-invasive diagnostic methods are a priority in oncology. In immunocompetent genetically-engineered mouse models, Kirkpatrick et al. [ 92 ] utilized nanosensors with urine tests to detect protease activity in diverse types of cancer, including lung cancer, achieving 100% specificity and 81% sensitivity. In this way, monitoring with nanosensors and clinical assays in animals has demonstrated that this technique can be an option for conducting accurate, radiation-free diagnostic tests.
These neural anticancer therapies in humans and animals indicate that while sympathetic nerves show cancer-promoting effects in prostate and breast cancer, and melanoma cases, the parasympathetic/vagal nerves are believed to trigger both reactions. For example, vagal nerves can promote prostate, gastric, and colorectal cancers, but suppress breast and pancreatic cancers, due to β-adrenergic and muscarinic effects that modify the behavior of cancer cells, angiogenesis, tumor-associated macrophages, and antitumor immunity [ 88 ]. The axonogenesis process in species such as mice, linked to the development of metastasis in breast cancer, showed through immunofluorescence that nerve twigs tend to be sympathetic-like, with no expression of parasympathetic fibers [ 91 ].
Another novel anticancer strategy involves managing nerve-tumor interaction [ 88 ] since tumor-specific denervation can suppress neoplasia growth [ 89 ]. A study by Kamiya et al. [ 90 ] with female Balb/c-nu mice and the use of xenografts in Hras128 rats in a model of chemically-induced breast cancer showed that sympathetic stimulation of the nerves in tumors accelerated cancer growth but that parasympathetic stimulation reduced growth and downregulated the expression of programmed death. In contrast, in the case of late-stage colorectal cancer, parasympathetic denervation via vagotomy and atropine administration in 150 male Wistar rats reduced the incidence of tumors and their weight and volume after eight weeks, as well as cell proliferation, angiogenesis, and regulated expression of the nerve growth factor [ 89 ].
The fact that the canine and human genomes share a high degree of similarity (75%) and that the risks of death due to neoplastic, congenital, and metabolic diseases are comparable means that the dog is an ideal translational model for studying human morbidity and mortality [ 75 87 ]. For example, the percentage of neoplasia is similar between dogs and humans (27.4 vs. 25.3%). However, because the types of cancer that affect each species correlate only marginally (Spearman rank= 0.661) [ 75 ], dogs have been replaced in many preclinical studies by genetically-modified pigs [ 87 ].
Koosha et al. [ 85 ] used diosmetin, an anti-tumorigenic, in colon cancer xenografts in 24 male nude mice. Results showed that tumor volume in the group treated with 100 mg/kg of diosmetin was significantly smaller than in the untreated group (264 ± 238.3 vs. 1428 ± 459.6 mm< 0.01). Promisingly, the drug did not produce toxicity even when administered at high doses. Studies of this kind show that laboratory animals allow researchers to test new drugs and better understand disease development but also aid in determining non-toxic doses that can be applied to humans or animals. Using these models as translational media for studying cancer has also revealed the importance of identifying the pain that animals may experience. Pain assessment is important in in human medicine and laboratory animal welfare. In this regard, recognizing degrees of cancer-induced bone pain has been studied by observing behavioral changes in rats and mice, where innate behaviors, such as burrowing, are reduced 9 days after inoculation when compared to control groups (< 0.05) as a result of the nociception associated with the degree of severity of cancer due to reduced bone density [ 86 ].
According to the World Health Organization [ 77 ] and the National Cancer Institute [ 77 78 ], the most common types of cancer in humans in 2020 were breast (2.26 million cases), lung (2.21 million), colorectal (1.93 million), prostate (1.41 million), skin (1.20 million), and stomach (1.09 million). These cancers cause 10 million deaths per year. Projections for 2022 estimate that around 1,918,030 new cancer cases will be diagnosed in the United States, with 350 cancer-induced deaths per day, making this disease a primary cause of mortality [ 79 ]. The pathogeny of these cancers and testing new treatment options is another field that extensively uses animal models. Over 95% of studies use rats and mice to inject cancer cell lines subcutaneously, study the primary cancer lesion, and follow its growth before excising tumors [ 80 81 ]. However, one disadvantage of this subcutaneous tumor model, is that injections in athymic nude mice may not accurately represent the interaction among tumor cells, local stroma, and the tumor’s microenvironment, depending on its precise location [ 82 ]. Contrarily, orthotopic murine models have been shown to replicate the tumor microenvironment –including metastasis– when inoculated in the original anatomical site of the tumor. In female BALB7c mice, inoculation of mammary cancer cell line 4T1 as a fat pad tumor model showed that 50% of the animals had metastasis to the ovaries, spleen, liver, and sternum. However, when compared to a heterotopic model, orthotopic tumors were smaller (1993.7 ± 197.15 mmvs. 1078.4 ± 300.26 mm< 0.05) and had a significantly lower percentage of infiltrating cells (< 0.05) [ 83 ]. Moreover, these orthotropic models, together with in vivo optical metabolic imaging, are proposed as an approach to studying how, for example, the fatty acid uptake by breast cancer cells increases accordingly to tumor aggressiveness and metastatic process (< 0.05) [ 84 ] Attacking this complication in tumor development is the principal objective of anticancer therapies, since most deaths from prostate cancer, for example, are due to metastasis into bone structures [ 80 ].
Techniques based on local anesthesia temporarily relieve pain by inhibiting nerve impulse transmission. However, when used to complement multimodal analgesia protocols, they can be associated with neurotoxicity in both human and veterinary patients [ 107 ]. Administration via polymer-based encapsulation is a new strategy designed to prevent toxicity and permit the prolonged release of the active ingredient to give a long-term analgesic effect for up to seven days [ 107 ]. A ketamine-polymer-based drug was applied transdermally to Wistar rats to determine its analgesic effects [ 98 ]. Results of the tail-flick test and readings from an analgesiometer led them to determine a significant analgesic effect (< 0.01) maintained for 24 h with a peak effect at 8 h and a response time on the test 5.72 s vs. a basal time of 2.44 s. The compound did not produce irritation when tested on rabbit skin. It prevented the secondary effects of intravenous, nasal, or oral administration, so it is a potential option for treating neuropathic pain [ 108 ].
Due to the adverse effects that NSAIDs can generate, especially for treating chronic afflictions such as arthritis and cancer, opioids are another therapeutic option [ 101 ]. However, since the long-term use of these drugs is also associated with complications, research has begun to new concepts and explore directions. The opioid-free anesthesia technique was introduced to prevent tolerance and hyperalgesia and reduce the use of these drugs in the postoperative period. This method uses agents such as alpha-2-agonists, ketamine, and local analgesics with distinct action mechanisms in multimodal analgesia [ 102 104 ]. Other new opioid-based pharmacological options are transdermal patches impregnated with morphine-like compounds. In 6–12-week-old C57BL/6JJmsSlc mice, patches synthesized with two new opioids (new-opioids 1 and 2, N1 at 3 mg/kg; N2 at 10 mg/kg) showed the same analgesic efficacy as morphine at 3 mg/kg. The effect remained constant, even under repeated administration (in contrast to fentanyl), and the cutaneous trans-permeability rate was greater, at 1.71 ± 0.35 and 3.94 ± 1.36 µg/cm/h [ 105 ]. The administration of opioid nanoparticles has also been suggested to prevent opioid tolerance and reduce the severity of adverse effects. Leucine-enkephalin hydrochloride-based nanoparticles with a size of 100–200 nm have been tested in male Sprague Dawley rats by applying them intranasally, reaching the brain directly. After dosing, high concentrations were found in the olfactory bulb and cerebrum between the first 60 min (approximately 80 ng/g and 160 ng/g, respectively), while plasma concentrations were not detected at any evaluation time (< 0.0001). This prevents the side effects of drug transit through peripheral pathways [ 106 ].
Parallel to the advances in our knowledge of the physiopathology of diverse conditions, developing and testing new therapeutic options is another field destined for animal models. Algology is a science in constant actualization to provide new and efficient drugs to prevent the consequences of pain by reducing the number and severity of secondary effects in both human and veterinary patients [ 97 98 ]. Adequate models are needed to evaluate analgesic efficacy accurately. In the case of treatments for open wounds, Parra et al. [ 99 ] applied carprofen (5 mg/kg) and buprenorphine (0.1 mg/kg) to the left hind paw of Sprague Dawley rats of both sexes using a punch biopsy to assess analgesia in an open wound model. Using four behavioral tests associated with aspects of nociception, mechanical and thermal stimulation, guarding behavior, and the weight-bearing test, they found that carprofen promoted recovery of the thermal response to basal levels after just 2 h. The same rat species were utilized to test the renal and gastrointestinal safety of non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen by administering single and multiple oral doses to pediatric patients. Furthermore, the necropsies performed on pigs of different ages (8-week-old and 6-to-7-months-old) in the study by Millecam et al. [ 100 ] revealed no severe lesions in the stomach after multiple doses of ibuprofen at 5 mg/kg. However, significant histological score differences (< 0.025) were observed in the duodenum (1.38 vs. 4) and jejunum (3.63 vs. 1.25) between the experimental and control group. Additionally, an increased clearance time for the drug after multiple doses was found, an effect similar to reports in human pediatric patients.
In this field, sustained release options such as nanoparticle-anchoring hydrogel scaffolds of the immunosuppressant tacrolimus allowed the localized release of the drug with tissue regeneration in nude female mice or those of the BALB/c line that were given the drug in the hind limb. Those combinations allowed the sustained release of 77% of the drug, without toxicity, within 28 days at <100 ng/mL [ 122 ]. Thus, refining these drugs in the future will make it possible to reduce the cases of organ rejection due to the immune response. This finding is significant because their benefits are not accompanied by systemic toxicity, complications, or dose reduction without pharmacological efficacy [ 123 ].
Observations on the immunosuppressor response to compounds such as anti-thymocyte globulin (20 mg/kg) and rituximab (20 mg/kg) demonstrate that, in addition to the use of transgenic animals, a strict immunosuppressor regimen is a critical element in allotransplants [ 119 ]. In this regard, drugs injected in nanoparticles such as mycophenolate mofetil allow low-water soluble compounds to be combined with other compounds and administered as solid lipid nanoparticles to improve their absorption and release by as much as 68% in acid media [ 121 ].
Due to the physiological similarity between nonhuman primates and humans, procedures for organ transplants are often tested in those species. Over seven years, Lee et al. [ 119 ] performed 22 xenotransplants using hearts from transgenic pigs eliminating alpha-galactosidase transferase knockout or expression of the regulatory proteins CD46, CD39, or CD73 in Cynomolgus monkeys (). Results showed that survival of the grafts was significantly higher in hearts with double or triple genetic manipulation (11.63 ± 11.29 days vs. 30.83 ± 20.34 days,= 0.03). This is similar to the report by Cui et al. [ 120 ] on triple knockout cells from pigs (that do not express any of the three carbohydrate xenoantigens). The complement-dependent cytotoxicity response and the amount of anti-pig IgG/IgM immunoglobulins (Ig) were evaluated in serum from 72 specific pathogen-free (SPF) baboons and in human serum. Results for humans and old-world monkeys showed similar antibody binding, but the cytotoxicity measured in IgM and IgG was lower in the humans (< 0.05 vs.< 0.01).
Another advance in biomedicine achieved thanks to experimental work with animal species such as pigs are based on animal-to-human organ transplants. On 7 January 2022, Bartley Griffith’s team performed the first heart transplant from a genetically-modified pig to a 57-year-old human patient with terminal heart disease [ 117 ]. Although the patient’s condition who received that xenotransplant deteriorated two months after surgery, and he died, the procedure set an important precedent. It showed the need to continue research on genetically-engineered animal organs and immunosuppressor drugs since the immune response and organ rejection are still the leading causes of transplant failure, especially when the organs come from other animal species [ 118 ].
Today, rodents are considered models for reimplanting extremities and restoring blood vessels because their vascularization is homologous to the human finger [ 112 ]. For example, developing heterotrophic osteomyocutaneus flap transplant protocols in Lewis rats furthered our understanding of the mechanisms and pathways involved in the immune response underlying tissue transplant rejection [ 113 ]. Likewise, in an experiment with five syngeneic mice and allografts—using a donor-supplied aorta and inferior vena cava—end-to-end anastomosis of those structures showed a 74% success rate as a technique for hind limb transplants [ 114 ]. In another study, Tee et al. [ 115 ] performed grafts of engineered cardiac muscle flaps in the epicardium of 8 rats. The flaps were transplanted by microsurgery to resolve one of the first limitations: failed vascular anastomosis. Those researchers performed successful end-to-end anastomosis of the carotid artery and jugular vein by placing the flap on the epicardium, achieving a survival rate of 75% during 4 weeks post-surgery, with viable cardiomyocytes and vascular connections between the flap and the epicardium by week 10 [ 115 ]. These techniques, tested first in animals, were later used with human patients with coronary artery disease caused by diseases such as squamous cell carcinoma, with a 96% survival rate of the flap in individuals subjected to neck and head surgery [ 116 ].
In addition to developing novel drugs, advances in surgical technology and techniques have opened fields in microsurgery in human and animal medicine since the 1900s when Carrel and Guthrie performed the first transplants in dogs [ 109 ]. Later, in 1950–1960, Buncke and Schultz tested the first microsurgery techniques using models of digital amputations and reimplantation in Rhesus monkeys, performing vascular microsurgery to restore circulatory connections successfully [ 110 ]. Anastomosis of 1-mm blood vessels in the ears of adult rabbits by reimplantation was the first demonstration of microsurgery in reconstructive medicine [ 111 ].
The field of neuroscience includes surgical and therapeutic procedures involving the central nervous system and conducts studies focused on specific diseases or pathologies of that system. With the discovery of neurological sequelae in COVID-19-infected patients, animal models have allowed researchers to observe the effects that the SARS-CoV-2 virus generates in sporadic cases, including epileptic seizures and encephalitis with a mortality rate of approximately 5.3% [ 124 ].
p
= 0.026 andp
= 0.045, respectively). In other research, models of medullar lesions are widely utilized with species such as rats, which are particularly important because between 236 and 1009 per million humans annually suffer a spinal cord injury [Estimates suggest that approximately 42 million people worldwide suffer brain injuries annually and that 80% of cases are classified as traumatic brain injury (TBI). Animal models based on rodent species are being used to improve our understanding of the physiopathology of TBI [ 125 ], though authors such as Vink [ 126 ] caution that neuroanatomical differences in the mouse’s lissencephalic brain can generate biomechanical responses distinct from those in humans. Moreover, the replication of trauma may be greater in rodents since traumatisms in these animals tend to generate focal instead of diffuse lesions [ 127 ]. Grovola et al. [ 128 ] used male Yucatán miniature pigs to analyze neurological dysfunction in animals with mild traumatism 1-year postevent. They found a persistent neuroimmune response in animals with morphological changes to the microglia, with increased branches and junctions per cell (= 0.026 and= 0.045, respectively). In other research, models of medullar lesions are widely utilized with species such as rats, which are particularly important because between 236 and 1009 per million humans annually suffer a spinal cord injury [ 129 ]. Although this species is the one most often employed to replicate medullar damage, Filipp et al. [ 129 ] affirm that between-species differences (quadrupeds, bipeds) must be considered when evaluating the neuroplasticity of the spinal neurons.
D. melanogaster
) because they manifest seizure-like behavior and share 70% of their genes with humans [Drosophilas
prevented induced seizures (p
< 0.0001). This led to the discovery that the action mechanism of their metabolites is not linked to the cannabinoid receptors but, instead, to transient potential receptors (TRP). This makes the fruit fly a suitable medium for studying this type of drug [Epilepsy is one of the most common neurological conditions, affecting over 50 million people worldwide [ 130 ] and 0.6–0.75% of the domestic canine population [ 131 ]. Recent studies of the physiopathology of this disorder and the testing of anti-seizure drugs have used fruit flies () because they manifest seizure-like behavior and share 70% of their genes with humans [ 15 ]. The use of the endocannabinoid anandamide (at 2, 20, and 200 µg/mL) inprevented induced seizures (< 0.0001). This led to the discovery that the action mechanism of their metabolites is not linked to the cannabinoid receptors but, instead, to transient potential receptors (TRP). This makes the fruit fly a suitable medium for studying this type of drug [ 132 ].
Drosophila
has been used to understand the neurobiological bases of processes still considered mysteries by biology, such as sleep, plasticity, and memory [D. melanogaster
, Toda et al. [Despite its nature and supposed organic simplicity,has been used to understand the neurobiological bases of processes still considered mysteries by biology, such as sleep, plasticity, and memory [ 133 ]. After studying 12,000 exemplars of, Toda et al. [ 134 ] reported the existence of the “nemuri” gene, a peptide with antimicrobial properties that favors sleep and helps these flies survive the infection. This suggests that its function could be linked to the immune competence of the sleep process in animals and humans. The association of sleep with long-term memory, known as post-learning sleep, was studied by Lei et al. [ 135 ], who found a neural circuit that excites the mushroom body neurons and a connection to the fan-shaped ventral neurons that promotes post-learning sleep during courtship. This finding underlined the association between the longer learning experience and the reinforcement of long-term memory, mechanisms sometimes found in mammals.
p
< 0.0001) due to deposits of amyloid plaques. Recognition of this physiopathology helped scientists establish the goal of preventing this process as a novel therapeutic approach [Neuroscience techniques applied to species such as nonhuman primates and transgenic models of those species have recently been proposed as useful for studying human evolution and the cerebral functioning of people with autism disorders and neurodegenerative diseases such as Alzheimer’s [ 136 ]. In humans, Alzheimer’s disease is considered the most common neurodegenerative disease accounting for around 80% of cases of dementia worldwide [ 137 ]. It is widely recognized that mitochondrial dysfunction is an event that precedes the onset of Alzheimer’s, and this has been studied in two lines of mice (APPswe/PSEN1 ∆E9 and C57BL/6J). There, the alteration of mitochondrial homeostasis and increased mitochondrial calcium levels caused damage and neuronal death (< 0.0001) due to deposits of amyloid plaques. Recognition of this physiopathology helped scientists establish the goal of preventing this process as a novel therapeutic approach [ 138 ].
p
< 0.001). Furthermore, no systemic effects were observed, presenting a condition similar to Parkinson’s [Another neurogenerative disease, Parkinson’s, has been studied primarily with murine models [ 139 ]. Recently, however, researchers recognized that the zebrafish shares more neuroanatomical traits with humans and that mutations of the PARK7 gene in adult fish were associated with the development of Parkinson’s in humans [ 140 141 ]. Exposure of zebrafish larvae to neurotoxins that act directly on the dopaminergic neurons constitutes a method to mimic the phenotype of Parkinson’s disease. Specifically, the MPP+ neurotoxin affected the locomotor function (total distance and velocity) of fish, reducing its performance by 80% and 85%, respectively (< 0.001). Furthermore, no systemic effects were observed, presenting a condition similar to Parkinson’s [ 142 ].
p
< 0.01) [p
< 0.01 when compared to non-treated fish) while reducing larval behavioral impairment measured as improvements in the total distance (peak distance around 180 cm) and velocity (peak values around 3.5 cm/s) (p
< 0.001) [Palliative treatments to control movement disorders such as dystonia, Huntington’s, and Parkinson’s disease have also been tested in zebrafish [ 143 ]. Treatment of Parkinsonian embryos with substances such as rosmarinic acid (RA) prevents the loss of dopaminergic neurons due to neurotoxicity. This acid has been proposed as a neuroprotector and antioxidant that reduces locomotor deficits measured, for example, by increasing the swimming distance in zebrafish treated with RA at concentrations of 10 or 100 µm (approximately 130 to 150 cm,< 0.01) [ 144 ]. Similarly, it has been suggested that herbal medicines based on Tongtian oral liquid have neuroprotective and antioxidant properties. The administration of Tongtian to zebrafish prevented neurotoxicity and the degeneration of dopaminergic neurons (< 0.01 when compared to non-treated fish) while reducing larval behavioral impairment measured as improvements in the total distance (peak distance around 180 cm) and velocity (peak values around 3.5 cm/s) (< 0.001) [ 145 ].
Oryzas celebensis
) [p
< 0.001,p
< 0.05), increased swimming speed under both light and dark conditions, and deficient social interaction, anxiety, and macrocephaly, all as consequences of neuronal cerebral cell proliferation. In a separate study, when applied to 28 neonate rat pups, this acid generated oxidative stress in the cerebellar hemispheres and reduced the count and nuclear size of the Purkinje cells [Aquatic models are also utilized to study other neurodevelopmental problems, such as autism spectrum disorder in zebrafish and Medaka fish () [ 146 ]. Chen et al. [ 147 ] found that prenatal exposure to valproic acid (at 5 and 50 µM) in AB lines of zebrafish produced embryos and larvae with signs similar to those seen in autistic humans, including hyperactivity, manifested in a greater frequency of tail-bending, greater distances traveled after touching of the dorsal tail (< 0.001,< 0.05), increased swimming speed under both light and dark conditions, and deficient social interaction, anxiety, and macrocephaly, all as consequences of neuronal cerebral cell proliferation. In a separate study, when applied to 28 neonate rat pups, this acid generated oxidative stress in the cerebellar hemispheres and reduced the count and nuclear size of the Purkinje cells [ 148 ]. These findings appeared, as well, in the brains of children with this condition. In the case of rats, administering grape seed extract served as a neuroprotector thanks to its antioxidant effect.
Referring to neurodegenerative disorders, a key strategy is to improve symptomatology through physiotherapy and rehabilitation protocols, another line of research that has increased in importance due to the prevalence of neurological conditions that can affect the quality of life of both humans and animals.