Creation Of Animal Models Research Articles

IPS cell technologies toward overcoming neurological diseases

Neurological diseases are often life threatening, with severely affecting an individual's quality of life. However, the disease mechanisms are still less understood, mainly because of lacking good disease models. Over the past decades, researchers developed many models using cell lines or animals, but most of them did not faithfully recapitulate the disease phenotypes. In particular, it is almost impossible to create animal models for multifactorial diseases or sporadic cases of unknown etiology. In these circumstances, it has come to be expected that induced pluripotent stem cells (iPSCs) can revolutionize neurological disease research as they retain patient's genetic information and provide an expandable source of disease-relevant neurons and glial cells. iPSC technologies are now widely used for disease modeling, and further for drug discovery and regenerative medicine. They are also enabling previously infeasible studies such as those uncovering how disease-associated single nucleotide polymorphism (SNP) and genetic variants increase the disease risk. This review describes a variety of iPSC technologies to produce various types of neurons and brain-like tissues (brain organoids) and summarize recent trends in iPSC technology-based neurological disease research. We also discuss the remaining challenges for understanding and overcoming brain disorders.

Unique roles of co-receptor-bound LCK in helper and cytotoxic T cells

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4–LCK and CD8–LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor–LCK interactions as promising targets for immunomodulation.

ИММУНОФЕНОТИПИЧЕСКИЕ ОСОБЕННОСТИ ПРОЛИФЕРАЦИИ И АПОПТОЗА ГАМЕТ САМЦОВ КРЫС ПОСЛЕ ЛОКАЛЬНОГО ОБЛУЧЕНИЯ

The “male factor” accounts for half of the causes of childlessness among married couples; in order to understand the etiopathogenetic mechanisms of these spermatogenesis disorders, it is necessary to create animal models, includ- ing with the use of radiation. In addition, in clinical practice, the methods of radiation therapy are successfully used in the treatment of malignant neoplasms. Given the increase in the number of testicular malignant neoplasms, it is necessary to improve existing methods and develop new ones.
 
 Aim of the study: molecular biological assessment of the proliferation and apoptosis of gametes in male rats after electron irradiation with doses of 2 Gy, 8 Gy and 12 Gy.
 
 Material and methods. Wistar males (220 ± 20 g; 9 – 10 weeks; n = 40) were randomly divided into control (I; n=10) and experimental (II – IV; n=10 in each) group depending on the dose of a single sighting irradiation with electrons of the pelvic segment in the projection of the testes: II – 2 Gy, III – 8 Gy, IV – 12 Gy, which were studied by immunohisto- chemical method using antibodies to Ki-67, caspase-3, Bcl-2 and p53
 Results. A week after irradiation with 2 Gy, 8 Gy, and 12 Gy, an increase in the level of the terminal phase of apop- tosis, caspase-3 (25.0±0.1, 55.0±0.2 and 74.0±0.1, respectively), and a decrease in the proliferation marker Ki-67 (18.0±0.1, 8.0±0.3 and 2.0±0.1, respectively), a decrease of degree of antiapoptotic Bcl-2 (27.0±0.7, 15.5±0.7, and 7.0±0.2, respectively) and an increase of proapoptotic p53 (33.0±0.1, 83.0±0.1, and 67.0±0.2, respectively) factors.
 Conclusion. In the testis, a single irradiation with electrons leads to a sharp shift in the proliferative-apoptotic bal- ance towards the death of germ cells, primarily spermatogonia.

The Neuroprotective Role of Acorus calamus in Developmental and Histopathological Changes in Autism-Induced Wistar Rats.

Introduction Autism spectrum disorder (ASD) is a neurodevelopmental disorder, and a tremendous increase in the incidence of autism poses challenges in identifying the different treatment modalities. Since the defined etiology, pathophysiology, and treatment of autism are unavailable, translational research is being done by creating animal models of autism. This study aimed to assess the effects of Acorus calamus on developmental and histopathological changes in autism-induced Wistar rats. Materials and methods A rat model of autism was created by administering sodium valproate on the 12th day of pregnancy, and rat pups of this group were considered autism-induced. Rat pups of pregnant rats who had received normal saline on the 12th day of pregnancy were consideredgroup I (negative control group). Neural reflexes were assessed in early postnatal days (PND) to confirm the development of autism. Autism-induced rat pups were divided into the following two groups: group II, autism (positive control group), and group III, autism + A. calamus (drug-treated group). On the 21st postnatal day (PND), group III was given an ethanolic extract of A. calamus (200 mg/kg), and group I and group II were given normal saline orally for 15 days. After 15 days of drug exposure, at 36thPND, the rats were sacrificed, and brain tissue was collected for histopathological analysis. Results When compared to the negative control group, autism-induced rat pups showed delayed appearance of neurological reflexes. Neurodegenerative changes were well appreciated in group II (autism-induced rats) than in group III (autism + A. calamus). In the histomorphometric analysis, group II showed a significant reduction in the number of neurons in the frontal cortex and Purkinje cells in the cerebellum. However, when compared to group II, group III (autism treated with A. calamus) did not show significant alteration. Conclusion Valproate exposure at mid-pregnancy creates autism by disturbing neural structures among rat pups. This was clinically represented as the delayed appearance of neural reflexes. Acorus calamus in the early postnatal period protects rat pups' brain morphology against autism pathology.

Cisplatin-Induced Kidney Toxicity: Potential Roles of Major NAD+-Dependent Enzymes and Plant-Derived Natural Products.

Cisplatin is an FDA approved anti-cancer drug that is widely used for the treatment of a variety of solid tumors. However, the severe adverse effects of cisplatin, particularly kidney toxicity, restrict its clinical and medication applications. The major mechanisms of cisplatin-induced renal toxicity involve oxidative stress, inflammation, and renal fibrosis, which are covered in this short review. In particular, we review the underlying mechanisms of cisplatin kidney injury in the context of NAD+-dependent redox enzymes including mitochondrial complex I, NAD kinase, CD38, sirtuins, poly-ADP ribosylase polymerase, and nicotinamide nucleotide transhydrogenase (NNT) and their potential contributing roles in the amelioration of cisplatin-induced kidney injury conferred by natural products derived from plants. We also cover general procedures used to create animal models of cisplatin-induced kidney injury involving mice and rats. We highlight the fact that more studies will be needed to dissect the role of each NAD+-dependent redox enzyme and its involvement in modulating cisplatin-induced kidney injury, in conjunction with intensive research in NAD+ redox biology and the protective effects of natural products against cisplatin-induced kidney injury.

Adeno-Associated Viruses for Modeling Neurological Diseases in Animals: Achievements and Prospects.

Adeno-associated virus (AAV) vectors have become an attractive tool for efficient gene transfer into animal tissues. Extensively studied as the vehicles for therapeutic constructs in gene therapy, AAVs are also applied for creating animal models of human genetic disorders. Neurological disorders are challenging to model in laboratory animals by transgenesis or genome editing, at least partially due to the embryonic lethality and the timing of the disease onset. Therefore, gene transfer with AAV vectors provides a more flexible option for simulating genetic neurological disorders. Indeed, the design of the AAV expression construct allows the reproduction of various disease-causing mutations, and also drives neuron-specific expression. The natural and newly created AAV serotypes combined with various delivery routes enable differentially targeting neuronal cell types and brain areas in vivo. Moreover, the same viral vector can be used to reproduce the main features of the disorder in mice, rats, and large laboratory animals such as non-human primates. The current review demonstrates the general principles for the development and use of AAVs in modeling neurological diseases. The latest achievements in AAV-mediated modeling of the common (e.g., Alzheimer’s disease, Parkinson’s disease, ataxias, etc.) and ultra-rare disorders affecting the central nervous system are described. The use of AAVs to create multiple animal models of neurological disorders opens opportunities for studying their mechanisms, understanding the main pathological features, and testing therapeutic approaches.

Kanta Subbarao—confronting influenza and COVID-19

Kanta Subbarao—confronting influenza and COVID-19

The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.

Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR–Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR–Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR–Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR–Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.

Theory of mind tested by implicit false belief: a simple and full-fledged mental state attribution.

About 40 years have passed since 'theory of mind (ToM)' research started. The false-belief test is used as a litmus test for ToM ability. The implicit false-belief test has renewed views of ToM in several disciplines, including psychology, psychiatry, and neuroscience. Many important questions have been considered via the paradigm of implicit false belief. We recently addressed the phylogenetic and physiological aspects of ToM using a version of this paradigm combined with the chemogenetic technique on Old World monkeys. We sought to create animal models for autism that exhibit behavioral phenotypes similar to human symptoms. The simultaneous manipulation of neural circuits and assessments of changes in phenotypes can help identify the causal neural substrate of ToM.

MODELING THE DEPRESSIVE STATE IN RATS BY STEREOTAXIC INJECTION OF LIPOPOLYSACCHARIDE INTO THIRD VENTRICLE OF THE BRAIN

Introduction: More than 4.4% of the world's population suffers from depression, which dramatically reduces both quality and life expectancy. For an accurate understanding of the links of pathogenesis and the possibility of correcting the depressive state, translational studies are widely used - in laboratory rodents. According to the modern concept of the inflammatory genesis of depression, the creation of animal models with nonspecific inflammation on the territory of the central nervous system is an urgent task. Purpose of the work: creation of a model of the depressive state in rats by injecting LPS into the third ventricle of the brain. Materials and methods: the study was carried out on female rats Wistar 280-300g. (n = 10), divided into 3 groups: 1 - with the introduction of LPS (n = 4), 2 - sham-operated (n = 3) and 3 - intact (n = 3). LPS was administered at a dose of 50 μg / animal in at volume of 4 μl in 15 minutes, according to the following stereotaxic coordinates: AP-0.8 ML-1.5 DV-3.5. Depression was assessed using the sucrose test and the behavior of animals in the open field tests, the elevated plus maze, the dark-light chamber, and the Porsolt test. Confirmation of the correct administration was carried out using MRI scanning (7 days) and postmortem on histological sections. Results: Animals treated with LPS showed a decrease in sucrose consumption, both compared to the sham-operated group and compared to the intact group. In behavioral tests, there was a decrease in locomotor activity in rats with the influence of LPS, but the differences between the group of sham-operated rats were insignificant. The experimental group had a sharp increase in the time of immobility in the Parsolt test (89.6 s VS 32.6 s VS 22.2 s) for the first, second and third groups, respectively. In the behavior of animals in the burrow chamber and in the open field, the presence of a neurological deficit in the experimental group was noted, consisting in a violation of gait in three out of four rats, in other groups this effect was not observed. When analyzing MRI images, the trace from the needle is traced exactly to the third ventricle. On a histological section in rats with neurological deficit, a demyelination area is visible (Fig. 1). Conclusion: The introduction of LPS into the cavity of the third ventricle causes depressive-like behavior in rats, a decrease in locomotor activity, but causes manifestations of neurological deficit as a result of local demyelination of the nervous tissue. The work was carried out within the framework of the state assignment of the Federal State Budgetary Educational Institution of Higher Education named after V.I. N.I. Pirogov for 2021–2023, state no. registration research work AAAA-A18-118051590108-1

Current Advancements in Animal Models of Postsurgical Lymphedema: A Systematic Review.

Significance: Secondary lymphedema is a debilitating disease caused by lymphatic dysfunction characterized by chronic swelling, dysregulated inflammation, disfigurement, and compromised wound healing. Since there is no effective cure, animal model systems that support basic science research into the mechanisms of secondary lymphedema are critical to advancing the field. Recent Advances: Over the last decade, lymphatic research has led to the improvement of existing animal lymphedema models and the establishment of new models. Although an ideal model does not exist, it is important to consider the strengths and limitations of currently available options. In a systematic review adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we present recent developments in the field of animal lymphedema models and provide a concise comparison of ease, cost, reliability, and clinical translatability. Critical Issues: The incidence of secondary lymphedema is increasing, and there is no gold standard of treatment or cure for secondary lymphedema. Future Directions: As we iterate and create animal models that more closely characterize human lymphedema, we can achieve a deeper understanding of the pathophysiology and potentially develop effective therapeutics for patients.

Toxin-induced aversive context conditioning: Assessing active aversive behaviors conditioned to the context of an automated activity monitor

Lithium chloride (LiCl) is an emetic drug that has been used to create animal models of anticipatory nausea and conditioned place aversion. In this study we examined escape behaviours from a context in which rats experienced the aversive effects of LiCl treatments. The experiment had two phases: acquisition of context conditioning, which consisted of pairing a distinct context with the pharmacological effects of a moderate dose of the toxin LiCl, and extinction of context conditioning, which consisted of placement in the distinct context in a drug free state. During context conditioning, 16 adult male Long-Evans rats were injected intraperitoneally with 96 mg/kg lithium chloride (LiCl; n = 8) or 0.9% saline (NaCl; n = 8) and placed individually in an automated locomotor activity apparatus for 30 min every other day for 4 days. During the extinction phase, rats were placed in the apparatus for 30 min every other day without injections during a 4 day extinction phase. A significant Drug x Trial interaction was found for the time spent in vertical position in the open field apparatus during trials 1–3 of the extinction phase. The LiCl treated rats exhibited significantly increased rearing behavior, relative to the control rats, indicative of conditioned aversion. The results of this study suggest that escape behavior (vertical activity) occurs in rats experiencing the aversive conditioned effects of LiCl in a distinct context. In the context of current theoretical accounts, the LiCl-conditioned increase in apparent escape behaviors can be considered a reflection of anticipatory nausea.

Akkermansia muciniphila Exerts Strain-Specific Effects on DSS-Induced Ulcerative Colitis in Mice.

Akkermansia muciniphila is a commensal bacterium of the gut mucus layer. Although both in vitro and in vivo data have shown that A. muciniphila strains exhibit strain-specific modulation of gut functions, its ability to moderate immunity to ulcerative colitis have not been verified. We selected three isolated human A. muciniphila strains (FSDLZ39M14, FSDLZ36M5 and FSDLZ20M4) and the A. muciniphila type strain ATCC BAA-835 to examine the effects of different A. muciniphila strains on dextran sulfate sodium-induced colitis. All of the A. muciniphila strains were cultured anaerobically in brain heart infusion medium supplemented with 0.25% type II mucin from porcine stomach. To create animal models, colitis was established in C57BL/6 mice which randomly divided into six groups with 10 mice in each group by adding 3% dextran sulfate sodium to drinking water for 7 days. A. muciniphila strains were orally administered to the mice at a dose of 1 × 109 CFU. Only A. muciniphila FSDLZ36M5 exerted significant protection against ulcerative colitis (UC) by increasing the colon length, restoring body weight, decreasing gut permeability and promoting anti-inflammatory cytokine expression. However, the other strains (FSDLZ39M14, ATCC BAA-835 and FSDLZ20M4) failed to provide these effects. Notably, A. muciniphila FSDLZ20M4 showed a tendency to exacerbate inflammation according to several indicators. Gut microbiota sequencing showed that A. muciniphila FSDLZ36M5 supplementation recovered the gut microbiota of mice to a similar state to that of the control group. A comparative genomic analysis demonstrated that the positive effects of A. muciniphila FSDLZ36M5 compared with the FSDLZ20M4 strain may be associated with specific functional genes that are involved in immune defense mechanisms and protein synthesis. Our results verify the efficacy of A. muciniphila in improving UC and provide gene targets for the efficient and rapid screening of A. muciniphila strains with UC-alleviating effects.

Telomere Length and Arsenic: Improving Animal Models of Toxicity by Choosing Mice With Shorter Telomeres.

Arsenic is both a chemotherapeutic drug and an environmental toxicant that affects hundreds of millions of people each year. Arsenic exposure in drinking water has been called the worst poisoning in human history. How arsenic is handled in the body is frequently studied using rodent models to investigate how arsenic both causes and treats disease. These models, used in a variety of arsenic-related testing, from tumor formation to drug toxicity monitoring, have virtually always been developed from animals with telomeres that are unnaturally long, likely because of accidental artificial selective pressures. Mice that have been bred in captivity in laboratory conditions, often for over 100 years, are the standard in creating animal models for this research. Using these mice introduces challenges to any work that can be affected by the length of telomeres and the related capacities for tissue repair and cancer resistance. However, arsenic research is particularly susceptible to the misuse of such animal models due to the multiple and various interactions between arsenic and telomeres. Researchers in the field commonly find mouse models and humans behaving very differently upon exposure to acute and chronic arsenic, including drug therapies which seem safe in mice but are toxic in humans. Here, some complexities and apparent contradictions of the arsenic carcinogenicity and toxicity research are reconciled by an explanatory model that involves telomere length explained by the evolutionary pressures in laboratory mice. A low-risk hypothesis is proposed which has the power to determine whether researchers can easily develop more powerful and accurate mouse models by simply avoiding mouse lineages that are very old and have strangely long telomeres. Swapping in newer mouse lineages for the older, long-telomere mice may vastly improve our ability to research arsenic toxicity with virtually no increase in cost or difficulty of research.

Expression of Semaphorin 3A after spinal cord injury

To investigate expression of Semaphorin 3A in rats after spinal cord injury and explore possible mechanism of inhibiting of axonal regeneration after SCI. Forty healthy female SD rats, 8 weeks old, weighing (210.00±9.88) g, were randomly divided into control group(20 rats in group A) and model group(20 rats in group B). In control group, removal of T10 lamina and partial removal of T9 and T11 lamina were performed, and no further operation was performed on spinal cord (pseudo operation). In model group, the total T10 and partial T9, T11 partial lamina were incised and the spinal cord transection was performed to create animal models of spinal cord injury. The rats were perfused and spinal cord tissue obtained at 3, 7, 14, 28 and 42 days after surgery (4 rats in each group at each time point), respectively, and then HE staining was performed. Meanwhile, the expression of Semaphoring 3A was detected in accordance with the protocol of SP kit. After a simple spinal cord transection injury, hemorrhagic necrosis, localized edema, neurodegeneration, necrosis, and cyst formation occurred in the injured area, and glial scar formation occurred in glial cells. Semaphorin 3A expression levels in control group was low in the gray matter area. There was no expression of Semaphorin 3A in the injured area of spinal cord injury in model group 3 days after operation. On the 14th day, the expression of Semaphorin 3A in the injured area of spinal cord injury increased significantly and was at a high level. On the 28th day, the expression of Semaphorin 3A was moderate. On the 42th day, the positive expression of Semaphorin 3A returned to normal level. The increased expression of Semaphorin 3A after spinal cord injury may be one of the mechanisms that inhibit axonal regeneration.

A systematic review on Valproate induced rat model of autism: Pathophysiology, treatment, developmental and neurobehavioral assessment of rat offsprings

Autism Spectrum disorder (ASD) is charecterised by certain degrees of disabilities in social communication, restrictive repetitive behaviour and altered motor and sensory perception. Translational research is carried out by creating animal models of autism to find out the correlation between behavioural changes and the pathology of brain tissue and for clinical trials of newer therapeutic formulations. Valproate induced animal model of autism has significant validity to demonstrate ASD manifestations. An early appreciation of ASD symptoms is needed for a better prognosis. So assessment of behavioural abnormalities and development stones in the first month of animal life are much important to study ASD.An extensive literature search was done on different databases. (PUBMED-MeSH, PMC, Webof Science, Google scholar and Research Gate). Original articles reported between the years 2000-2020 were selected. PRISMA protocol was followed. There are good quantities of studies on behavioural assessment of valproate induced animal model. This review explains the pathophysiology and various treatment modalities tried in valporate induced animal model and it also enlists the developmental and behavioural assessment methods of rat off springs. It will be useful to demonstrate all signs of autism.

Streptozotocin-Induced Diabetic Models in Mice and Rats.

Streptozotocin (STZ) is an antibiotic that causes pancreatic islet β-cell destruction and is widely used experimentally to produce a model of type 1 diabetes mellitus (T1DM). Detailed in this article are protocols for producing STZ-induced insulin deficiency and hyperglycemia in mice and rats. Also described are protocols for creating animal models for type 2 diabetes using STZ. These animals are employed for assessing the pathological consequences of diabetes and for screening potential therapies for the treatment of this condition. © 2021 The Authors.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Advances in the creation of animal models of paroxysmal nocturnal hemoglobinuria

ABSTRACT Paroxysmal nocturnal hemoglobinuria (PNH) is a disease caused by a phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) mutation in hematopoietic stem cells. There are three theories about the possible mechanism of the pathogenesis of PNH: immune escape, anti-apoptotic mechanism, and secondary gene mutation. There has been little gain in the knowledge regarding its pathogenesis during the last decade owing to the lack of representative cell lines and animal models. There have been recent reports about the successful creation of PNH mouse and PNH rhesus macaque models. The detection of glycosylphosphatidylinositol-anchor protein (GPI-AP)-deficient cells and/or fluorescently labeled variant of aerolysin (FLAER) test, estimation of erythrocyte life span, and hemolysis-related experiments demonstrated that these animal models of PNH had GPI-AP-deficient blood cells with shortened lifespans and increased sensitivity to complement-activated hemolysis. However, there were no clinical manifestations such as hemolysis and thrombosis in these animal models. This suggested that the PIG-A mutation is one of the several conditions required for PNH, but it alone is not enough to cause PNH.