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Comparison between a normal marmoset brain and a marmoset brain model of Parkinson's disease induced by subcutaneous administration of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Both coronal sections appearing the largest area of the striatum.
Brief Note
We believe that the marmoset model of Parkinson's disease induced by the administration of the neurotoxin MPTP possesses high disease validity (similarity to the human disease) and offers greater utility compared to similar models using large macaques.
Contents of this page
1. Introduction
1. Introduction
When the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is administered subcutaneously or peripherally to the common marmoset, a small monkey, behavioral changes such as tremors, immobility, impaired posture, and muscle rigidity are observed for several months or more. When the brains of these marmosets are examined by in vivo (living) Positron Emission Tomography (PET) or in vitro (postmortem) immunohistological methods, degeneration or loss of dopamine neurons in the substantia nigra-striatum is observed (Ando et al., 2012).
The behavioral syndrome and neurodegeneration of the brain of such MPTP-treated marmosets are very similar to those of Parkinson's disease (PD). Therefore, this is considered to be an experimental animal model with high disease validity for PD, and has been used by many researchers (Jenner and Marsden, 1986, Nomoto, 1995). MPTP-based PD models have also been used in macaques and squirrel monkeys, and historically, these monkeys have been used more than marmosets (Langston et al., 1984).
However, we believe that the MPTP-treated marmoset PD model has advantages that are different from, and even surpass, those of macaques. Here, we describe the practical aspects of experiments using MPTP-treated marmosets by items below (Ando, 2018).
See this website page: Marmoset as Experimental Primate
For the author's Japanese commentary PDF on the marmoset MPTP treatment model, see the following URL: https://researchmap.jp/read0179769/published_papers/19447809
2. Selection of experimental animal species for creating MPTP-treated PD model
2. Selection of experimental animal species for creating MPTP-treated PD model
First, as a prerequisite for this project, we would like to state that the neurotoxicity of MPTP is expressed in humans and monkeys. In rats and mice (excluding some strains), the toxicity cannot be observed due to species-specific differences in drug metabolism. Therefore, monkeys are basically the first choice for creating a PD model using MPTP.
3. Ease of handling as an experimental animal
3. Ease of handling as an experimental animal
Marmosets are smaller and less aggressive than large macaques, making them easier to handle as experimental animals. Furthermore, we believe that the use of marmosets has a significant advantage over macaques in terms of safety from the toxicity of MPTP. The ammount of MPTP use in the experiment is smaller in the marmosets than the macaque monkeys.
4. Storage, use, and management of MPTP pure powder and solution
4. Storage, use, and management of MPTP pure powder and solution
This neurotoxin requires extremely careful handling. This is natural considering the history of how MPTP was originally ingested by humans and the manifestation of severe PD-like syndrome led to its use in experiments involving monkeys. However, a distinction should be made between cases where a drug abuser actively and voluntarily ingested large amounts of MPTP at that time and cases where an experimenter carefully and cautiously handles MPTP in an experimental setting. That being said, the processes of storing MPTP pure powder for experimental use, preparing its solution, storing the prepared solution, administering the prepared solution, and disposing of animal waste after administration should be handled extremely carefully for the safety of the experimenter. A series of these procedures must be defined in a standard operating procedure (SOP). Furthermore, it is necessary to monitor whether the actual work is being carried out according to the procedure. In addition, sodium hypochlorite solution is known to weaken the toxicity of MPTP at the appropriate concentration and over time (Przedborski et al., 1996), and it is important to use it appropriately. A sample of the standard operating procedure is described in Additional Note 1 below.
5. Safe MPTP administration procedure for creating a PD model in marmosets
5. Safe MPTP administration procedure for creating a PD model in marmosets
The creating a PD model in marmosets has been established through much trial and error. MPTP is dissolved in saline or other liquids, and MPTP HCl is purchased and used. The administration schedule is as follows: MPTP is administered subcutaneously at 2 mg/kg, 2 mg/kg, and 1 mg/kg (2 mg/kg on the third day depending on the marmoset's syndorome) for three consecutive days (Ando et al., 2020). However, this dose is calculated based on MPTP as the base dose. When calculating the doses as MPTP HCl including salt, 1.2 times the dose of MPTP HCl above should be used. Just to be clear, MPTP is obtained as MPTP HCl, not as MPTP base powder, which is not water-soluble.
After MPTP administration, marmosets hardly eat or drink spontaneously for more than two weeks. Therefore, it is necessary to replenish water and nutrients, electrolytes, and glucose, multiple times a day, including on holidays. If this effort is not taken care of, the marmoset will become weak and die. This is one of the most important points when conducting MPTP administration experiments.
On the other hand, in the case of cynomolgus macaques, MPTP HCl is administered once only subcutaneously, intramuscularly or intravenously, and the condition is observed. The same dose of MPTP is administered repeatedly at intervals of 1 to 2 weeks for several months. During this time, as in the case of marmosets, the experimenter must replenish nutrients multiple times a day, including on holidays. In addition, the treatment of large amounts of excrement containing MPTP is also required, which requires more work and effort than marmosets. Moreover, in the case of cynomolgus monkeys, the individual differences in sensitivity to MPTP are greater than in marmosets, and while some monkeys show clear syndrome, other monkeys may not show any changes even under the same administration conditions. In some cases, the frequency of sudden death due to sudden physical debilitation without any clear reason is higher than in the marmosets.
For these reasons, we believe that creating a PD model in marmosets has advantages over macaques in terms of the safety of the experimenter, the efficiency of the experiment, and the reliability of the data. As for the reason for the low inter-individual variation in susceptibility to MPTP in marmosets, we believe that marmosets are bred and raised for many generations under controlled conditions for many years, which maintains homogeneity among individuals. This also leads to relatively small data variability, which is related to the reliability of the data.
6. MPTP-induced PD-like syndrome and their measurement
6. MPTP-induced PD-like syndrome and their measurement
To be a PD model, the behavioral changes after MPTP administration must manifest as PD-like syndrome and persist. Immediately after administration, acute MPTP toxicity appears, and the marmoset loses spontaneous food and water intake and becomes weak. After about two weeks of recovery, PD-like ssyndorme such as moving tremor, impaired postural control, and muscle rigidity appear, as described above. In addition, PD like immobility is observed in a different form to the immobility caused by acute toxicity and weakness immediately after MPTP administration.
Therefore, the issue of how to accurately measure these PD-like syndrome is an issue. First, it is necessary to grasp the overall picture of the syndrome. Using a Dysfunction score, which is a set of items related to motor function, an experienced observer observes and records the presence or absence of singn in MPTP-treated marmosets and how they have changed compared to before treatment (Ando et al, 2008, Ando et al, 2012). Such macroscopic observations are extremely important for understanding the behavior of marmosets from a bird's-eye view. When doctors diagnose PD patients and understand the effectiveness of treatments such as medication, they use the Unified Parkinson's Disease Rating Scale (UPDRS), Hoehn and Yahr's severity classification (Yahr, 1993), and other scores. These score observations are the starting point for understanding the syndrome (signs and symptom) of PD patients as a whole, and the goal for observing the effectiveness of treatment, so they are extremely important observations. A motor dysfunction score sheet for syndrome observation of the Parkinson's disease model marmoset is described in Additional Note 2 below.
On the other hand, in the experiment with marmosets, it is important to objectively and quantitatively measure the PD-like syndrome of marmosets in addition to the scores obtained by macroscopic observations. This is because it is necessary to clearly, objectively and quantitatively determine whether treatments such as drug candidate substances and neuronal transplants have an effect of improving syndorme. Therefore, the locomotion or spontaneous motor activity of marmosets in individual cages is continuously recorded for several months using a motion-sensing (proximity) sensor (Ando et al., 2020). The decrease in locomotion 1 or 2 weeks after MPTP administration is an objective, quantitative indicator of immobility, which is one of the important indicators of PD-like syndorme. There is a major reason for using this indicator in assessing the effectiveness of treatments such as drugs and neuronal cell transplantation, because it can clearly and sensitively capture whether immobility improves (Ando et al., 2008).
Tremors can also be objectively and quantitatively grasped using computer image analysis. However, this behavior cannot be grasped as a holistic and integrated behavior like spontaneous locomotion, but rather appears as an episodic and partial behavior that is sometimes seen. Therefore, it cannot be said that measuring tremors alone is sufficient to grasp the effects of treatments such as drug administration and neuronal cell transplantation from a bird's-eye view and global perspective. Other syndorme such as postional dysfunction and muscle rigidity, when measured individually, can be said to only observe partial aspects, like tremor. However, accurately measuring and understanding these objectively and quantitatively is an important academic research topic. Preclinical research aimed at unambiguously determining the effectiveness of drugs and neural cell transplants and academic research to clarify pathology have different ultimate goals, and it is necessary to distinguish between them when conducting research.
For the above reasons, the PD model marmoset must be useful in preclinical research to determine the effectiveness of drugs and neural cell transplants. In that sense, we believe it is appropriate to grasp the overall symptoms related to PD-like syndrome by scoring them with the naked eyes of experimenters, and at the same time, to objectively and quantitatively grasp the amount of spontaneous movement using a sensor. We believe that this will provide basic scientific data from animal experiments in preclinical research as a basis for determining the appropriateness of treatment for human PD.
7. MPTP-induced dopamine neurodegeneration in the nigrostriatal system and its detection method
7. MPTP-induced dopamine neurodegeneration in the nigrostriatal system and its detection method
The above syndrome caused by MPTP administration are thought to be mainly due to degeneration and loss of dopamine neurons in the nigrostriatal system of the brain. In fact, many studies have already published evidence supporting neurodegeneration histopathologically, biochemically, and neurocytologically. In addition, dopamine neurodegeneration and other syndrome have been observed in parallel with the onset of syndrome by in vivo image analysis using Positron Emission Tomography (PET) and other methods in PD model monkeys (see Marmoset Brain Imaging on this website).
PET measurements were also performed in preclinical evaluation studies using PD model marmosets (Ando et al, 2012). As a result, a high correlation of r = 0.98 was observed between the binding potential of [11C]PE2I, a ligand for the dopamine neurotransporter, in the striatum (putamen) and the decrease in spontaneous locomotor activity, which is an indicator of immobility. This shows that in the marmoset PD model, dopamine neurodegeneration and syndrome manifestation are almost parallel. In addition, histological examination of marmoset postmortem brains using tyrosinhydroxilase (TH) immunohistilogy showed that the TH staining area in the MPTP-treated marmoset brain was significantly reduced, clearly indicating that catecholamine neurons, including dopamine neurons, had been lost. In a quantitative examination of the staining area using NIH Image J, there was a correlation between the reduction in the TH staining area in the striatum and the reduction in spontaneos locomotor activity, with a coefficient of 0.83 or 0.93 (see Marmoset Brain Imaging on this WEB site).
In preclinical medical research such as pharmaceuticals and cell transplantation, syndrome improvement and its neurological support are important, so it is necessary to quantitatively and clearly grasp the neurodegeneration in the PD model marmoset and its protective effects and drug efficacy. In the marmoset PD model, an experimental system has been completed that can clearly detect the loss and protective effects of neurodegeneration as well as the syndrome. This model is similar to human PD in terms of syndrome expression and neurodegeneration, and is useful in preclinical research on drug development and other treatments. In this respect, this model is one of the few useful experimental animal models with high disease validity in the field of neuropsychiatric disease research. This is because the PD model is related to motor dysfunction that is highly common between humans and monkeys. In this respect, it is necessary to point out the fact that there are facts that differ from animal models of psychiatric disorders related to higher brain functions, such as schizophrenia and bipolar disorder.
8. References
8. References
Ando K, Inoue T, Hikishima K, Komaki Y, Kawai K, Inoue R, Nishime C, Nishinaka E, Urano K, Okano H (2020) Measurement of baseline locomotion and other behavioral traits in a common marmoset model of Parkinson's disease established by a single administration regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: providing reference data for efficacious preclinical evaluations. Behav Pharmacol 31:45-60.
Ando K, Maeda J, Inaji M, Okauchi T, Obayashi S, Higuchi M, Suhara T, Tanioka Y (2008) Neurobehavioral protection by single dose l-deprenyl against MPTP-induced parkinsonism in common marmotsets. Psychopharmacology 195:509-516.
Ando K, Obayashi S, Nagai Y, Oh-Nishi A, Minamimoto T, Higuchi M, Inoue T, Itoh T, Suhara T (2012) PET analysis of dopaminergic neurodegeneration in relation to immobility in the MPTP-treated common marmoset, a model for Parkinson's disease. PLoS One 7:e46371.
Jenner P and Marsden CD. (1986) The actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in animals as a model of Parkinson's disease. J Neural Transm Suppl 20:11-39.
Langston JW, Forno LS, Rebert CS, Irwin I (1984) Selective nigral toxicity after systemic administration of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyrine (MPTP) in the squirrel monkey. Brain Res 292:390-394.
Przedborski S., Jackson-Lewis V., Yokoyama R., Shibata T., Dawson V. L. & Dawson T. M. (1996) Role of neuronal nitric oxide in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced dopaminergic neurotoxicity. Proc Natl Acad Sci USA 93, 4565–4571.
Yahr MD (1993) Parkinson's disease: new approaches to diagnosis and treatment. Acta Neurol Scand Suppl 146:22-25.
Following Articles in Japanese
Ando K (1918) A common marmoset model of Parkinson's disease induced by administration of the neurotoxin MPTP – Preclinical evaluation using behavioral analysis – Obelisk Vol.23,1:14-22.
安東潔 (1918) 神経毒MPTP 投与によるコモンマーモセットのパーキンソン病モデル – 行動解析による前臨床評価を中心として – オベリスク Vol.23,1:14-22. https://researchmap.jp/read0179769/published_papers/19447809
Nomoto M (1995) Application of marmosets (small monkeys) to pharmacological research. Japanese Journa of Pharmacology, 106:1, 11-18. Download PDF (6345K)
野元正弘 (1995) マーモセット(小型のサル)の薬理学研究への応用. 日本薬理学雑誌106:1, 11-18. PDFをダウンロード (6345K)
Additional Note 1
Additional Note 1
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Standard Operating Procedure (SOP) for the use of MPTP in experiments
Standard Operating Procedure (SOP) for the use of MPTP in experiments
Experimental facility XXX
1. Purpose of this SOP:
This SOP stipulates how to administer the neurotoxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) to marmosets appropriately when using the chemical in an experiment, and how to use the chemical so that it does not come into contact with the human body.
2. Purchase of MPTP:
As a rule, MPTP HCl from Sigma Aldrich is purchased as MPTP. In addition, when purchasing, the amount purchased and other necessary information are recorded in the MPTP ledger.
3. Storage and use of MPTP:
After designating a person responsible for MPTP storage and management, purchased MPTP is stored in a designated chemical storage cabinet under lock and key. In addition, when using, the amount used and other necessary information are recorded in the ledger.
4. Preparation of MPTP solution:
When preparing MPTP, the necessary information should be recorded in the preparation record. The weight of MPTP should be measured using an electronic scale for MPTP in a designated room. When preparing the solution, wear the designated disposable hat, mask, gloves, and protective clothing, and immerse these, along with the equipment used in preparation, in 1% sodium hypochlorite solution for at least 10 minutes to detoxify them. After that, entrust the disposal to a contractor as industrial waste as described below. For sodium hypochlorite, use 6% Purax-S (Oyalux Co., Ltd.) or similar. To prepare a 1% concentration solution, dilute 100 ml of Purax with 500 ml of water.
5. Storage of MPTP preparation:
The preparation storage container should be double wrapped in a plastic bag or similar, labeled as MPTP preparation and hazardous, and stored in a designated refrigerator.
6. Administration of MPTP preparation:
When administering the preparation to marmosets, only the designated person administering the preparation should administer it correctly through the designated administration route. When administering, the designated disposable hat, mask, gloves, arm covers, protective clothing, apron, protective face mask, leather gloves, and boots should be worn.
7. Disposal of remaining MPTP preparation and administration equipment:
The remaining preparation is soaked in moisture-absorbing material and disposed of as industrial waste together with the used administration equipment (disposable syringes and needles), disposable hat, mask, gloves, protective clothing, hood, and arm covers after the above-mentioned detoxification process. In addition, the protective face mask and leather gloves used during administration are detoxified with 1% sodium hypochlorite solution or sprayed with this solution before reuse during the MPTP administration period, but are detoxified as described above and disposed of as industrial waste after the end of the administration period. The apron and boots are reused after detoxification process.
8. Management of administered animals:
The marmosets receiving MPTP should be kept in cages isolated from other marmosets, and their general condition should be observed during the administration period. The feces and urine of the animals from the start of the administration period to the end of the final administration should be absorbed in moisture-absorbing material laid under the floor of the cage. On the day after the final administration and at least three days after the final administration, the material laid under the floor should be placed in a double-sealed plastic bag, labeled as MPTP waste and toxic, and disposed of as industrial waste. After three days or more have passed since the final administration, the perches should be sufficiently detoxified or replaced with new ones. The feeding boxes should be detoxified in the relevant breeding room each time during the administration period and then reused. After three days or more have passed since the final MPTP administration, the feeding boxes should be disposed of or sufficiently detoxified and reused. After three days or more have passed since the final administration of MPTP, the marmosets should be temporarily removed, the inside of the cage should be sprayed with 1% sodium hypochlorite solution, and after leaving it for at least 10 minutes, the cage should be wiped off with a cloth soaked in water or a Kimtowel. During general condition observation, the designated hat, mask, gloves, and protective clothing are worn, which are detoxified after use and disposed of as industrial waste. If an animal dies during the administration period or within 3 days of the end of the final administration, only the weight is measured, and the carcass is detoxified by immersing it in 1% sodium hypochlorite solution for more than 10 minutes, placed in a double-sealed plastic bag, and a note is written on it that it is an animal administered MPTP and that it is harmful to humans. It is stored in the designated animal carcass disposal freezer on the fixed place, and disposal is entrusted to a contractor. The necessary information is written in the animal death record.
9. Treatment of the MPTP administration experiment site:
In the room where the MPTP preparation solution was administered to the marmosets, 1% sodium hypochlorite solution is sprayed on the walls, etc. to detoxify them, as in the cages, and then wiped off with water after 3 days after the end of administration. In addition, 1% sodium hypochlorite solution is sprayed on the areas along the air flow in the cage to detoxify them.
10. Disposal of the waste containing MPTP:
The above MPTP-containing waste incineration treatment will be outsourced to a contractor based on a contract concluded between the experimental institution and the contractor. Until the materials to be treated are removed, bags labeled with a hazard mark and labeled as MPTP-containing materials will be stored in a specific location. When the experimental institution concludes a normal contract with these contractors, it is agreed in writing that the treatment of MPTP-containing materials will also be included.
11. Response to excessive MPTP exposure to humans just in case:
Ultimate care must be taken when handling MPTP, but in the unlikely event that an experimenter is exposed to a significant amount of MPTP or its preparation, the Principal Investigator (PI) of the research using MPTP will be contacted and the response will be discussed immediately. The PI will request treatment from a neurologist at a university hospital in Tokyo who has given prior consent. The PI will be appointed by the head of the institution before the start of the experiment.
Additional Note 2
Additional Note 2
A motor dysfunction score sheet for syndrome observation in Parkinson's disease model marmosets
A motor dysfunction score sheet for syndrome observation in Parkinson's disease model marmosets
Explanation of the motor dysfunction score sheet
MPTP-treated marmoset Parkinson's disease model:
The model marmoset shows immobility, moving tremors, impaired postural reflexes, and muscle rigidity, which are similar to the main syndrome of human Parkinson's disease, and these syndrome are continuously and stably expressed for several months or more. The motor dysfunction score is a rough observation of the overall profile of the syndrome, by the experimenter macroscopically/subjectively. On the other hand, the main observation is a quantitative index measured by a proximity sensor for the spontaneous motor activity (locomotion) of the model marmoset in an individual living cage. This is an index that can objectively and quantitatively measure one of the main syndrome "immobility", and is the most important index for determining the stability of the model and the presence or absence of an improving effect of the test substance or implanted neural cells on the model.
Therefore, the motor dysfunction score value described here can be considered as a supplementary index that supports the spontaneous motor activity, which is the above-mentioned objective index. However, just because it is a supplementary index, it should never be overlooked. It is extremely important to understand the overall profile behind the amount of spontaneous motor activity.
Observation of motor dysfunction and recording on the score sheet:
Motor dysfunction, including the above-mentioned major syndorme, is observed and recorded by the experimenter with the naked eye in the model marmoset. This score sheet contains items related to various motor dysfunctions, including major Parkinson's disease-like syndrome (items 1 to 11), and the experimenter records only the presence or absence of each item with 1 or 0. The total score is used as the motor dysfunction score.
Item 12 dyskinesia and item 13 hyperexcitability are reference items to confirm the presence or absence of side effects when the positive control drug such as L-DOPA is repeatedly administered, and are not included in the above score.
Scoring method for motor dysfunction score:
For the score, the experimenter records whether or not each item is applicable with 1 or 0. Many other scores have a method of adding points such as 1, 2, 3, etc. depending on the severity. Here, we did not adopt such a method. The reason is to record only clear changes and minimize subjective differences between experimenters. In addition, the motor dysfunction score value is calculated by adding up the frequency of occurrence for each item for each marmoset, which is a ratio scale value data and is a value that can withstand statistical analysis between the control group and the test substance administration group. On the other hand, the value obtained by adding points such as 1, 2, 3, etc. is an order scale value data, that is, a score of 2 is not guaranteed to be twice as severe as a score of 1 in terms of the degree of motor dysfunction. Therefore, we believe that it cannot withstand general statistical analysis except for statistical analysis that takes into account the order scale. Statistical analysis is extremely important in determining the presence or absence of an improvement effect of the test drug, etc.
Syndrome observation points for motor dysfunction score:
First of all, it is important to confirm the baseline of how clearly and stably the model marmoset is expressing syndrome as a pathological condition before administering the test drug, etc. If this is insufficient, there is a problem with the administration conditions of the neurotoxin MPTP used to create the model or with the marmosets used, and reexamination and re-creation of the model are necessary.
On the other hand, even if observations are performed based on the motor dysfunction score for model marmosets that clearly exhibit Parkinson's disease-like syndrome, tremors, postural reflex disorders, muscle rigidity, etc. are not always observed at the time of observation, and the onset is often episodic.
Therefore, as mentioned above, in this model, it is important to observe and record the spontaneous motor activity of marmosets in individual living cages for a long period of time, such as 24 hours, from before MPTP treatment. This spontaneous motor activity decreases continuously due to MPTP treatment. As mentioned above, this is an objective quantitative indicator of immobility, one of the main syndrom of Parkinson's disease. In other words, after confirming the stable high-frequency spontaneous motor activity before MPTP treatment as a baseline, the sustained decrease in the motor activity after MTPT administration and the recovery from the decrease in the motor activity as an improvement effect of the treatment of the test drug, etc. are confirmed, and the presence or absence of the improvement effect of the test drug, etc. is objectively and quantitatively judged through the main syndorme of immobility. In preclinical trials, we believe that the significance of unambiguous effect judgment by such objective quantitative indicators is extremely important.
Preclinical trials require a clear conclusion:
For the above reasons, when a preclinical trial is conducted, a clear conclusion is required as to whether or not the test substance had an improvement effect on the syndorme of the model marmoset. Even if a clear conclusion is not obtained, it is necessary to clearly state which part of the test metod had a problem and was unclear. In this sense, we believe that it is important to first grasp the improvement of the syndrome of the model marmoset by capturing the improvement of objective quantitative indicators of spontaneous motor activity, and to understand the various syndrome conditions behind it as a supplementary but overall picture by the motor dysfunction score, removing as much subjectivity across experimenters as possible.
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