Method for diagnosing bladder inflammation based on results of scanning electron microscopy and elemental spectrometry

Abstract

FIELD: urology.SUBSTANCE: used to diagnose bladder inflammation based on the results of scanning electron microscopy (SEM) and elemental spectrometry (ES). During endoscopic cystoscopy, a bladder biopsy is performed. Layer-by-layer scanning electron microscopy of tissue is performed in combination with elemental spectrometry of the surface of cells of the mucous membrane, submucosal and muscular layers. If the percentage predominance of atomic oxygen over atomic carbon is determined in each of the three layers of the bladder wall, inflammation of the bladder is diagnosed. If the percentage predominance of atomic carbon over atomic oxygen is determined in each of the three layers of the bladder wall, then the absence of bladder inflammation is diagnosed.EFFECT: diagnosing bladder inflammation, evaluation of the ultrastructural state of specialized bladder wall cells, as well as the percentage ratio of atomic oxygen and carbon on their surface.1 cl, 7 dwgs, 7 tbls, 3 ex

Description

[0001] The invention relates to medicine, namely to urology, and can be used to diagnose bladder inflammation based on the results of scanning electron microscopy (SEM) and elemental spectrometry (ES) of the surface of bladder wall cells.

[0002] The main cause of inflammation of the bladder wall (cystitis), regardless of the reasons causing it, is considered to be the destruction of the biological membranes of specialized cells that come into contact with the aggressive biochemical composition of urine, which has been convincingly demonstrated in experimental studies on animals using dyes with varying degrees of impregnation of bladder tissues during inflammation (Levin RM, et al 1990; Monson FC, Wein AJ, et al 1991).

[0003] It is believed that inflammation begins with the destruction of carbon bonds of the glycosaminoglycan layer on the surface of the bladder mucosa (urothelium) with the subsequent involvement of specialized (stromal) cells of the submucosal layer, and a decrease in bladder capacity is associated with the development of secondary inflammation and fibrosis of muscle layer cells (myocytes) (Mirkin Ya.B., Karapetyan A.V., Shumov S.Yu. Interstitial cystitis: a discussion of pathogenesis, diagnosis and treatment. Part 1 - pathogenesis / / Experimental and clinical urology. - 2017. - No. 4. - p. 96-100; Hepner KA, Watkins KE, Elliott MN, Clemens JQ, Hilton LG, Berry SH. Suicidal ideation among patients with bladder pain syndrome / interstitial cystitis. Urology 2012;80(2): 280-285. doi: 10.1016 / j.urology.2011.12.053).

[0004] A number of researchers believe that the process is initially formed in the cellular structures of the submucosa, or even in the muscular layer of the bladder with secondary involvement of mucosal cells. Painful bladder syndrome / interstitial cystitis: prognostic factors for the clinical course of the disease (Zaitsev AV, Arefieva OA, Pushkar DY / / Urology 2018.-№6(3).-p 26-35. https: / / doi.org / 10.21886 / 2308-6424-2018-6-3-26-35; Theoharides TC, Kempuraj D, Sant GR. Mast cell involvement in interstitial cystitis: a review of human and experimental evidence. Urology 2001;57(6 Suppl 1): 47-55). There is no consensus on this issue yet, and the diagnosis of bladder inflammation (cystitis) is established clinically, based on the results of laboratory tests and visual examination of the bladder during instrumental visual cystoscopy (Clinical guidelines. Cystitis. ICD 10: N30. Age category: adults ID: KR14. Year of approval: 2016 (revised annually).Urine biochemical markers, as non-invasive diagnostic tools, are generally non-specific and reflect the general response to bladder tissue injury. In clinical laboratories, the study of the oxidative status of urine is most often carried out by testing the content of membrane lipid peroxidation products in it, which appear during the flushing process from the surface formations of specialized bladder cells and the total antioxidant activity of urine (TAO), which levels out their toxic effect (Kurutas, E.B. The importance of antioxidants playing a role in the cellular response to oxidative / nitrosative stress: current status. Nutr J 15, 71 (2015). https: / / doi.org / 10.1186 / s12937-016-0186-5; Svetlova Z.V., Smirnova N.N. Free radical oxidation of lipids and proteins in children with chronic pyelonephritis. / / Nephrology. 2003; 7 (3): 44-47. https: / / doi.org / 10.24884 / 1561-6274-2003-7-3-44-47).Based on the increase or decrease of these indicators, indirect conclusions are made about the increase or decrease in the role of atomic oxygen, the initiator of free-radical toxic effects on cell membranes in the process of inflammation in the wall of the bladder (cystitis). Oxygen is present in the form of three allotropic modifications: atomic oxygen, dioxygen (oxygen itself), and trioxygen (ozone) (Zakharova I.N., Osmanov I.M., Kasyanova A.N., Mumladze E.B., Machneva E.B., Lupan I.N. Protective factors of the bladder mucosa are the key to new approaches to the treatment of urinary tract infections. Russian Bulletin of Perinatology and Pediatrics -2018- №2. - pp. 16-21. https: / / doi.org / 10.21508 / 1027-4065-2018-63-2-16-21; Richard K. Petersen. Free radicals and complex chemical processes involved in the organization of the cell membrane affect oxygen diffusion and the treatment of pathologies [J]. AIMS Biophysics, 2017, 4(2): 240-283. doi:10.3934 / biophy.2017.2.240).

[0005] Currently, no pathognomonic (characteristic) histological manifestations of any particular type of inflammation have been identified, visualized by light microscopy at cell magnifications up to 100x. However, a biopsy is indicated to exclude tuberculosis and bladder cancer. A definitive diagnosis also requires histopathological examination. Diagnosis of classic interstitial cystitis (IC) requires a deep biopsy, including bladder muscle tissue, as the pathological process affects both the superficial and deeper layers of the bladder wall. Important features to consider when establishing a histopathological diagnosis include denudation, ulceration, granulation tissue, fibrosis, and mast cells in the urothelium, as well as a cell count in the detrusor muscle.Thus, cystoscopy and bladder biopsy are currently the most acceptable methods for making a correct diagnosis (Zaitsev AV, Pushkar DY, Korsunskaya IL, Kovylina MV, Tsybulya OA. Modern aspects of diagnostics and treatment of painful bladder syndrome / interstitial cystitis. RMJ. 2010;17:1084; Kåbjörn Gustafsson C, Peeker R. Clinical features and histopathological findings in BPS / IC with and without Hunner lesions. Version: 03.09.2018. Urogenital infections and inflammations. Berlin: German Medical Science GMS Publishing House; 2017. DOI: 10.5680 / lhuii000031).

[0006] Elemental analysis of various tissues, including methods of spectrometric determination of chemical elements at the level of individual cells, is a new area of ​​clinical medicine (Skalny A.A. Comparative assessment of the information content of determining the content of chemical elements in blood serum, hair (wool), organs and tissues of experimental animals. Issues of biological, medical and pharmaceutical chemistry. 2021; 24 (11): 54-61. https: / / doi.org / 10.29296 / 25877313-2021-11-09). A separate direction is being developed for the elemental analysis of the oxidative status in the cell based on the balance of production of reactive oxygen species and angiooxidant defense molecules (Kurutas, E.B. The importance of antioxidants playing a role in the cellular response to oxidative / nitrosative stress: current status. Nutr J 15, 71 (2015). https: / / doi.org / 10.1186 / s12937-016-0186-5).

[0007] A promising direction is the combination of methods of elemental visualization and ultrastructural analysis of tissues, since this allows for valuable spatial analysis of both the atomic and structural molecular composition of the cell surface. And one of the leading tasks for the development of atomic spectrometry is the development of methods that allow obtaining surface multimodal information of tissues in contact with biological fluids in the body (Mochalov K.E., Korzhov D.S., Altunina A.V., Agapova O.I., Oleynikov V.A. Ultrastructural 3D microscopy for biomedicine. Principles, application, prospects / / Acta Naturae. - 2024. - Vol. 16. - No. 1. - P. 14-29. doi: 10.32607 / actanaturae.27323). Thus, spectrometry, based on the registration of the emission or absorption of quanta of electromagnetic radiation by atoms of the substance being studied, makes it possible to determine which atoms a specialized cell consists of (Khmelevskaya E.S., Perina E.A., Buiko E.E., Ufandeev A.A., Kaidash O.A., Ivanov V.V., Baikov A.N., Parochkina E.V., Udut E.V. Precision medicine in oncology: the role and prospects of mass spectrometry. Bulletin of Siberian Medicine. 2024; 23(2):162-182. https: / / doi.org / 10.20538 / 1682-0363-2024-2-162-182). It allows to determine in a specific specialized cell the presence of the percentage of the main chemical elements of living matter, carbon (C) and oxygen (O). Unlike existing standard methods of tissue research, elemental spectrometry of individual cells makes it possible to link their dynamics with the manifestations of inflammation at the ultrastructural level. (Konstantinov M.A., Zhdanov D.D., Toropygin I.Yu. Quantitative mass spectrometry with a label. 18As an alternative approach to determining protease activity using trypsin as an example. Biopreparations. Prevention, diagnostics, treatment. 2024; 24(1):46-60. https: / / doi.org / 10.30895 / 2221-996X-2024-24-1-46-60). For this purpose, a number of scientific centers began to use the results of combined scanning electron microscopy (SEM) in combination with a special probe analytical attachment, which allows, at a magnification of thousands of times, to obtain information on the morphology of the tissue surface and analyze its chemical elemental composition (Faizullin D.A., Kobelev A.V., Klementyev S. "Application of scanning electron microscopy and IR spectroscopy for rapid assessment of the morphology and chemical composition of bacterial films during periodic cultivation" News of universities. Applied chemistry and biotechnology, vol. 12, no. 3 (42), 2022, pp. 406-416).

[0008] Scanning electron microscopy (SEM) of cellular structures enables detailed visualization of their ultrastructural morphology and intercellular interactions. Its combination with elemental spectrometry (ES) of the content of atomic carbon (C) on the cell surface, which forms the carbon skeleton, allows for the study of structural changes during exposure to an aggressive environment. A decrease in the percentage of carbon on the cell surface can serve as a marker of its destruction. The presence of atomic oxygen (O) on the surface of specialized cells, including in the form of free radicals, is necessary for maintaining the oxidative status of the cell membrane during physiological renewal. However, excessive accumulation of free oxygen radicals on the surface can lead to the rupture of carbon bonds, leading to the loss of their structural and functional integrity.(Pozhilova Elena Vasilievna, Novikov Vasily Egorovich, and Levchenkova Olga Sergeevna. "Reactive oxygen species in cell physiology and pathology." Bulletin of the Smolensk State Medical Academy, vol. 14, no. 2, 2015, pp. 13-22.). Moreover, the results of spectrometry showing the content of atomic oxygen in the composition of oxygen functional groups with carbon-containing structures on the surface of a specialized human cell reflect its redox stability and allow us to improve the clinical understanding of the chemical mechanisms of inflammation at the atomic level (Dang Y, Liu Y, Xiang P, Tan Z, Tian Z, Greiner M, Heumann S, Ding Y, Qiao ZA. Carbon Surface Chemistry: Benchmark for the Analysis of Oxygen Functionalities on Carbon Materials. Adv Mater. 2025 Mar;37(11):e2418239. doi: 10.1002 / adma.202418239. Epub 2025 Feb 7. PMID: 39916535).

[0009] It is known that the cell membrane consists of a phospholipid bilayer in which specific membrane proteins are embedded. They are not firmly attached by ionic bonds to the bilayer surface and carry side carbon chains of complex sugars on their outer surface, which are in direct contact with the environment surrounding the cell (Radyukhin V.A., Baratova L.A. Molecular mechanisms of formation of rafts of biological membranes / / Bioorganic Chemistry. - 2020, - Vol. 46, No. 3, pp. 227-238. DOI: 10.31857 / S0132342320030264). The content of atomic carbon and oxygen in specialized cells of living organisms is about 98% of the cell mass. These elements are part of organic substances and perform important biological functions. The main chemical composition of protein molecules is represented by 50-59 percent carbon and 21-24 percent oxygen (Sadchikova E.V., Selezneva I.S. Chemical composition of the cell: Textbook. - Ekaterinburg: State Educational Institution of Higher Professional Education USTU-UPI, 2005. - 42 p.; Shugaley, I.V.Protein chemistry: a textbook / I.V. Shugaley, A.V. Garabadzhiu, I.V. Tselinsky. - St. Petersburg: Prospect Nauki, 2010. - 200 p.).

[0010] It is known that the intercellular spaces separating the cells of the mucous layer from each other, as well as the elements of the lamina propria of the submucosal and muscular layers, are extremely narrow and are not accessible for visual analysis during examination cystoscopy (SC) and routine optical light microscopy (LM). This is possible only through scanning electron microscopy (SEM) at a magnification of thousands of times (Sholand R.F., Gasymov E.K., 2020. Electron microscopic characteristics of the bladder mucosa in interstitial cystitis / painful bladder syndrome in an experiment. / / Urology 2020. - No. 4. - pp. 14-17.). Elemental analysis of the cellular structures of various tissues is a crucial area of ​​clinical medicine and bioanalytics. An interesting approach is to combine elemental visualization and ultrastructural analysis methods, as it allows for spatial analysis of both the atomic composition and volumetric components of cell samples.One of the promising tasks for the development of atomic spectroscopy is the development of methods that allow obtaining multimodal information (Davison, K., Beste, D., Bailey, M., et al. Expanding the boundaries of atomic spectroscopy at the single-cell level: a critical review of the advances of SP-ICP-MS, LIBS, and LA-ICP-MS for the elemental analysis of tissues and single cells. Anal Bioanal Chem 415, 6931–6950 (2023). https: / / doi.org / 10.1007 / s00216-023-04721-8).

[0011] The closest to the declared method is the method described in the article by Sholan R.F., Gasimov E.K. (Electron microscopic characteristics of the bladder mucosa in interstitial cystitis / painful bladder syndrome in the experiment. / / Urology 2020. - No. 4. - pp. 14-17.) and Sholand R.F. / / Cytology 2020. - No. 9. - T62. - pp. 678-684. The disadvantage of this method is the lack of a comprehensive description of the ultrastructural state and elemental composition of all layers of the bladder wall in the experiment.

[0012] In the article Persche M, Injac R, Erman A "Oxidative status and lipofuscin accumulation in bladder urothelial cells from aging mice" in PLoS One. 2013;8(3):e59638. doi: 10.1371 / journal.pone.0059638 the oxidative status in bladder urothelial cells of young and aging mice was studied and described using different methods. It was found that healthy young urothelium has a powerful antioxidant defense system, which functions as a reliable barrier against reactive oxygen species. In contrast, urothelial cells of the aging bladder demonstrate a significant decrease in total antioxidant activity and a significant increase in the level of lipid peroxides (MDA). Similar changes in the oxidative status and structure of surface urothelial cells can be observed in other long-lived postmitotic cells.

[0013] The disadvantage of this method is that it is an experiment and does not provide a comprehensive description of the ultrastructural state of all bladder wall cells and their elemental composition.

[0014] In this study, we found that scanning electron microscopy (SEM) of bladder tissue, including all layers at 6000x magnification, allows for the visualization of closed intercellular spaces, unlike the visual image of the mucosa obtained during minimally altered cystoscopy. During inflammation, the intercellular spaces in the mucosal layer expand, forming multiple intercellular channels. Similar changes are observed in the submucosa and muscular layers, which tend to close after effective treatment. This method allows for visual assessment of the ultrastructural state of specialized bladder wall cells, as well as the percentage of atomic oxygen over carbon on their surface, which is characteristic of inflammation and correlates with the clinical manifestations of interstitial cystitis / bladder pain syndrome.

[0015] The objective of the invention is the clinical diagnosis of the ultrastructural state of the bladder without signs of inflammation and its features in the process of clinical manifestations of bladder inflammation using the example of interstitial cystitis / painful bladder syndrome (IC / PBS).

[0016] The technical result of the proposed method is the diagnosis of bladder inflammation based on the results of scanning electron microscopy (SEM) and elemental spectrometry (ES) of a bladder biopsy.

[0017] The technical result is achieved in that in the method for diagnosing inflammation of the bladder based on the results of scanning electron microscopy and elemental spectrometry, a biopsy of the bladder is performed on patients during endoscopic cystoscopy, layer-by-layer scanning electron microscopy of the collected tissue is carried out in combination with elemental spectrometry of the surface of cells of the mucous membrane, submucosal and muscular layers, and in the event that in each of the three layers of the bladder the percentage predominance of atomic oxygen over atomic carbon is determined, then inflammation of the bladder is diagnosed, and in the event that in each of the three layers of the bladder the percentage predominance of atomic carbon over atomic oxygen is determined, then the absence of inflammation of the bladder is diagnosed.

[0018] The method is based on identifying the percentage predominance (digital predominance in percentage terms) of the content of atomic carbon (C) over atomic oxygen (O) on the surface of specialized cells of the bladder wall as an indicator of its inflammation.

[0019] The essence of the claimed invention is that during endoscopic cystoscopy, a biopsy of the bladder is performed on patients, followed by layer-by-layer scanning electron microscopy in combination with elemental spectrometry of the surface of specialized cells - urothelial cells of the mucous membrane, stromal cells of the submucosal layer, muscle cells of the muscular layer of its wall, and based on the percentage predominance of atomic oxygen over carbon, a conclusion is made about the ultrastructural changes characteristic of inflammation.

[0020] The claimed method is explained and illustrated by the following figures:

[0021] Fig. 1. Scanning electron microscopy of specialized urothelial cells of the bladder mucosa (A) and elemental spectrometry of their surface (B) of patient O., 28 years old, without clinical manifestations of bladder inflammation.

[0022] Fig. 2. Scanning electron microscopy of specialized stromal cells of the submucosal layer of the bladder (A) and elemental spectrometry of their surface (B) of patient O., 28 years old, without clinical manifestations of bladder inflammation.

[0023] Fig. 3. Scanning electron microscopy of specialized muscle cells (myocytes) of the muscular layer of the bladder (A) and elemental spectrometry of their surface (B) of patient O., 28 years old, without clinical manifestations of bladder inflammation.

[0024] Fig. 4. Scanning electron microscopy of specialized urothelial cells of the bladder mucosa (A) and elemental spectrometry of their surface (B) of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) before treatment.

[0025] Fig. 5. Scanning electron microscopy of specialized stromal cells of the submucosal layer of the bladder (A) and elemental spectrometry of their surface (B) of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / PBS) before treatment.

[0026] Fig. 6. Scanning electron microscopy of specialized muscle cells (myocytes) of the muscular layer of the bladder (A) and elemental spectrometry of their surface (B) of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / CB) before treatment.

[0027] Fig. 7. Scanning electron microscopy of specialized A cells (A - mucous, B - submucous, C - muscular layer) of the bladder and elemental spectrometry of their surface in patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) after treatment.

[0028] The proposed method for diagnosing bladder inflammation based on the results of scanning electron microscopy and elemental spectrometry is carried out as follows.

[0029] Patients with suspected bladder inflammation undergo ultrastructural tissue biopsy under general anesthesia. This tissue is then examined using layer-by-layer scanning electron microscopy (SEM) and elemental spectrometry (ES) of the surfaces of specialized cells—the mucosa (urothelial cells of the bladder mucosa), the submucosa (stromal cells of the submucosa of the bladder), and the muscle cells (myocytes) of the muscular layer of the bladder. This includes visual assessment of the cell's ultrastructural state and the percentage ratio of atomic carbon (C) and atomic oxygen (O) on its surface.

[0030] Next, based on the analysis of the obtained data on the percentage predominance of atomic carbon (C) on the surface of a specialized cell, a conclusion is made about its structural and functional integrity and the absence of inflammation, and based on the percentage predominance of atomic oxygen (O) over carbon (C), a conclusion is made about the presence of ultrastructural changes corresponding to inflammation (changes in the size, shape of specialized cells and expansion of their intercellular spaces, accompanying clinical manifestations and changes characteristic of inflammation (frequent painful urination).

[0031] To date, 5 patients (three women and two men) with bladder inflammation have been examined using the proposed method; all of them had their diagnosis clarified and confirmed by the results of clinical, laboratory, and morphological ultrastructural studies before and after adequate treatment.

[0032] The method was developed at the Department of Pediatric Surgery with a course in urology and andrology and has undergone clinical testing with positive results at the urology department of the State Budgetary Healthcare Institution of the Tyumen Region “Regional Clinical Hospital No. 2” in the city of Tyumen.

[0033] The proposed method has a number of advantages over known ones, the main one being the high accuracy of ultrastructural and spectrometric diagnostics of inflammatory diseases of the bladder, as well as the assessment of the effectiveness of the treatment at the atomic level.

[0034] The claimed invention is illustrated by the following clinical examples.

[0035] Thus, a patient without a urological history (traumatic rupture of the bladder) and a patient with clinical manifestations of bladder inflammation interstitial cystitis / bladder pain syndrome (IC / BPS) were selected as an example for comparison.

[0036] Example 1.

[0037] Patient O., 28, with no urological history, was admitted to the urology department as an emergency patient an hour after a bicycle accident, complaining of lower abdominal pain and blood in her urine. A cystogram (an X-ray of the bladder with contrast) revealed contrast leakage into the abdominal cavity.

[0038] Patient examination results: Renal ultrasound revealed no abnormalities. Urinalysis revealed 1-2 leukocytes per high-power field, specific gravity 1022, pH 6.0, 1-2 epithelial cells per high-power field, 10-15 erythrocytes per high-power field. Bacteriological examination of urine revealed no microbial growth.

[0039] During surgical exploration of the bladder and suturing of a rupture in its wall (linear defect measuring 2.2 by 2.4 cm), a gentle excision of the rupture edges was performed with a double-layer absorbable suture and transurethral drainage of the bladder with a catheter after surgery. Bladder tissue was sent for biopsy. The postoperative period was uneventful, and on the 14th day after surgery, suture removal, and recovery, the patient was discharged for outpatient treatment.

[0040] SEM biopsy results. Scanning electron microscopy of specialized urothelial cells of the bladder mucosa (Fig. 1A). The cells of the mucosa (urothelium) are polygonal in shape and uniform in size, with tightly closed intercellular spaces. The surface of the facet cells is covered with short microvilli due to the folding of the surface membranes. Elements of the lamina propria, including epithelial cells, stromal cells with fibroblast-like dendritic cells, and basement membranes are structurally preserved.

[0041] Elemental spectrometry of the surface of specialized urothelial cells of the bladder mucosa (Fig. 1B), the obtained summary results of elemental spectrometry of the surface of specialized urothelial cells of the bladder mucosa of patient O., 28 years old, without clinical manifestations of bladder inflammation are presented in Table 1:

[0042] Table 1 - Summary results of elemental spectrometry of the surface of specialized urethelial cells of the bladder mucosa of patient O., 28 years old, without clinical manifestations of bladder inflammation

[0043] Element Atoms % Carbon 60,789 Oxygen 36,424

[0044] Scanning electron microscopy of specialized stromal cells in the submucosa of the bladder (Fig. 2A). Collagen fibers and individual bundles synthesized by the stromal cells are visualized on the surface, and the structured muscle fibers of the detrusor have narrow intercellular spaces.

[0045] Elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder (Fig. 2B), the obtained summary results of elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder of patient O., 28 years old, without clinical manifestations of bladder inflammation are presented in Table 2.

[0046] Table 2 - Summary results of elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder of patient O., 28 years old, without clinical manifestations of bladder inflammation

[0047] Element Atoms % Carbon 63,267 Oxygen 35,057

[0048] Visual graphical assessment of the elemental composition of the surface of specialized cells revealed a percentage predominance of atomic carbon over oxygen, indicating the preservation of the ultrastructural state of the mucosa. For specialized stromal cells of the submucosa, these values ​​were significantly lower, confirming the mucosa's protection from urine penetration into the submucosa of the bladder.

[0049] The percentage predominance of carbon over oxygen on the surface of muscle layer cells (myocytes) also indicated their preservation (Fig. 3B). Scanning electron microscopy of specialized muscle cells (myocytes) of the bladder muscular layer (Fig. 3A), elemental spectrometry of their surface (Fig. 3B), and the obtained summary results of elemental spectrometry of the surface of muscle layer cells (myocytes) of the bladder of patient O., 28 years old, without clinical manifestations of bladder inflammation are presented in Table 3.

[0050] Table 3 - Summary results of elemental spectrometry of the surface of muscle layer cells (myocytes) of the bladder of patient O., 28 years old, without clinical manifestations of bladder inflammation

[0051] Element Atoms % Carbon 71,586 Oxygen 28,414

[0052] Summary: Based on the data obtained, patient O., 28, has no ultrastructural or elemental manifestations of inflammation in the bladder wall, which could have caused bladder fragility following a fall. The patient is recommended to undergo follow-up with a urologist at the clinic.

[0053] The presented clinical example demonstrates that when the percentage of atomic carbon (C) content is higher than that of atomic oxygen (O) on the surface of specialized cells of the bladder wall, when performing layer-by-layer scanning electron microscopy in combination with elemental spectrometry, a conclusion is made about the structural and functional integrity of the cell and the absence of changes characteristic of inflammation.

[0054] Example 2.

[0055] Patient I., 42, with suspected bladder inflammation and ineffective treatment, was admitted to the urology department for a biopsy. During an instrumental cystoscopy under general anesthesia, engorgement of the mucosal vessels, turbidity, and ulceration were visualized.

[0056] SEM biopsy results. Scanning electron microscopy of specialized urothelial cells of the bladder mucosa (Fig. 4A). In contrast to the results of a visual ultrastructural assessment of the condition of specialized bladder cells without pathology, clinical manifestations of inflammation were characterized by deformation of urothelial cells with the appearance of roughness and expansion of intercellular spaces.

[0057] Elemental spectrometry of the surface of specialized urothelial cells of the bladder mucosa (Fig. 4B), the obtained summary results of elemental spectrometry of the surface of specialized urothelial cells of the bladder mucosa of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) are presented in Table 4.

[0058] Table 4 - Summary results of elemental spectrometry of the surface of specialized urothelial cells of the bladder mucosa of patient I., 42 years old, with clinical manifestations of bladder inflammation before treatment

[0059] Element Atoms % Carbon 38,187 Oxygen 49,316

[0060] In contrast to the results of elemental spectrography of the surface of specialized cells of the bladder without inflammation, for clinical manifestations of cystitis accompanied by the appearance of pain in the projection of the bladder and frequent urination, there was a characteristic percentage predominance of atomic oxygen (O) over carbon (C) on the surface of urothelial cells of the mucous membrane, which coincided with a decrease in cell size, expansion of intercellular spaces increasing the possibility of urine penetration into the submucosal layer.

[0061] Scanning electron microscopy of specialized stromal cells in the submucosa of the bladder (Fig. 5A). In areas with a completely absent epithelial covering, the surface of damaged stromal cells with smooth muscle is visualized.

[0062] Elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder (Fig. 5B), the obtained summary results of elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) are presented in Table 5.

[0063] Table 5 - Summary results of elemental spectrometry of the surface of specialized stromal cells of the submucosal layer of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation before treatment

[0064] Element Atoms % Carbon 11,812 Oxygen 84,916

[0065] At the level of specialized stromal cells of the submucosa, the percentage predominance of oxygen over carbon indicated ultrastructural changes characteristic of inflammation.

[0066] Scanning electron microscopy of specialized muscle cells (myocytes) of the muscular layer of the bladder (Fig. 6A). The ultrastructural state of the myocytes of the muscular layer is also subject to changes in the size and shape of the cells, expansion and layering of the intercellular spaces between them.

[0067] Elemental spectrometry of the surface of specialized muscle cells (myoites) of the muscular layer of the bladder (Fig. 6B), the obtained summary results of elemental spectrometry of the surface of specialized muscle cells (myoites) of the muscular layer of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) are presented in Table 6.

[0068] Table 6 - summary results of elemental spectrometry of the surface of specialized muscle cells (myoites) of the muscular layer of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation before treatment

[0069] Element Atoms % Carbon 29,407 Oxygen 53,983

[0070] On the surface of muscle cell layers (myocytes), the percentage predominance of oxygen over carbon indicated ultrastructural changes characteristic of inflammation.

[0071] Summary: Based on the data obtained in patient I., 42, one of the causes of bladder inflammation and the low effectiveness of standard treatment was ultrastructural failure of the bladder wall barrier. The patient underwent an 8-week course of intravesical bladder irrigation (instillations) with a solution containing hyaluronic acid as a viscoelastic protector for the bladder mucosa. The patient was recommended to undergo follow-up with a urologist and undergo a control cystoscopy and bladder wall biopsy after completion of treatment.

[0072] The presented clinical example demonstrates that when the percentage of atomic oxygen (O) content is higher than that of atomic carbon (C) on the surface of specialized cells of the bladder wall, when performing layer-by-layer scanning electron microscopy in combination with elemental spectrometry, a conclusion is made about the ultrastructural changes in the cell characteristic of inflammation and inflammation of the bladder is diagnosed.

[0073] Example 3.

[0074] In a urology hospital, after completing a course of therapy, a 42-year-old patient I. with IC / BPS underwent a cystoscopy, where she underwent pincer sampling of material and scanning electron microscopy in combination with elemental spectrometry of the surface of specialized cells: urothelial cells of the bladder mucosa (Fig. 7A), stromal cells of the submucosal layer of the bladder (Fig. 7B), and muscle cells (myoites) of the muscular layer of the bladder (Fig. 7C).

[0075] The obtained summary results of elemental spectrometry of the surface of specialized cells of the mucous membrane, submucosal and muscular layers of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation (IC / BPS) after treatment are presented in Table 7.

[0076] Table 7 - summary results of elemental spectrometry of the surface of specialized cells of the mucous membrane, submucous and muscular layer of the bladder of patient I., 42 years old, with clinical manifestations of bladder inflammation after treatment

[0077] Element Atoms % A IN WITH Carbon 65,838 63,533 65,339 Oxygen 34,162 33,119 34,661

[0078] The results of SEM image analysis of specialized bladder wall cells in patient I., 42, with IC / PBS, after an 8-week course of standard therapy showed that a reduction in the severity of clinical and laboratory manifestations of inflammation was associated with changes in the ultrastructural state of specialized bladder wall cells. Elemental spectrometry revealed a decrease in the percentage of atomic oxygen (O) on the surface of specialized cells and an increase in atomic carbon (C) levels, which coincided with a reduction in pain and urinary frequency to an acceptable level.

[0079] Abstract: A course of standard anti-inflammatory treatment, including instillation of the bladder with a viscoelastic protector of its mucous membrane, administered to patient I., 42 years old, was accompanied by a tendency to restore the size and shape of cells, a reduction in the intercellular spaces that limit the flow of urine into the submucosal and muscular layers, an increase in the percentage of carbon on the surface of all specialized cells, which coincided with a decrease in the manifestation of pain and the frequency of urination to an acceptable level.

[0080] The presented clinical example demonstrates that the percentage predominance of atomic carbon (C) over atomic oxygen (O) on the surface of specialized cells of the bladder wall during layer-by-layer scanning electron microscopy in combination with elemental spectrometry leads to the conclusion about the absence of ultrastructural changes in the cell characteristic of inflammation.

[0081] The presented clinical examples demonstrate the following. Normally, the cells of the superficial layer of the bladder are polygonal in shape, uniform in size, and have clearly defined boundaries between them. An inflammatory reaction in the bladder wall was characterized by cell polymorphism with a large number of villi and roughnesses forming intercellular channels. After standard therapy, ultrastructural examination revealed a decrease in the number of villi and roughnesses, with a tendency toward closure of the intercellular channels. Normally, the stromal cells of the submucosal layer are represented by bundles of collagen fibers enveloping fibroblasts. Inflammatory reactions in the bladder wall were characterized by the presence of intercellular channels with a large number of plasma cells in contact with macrophages, fibroblasts, and lymphocytes, surrounded by loosened collagen and elastic fibers.Standard therapy resulted in a reduction in tissue porosity at the ultrastructural level due to the restoration of collagen and elastic fiber structure. Normally, the detrusor muscle is composed of three layers of muscle cells. During inflammation, myocytes lose their structure due to interfibrillar and interfascicular edema, connective tissue proliferation with elements of intercellular fibrosis, and increased volumetric density. Standard therapy resulted in restoration of the detrusor muscle cell structure, with a tendency toward a reduction in intercellular spaces.

[0082] Thus, the proposed method for diagnosing bladder inflammation based on scanning electron microscopy and elemental spectrometry of the bladder wall cell surface during clinical and laboratory manifestations of inflammation (using IC / PBS as an example) is recommended for implementation in clinical practice as a user-friendly visualization tool for urologists. The method can be recommended both for widespread use in urological practice and for scientific research into new methods for monitoring the effectiveness of treatment for this type of pathology.

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