Histological structure of intercellular fluid circulation pathways

Keywords: nervous system, glymphatic system, methylene blue, in vivo staining technique.


Studies of the nervous system today are quite relevant and important. There are a large number of methods of studying and researching the brain, among which the histological method is widely used. Despite the variety of methods, in the possible practical application of histological examination of the central nervous system and brain in particular there are problems encountered by researchers: the complexity of the method, a large number of conventions to consider when working with nerve tissue, methods of fixation. Among other research methods, there is a group of histological methods, united by a common feature – in vivo staining of the nervous system, among which there is also a method of in vivo staining with methylene blue. The aim of the study was to establish the morphological features of the structure of the intercellular fluid circulation of the brain in experimental animals. The article describes a new method of injectable in vivo staining of the brains of laboratory animals with methylene blue. It is relevant for studying the morphology of the intercellular fluid circulation of the brain and the study of the structure of the microcirculatory tract. In our work it is offered to combine already known methods of perfusion fixation and a technique of supravital staining with methylene blue. Since most scientific studies of the brain use conventional research methods without a comprehensive study of the entire microcirculatory tract and intercellular fluid circulation, there is a need for more detailed study of the histological structure and topography of microcirculatory brain complexes to establish their normal structure. The results of the study confirm the researchers' observation that methylene blue has a high affinity for nerve fibers in the brain. In addition, it is obvious that the additional use of formalin as a solvent increases the resistance of methylene blue to leaching and the ability to stain the structures of the microcirculatory tract. In addition, the lifetime application of this technique allows you to visualize the morphological features of the microcirculation pathways of the intercellular fluid of the brain, Virchow-Robin space and capillary walls.


Abbott, N. J., Pizzo, M. E., Preston, J. E., Janigro, D., & Thorne, R. G. (2018). The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic’system?. Acta Neuropathologica, 135(3), 387-407. doi: 10.1007/s00401-018-1812-4

Benveniste, H., Liu, X., Koundal, S., Sanggaard, S., Lee, H., & Wardlaw, J. (2019). The glymphatic system and waste clearance with brain aging: a review. Gerontology, 65(2), 106-119. doi: 10.1159/000490349

Fawcett, J. W., Oohashi, T., & Pizzorusso, T. (2019). The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. Nature Reviews. Neuroscience, 20(8), 451-465. doi: 10.1038/s41583-019-0196-3

Gage, G. J., Kipke, D. R., & Shain, W. (2012). Whole animal perfusion fixation for rodents. JoVE (Journal of Visualized Experiments), (65), e3564. doi: 10.3791/3564

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. (2016). Methylene blue. In: Some drugs and Herbal Products. Lyon: International Agency for Research on Cancer.

Ikeda, K., Nara, Y., & Yamori, Y. (1991). Indirect systolic and mean blood pressure determination by a new tail cuff method in spontaneously hypertensive rats. Laboratory Animals, 25(1), 26-29. doi: 10.1258/002367791780808176

Iliff, J. J., & Nedergaard, M. (2013). Is there a cerebral lymphatic system?. Stroke, 44(6, Suppl. 1), S93-S95. doi: 10.1161/STROKEAHA.112.678698

Jonkers, B. W., Sterk, J. C., & Wouterlood, F. G. (1984). Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device. Journal of neuroscience methods, 12(2), 141-149. doi: 10.1016/0165-0270(84)90013-X

Kasukurthi, R., Brenner, M. J., Moore, A. M., Moradzadeh, A., Ray, W. Z., Santosa, K. B., ... & Hunter, D. A. (2009). Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration. Journal of Neuroscience Methods, 184(2), 303-309. doi: 10.1016/j.jneumeth.2009.08.019

Kondor, Y., Tykholaz, V., & Huminskyi, Y. (2019). Morphology of the pathways of intracellular circulation in the brain. Pain Medicine, 4(4), 61-70. doi: 10.31636/pmjua.v4i4.4

Mayer, B., Brunner, F., & Schmidt, K. (1993). Inhibition of nitric oxide synthesis by methylene blue. Biochem. Pharmacol., (45), 367-374. doi: 10.1016/0006-2952(93)90072-5

McFadden, W. C., Walsh, H., Richter, F., Soudant, C., Bryce, C. H., Hof, P. R., … & McKenzie, A. T. (2019). Perfusion fixation in brain banking: a systematic review. Acta Neuropathologica Communications, 7(1), 146. doi: 10.1186/s40478-019-0799-y

Mestre, H., Mori, Y., & Nedergaard, M. (2020). The Brain’s Glymphatic System: Current Controversies. Trends in neurosciences, 43(7), 458-466. doi: 10.1016/j.tins.2020.04.003

Mueller, T. (1998). Methylene blue supravital staining: an evaluation of its applicability to the mammalian brain and pineal gland. Histology and histopathology, 13(4), 1019-1026. doi: 10.14670/HH-13.1019

Mulisch, M. (2015). Fixierungen für Licht-und Elektronenmikroskopie. In: Romeis-Mikroskopische Technik (pp. 87-98). Springer Spektrum, Berlin, Heidelberg. doi: 10.1007/978-3-642-55190-1

Yankova, G., Bogomyakova, O., & Tulupov, A. (2021). The glymphatic system and meningeal lymphatics of the brain: new understanding of brain clearance. Reviews in the neurosciences, 32(7), 693-705. doi: 10.1515/revneuro-2020-0106

Zwienenberg, M., Gong, Q. Z., Lee, L. L., Berman, R. F., & Lyeth, B. G. (1999). ICP monitoring in the rat: comparison of monitoring in the ventricle, brain parenchyma, and cisterna magna. Journal of neurotrauma, 16(11), 1095-1102. doi: 10.1089/neu.1999.16.1095

How to Cite
Kondor, Y. Y., Tykholaz, V. O., & Guminskyi, Y. Y. (2022). Histological structure of intercellular fluid circulation pathways. Reports of Morphology, 28(1), 59-63. https://doi.org/10.31393/morphology-journal-2022-28(1)-09