The rats that root for us

A special purpose rodent

Tuyana Malankhanova, "Biomolecule"

Prevention and treatment of human diseases are the main tasks of modern biomedicine. Laboratory animals, especially rats, are indispensable assistants of scientists to solve them. Rats are very similar to humans in blood composition, tissue structure, physiological reactions in response to various influences, etc. This makes laboratory rats an excellent object for modeling a large number of human diseases, studying pathological processes, creating medicines and developing preventive measures. The article will focus on these "workers" of science, who at the cost of their lives contribute to progress in science and medicine.

For young children, rats are cute cartoon characters who do nothing else but perform funny songs and dances. For adults, rats are malicious pests that destroy crops and stocks, damage electrical networks and property. In addition, they can carry dangerous infectious diseases, including cholera, bubonic plague, typhus. Separately, it is worth noting that many women are terrified of rats for no particular reason at all, copying the mother's reaction captured in childhood. All these facts emphasize only the negative aspects that have fallen to the lot of rats. However, paradoxically, the role of the rat in modern science should not be underestimated.

Over the past century, the role of the rat in medicine has transformed from a carrier of infectious diseases into an indispensable tool of experimental medicine. Now rats are used in testing new techniques of surgery and transplantology, as well as in research on cancer, diabetes, psychological disorders, neuron regeneration and in many other areas of medicine. These rodents are also valuable experimental animals in assessing the therapeutic efficacy and toxicity of new medicinal compounds before clinical trials on humans.

Why exactly did rats become so popular? Firstly, they are relatively inexpensive to maintain. Secondly, they multiply rapidly, which allows biologists to study the genetic effects after several generations of descendants within a reasonable period of time. Thirdly, the rat genome has more than 90% similarity with the human genome [1]. (We talked about the Human Genome program in the article "Human Genome: how it was and how it will be" [2] – Ed.) In addition, compared with another convenient laboratory object – a mouse – it is easier to perform operations on rats due to their larger size; moreover, they are more resistant to various diseases.

There are many types of rats, but Rattus norvegicus is mainly used as a laboratory object. This is the first mammal species that has been the object of scientific research since 1828.

(They also started working with mice back in the XIX century, but the official year of the "birth" of a laboratory mouse is considered to be 1909, because it was in this year that the first clean line was obtained [4]. In Novosibirsk Akademgorodok in 2013, a monument was erected in gratitude to this animal (Fig. 1, from Wikipedia. – Ed.)

Over time, science has developed, and questions have arisen that require increasing the accuracy and reproducibility of experiments by reducing the influence of genetic differences between individuals. Puzzled by this problem, scientists using breeding methods began to breed genetically homogeneous animals – the so-called clean lines. Already in 1909, Dr. Helen King, an employee of the Vistar Institute (Pennsylvania, USA), obtained the first pure line of King Albino rats, which was then renamed the RA line [3]. They were ordinary albinos. To date, about five hundred lines of rats have been created for various studies.

With the development of genetics and molecular biology, rats have become objects of study of genetic diseases and the molecular processes underlying them. It is known that more than 1,000 hereditary human diseases are associated with mutations in certain genes. And since rats have a great genetic similarity with humans, scientists have created lines of rats that simulate human genetic diseases. Now a huge number of such lines have been derived, but we will focus only on some of the most interesting for modern medicine.

Eating a lot is not goodZucker rats are a classic model for the study of obesity, hypertension, type II diabetes mellitus and cardiac dysfunction (Fig. 2).

They are so named, oddly enough, not in honor of diabetes, but in honor of pathologists Louis and Theodore Zucker from Columbia University, who first discovered the gene responsible for obesity in rats. In representatives of the 13M line in 1961, the Zuckers revealed a spontaneous recessive mutation fa (from the English fatty – fatty) in the Lepr gene encoding the receptor for the satiety hormone – leptin. This mutation leads to the replacement of the amino acid glutamine with proline in the receptor. The cascade of events leading to obesity looks like this: synthesis of a mutant receptor that is embedded in the membrane of hypothalamus cells → leptin cannot communicate with its "destination", because the conformation of the receptor has been changed → saturation signals are not received in the brain → the animal eats the entire waking period [5]. With a weight of one kilogram, these rats are real giants compared to their "ordinary" 500–gram brothers and sisters. However, it usually does not reach the development of diabetes – insulin resistance is compensated by increased hormone synthesis.

Figure 2. Rat of the Zucker line. Figure from [5].

Interestingly, Zucker rats have a suppressed physiological state of "fight or flight". Normally, the sympathetic nervous system activates this condition, in particular, it is responsible for an increased pulse rate. When the heart of a Zucker rat is stimulated by the sympathetic nervous system hormone norepinephrine, the heart rate does not change. This may indicate a violation in the interaction of norepinephrine with its receptor. Consequently, Zucker rats can be used not only to study the effect of various hormones and drugs on the frequency and strength of heart contractions, but also to study diseases caused by disorders at the level of hormone–receptor interaction.

Why are you standing there, swaying, skinny divchina? There is no rilyn in the brain – that's the whole reasonThe shaking rat Kawasaki (SRK) was first described in 1988 in the Land of the Rising Sun [6].

Rat trembling is caused by a mutation in the gene encoding the protein rilin. This protein is responsible for the migration of neural stem cells – future neurons – during fetal maturation, as well as some time after birth. In the adult brain, rilyn plays an important role in memory formation.

In Kawasaki rats, due to the mutation, the level of rilin is lowered. Its deficiency leads to brain disorders, abnormal behavior and trembling gait (Fig. 3). Interestingly, humans have a homologous protein that performs the same functions as rats. Therefore, rodents with the described mutation are a good model for studying the role of rilin in the pathogenesis of various brain malformations due to impaired neuronal migration. So, it has already been shown that schizophrenia and autism are associated with a lack of rilin; Alzheimer's disease, on the contrary, is accompanied by an excess of it. This makes it possible to predict future human pathologies already at the early stages of development and begin disease prevention.

Figure 3. Comparison of the organization of the layers of the cerebral cortex in normal and pathological conditions.
Drawing from Wikipedia.

Blind HelpersThe RCS rat (from the Royal College of Surgeons) is the first known animal with hereditary retinal degeneration.

This line was developed more than sixty years ago at the Royal College of Surgery in Edinburgh. For the first time in 2000, a genetic defect was identified that causes this disease. Scientists have managed to identify a mutation in the Mertk gene in RCS rats, as a result of which photoreceptors die [7]. By the age of three months, the animals are completely blind [8].

Figure 4. Structure of the mammalian retina. Sops, rods – cones and rods; RPE (Retinal Pigment Epithelium) – pigment epithelium cells; 1 – inner segment of the photoreceptor; 2 – outer segment of the photoreceptor. Figure from [9].

It is known that the outer segments of photoreceptor cells gradually die off, and new segments come in their place according to the type of conveyor (Fig. 4). The dead segments are absorbed by the cells of the pigment epithelium. In order for these cells to be able to "eat" the segments, it requires the launch of a molecular cascade, the main activator of which is the tyrosine kinase receptor Mertk. In RCS rats, due to the mutation, the aforementioned cascade does not start, "garbage" segments accumulate in the retina, which contribute to the death of photoreceptors.

In 2001, a group of American scientists managed to correct the mutant phenotype of rats by injecting recombinant adenoviruses with a healthy Mertk gene directly into the retina.

As a result, the researchers managed to stop the degradation of the retina of sick rats. This suggests that the healthy Mertk gene was embedded in the rat genome and was normally expressed [10]. In 2010, it was found that a homologous mutant gene was also found in patients (people) with a similar retinal disease – retinitis pigmentosa [11]. All these results emphasize the importance of RCS rats as a model for the analysis of molecular processes in retinal degeneration and gene therapy of retinal diseases. (Read about cybernetic retinal prostheses in the article "Optogenetics + holography = epiphany?" [12]. – Ed.)

Old age is not a joyThe first model for the study of diseases of old age [13, 14] were rats of "domestic production" of the OXYS line, obtained in 1975 at the Institute of Cytology and Genetics SB RAS.

Initially, scientists wanted to get a line with hereditary cataracts. To do this, they used selection methods and a load of galactose, which has cataractogenic properties. However, in addition to cataracts, rats developed other pathologies: emphysema, cardiomyopathy, memory impairment, decreased immunity, osteoporosis, precancerous conditions, as well as decreased fertility and life expectancy. It should be emphasized that these signs were analyzed either against the background of galactose loading, or in the next subsequent generations. Thus, hereditary galactosemia (inability to assimilate galactose) was obtained in these animals. In the future, the selection was carried out for early spontaneous cataract. The first signs of cataracts are observed by the second month of the animal's life, and by six months cataracts develop in 100% of individuals (and only 5% of "normal" rats). By the year of life, both eyes of the OXYS rat are affected by cataracts (Fig. 5).

Figure 5. Photos of the fundus of rats.
a – normal blood supply to the retina of healthy rats; b – blood supply to the retina of 3-month-old OXYS rats;
c – reduced blood supply to the retina of OXYS rats at the age of 12 months. Figure from [15].

With the help of ophthalmological and other studies, it was shown that the cataract of OXYS rats by nature and molecular processes corresponds to age-related cataract in humans. The remaining diseases of old age of rodents also had a common nature with the corresponding human pathologies. These animals can help to investigate the mechanisms of development of senile diseases, develop ways to prevent and treat them, and probably create drugs that will prolong life. In addition, OXYS rats can be considered as a universal model for studying the aging process of the eye [16].

Diabetes is not from sweetsIn early 1961, an assistant at the West Brattleboro Scientific Laboratory (Vermont, USA) noticed that the drinking bottle was almost always empty in only one rat cage out of several hundred.

In this cage lived a female rat with seventeen cubs. Then it was discovered that some cubs drank a lot of water, and when they were injected with vasopressin – an antidiuretic hormone – water consumption returned to normal. As a result, it was shown that these baby rats were suffering from sugar–free hypothalamic diabetes caused by a spontaneous mutation – the loss of one nucleotide (guanine) - in the sequence of the vasopressin gene (Fig. 6) [17]. This mutation is recessive and is designated di (abbreviation of the Latin name of the disease – diabetes insipidus). Vasopressin is synthesized by certain cells of the hypothalamus, and is secreted from the pituitary gland. With a lack of water in the body or with an increase in the concentration of salts in the blood plasma, it stimulates the reverse absorption of water in the kidneys. In addition, it increases blood pressure due to its ability to cause blood vessels to contract [18].

Figure 6. A section of the nucleotide sequence of the vasopressin gene with a mutation in Brattleboro rats.
The picture is provided by the Laboratory of Epigenetics of development of ICIG SB RAS.

Brattleboro rats are the record holders among animals in terms of the volume of water they can drink, based on body weight. The absence of vasopressin in the blood leads to constant diabetes and water consumption, reaching the level of 25-100% of body weight per day. They also have impaired overall resistance to infections. Behavioral tests have shown that Brattleboro rats exhibit accelerated development of motor activity, inflexible, stereotypical behavior, therefore, in some works it is proposed to use these rats as a model object in the study of schizophrenia [19]. In addition, in Brattleboro rats, when cancer cells are injected, there is a stable slowdown in tumor growth compared to normal rats. In contrast to the continuous growth of the tumor, typical for ordinary rats, in mutants in the experiment it was insignificant and was observed only during the first 15-18 days after transplantation, then the tumor shrank and completely disappeared. With vasopressin injections, tumor growth temporarily increased, but in the future it still decreased and resorbed [20].

Brattleboro rats are an irreplaceable model for studying all processes occurring in kidneys devoid of vasopressin regulation. Now these rodents are also used as model animals for working out methods of correction of hereditary diseases.

* * *

Thus, rats are indispensable assistants to researchers in the struggle to save humanity from all kinds of ailments. They are fighting shoulder to shoulder with scientists to solve the tasks set by nature for people. Only a small part of the existing lines was presented in the article.

A lot has already been achieved, and with the use of technologies for obtaining pluripotent cells and the latest genomic engineering tools – TALEN [21] and CRISPR/Cas9 [22] – revolutionary approaches to the correction of hereditary human diseases will be developed, which can be safely introduced into the healthcare system in the future. But the "first blow", of course, will be taken by laboratory animals...

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Portal "Eternal youth" http://vechnayamolodost.ru14.04.2015