Experimental models provide a deeper understanding of the different pathogenic mechanisms involved in Demyelinating disorders. The development of new in vitro and in vivo models or variations of existing models will contribute to a better understanding of these diseases and their treatment. Experimental models help to extrapolate information on treatment response. Indeed, the choice of the experimental model strongly depends on the research question and the availability of technical equipment. In this chapter, the current in vitro and in vivo experimental models to examine pathological mechanisms involved in inflammation, demyelination, and neuronal degeneration, as well as remyelination and repair in demyelination disorders are discussed. We will also point out the pathological hallmarks of demyelinating disorders, and discuss which pathological aspects of the disorders can be best studied in the various animal models available.
- Demyelinating disorders
- In vitro model
- In vivo model
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system [1, 2, 3, 4, 5]. Experimental animal models are one of the useful tools because they can increase our knowledge about the central system disorders . Unfortunately in MS, there is no model that can reflect all of the pathological features .
The use of experimental animal models, including MS models, has recently been the focus of several reports . These subjects are mentioned in the ARRIVE guidelines as well . Adherence to the guidelines on reporting and referring papers using experimental models of MS will be key for the translation from the bench to experimental models and eventually to the bedside of MS patients .
Animal models are the advantageous way to identify the immunopathological mechanisms involved in MS . Animal models help scientists to develop novel therapeutic and in regenerative medicine approaches . Animal models of MS have provided a beneficial platform for evaluating its efficacy in MS treatment and how this may be targeted for therapy lastly . Indeed, choosing an appropriate animal model to study a complex disease like MS presents several challenges, chiefly associated with heterogeneity [14, 15]. It is well established that MS is highly heterogeneous in terms of its genetic basis, environmental triggers, clinical course, pathology, and therapeutic responsiveness in each treatment . Important factors such as genetic and environmental contribute toward MS development; however, etiology is complex and not completely assumed. Ideally, an advanced animal model has to include this heterogeneity [15, 17].
Certainly, studies in MS model need to be carefully covered the pathogenesis of the disease. A high degree of consistency between models and experimental conditions makes possible translation into therapeutic achievement .
Not remarkably, most of our present facts of MS have been derived from the EAE model . Although EAE must be induced by artificial immunization against myelin, most therapies tested in MS patients are based on concepts derived from the EAE model, which continues to be the model system of choice. EAE models are vital for studying general concepts other than specific processes of autoimmunity; however infrequently, they predict success in clinical trials .
There are many mismatched aspects of pathology and immunology between EAE and multiple sclerosis. These differences are significant. For example, persistent imbalances in immune regulation are vital to the progression of multiple sclerosis, but these orders of complexity have not yet been summarized in the MS models . This, in combination with a diversity of animal models that mimic specific features and processes of MS, has contributed to filling the gap of knowledge in the cascade of events underlying MS pathophysiology .
Until now several different EAE models have been developed, differing in the immunological reaction, inflammatory processes, and the neuropathophysiology in the CNS.
Access to up-to-date knowledge of the dynamic responses of neural cells, such as microglia in the commonly used animal models of MS, specifically the immune-mediated experimental autoimmune encephalomyelitis (EAE) model, and the chemically induced cuprizone and lysolecithin EAE models can be really helpful . It is essential to elucidate the spectrum of microglial functions in these models, from harmful to protective roles, to identify emerging therapeutic targets and guide drug discovery efforts .
In all models, it can be observed that the harmful activation of microglial cells is in the acute stage of diseases such as encephalitis, cuprizone-induced demyelination, PCL, and FAE. However, in subacute and chronic stages, regenerative healing by microglial cells may be observed . The role of T lymphocytes in EAE, CPL, and cuprizone models is important too; however, they cannot impact like microglial cells . This makes these models the cleanest method for studying microglial mechanisms in innate immune systems and also all aspects of oligodendrocytes such has proliferation, differentiation, and especially the cause of remyelination [27, 28].
Briefly, this overview of the
2. Fundamental pathogenesis factors change during the progression of the demyelinating disorders
As MS disease progressed, two fundamental changes occurred . First, the activity of the adaptive immune system decreases leading clinically to a decrease in the incidence of clinically detectable relapses. It is currently unclear why the adaptive (and perhaps also innate) immune system becomes less active as the disease progresses, but immunosenescence likely plays an important role . Second, the slow-burning degenerative process reaches a certain threshold and becomes clinically apparent.
Two possible mechanisms play a role in the retarding clinical performance . One of them is a compensation of damaged or degenerated neurons by nearby neurons known as neural plasticity. Another one is the destruction of neurons without obvious clinical signs and symptoms . Likely, this condition can be seen in 80% of Parkinson’s patients. Many dopaminergic neurons in their brain may be lost before any clinical deficits . For example, imagine that across from you is a container with 1,000 bullets. If someone removes a single bullet, it is not likely that it will be recognized. However, if only two bullets remain, most people will certainly recognize if another bullet has left the container. At the RRMS disease stage, the initial loss of neuronal structures is neither recognized by the patient nor, in the other words, does not lead to obvious clinical deficits . Subsequently, during the disease, when numerous neurons are already dying at the transition phase from RRMS to SPMS, evident clinical deficits can be seen obviously .
However, it is not difficult to find primary CNS cell cultures from rodents such as mice and rats, embryonic cells, mainly neurons, and oligodendrocytes are limited.
It can be more difficult for isolating cells from adult animals and also from the human brain . Because of these limitations, cell lines are used in most of the studies . Among many cell lines, glial cell lines are used frequently because of a key role in the explanation of the mechanisms involved in health and disease; however, it is important to ensure the cell lines have properties like
Microglial cell activation may be seen in active demyelinating lesions. Also, it can be seen in pre-active lesions, remyelination areas, and the normal-looking white matter . For finding the functions of these cells in MS, isolation of microglia from embryonic or early post-natal animals was done before . However, it must be mentioned that in the field of MS and other neurodegenerative disorders, using young animal cells may not be relevant to study chronic diseases that happened in older animals and humans [41, 42]. Thus, few studies are using aged microglial cells isolated from adult animals such as rhesus monkeys and humans. Notably, because of many limitations such as ethical aspects, using cells isolated from post-mortem are often in humans and primates [42, 43].
An important issue with respect to the use of microglia cell cultures is what it tells us about the pathogenesis of MS. Based on previous studies, primary human microglia cultures derived from MS brain tissue versus healthy brain have the major advantage of revealing pathways involved in the disease process . Another major problem is microglial cells like other cells of the innate immune system, which reacts quickly to any danger or foreign signals. Therefore, the use of fast isolation methods for revealing the initial trigger of microglia activation is critical . Studies using a detailed transcriptomic analysis in the cuprizone animal model of MS found that in the early stages of demyelination, a microglia phenotype supportive of regeneration is observed . It stays to be determined if this is also observed in tissues affected by multiple sclerosis, which are normally available only after a very long continuance of the disease. Nevertheless, multiple sclerosis lesions disappear throughout the disease and early stages of lesion development (microglia clusters considered pre-active lesions) are honored even after a long continuance of the disease. Detailed studies using animal models may provide important facts about the role of microglia in lesion development in MS .
Oligodendrocytes are diligent in the production of myelin in the CNS . In CNS tissues, various development stages of oligodendrocytes can be observed, including the pre-progenitor, progenitor, pro-oligodendrocyte, and juvenile oligodendrocytes. Each of these steps can be identified
In general, isolation of primary oligodendrocytes is dependent upon their ability to not adhere to culture plates. This feature has benefits because by gently shaking isolated cells from the CNS, microglia and adherent atrocities can be separated from floating oligodendrocytes . A disadvantage of the primary cell culture of oligodendrocytes is that they are usually only available in small numbers. However, mature oligodendrocytes can be obtained when glia progenitors are cultured in serum-free media or by differentiating stem cells .
Other important cells that change with MS include astrocytes. In the injured brain, the shape of astrocytes turns into hypertrophic form and the scar tissue as typical of chronic MS lesions generate [53, 54]. When astrocytes are damaged, they are at the risk of losing the ability to maintain the blood–brain barrier (BBB), thus contributing to additional damage . Astrocytes also contribute to the repair process by secreting growth factors, so they have the ability to promote regeneration [56, 57]. By now, several human and animal cell lines are available, as well as primary astrocyte cultures. Primary astrocyte cultures often grow slowly. Therefore, it is the advantage of astrocytes not only being suitable for repeated subcultures, but also suitable for cryopreservation . Many studies using the cell lines of astrocytes are available. Usually, they have been derived from rodents or isolated from human brain tissue with astroglioma. A weakness of astrocytic cell lines is that they respond in a different way in comparison with primary cultures . Several protocols allow obtaining primary astrocytes from adult tissues or post-mortem fetal as well as from biopsies or resected brain tissue in neurosurgery cases. While primary human astrocytes are attractive to culture, care must be taken to ensure the adequate removal of microglia that frequently contaminate primary astrocyte cultures and may influence responses in culture .
In MS research, the increasing awareness that axonal damage and neurodegeneration contribute to the progression of the disease has prompted researchers to use neuronal cells. One ordinary cell line is the neuroblastoma cell line is SH-SY-5Y that can be differentiated with retinoic acid (RA), while the HCN and the NT2 cell lines are differentiated with brain-derived growth factor (BDNF). But it has to be considered that these cells cannot express many markers of mature neuronal cells. These cell lines are also slow to proliferate
Auto-immune or inflammatory patterns, such as examples of experimental allergic encephalitis (EAE) and viral patterns.
Demyelinating or remedial models, including models for chemical damage induced by cuprizone, lysolecithin and ethidium bromide.
4.1 Experimental autoimmune encephalomyelitis (EAE)
EAE is a spectrum of neurological disorders. EAE can be induced in laboratory animals such as rats and mice after immunization with CNS antigens emulated in an adjuvant to enhance immune response .
Usually, in these models, purified myelin, recombinant proteins, or peptides related to encephalitogenic myelin protein are applied. Notably, myelin lesions and inflammation in MS are common features; however, neurodegeneration is a major characteristic as well. Therefore, the secondary progressive EAE was developed with main characteristics such as cortical demyelination, experimental inflammatory neurodegenerative, and spastic diseases [22, 65].
The clinical course of EAE depends on several factors, including the immunization protocol, the antigen, and age, gender species, and strain of the animals. With the active immunization protocol, the first signs of neurological illness are usually weight and activity loss and can be observed between 10 and 17 days. When the adoptive transfer method is used, signs are seen a little earlier, starting 5–7 days after cell transfer [21, 24].
The EAE clinical signs are typically rated based on a muscle force scale (0 to 5) that reflects increasing degrees of paresis, with grade zero being normal and graded 5 being moribund. There are other scoring systems depending on the species used and focus on other clinical signs than just paresis (muscle weakness). Neurological disease can also be monitored using a rotor rod to measure dexterity, and as well as by monitoring behavioral changes .
4.2 Toxin models
A further possibility of investigating demyelination with subsequent remediation is the use of toxin models . In these models, demyelination is induced after focal application or systemic administration of the toxin. Copper-chelating cuprizone is the most common toxin used to induce demyelination in the CNS using systemic administration .
Cuprizone or oxalic acid bis cyclohexylidene hydrazide is a selective and sensitive copper-chelating agent. Shortly after its discovery, it was used to detect copper in serum. W. Carlton was one of the first ones to systematically study and describes disabling changes in cuprizone-fed animals, including sponginess, edema formation, hydrocephalus, demyelination of the central nervous system, and liver damage . Based on Carlton’s findings, we can find several studies using 6–9-week-old mice with a diet containing 0.2–0.3% cuprizone. In older animals, the concentration of comparison must be high in the feeding to guarantee adequate demyelination. In addition, vulnerability to cuprizone that depends on the strain has been reported. For example, there is a difference in the anatomical distribution of demyelinated foci between SJL and C57BL/6 mice. Based on previous studies, we cannot observe demyelination at the brain midline within the corpus callosum in SJL mice; however, demyelination can be seen immediately from the lateral part of mice brain . Therefore, focusing on these variables can be very important before starting any studies.
4.3 Viral models
Viral myelin damage models have been used to investigate the relationship of viruses in multiple sclerosis, and they have led to major breakthroughs in our understanding of the pathology of multiple sclerosis . Both genetic and environmental factors have been implicated in MS, with greater importance attributed to the latter .
A possible role of viruses in the pathology of MS is suggested by epidemiological studies by the detection of viral antigens and virus-specific antibodies in the greater part of MS patients . Several mechanisms can explain how viruses can induce demyelination and also involved in MS . Damage may consequence either from an effect on neurons directly, known as a secondary event (the so-called inside-out model), or from a direct attack on myelin, in which case neurons die due to the lack of trophic support by myelin (the so-called outside-in model).In brief, the virus can damagethe CNS through direct infection of oligodendrocytes, which can be seen in progressive multifocal leukoencephalopathy and lately in MS patients following immunomodulatory therapies . Moreover, viruses can irritate infected oligodendrocytes to attack the CNS. Through these direct effects, virus infection can affect myelin and neurons
4.3.1 Theiler’ s murine encephalomyelitis virus (TMEV)
Theiler’s Murine Encephalomyelitis Virus, first named by Max Theiler, is a natural pathogen of black eyes, causing paralysis and encephalomyelitis. Unlike Semliki Forest virus (SFV), Theiler’s murine encephalomyelitis virus infection causes clinical neurological disease in immunocompetent mice, as well as atrophy of the brain and spinal cord. In addition, myelin damage is seen in bare/bare athymic mice, indicating a direct effect of viral infection independent of immune response .
4.3.2 Murine hepatitis virus (MHV)
Like TMEV, MHV is a natural pathogenic agent of mice that infects all types of CNS cells (neurons, astrocytes, and …). Specific strains of MHV, such as John Howard Mueller (JHM), have a distinctive tropism of CNS leading to severe acute encephalitis . Strains with a less pronounced neurotropism, such as the gliatropic MHV-A59 strain, generally establish a persistent CNS infection, contributing to chronic inflammation and demyelination. Mice inoculated intra-nasally or intra-cerebrally with the JHM virus or MHV-A59 strains mount a robust immune response leading to an influx of immune cells that largely clear the virus, although low-level viral infection persists in animals surviving from the acute infection . In contrast to TMEV, infected MHV-susceptible mice develop a single major symptomatic episode such as hind limb laziness, ataxia, and paralysis, most of which recover. Demyelination begins about a week after infection, with the peak at week 3–4, after which lesion repair and remyelination can occur .
4.3.3 Semliki Forest virus (SFV)
SFV is a neurotropic alphavirus of the family Togaviridae that infects neural cells in the CNS such as neurons and oligodendrocytes. In adult C57BL/6 and BALB/c mice, the virus is largely cleared from the CNS by 6 days post-infection. Demyelination peaks around day 14 and subsequently wanes, with sporadic and mild clinical symptoms . The CNS demyelination observed in SFV-infected mice appears to involve T cells, as demyelination does not occur in nude or SCID mice. Indeed, in BALB/c mice, depletion of CD8+ but not CD4+ T cells abolishes demyelinating lesions. Demyelination may also occur following cytolytic damage of virus-infected oligodendrocytes. In this model, in C57BL/6 mice, molecular mimicry may also take on a role in demyelination, as infected mice exhibit proliferative T cell responses to myelin basic protein (MBP), and antibodies (Abs) reactive to MBP and myelin oligodendrocyte glycoprotein (MOG). Indeed, it was suggested that demyelinating lesions are mainly made by antibody responses, which have cross reaction to MOG and the SFV E2 protein .
4.3.4 Sindbis virus (SV)
The VS infection in LSU mice is another demyelinating model. This model has not been extensively studied by investigators; however, recent findings indicate that pathogenic infection may cause autoimmune disease . Infected LSU mice develop EAE-like palsy that begins at 6 dpi and lasts up to 8 weeks after infection. Treatment with cyclophosphamide improves the signs of neurological deficits despite the increase in CNS viral load, indicating that paralysis in these mice is mediated by the immune response. CNS lymphocytes taken at 7 dpi are specific to VC, but not to MBP. Interestingly, MBP-specific T cell and Ab responses are found in the peripheral tissues at 8-week post-infection, indicating that anti-myelin responses arise due to bystander damage
4.4 Transgenic, mutant, and parabiotic mice
The accessibility of mutant, transgenic, and probiotic experimental animal models could increase our knowledge about the mechanisms of myelin and axonal damage . These models include mutant mice in which defects in myelin assembly occur. Another one is transgenic mice with deleted or inserted genes coding for immune components, and the other one is conditional knockout mice with genes manipulations in mature animals . The generation of parabiotic mice has come about recently. The role of specific myelin proteins in myelin synthesis and remyelination has been examined in mutant mice in which the CNS myelin is affected. These include the Shiverer mouse in which the MBP gene is duplicated and inverted. Also, these consist of the Rumpshaker mouse with mutated proteolipid protein (PLP) Plp1 and the Jimpy mouse with the PLP gene mutation. These genetic mutations result in dysmyelination due to, for example, oligodendrocyte apoptosis and lastly neurodegeneration because of myelin damage. The importance of the role of immune responses in multiple sclerosis has led to the development of transgenic mice by focusing on myelin proteins or specific immune molecules. MOG-knockout mice have no recognizable phenotype. However, the use of its for EAE studies has revealed a major role for autoimmunity to MOG in chronic EAE in both mice and marmosets .
The parabiotic model is the act of living side-by-side . Parabiotic studies involve suturing two mice together so blood vessels can connect, creating mice that share a common blood supply. Thus, the effect of the treatment of one mouse can be considered in the other mouse. Previous studies by using irradiation and separation of parabiotic mice indicated the importance of infiltrating monocytes in the induction of clinical signs of EAE . Such surveys have been performed using transplantation of donor cells, although it has often been impossible to distinguish between donor cells and activated microglia from the host. Another study using isochronic (same age) or heterochronic (same age) parabiotic mice indicates that exposure of young mice to the blood flow of old mice interferes with neurogenesis. This parabiotic study revealed age-related chemokines, suggesting the presence of rejuvenation factors. Heterochronic parabiotic mice are useful for understanding this point that remyelination is not merely more efficient in juvenile mice, but also some factors from juvenile mice can even restore the potential of remyelination in old mice .
There is no one specific model showing all the factors involved in the pathogenesis of multiple sclerosis, so the researchers attempt to acquire a spacious range of examples that can imitate various MS features. Extremely, the development of alternative and mixed models will contribute to an improved understanding of multiple sclerosis and its treatment. With the help of animal models, information on response to treatment can be extrapolated. The development of models better able to reproduce the pathological changes of MS constitutes the beginning stage in the development of novel treatments; however, we are most likely to achieve a genuine understanding of MS through data from patients. The existing examples have shown useful in the evolution of MS treatment, and further integration of these examples will help further our understanding of the etiology.
Conflict of interest
The authors declare no conflict of interest.
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