preparation of mog35-55 emulsion for immunization of mouse

Induzione e valutazione dell'encefalomielite autoimmune sperimentale nel topo

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It shows the presence of perivascular infiltration of inflammatory cells, demyelination, axonal loss, and gliosis1. Its etiology remains unknown, and its clinical aspects, radiographic, and pathological features suggest remarkable heterogeneity in the disease2.
Due to its unknown etiology and complexity, at present, no animal model recapitulates all the clinical and radiological features displayed in human MS3,4. However, various animal models are employed to study different aspects of MS3,4. In these models, disease initiation is typically extremely artificial, and the timeframe of the onset of clinical signs is different between humans and mice. For example, in humans, the pathophysiological processes underlying the disease are undetected for years before the onset of clinical manifestations. Conversely, the experimenters can detect symptoms in animal models within weeks or even days after MS induction4.
Three basic animal models produce the features of demyelination that are characteristic of MS: those that are virus-induced (e.g., Theiler&#39;s murine encephalomyelitis virus), those that are induced by toxic agents (e.g., cuprizone, lysolecithin), and the different variants of experimental autoimmune encephalomyelitis (EAE)5. Each model helps study some specific facets of the disease but none replicates all the features of MS6. Thus, it is critical to choose the correct model considering the specific experimental needs and the scientific questions to be addressed.
Thanks to immunization procedures against myelin-derived antigens, EAE is induced by triggering an autoimmune response to CNS components in susceptible mice. The interplay between a wide range of immunopathological and neuropathological mechanisms causes the development of principal pathological traits of MS (i.e., inflammation, demyelination, axonal loss, and gliosis) in the immunized mice7,8. Mice begin to show clinical symptoms around the second week after immunization and generally show ascending paralysis from the tail to the limb and forelimb. The clinical score (i.e., quantification of the accumulation of disease-related deficits) is generally assessed using a 5-point scale7.
Active immunization with protein or peptide or passive transfer of encephalitogenic T cells can be used to induce EAE in mice with different genetic backgrounds (e.g., SJL/J, C57BL/6, and non-obese-diabetic (NOD) mice). Myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG) are examples of self-CNS proteins from which immunogens are usually produced. Particularly, SJL/J mice immunized with the immunodominant epitope of PLP (PLP139&#8722;151) develop a relapsing-remitting (RR) disease course, while C57BL/6J mice immunized with the immunodominant MOG35-55 peptide show EAE of a chronic nature1. Despite some limitations, such as providing very little information about MS progression, the role of B cells in the disease, the inside-out mechanisms, or difficulties in studying remyelination, the EAE models have hugely contributed to the understanding of autoimmune and neuroinflammatory processes, increasing the knowledge in the MS field and thus allowing the development of novel therapeutic approaches for this disease4,6.
In the present work, we focused on a particular form of active EAE, the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced form9,10,11,12. The MOG35-55-induced EAE models a chronic form of MS. After immunization, the mice undergo an asymptomatic phase within the first week after immunization, then the disease typically arises during the second week after immunization, while between the third and fourth weeks after immunization, the disease becomes chronic, with no possibility of full recovery from the accumulated deficits7,8,13. Interestingly, no differences between males and females in incidence, disease onset, course, or progression are observed in most of the studies present in the literature14, even if fewer studies compare the disease in males and females.
In contrast, in humans, these parameters are known to be strongly sexually dimorphic2. MS affects more women than men; however, men generally develop a more aggressive form of the disease2. This evidence has suggested an essential, as well as complex, role of the gonadal hormones15; nevertheless, the role and the mechanism of action of sex hormones in the pathology remain unclear. Moreover, data from animal models support the idea that both estrogens and androgens exert positive effects on different tracts of the pathology in a sex-specific manner16,17.
Some studies also suggest neuroprotective, promyelinating, and anti-inflammatory effects of progesterone18 and, although evidence in MS patients is scarce18, neuroactive steroids (i.e., de novo synthetized steroids by the nervous system, such as pregnenolone, tetrahydroprogesterone, and dihydroprogesterone) might also affect the pathological course19. Collectively, these data support the idea that sex hormones produced both peripherally and inside the CNS have an important and sex-specific role in disease onset and progression. Therefore, in the present work, we urge the collection of separate data from both male and female animals.
From the histopathological point of view, the white matter of the spinal cord serves as the principal site of CNS injury in this model, which is characterized by multifocal, confluent regions of mononuclear inflammatory infiltration and demyelination8. Thus, in describing this protocol for the induction of MOG35-55-induced EAE in C57BL/6J mice, we will take into account the disease outcome in the two sexes and provide some histopathological insights regarding the spinal cords.

Autore in primo piano: Creazione di un modello sperimentale versatile di encefalomielite autoimmune rilevante sia per i topi maschi che per le femmine 

Modellazione della sclerosi multipla nei due sessi: encefalomielite autoimmune sperimentale indotta da MOG35-55

Our research focuses on the experimental autoimmune encephalomyelitis, or EAE model, which can significantly announce the understanding of peripheral immune-mediated mechanism and evaluate neuroinflammatory and partially delineating processes. This model has also been widely used to develop and test a wide range of therapeutics drugs. Due to the unknown etiology and complexity of multiple sclerosis, no animal model currently recapitulates all the clinical and radiological features displayed in humans.Thus, it is quite challenging for the researchers to select the animal model most fitting for a specific experimental question. EAE is a widely used model of multiple sclerosis. However, to do several issue, only some studies performing this model considered both sexes.With our protocol, we want a light possibility of including both sexes in the study conducted in this model. Specifically, our protocol proposed a clinical evaluation based of two methods:the clinical score assessment and the rotator test, leading a more quantitative, less subjective and more precise clinical assessment of the disease course. Even though this model does not entirely reproduce the sexual dimorphism of multiple sclerosis, it has potential advantages.The combination of the induction with other possible risk factor, especially the environmental ones, can end in understanding specific effects of such factors and identifying the specific role in some sexually dimorphic aspect of the disease.

Watch this Scientific Journal Video about Preparation of MOG35-55 Emulsion for Immunization of Mouse at JoVE.com

Preparation of MOG35-55 Emulsion for Immunization of Mouse  

Preparazione dell'emulsione MOG35-55 per l'immunizzazione del topo

Per iniziare, preparare il peptide glicoproteico oligodendrocitario della mielina a 35-55, o MOG 35-55 soluzione, in un bicchiere di vetro. A tale scopo, aggiungere la componente liquida al becher, quindi aggiungere il ceppo di mycobacterium tuberculosis appropriato. Mettere il bicchiere di vetro contenente la soluzione in un bagno di ghiaccio.Utilizzando una siringa di vetro con un ago calibro 18, avviare il processo di emulsione e continuare ad emulsionare la soluzione per un minimo di 15 minuti. Per valutare la qualità dell'emulsione, erogare una goccia di emulsione in un contenitore trasparente riempito d'acqua. Considerare l'emulsione pronta se la goccia mantiene la sua struttura e rimane intatta.Trasferire l'emulsione direttamente nelle siringhe da un millilitro designate per l'immunizzazione. Conservare le siringhe riempite a quattro gradi Celsius fino a quando non sono pronte per l'uso.

preparation-of-mog35-55-emulsion-for-immunization-of-mouse

Research

JoVE Journal

Neuroscience

Preparation of MOG35-55 Emulsion for Immunization of Mouse

93 Views.  Neuroscience Institute Cavalieri Ottolenghi (NICO). Per iniziare, preparare il peptide glicoproteico oligodendrocitario della mielina a 35-55, o MOG 35-55 soluzione, in un bicchiere di vetro. A tale scopo, aggiungere la componente liquida al becher, quindi aggiungere il ceppo di mycobacterium tuberculosis appropriato. Mettere il bicchiere di vetro contenente la soluzione in un bagno di ghiaccio.Utilizzando una siringa di vetro con un ago calibro 18, avviare il processo di emulsione e continuare ad emulsionare la soluzione per un minimo ...

Video: Preparation of MOG35-55 Emulsion for Immunization of Mouse  

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Ricerca

Neuroscienze

To begin, prepare the myelin oligodendrocyte glycoprotein peptide at 35 to 55, or MOG 35 to 55 solution, in a glass beaker. To do so, add the liquid component to the beaker, followed by adding the appropriate mycobacterium tuberculosis strain. Place the glass beaker containing the solution in an ice bath.Using a glass syringe with an 18-gauge needle, initiate the emulsification process, and continue emulsifying the solution for a minimum of 15 minutes. To assess the quality of the emulsion, dispense a drop of the emulsion into a transparent container filled with water. Consider the emulsion ready if the drop maintains its structure and remains intact.Transfer the emulsion directly into the one-milliliter syringes designated for immunization purposes. Store the filled syringes at four degrees Celsius until they're ready for use.

Immunization of Mouse with MOG35-55 to Induce Experimental Autoimmune Encephalomyelitis

Immunization of Mouse with MOG35-55 to Induce Experimental Autoimmune Encephalomyelitis  

Begin the immunization of the mouse by performing a PT intravenous injection into its lateral caudal vein. To do so, gently plug the tail of the properly anesthetized mouse with an ethanol solution, which acts as a vasodilator to make the veins visible. Focusing on one of the two lateral veins of the tail, use 0.5 milliliter syringe fitted with a 30 gauge needle to inject 500 nanograms of PT.Next, proceed to administer subcutaneous injections of MOG 35 to 55 emulsion previously prepared in one milliliter syringes.Using the syringes with 26 gauge needles, perform two subcutaneous injections of 100 microliters each under the rostral part of the flanks. Then administer one injection of 100 microliters at the base of the tail.

Immunizzazione del topo con MOG35-55 per indurre encefalomielite autoimmune sperimentale

Clinical Score and Motor Performance Evaluation of Mouse Induced with Experimental Autoimmune Encephalomyelitis

After inducing the experimental autoimmune encephalomyelitis in the mouse, have a blinded investigator perform daily assessment of the animal's clinical scores. Score each animal on a scale from 0 to 5 to evaluate the progression of the disease. To evaluate motor performance, initiate Rotor-Rod sessions for the mice daily, starting at 1 DPI until the study requires.Conduct each session for 300 seconds, during which, increase the rotation speed of the rod linearly from 4 to 40 rotations per minute. When the animal loses its balance and falls from the Rotor-Rod device, it lands on the ground triggering a sensor that records the time. Record the animal's performance and score it as a latency to fall.A significant increase in the clinical score or CS with time was observed in both male and female mice, starting from 10 DPI until the endpoint. Females displayed higher CS than males, but not significantly. A tendency for earlier disease onset was also observed in females compared to males.Furthermore, females showed a significantly higher cumulative CS compared to males. A significant decrease in the Rotor-Rod performance with time was observed in both male and female mice. Particularly, males displayed the minimum performance at 16 DPI, while the females at 17 DPI.Males performed better than females, especially during the chronic phase of the disease, probably as a consequence of lower CS.