RU2845390C1 - Method for administering the solutions into small laboratory animals into a cerebellomedullary cistern - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to experimental medicine, particularly to a method for introducing solutions into a cerebellomedullary cistern in small laboratory animals. A method involves anaesthesia, fixing an animal in a position of maximum head flexion, a needle puncture into a greater cerebral cistern and introduction of a solution. Anaesthesia is carried out with using inhalation anaesthetic. After the head is bent as much as possible in the atlantooccipital joint, navigation is carried out to locate the prick-in point by marking three points along the median line with a rod with a sterile colouring solution: greater occipital tubercle, spinous process of the II cervical vertebra and the middle of the distance between them, which is defined as the stick in point, then a disposable needle having a limiter is used to puncture the skin, soft tissues, atlanto-occipital membrane and dura mater, and the analysed solution is administered.

EFFECT: declared method enables the fast introduction of the aqueous solutions into the cerebellomedullary cistern with a low risk of post-injection complications.

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Description

The invention relates to experimental medicine and can be used for the introduction of aqueous solutions for the purpose of modeling and pharmacological correction of pathologies of the central nervous system.

The creation of models of diseases of the central nervous system is one of the important aspects of studying their pathogenesis. The introduction of pharmacological agents into the cisterna magna cerebral is a method that is often used to study the delivery of pharmaceutical vectors that are used as gene therapy (Melnikova, E.V. Gene therapy of neurodegenerative diseases: achievements, developments, problems of implementation in clinical practice / Melnikova E.V., Merkulov V.A., Merkulova O.V. // BIOpreparations. Prevention, diagnostics, treatment. - 2023. - No. 2. - P. 127-147).

The development of a method for injection into the cisterna magna of the brain in mice is an important tool for modern research, in particular for modeling neurodegenerative diseases such as Alzheimer's disease (Experimental models of Alzheimer's disease: advantages and disadvantages / Iptyshev AM, Gorina YaV, Lopatina OL, Komleva YuK, Salmina AB // Siberian Medical Review. - 2016. - No. 4. - P. 100) or Parkinson's disease (Migration of multipotent mesenchymal stromal cells upon systemic and local administration in an experimental model of parkinsonism / Zafranskaya Marina Mikhailovna, Nizhegorodova DB, Aleinikova NE, Kuznetsova TE, Vanslav MI, Ignatovich TV, Boyko AV, Ponomarev VV // Annals of Clinical and Experimental Neurology. - 2019. - No. 2. - P. 32-40), autoimmune diseases associated with brain damage, such as neuromyelitis optica (Neuromyelitis optica (NMO) - an autoimmune disease of the central nervous system (CNS) / Asgari N., Owens T., Frokiaer J., Stenager E., Lillevang S.T., Kyvik K.O. // Acta Neurol Scand. - 2011. - Vol. 123, No. 6. - P. 369-384). This makes it possible to study the pathogenesis of diseases, as well as to assess the influence of various factors on the development of pathologies.

An equally important reason for the relevance of this method is the ability to overcome the blood-brain barrier. The blood-brain barrier is a serious obstacle to the delivery of many therapeutic substances to the brain. Intrathecal injection allows you to deliver drugs directly into the cerebrospinal fluid, bypassing the blood-brain barrier. This is especially important for the development and testing of new treatments using gene therapy (Targeted gene silencing in the nervous system with CRISPR-Cas13 / Powell J.E., Lim C.K.W., Krishnan R., McCallister T.X., Saporito-Magriña C., Zeballos M.A., McPheron G.D., Gaj T. // Science Advances. - 2022. - Vol. 8, No. 3. - P. 2485).

Preclinical studies using the intrathecal injection method can be used to study the effectiveness of therapy with pharmacological agents. The method allows for an accurate assessment of the effectiveness of new methods for correcting central nervous system diseases, such as treatment of spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection (Romero-Ramírez L., Wu S., de Munter J., Wolters E.C., Kramer B.W., Mey J. // Journal of Biomedical Science-2020. - Vol. 27, No. 1. - P. 35). This is especially relevant for the development of personalized treatment approaches.

Of particular importance is the minimally invasiveness and high accuracy of this injection method, which reduces the risks of adverse pathologies in laboratory animals and allows for long-term studies (A minimally invasive endovascular approach to the cerebellopontine angle cistern enables broad CNS biodistribution of scAAV9-CB-GFP / Benatti H.R., Anagnostakou V., Taghian T., Hall E.F., Nath S., Heilman C.B., Beneduce B.M., Leporati A., Raskett C., Epshtein M., King R., Gounis M.J., Malek A.M., Gray-Edwards H.L. // Molecular Therapy. - 2024. - Vol.32, No. 10. - P. 3346-3355). The method is also useful for studying the role of cerebrospinal fluid in maintaining brain homeostasis, immune responses in the central nervous system, and the interaction between the nervous and immune systems (In Vivo Imaging of Lymphatic Drainage of Cerebrospinal Fluid in Mouse / Mathieu E., Gupta N., Macdonald R.L., Ai J., Yücel Y.H. // Fluids and Barriers of the CNS. -2013. - Vol. 10, No. 1 - P. 35).

Thus, the development and improvement of the method of injection into the cisterna magna of the brain in mice opens up wide opportunities for fundamental and applied research in the field of neurobiology, pharmacology and gene therapy, contributing to the development of new approaches to the treatment of diseases of the central nervous system.

The closest analogues to the claimed invention are several methods.

A method for repeated collection of cerebrospinal fluid from the cisterna magna of the brain of anesthetized strain 13 guinea pigs is known (Liu, C.T. Technique for repeated collection of cerebrospinal fluid from cisterna magna of anesthetized strain 13 guinea pigs / Liu C.T., Guo Z.M. // Proc Soc Exp Biol Med. - 1991. - Vol. 197, No. 4. - P. 400-403). The method was carried out as follows: the animal's head was fixed in a stereotaxic device at an angle of 30 degrees, and cisternal puncture was performed using a 23-gauge L-shaped needle through shaved skin.

A method of percutaneous puncture of the cisterna magna for collecting cerebrospinal fluid in rats is known (Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier / Daniels B.P., Cruz-Orengo L., Pasieka T.J., Couraud P.O., Romero I.A., Weksler B., Cooper J.A., Doering T.L., Klein R.S. // Journal of Neuroscience Methods. - 2013. - Vol. 212, No. 1. - P. 173-179), a device for collecting cerebrospinal fluid was designed using a 1 ml syringe, a silicone tube, a disposable 21G needle and water. The animal was placed on an elevated platform above the base of the stereotaxic apparatus, and the puncture site was determined using stereotaxic coordinates.

A method of percutaneous punctures for collecting cerebrospinal fluid in rats is also known (Safety assessment of multiple repeated percutaneous punctures for the collection of cerebrospinal fluid in rats / Wang D., Zhao Y., Yang Y., Xie H. // Brazilian Journal of Medical and Biological Research. - 2021. - Vol. 54, No. 6 - P. 10032). The method was carried out by inserting a needle approximately 0.5 cm into the occipital crest depression to reach the large cistern. Then the needle insertion was immediately stopped, and the other side of the collecting needle was connected to a pressure sensor. A BL-420 biological experiment system was used to monitor the pressure in the large cistern in each group.

The disadvantages of these methods are: firstly, they were used to collect cerebrospinal fluid, not to administer solutions; secondly, to conduct the experiment, additional complex devices were required, namely, stereotaxis, a pressure sensor, and high time costs. In addition, it should be noted that performing a puncture in this way requires significant technical training and surgical skills from the researcher. It is also difficult to assess the applicability of these methods in mice.

A method for intrathecal drug delivery to rats and mice for experimental research is known (Epidural and Intrathecal Drug Delivery in Rats and Mice for Experimental Research. Fundamental Concepts, Techniques, Precaution, and Application / Rahman M.M., Lee J.Y., Kim Y.H., Park C.K. // Biomedicines. - 2023. - Vol. 11, No. 5. - P. 1413). After anesthesia, the thoracolumbar region is shaved. Needles are inserted into the intervertebral space, where there are no bone parts and which is closed by a ligament, at an angle of 70-80 °, and then the angle is reduced to 30-45 ° during drug administration in order to easily distribute the injected drug from the narrow needle and prevent the release of cerebrospinal fluid during needle removal. It was reported that the depth of needle insertion is about 0.30 cm in rats after skin and muscle incision and 0.30-0.40 cm without skin and muscle incision in mice. The disadvantages of this development are: technological complexity of detecting the anatomical landmarks specified in the article and a high risk of error in their identification. Also, as the authors themselves note, the procedure requires high skill of the researcher. It should be noted that this technique is associated with a high risk of complications: the needle can easily injure the spinal cord and / or its vascular membrane with the formation of a spinal hematoma. In addition, as noted by the authors, after the injection of the drug, temporary motor paralysis occurs, which complicates the assessment of the neurological status and may probably be irreversible.

The closest to the claimed solution is the method of intrathecal administration of type 9 AAV vector encoding arylsulfatase A in adult mice (Treatment of adult metachromatic leukodystrophy model mice using intrathecal administration of type 9 AAV vector encoding arylsulfatase A / Miyake N., Miyake K., Sakai A., Yamamoto M., Suzuki H., Shimada T. // Scientific Reports. - 2021. - Vol. 11, No. 1. - P. 20513). Mature mice were deeply anesthetized with pentobarbital, placed in a stereotaxic frame and bent in a supine position, after which the back of the neck was cleaned with povidone-iodine. Then a skin incision was made on the back of the head. The dura mater was exposed between the occipital bone and cervical vertebrae and a needle was inserted into the cistern through a suboccipital puncture. Ten microliters of vector solution were injected over 1 minute using a Hamilton syringe with a 33G beveled needle.

The disadvantage of this method is the need for long-term anesthesia using pentobarbital, which increases the recovery period of animals after the procedure and the risk of neurotoxic reactions. In addition, it requires the use of a stereotaxic frame and processing of the surgical field, which requires additional time, experience in working with the specified equipment, as well as the possibility of inactivating some pharmacological agents or genetic constructs with a povidone-iodine solution. The main disadvantage of the method is the need to incise the skin with underlying tissues and expose the dura mater, which increases the time of the procedure, the recovery time of the animal and increases the risk of infectious complications. It should also be noted that a Hamilton-type syringe was used to perform the intervention, which requires its processing before and after the injection, since these syringes are not disposable.

The objective of the invention is to expand the range of methods for introducing solutions into the large cerebellar-cerebral cistern of small laboratory animals.

The technical result of the proposed invention is a method for introducing solutions into the large cerebellomedullary cistern of small laboratory animals by suboccipital injection, which allows for the rapid introduction of aqueous solutions into the large cerebellomedullary cistern by suboccipital injection of small laboratory animals, with a low risk of post-injection complications. The method is also distinguished by its simplicity of implementation and rapid recovery of animals.

The stated objective is achieved by proposing a method for administering solutions to small laboratory animals in the large cerebellar-cerebral cistern, which includes anesthesia, fixing the animal in a position of maximum head flexion, after which a needle is punctured into the large cerebral cistern and a solution is administered, which includes the following new features:

- anesthesia is performed using an inhalation anesthetic,

- after maximum flexion of the head at the atlanto-occipital joint, navigation is performed to determine the injection site, for which three points along the midline are marked with a stick with a sterile dye solution: the greater occipital tubercle, the spinous process of the second cervical vertebra and the middle of the distance between them, which is determined as the injection point,

- then, using a disposable needle with a limiter, a puncture is made in the skin, soft tissues, atlanto-occipital membrane and dura mater, after which the test solution is injected.

The advantage of the claimed method is that it does not require removal of the hair coat and incision of the skin and soft tissues, which reduces the risk of infectious complications, anesthesia is carried out using an inhalation anesthetic, which reduces the recovery time of the animal, and navigation to determine the injection site is carried out without the use of stereotaxic equipment, in addition, the injection is performed with a disposable syringe with a limiter on the needle at a level of 3 mm for mice and 5 mm for rats, which allows you to pre-set the permissible amount of penetration of the needle from the skin into the depth.

The invention meets the criteria of “novelty” and “inventive step” and allows for increased repeatability of results in modeling and pharmacological correction of central nervous system pathology.

THE METHOD IS IMPLEMENTED AS FOLLOWS

Small laboratory animals are anesthetized by inhaling evaporated isoflurane in a mask at a concentration of up to 4% at the induction stage, 1.5% during acute intervention. The animal is fixed in a position of maximum flexion of the head in the atlanto-occipital joint. A stick with a sterile dye solution is used to mark three points along the midline: the greater occipital tubercle, the spinous process of the second cervical vertebra and the middle of the distance between them - the needle puncture point. A syringe with a removable 30G (0.3 × 4 mm) needle with a limiter is used to puncture the skin, soft tissues, atlanto-occipital membrane and dura mater. The limiter allows you to set the permissible depth of penetration of the needle from the skin into the depth. The test solution is administered. If the needle is correctly inserted into the large cerebral cistern, the solution is injected freely, without resistance. After the solution is administered, the needle is removed. The supply of inhalation anesthetic is stopped. The animal is returned to the cage for observation.

When implementing the method, a disposable syringe with a removable 30G (0.3×4 mm) needle with a limiter at 3 mm is used for mice, and a disposable syringe with a removable 30G (0.3×6 mm) needle with a limiter at 5 mm is used for rats.

The invention is characterized by the following figures:

Fig. 1 - Photographs of biomaterial from the central nervous system of mice 1 hour after the suboccipital administration of an aqueous solution of methylene blue.

Fig. 2 - Photographs of biomaterial from the central nervous system of mice 24 hours after suboccipital administration of an aqueous solution of methylene blue.

Fig. 3 - Photographs of biomaterial from the central nervous system of mice 7 days after the suboccipital administration of an aqueous solution of methylene blue.

Fig. 4 - Photographs of biomaterial from the central nervous system of mice 14 days after the suboccipital administration of an aqueous solution of methylene blue.

EXAMPLE OF SPECIFIC IMPLEMENTATION

The experimental animals used were 3-4 month old male C57BL/6 mice weighing 30-35 g. The animals were kept in accordance with the recommendations of the Board of the Eurasian Economic Commission of November 14, 2023 N33 "On the Guidelines for Working with Laboratory (Experimental) Animals in Preclinical (Non-Clinical) Studies" in compliance with the ethical principles of handling laboratory animals in accordance with Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals used for scientific purposes.

The experiment involved 40 sexually mature male mice of the C57BL/6J line weighing 30-35g. The animals were kept at the Experimental Biological Clinic in the vivarium of the National Research University "BelSU". To facilitate detection and distribution kinetics in anatomical structures, a methylene blue solution was used as an aqueous solution, which was administered in a constant volume of 20 μl suboccipitally with one injection.

Mice were anesthetized by inhaling vaporized isoflurane in a mask at a concentration of up to 4% at the induction stage and 1.5% during the acute intervention. The animal was fixed in a position of maximum head flexion at the atlanto-occipital joint. A stick with a sterile dye solution was used to mark three points along the midline: the greater occipital tubercle, the spinous process of the second cervical vertebra, and the middle of the distance between them, the needle puncture point. A syringe with a removable 30G needle (0.3×4 mm) with a limiter at the level of 3 mm was used to puncture the skin, soft tissues, atlanto-occipital membrane, and dura mater, and overcoming the dense-elastic resistance was clearly felt. The test solution was administered suboccipitally in a volume of 20 μl in a single injection. If the needle is correctly inserted into the cisterna magna, the solution is injected freely, without resistance. After the solution was administered, the needle was removed. The inhalation anesthetic was discontinued. The animal was returned to the cage for observation.

The animals were observed for the next 14 days: their general condition, the presence of focal neurological symptoms, and lethal outcomes were recorded.

In 35 animals, no decrease in activity or any pathological symptoms were observed after recovery from anesthesia. After 1 hour, 24 hours, 7 days and 14 days, 4 mice from the animals that had no complications after the suboccipital puncture were euthanized, followed by isolation of the brain and spinal cord and macroscopic examination of their sections to assess the distribution of the injected dye.

During dynamic assessment of dye distribution it can be noted that with suboccipital injection the dye was detected in all parts of the cerebrospinal fluid system: subarachnoid space of the brain and spinal cord, cerebral ventricular system, Sylvian aqueduct and central canal of the spinal cord. The most pronounced staining was observed during the first day, after which the intensity decreased, but the dye remained until the 14th day of observation. The distribution of the dye in the cerebrospinal fluid systems 1 hour after the injection of an aqueous solution of methylene blue is shown in Fig. 1, after 24 hours - Fig. 2, after 7 days - Fig. 3, after 14 days - Fig. 4.

As a result of intrathecal administration of aqueous methylene blue solution by the specified method, the following complications were observed in 5 out of 40 animals: one mouse died from respiratory arrest immediately upon dye administration, 4 animals showed changes in neurological status after recovery from anesthesia, 2 of which showed arena movements, and 2 animals showed a decrease in general activity and muscle weakness in the limbs. All animals with pathological symptoms were withdrawn from the experiment with subsequent macroscopic assessment of the brain and spinal cord in order to establish the causes of the complications that developed. During examination, the needle puncture site was found at the level of the medulla oblongata or cervical spinal cord, which was probably the cause of the negative outcome.

Thus, it was established that with suboccipital administration of methylene blue solution by the specified method, 87.5% of animals had no pathological symptoms, only 12.5% had post-injection complications. The obtained results of this method of performing intrathecal injection demonstrate a low probability of complications in laboratory animals.

Therefore, the proposed method allows for the rapid introduction of aqueous solutions into the large cerebellomedullary cistern by suboccipital injection into small laboratory animals, with a low risk of post-injection complications, and is also distinguished by its ease of implementation and rapid recovery of animals.

Claims (1)

A method for administering solutions to small laboratory animals in the large cerebellomedullary cistern, including anesthesia, fixing the animal in a position of maximum head flexion, after which a needle is punctured into the large cerebellar cistern and the solution is administered, characterized in that the anesthesia is administered using an inhalation anesthetic, after maximum head flexion in the atlanto-occipital joint, navigation is performed to determine the injection site, for which three points are designated along the midline with a stick with a sterile dye solution: the greater occipital tubercle, the spinous process of the second cervical vertebra and the middle of the distance between them, which is determined as the injection point, then a disposable needle with a limiter is used to puncture the skin, soft tissues, atlanto-occipital membrane and dura mater, after which the test solution is administered.