Animal Models Studying  Human Neuropsychiatric Disease

A brief note

In research involving human disease models using experimental animals, the validity of the model (its similarity to the human disease) and its utility for developing treatments for the disease serve as crucial benchmarks.

1. Animal models of neuropsychiatric dieases - Framework for creating models and conducting test measurements -

The above theme has been taken up many times as a symposium topic at the Japanese Society of Pharmacology and the Japanese Society of Neuropsychopharmacology. In these societies, research using experimental animals is premised on a link with clinical medicine. Therefore, measurement data produced using animal models are subject to strict scrutiny for validity and usefulness from a clinical perspective.

For many years, the author has created various models of neuropsychiatric diseases and conducted research using mice, rats, marmosets, cynomolgus monkeys, and rhesus monkeys. It is true that some of these studies, despite the expenditure of many resources, were merely trial and error and of little significance. However, on the other hand, we can recall that there were also studies that were extremely useful. That is, the preclinical research on Parkinson's disease models, which were treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmosets, and the drug dependence research using intravenous drug self-administration in rhesus monkeys are examples of experimental animal models that are extremely valid and useful. These two examples are very helpful in thinking about what experimental animal models are. Therefore, based on these examples, the author would like to present the framework of an animal model that can serve as a reference for future research development. the author hopes that the content of animal model creation will be organized and that research using more valid and useful models will develop (see the following Table 1).

Table 1. Framework for creating animal models of neuropsychiatric disorders and conducting measurements using them. This section describes the importance of clarifying the purpose and scope of the research, understanding the validity of the model created, and the standards for conducting test measurements using the model. In addition, we would like to emphasize the importance of fully understanding the usefulness and limitations of each model and measurement method.

2. Purpose and scope of creating animal models of neuropsychiatric diseases and conducting tests and measurements

When creating disease model in animals, it is important to clearly distinguish whether the research is basic disease research or applied research. This is because the goals and emphasis of each research are different. In other words, in basic research, it is important to thoroughly use analytical skills and delve into the essence of the pathophysiology.

On the other hand, in applied research, the goal is to determine the effectiveness of drug administration, cell transplantation, genetic manipulation, experimental surgery, etc. at the preclinical medical level. Here, it is important to use an animal model whose validity has already been established and to use a measurement method that clearly shows the effectiveness of the experimental treatment. After the experiment, it is important to be able to clearly state one of the following: 1) an improving effect was observed at the animal level, 2) an improving effect was not observed at the animal level, or 3) the effect was not determind in the experimental conditions used, and the reason for the uclear results should be described.

3. Validity of the model in terms of pathophyisology of the disease

Next, depending on the pathophysiology targeted in light of the research objective, similarity to either the disease syndrome, disease pathophysiology, or disease etiology of the model used is assumed. With this in mind, it is important to understand the utility and limitations of the model.

As an example, let us take a Parkinson's disease model in which the neurotoxin MPTP  (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is administered subcutaneously or other peripheral route to monkeys. In this model, the persistent motor dysfunction and other syndrome are extremely similar to those of human Parkinson's disease. In addition, the site of action of this neurotoxin is in the dopamine nerves of the substantia nigra, and it is similar to the disease in that it manifests neuronal destruction there. In other words, this model can be said to have similarities in disease syndrome and disease pathophysiology. Regarding disease etiology, it may be possible to use it as a model of Parkinson's-like disease caused by long-term exposure to certain chemicals. However, the similarity to the etiology of essential Parkinson's disease is limited in the case of MPTP administration. To explore the etiology of Parkinson's disease, we may establish a model in which some genes known to be involved in familial Parkinson's disease are introduced into mice or marmosets.

See this website Parkinson's Model of Marmoset

Reference in Japanese: Kiyoshi Ando (2018) A common marmoset model of Parkinson's disease induced by administration of the neurotoxin MPTP – Focusing on preclinical evaluation by behavioral analysis –. Obelisk Vol. 23,1:14-22. https://researchmap.jp/read0179769/published_papers/19447809

Diseases related to motor dysfunction such as Parkinson's disease are cases in which there are many commonalities between humans and monkeys, and therefore animal models have high disease validity. On the other hand, it is extremely difficult to create an animal model of a disease related to a higher brain function unique to humans that is separate from motor function. For example, consider schizophrenia. The core syndrome of this disease are hallucinations and delusions. It is difficult to reproduce this in an animal model, and even if an animal model is created, there is a problem of how to detect hallucinations and delusions. Here, there are animal models using repeated administration of stimulants such as methamphetamine, and behavioral science and molecular biological searches are being conducted. However, in terms of syndrome and pathophysiology, it must be said that there is a large distance between human schizophrenia and model schizophrenia. In reality, it is understandable that research must proceed based on only a few similar aspects between the model and the disease.

There is also research comparing the behavioral characteristics of a mouse genetically modified model in which several genes thought to be specific to human schizophrenia patients have been introduced with the wild type. Doctors who face schizophrenia patients day and night and struggle to treat them also deeply understand the importance of biological research using experimental animals. However, the author has heard from psychiatrists that they feel uncomfortable calling models such as mice schizophrenia models. Furthermore, research on genetic modification of mice may be important for elucidating the pathology and etiology of schizophrenia. However, it is necessary to fully understand the great distance from the disease and its limitations. The author thinks it is inappropriate to call this a schizophrenia model just because a gene that is thought to be related to schizophrenia has been introduced into a mouse. The author would like it to be called a schizophrenia gene-introduced model at the very least.

4. Criteria for test measurements using animal models of diseases

It is important to determine what aspect of the disease is measured using the disease model animal created. Even if a highly valid disease model animal is created and used, good experimental results will not be obtained if the measurement method is off-target. Therefore, the author thinks it is important to check the following measurement points one by one.

4.1. Validity of test measurement index data

Since a model with high disease validity has been created, it is necessary to set appropriate measurement methods and indices that correspond to it. We would like to obtain meaningful data that matches the research purpose by using measurement indices that are closely related to the syndrome, pathophysiology, or etiology expressed by the created model.

As already mentioned, the MPTP-administered Parkinson's disease model of monkeys has excellent disease validity as a characteristic of the model in terms of syndrome and pathophysiology. Furthermore, behavioral analysis such as spontaneous motor activity (or locomotion) measurements and macroscopic and gross observation of syndrome have enabled detection of Parkinson's disease-like motor impairments. In addition, dopamine neurodegeneration has been clearly detected in the brains of these model animals through in vivo measurements such as Positron Emission Tomography (PET) / Magnetic Resonance Imaging (MRI) and in vitro measurements such as tyrosine hydroxilase histopathological examinations.

See this WEB site Parkinson's Model of Marmoset

4.2. Objectivity/quantity of test measurement data

Objectivity of measurement indices is the basis of scientific observation. In the MPTP marmoset Parkinson's disease model experiment, the spontaneous motor activity or locomotion of the marmoset in its individual cage was detected and quantified by a moving sensor. This is an objective, quantitative indicator of immobility, one of the most important Parkinson's disease-like syndrome, and is extremely sensitive and useful in detecting the therapeutic effects of drugs. However, in this study, in addition to these indices, the experimenter also observed the marmosets' syndrome with the naked eye. Here, we set up observation items that left as little room for the experimenter's subjectivity as possible. In other words, the observer recorded only whether a specific observation item was present or absent, with a score of 1 or 0. A total score was then obtained for several items (CIEA Dysfunction Score). This quantitatively supplemented the overall picture of the marmoset's syndrome, which is not sufficient to measure spontaneous motor activity alone.

See this WEB site Parkinson's Model of Marmoset (A motor dysfunction score sheet for syndrome observation in Parkinson's disease model marmosets in Additional Note 2) 

4.3. Reliability/reproducibility of test measurement data

The reliability of measurement data requires a stable model, a sufficient number of animals, and small variance in the data obtained. We believe that it is appropriate to use the standard deviation (SD) as the variance of the sample values ​​obtained in the experiment. Standard Error of Mean (SEM) is also used, but this is the standard deviation divided by the square root of the number of animals used (n) (SD/√n), which makes the variability appear smaller. However, this standard error refers to the variability of the mean value of each sample in the population. In other words, if an experiment under the same conditions is conducted in, for example, many facilities, the distribution of the mean values ​​at each facility is described. In that sense, the author thinks it is appropriate to express the variability of the distribution of data obtained in one experiment in one facility with SD rather than SEM. The author thinks that it is a little different to use SEM, which is a distribution that collects many mean values ​​obtained under the same experimental conditions from various facilities. Some may think that it is fine as long as it is defined as either SD or SEM. However, the author thinks that the positive and negative units of each variability are theoretically linked to the probability on the distribution, and the meaning of the variability is different between SD and SEM. The author is not an expert in statistics, so please let the autho know if the author is wrong.

Reproducibility means that the same experimental results will be obtained even if the same researcher repeats the experiment or if a researcher at another facility performs the experiment under the same conditions. Proof of reproducibility is especially necessary for research results by researchers with a certain level of mastery. One of the measures of the reproducibility may be a correlation coefficient.

4.4. Clinical predictability/validity of test measurement data

Even if the data obtained is objective/quantitative, reliable/reproducible, if the data does not have clinical predictability or clinical meaning (validity), what is the point of the research? Preclinical drug efficacy evaluation using MPTP-treated monkey Parkinson's disease models has extremely high predictability/validity as a treatment for the disease. This is because MPTP destroys dopamine neurons in the nigrostriatal system, and there is a commonality between motor functions, including limbs, between monkeys and humans, and when degeneration is observed in these functions and they are impaired, the disease pathophysiology and syndrome of Parkinson's disease are extremely similar.

As another example, we will also describe the intravenous drug self-administration method using rhesus monkeys. This method has shown extremely sensitive prediction of drugs that cause dependence in humans (dependent drugs/drugs of abuse). Furthermore, it has been proven that this method can also be used to voluntarily and compulsively ingest most drugs that cause psychic dependence in humans by lever pressing in monkeys, leading to a state of psychic dependence. It is extremely significant that the drug self-administration method has been shown to cause drug-seeking behavior, a behavioral aspect of psychic dependence, in laboratory animals. In addition, the drug self-administration method in rhesus monkeys can be said to be an excellent method for predicting whether or not a new compound will be dependence-producing potential in humans.

See this website: Drug Dependence Concept

4.5. Test measurements that consider the balance of macro/micro perspectives on pathophysiology

In order to study psychic dependence in drug dependence studies, experiments are conducted using the drug self-administration behavior of laboratory animals. This can be said to capture the behavioral syndrome of drug-seeking behavior related to drug dependence at a macro level. The mechanistic changes in the brain caused by drug dependence at this time will be elucidated in detail at the molecular biological level. The author has seen that experimental animals were forced to repeatedly administer drug dependence producing drugs at doses highe than those used in actual drug self-administration experiments, and the results of detailed molecular biological analysis of the brain were linked to drug dependence. The author believes that drug dependence producing drugs have the dependence effects in addition to their general pharmacological effects. The author thinks that micro-level analysis that mixes the two may be dissociated from the essence of drug dependence at the macro level. Here, the author thinks that an integrated macro- and micro-analysis that separates the general pharmacological effects and the drug dependence producing effects, keeping in mind the conditions of drug-seeking behavior, is necessary.

4.6. Feasibility of model creation and test measurement

When creating a model using experimental animals, it must be realistically feasible in the laboratory. In experiments using the neurotoxin MPTP to create a Parkinson's disease model, it is necessary to have a system in place that can properly manage MPTP. Otherwise, the safety of the experimenter cannot be ensured. In addition, the best way to evaluate the psychic dependence of a drug is to use rhesus monkeys.

The currently ongoing research described above did not begin with everything in place. Supported by the research objectives and strong motivation to carry them out, it started from scratch and gradually developed. Using the previous example, for safety reasons, it might be better to start with a neurotoxin such as 6-hydroxydopamine administered intracerebrally rather than MPTP. Furthermore, if there are no facilities for rhesus monkeys, it might be possible to start with rats or marmosets for drug self-administration experiments.

Ultimately, the feasibility of creating models and conducting test measurements depends on the researchers' clear understanding of the research topic and strong motivation regarding the significance of conducting these studies, combined with the practical situation (resources: experts, personnel, facilities, time, research funds, etc.).