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10MEDICAL SCIENCES
MORPHOLOGICAL DEFORMATION OF THE ARTERIAL VESSELS OF SENSORY GANGLIA DURING ACUTE EXPERIMENTAL ENDOTOXEMIA
Aliyarbayova A.
Senior teacher of department Cytology, embryology and histology, Doctor of philosophy (PhD) in Medicine
Aliyeva I.
Senior teacher of department Cytology, embryology and histology, Doctor of philosophy (PhD) in Medicine
Nacafova T.
Senior teacher of department Cytology, embryology and histology
Huseynova Sh.
Assistant of department Cytology, embryology and histology
Sadiqi I.
Assistant of department Cytology, embryology and histology Azerbaijan Medical University, Baku
Abstract
The aim of the present study was to investigate the role of arterial vessels supplying (extra organic vessels) dorsal root sensory ganglion at lipopolysaccharide (LPS) induced inflammation. As we know vascularization of each organ appointed its proper function. That's why morphological investigation of extraorganic vessels at different pathological situation is necessary. Morphological deformation of endothelial cells and arterial blood vessels vascularizing dorsal root sensory ganglia at acute endotoxemia caused to decrease luminal diameter of vessel. Thus flow of blood to organ decreased and elements of sensory ganglion exposed to hypoxia, as well as to mechanical force of formed edematous fluid around them.
Keywords: vessels, arterioles, sensory ganglion, acute endotoxemia, ultrastructure.
Introduction. Dorsal root sensory ganglia play an essential role in the transduction of the sensory signals, also pain signals from the periphery to the central nervous system (1, 2). Pathological circumstances such as inflammation, mechanical or chemical nerve injury, hy-poxia can sensitize components of dorsal root sensory ganglion and alter their functions (3, 4). LPS is an endotoxin that found on the outer membrane of pathogens and non-pathogen gram-negative bacteria. Lipopoly-saccharides are molecules composed of lipids and pol-ysaccharides. Lipopolysaccharide (LPS)-induced inflammation and dysfunction in the sensory ganglion the major risk factors for subsequent neuropathic pain in neuropathy (5). LPS directly influenced endothelial cell of blood vessels. Endothelial cells are dynamic components of the vessels that maintain homeostasis of all organs of body (6, 7). Vascular endothelial dysfunction is an initiation factor of different neuropathic situation.
The purpose of the research work was to study alterations in dorsal root sensory ganglion vessels, especially in arterial vessels which participated in vascularisation of organ, during acute experimental endotoxemia.
Materials and methods. The objects of research were dorsal root sensory ganglions and surrounding tissue elements taken from 20 white male rats with weight of 180-220 gr. The animals grew up in scientific laboratory with a special pathogen-free condition. All procedures complied with the Principles of Laboratory and Animal Care established by the Azerbaijan Medical University. Research objects (rats) divided into 2 groups: control (intact) and experimental.
Firstly, the animals in control groups (10 rats) laid down in a supine position on a pad. Then thoracic cavity of animals had opened with parasternal incisions under deep ketamine / xylazine (100/10mg/kg) anesthesia. Afterward the vessels of objects had washed 1 minute at a pressure of 80 mm Hg with Hank's solution. Hanks solution, containing 500 ml physiological salt solution +50V heparin +1 ml 1% papaverine hydro-chloride administered by catheter to the initial part of aortic arch; then research animals fixated with vascular perfusion with 2,5% glutaraldehyde solution prepared in 0,1M phosphate buffer (pH=7,4). The perfusion lasted 25 minutes under a pressure of 80 mm Hg. After intravascular fixation, a spinal canal had opened and removed dorsal root ganglions with surrounding soft tissue.
Other groups of animals (10 rats) used for experiment. Acute experimental endotoxemia established by intravenous injection into the tail vein of rats a nonpu-rified LPS from Escherichia coli (Serotype 0111:B4 InvivoGen, San Diego, CA 92121) at a dose of 1,0mg/kg dissolved in saline. After 2 hours of the intravenous administration, the animals decapitated under ketamine anesthesia; the abdominal and thoracic cavity of the rats had opened, internal organs taken out and the vertebral bodies were cut. Later by the help of special lancet, a spinal canal carefully opened and dorsal root ganglions with surrounding soft tissue removed from the intervertebral foramen. The specimen had been fixated in solution that contains 2% paraformaldehyde, 2% glutaraldehyde and 0.1% picrin acid prepared in phosphate buffer (pH 7.4).
The ganglions with surrounding soft tissue purchased from both groups animals then postfixed in 1% osmium acid solution during 2 hours in phosphate buffer. After specimens washed in distilled water, dehydrated in alcohol, embedded in Spurr, Epon - Araldit medium and prepared blocks according to general methods accepted in electron microscopy (8).
Obtained from blocks on ultratome Leica EM UC7 the semithin (1-2 ^m) sections were stained with methylene blue, azure II and basic fuchsine accordingly. After examination with Latimet (Leitz) microscope necessary parts of images were taken on Pixera (USA) digital camera. In addition, gained silver and gold ultrathin
sections from same blocks were stained with 2% uranyl acetate solution, then in 0.6% lead citrate made in NaOH 0.1N solution; each section was carefully examined under the 80-120 kV in Transmission Electron Microscope JEM-1400 (Jeol, Japan), photographed at different magnification and received the electron micrographs. This procedure continued until the block had used.
Results and discussion. in figure 1A (control groups specimen) demonstrated connective tissue elements surrounding dorsal root ganglia and ventral root of spinal nerve.
Figure 1. Histological section of extraorganic vessels situated in surrounding connective tissue elements of dorsal root ganglia in control (A) and acute endotoxemia (B). Explanation given in text. Semithin section. Stain:
methylene blue, azure II and basic fuchsine. Scale bar: A 50¡m, B 50 ¡m. Abbreviations: A-arterial vessels, CTE-connective tissue elements, DRG-dorsal root ganglion, VR-ventral root, NB- nerve bundles, by two arrow shown destruction of covering layer of nerve bundles in 1B.
In the middle of figure shown extraorganic arterial vessels (shown by A) that participated in vascularisation of sensory ganglion and lymphatic vessels which wall contains single layer of endothelial cells (shown by red arrow). On the left upper corner on histological slide seen neurons of the sensory ganglion and between them visible branched intraorganic vessel (free luminal spaces). Dorsal root ganglion izolated from surrounding connective tissue elements by thick capsule.
It's easily detected difference between arterial and other (venous, lymphatic) vessels by thickness of their wall. Thickness to vascular wall in arterial vessels is given by smooth muscle cells. Luminal diameter of extraorganic arterial vessels that demonstrated on slide 1A is more than 50^m. At the right down corner visible large sized myelinated nerve fibers (without any destruction) composing the ventral spinal roots.
In figure 1B shown specimen from experimental groups which obtained after 2 hours of intravenous injection LPS from E. Coli. At acute experimental endotoxemia, firstly attracted attention the structurally (shape and size) changes in the extraorganic vessels of the spinal ganglia. The altering accompanied by acute deformation, which is observed with narrowing of their lumen more than 2 times as a result of contraction (spasm) of smooth muscle fibers participated in the formation of the middle layers of the arterial vessels.
The thickness (due to contraction of smooth muscle cells) and staining (more osmiophilic) of the arterial vessels wall also was changed.
In addition, the bundles of nerve fibers deformated and destructed (on the right corner shown by two red arrow). The detection of edematous fluid (shown by red star) around the arterial vessels, bundle of nerve fibers and within connective tissue elements, as well as surrounding dorsal and ventral spinal roots, also indicates a violation of the permeability of vessels walls.
All information given above in comparasion with endotoxemia was gained at histological slide, but detailed information were taken only under electron microscopic study.
In the control group on figure 2A only observed a protrusion on the luminal surfaces of the nucleus-located parts of endothelial cells; and thin without any fragmentation (interruption) internal elastic membrane isolated endothelial cell from the smooth muscle cells in tunica media. As well as detected the arrangement of smooth muscle cells in three layer in tunica media, also visible effector nerve terminal (ENT) in tunica adventitia of arterial vessel. Endothelial -smooth muscle cells connection unvisible, but between smooth muscle cells which arranged minimum in three layer that composing tunica media recognized junction.
Figure 2. Ultrastructural study of vascular elements of the extraorganic arterial vessels wall in intact groups animals (A) and deviation of it at experimental endotoxemia (B). Stain: uranil acetat and lead citrate. Scale bar on both electronograms 2 ¡m. Abbreviations: LEAV - lumen of extraorganic arterial vessels; IEM - internal elastic membrane; E1, E2, E3 - endothelial cells; SMC - smooth muscle cells; 1, 2, 3 smooth muscle cells arranged in 3 layer. The explanation is given in text.
On figure 2B (at endotoxemia model) at ultrastructural level wasnt established type of intercellular junction between endotelial cells, because its acute deformated. As well as accumulation of oedematous fluid in cytoplasm and between of endothelial cells mechanically influenced and destroyed structural peculiarities of it. On slide demonstrated three in different shape and size endothelial cells (E1, E2, E3) that lined in tunica intima of arterial vessel. Destruction and breaking down of cytoplasm of E2 cell sharply visible as well as shown decreased volume of E3 cell (9).
During acute endotoxemia, as a result of the sharp contraction of the muscle layer of the extra organic arteries, the nucleus-located central part together with perinuclear parts of the endothelial cells swell towards the vascular cavity, causing a sharp narrowing of their lumen and hypoxia, that reducing the volume of arterial blood entering the vascular network. The destructive changes occur in the endothelial cells located on the inner layer of the arteries, as well as in inner elastic membrane that separated tunica intima from tunica media. Also the protrusions on the abluminal surfaces of the endothelial and smooth muscle cells facing each other divided the inner elastic membrane into separate fragments in longitudinal size 1,2 - 4,5 ^m. A violation (fragmentation) of internal elastic memrane between tunica intima and tunica media demonstrated in figure 2B.
Conclusion. In acute experimental endotoxemia morphological alteration on structure of endothelial cells of extraorganic arterial vessels of the sensory dorsal root ganglion result in increasing in the permeability of the vessels, as a result of the formation of the edema fluid caused by E. coli endotoxin. The mechanical pressure of edematous fluid altered not only connective tissue elements surrounding vessels, nerve roots, also nerve elements (neuron, glial cells) in ganglia, that event called destructive reactive changes.
Since the mentioned changes, mechanical and destructive disorders occurring in other cellular and non-cellular structures located within and around the ganglia, as well as structural disintegration of the spinal sensory ganglion capsule influence to alteration function of it.
This work is implemented basing on financial support of the Science Development Foundation under the President of the Republic of Azerbaijan. Qrant № EiF-2011- 1(3)-82/44/3-M-6.
References
1. Ti-Yen Yeh, I-Wei Luo, Yu-Lin Hsieh, To-Jung Tseng, Hao Chiang, Sung-Tsang Hsieh. Peripheral neuropathic pain: from experimental models to potential therapeutic targets in dorsal root ganglion neurons / Cells. 2020 Dec 21;9(12):2725. doi: 10.3390/cells9122725.
2. Michael F Esposito, MD, Rudy Malayil, MD, Michael Hanes, MD, and Timothy Deer, MD Unique characteristics of the dorsal root ganglion as a target for neuromodulation / Pain Med. 2019 Jun; 20(Suppl 1): S23-S30
3. Cheryl L Stucky, Alexander R Mikesell. Cutaneous pain in disorders affecting peripheral nerves /Neurosci Lett. 2021 Nov 20;765:136233. doi: 10.1016/j.neulet.2021.136233. Epub 2021 Oct 1
4. Markus Leo, Linda-Isabell Schmitt, Patricia Küsterarent, Andrea Kutritz, Tienush Rassaf, Christoph Kleinschnitz, Ulrike B Hendgen-Cotta, Tim Hagenacker. Platinum-based drugs cause mitochondrial dysfunction in cultured dorsal root ganglion neurons /Int J Mol Sci. 2020 Nov 16;21(22):8636. doi: 10.3390/ijms21228636
5. Anita Mikolajczyk, Dagmara Zlotkowska. Subclinical lipopolysaccharide from salmonella enter-itidis induces neuropeptide dysregulation in the spinal
cord and the dorsal root ganglia/ BMC Neurosci. 2019 Apr 25;20(1):18. doi: 10.1186/s12868-019-0502-z
6. Xuhui Hou, Songbai Yang, Jian Yin. Blocking the REDD1/TXNIP axis ameliorates LPS-induced vascular endothelial cell injury through repressing oxida-tive stress and apoptosis /Am J Physiol Cell Physiol. 2019 Jan 1;316(1):C104-C110. doi: 10.1152/aj-pcell.00313.2018.
7. Juan M Jimenez-Andrade, Monica B Herrera, Joseph R Ghilardi, Marina Vardanyan, Ohannes K Melemedjian, and Patrick W Mantyh. Vascularization of the dorsal root ganglia and peripheral nerve of the
mouse: Implications for chemical-induced peripheral sensory neuropathies/ Mol Pain. 2008; 4: 10
8. Electron microscopy. Methods and protocols. Edited by John Kuo. // USA, Totowa, New Jersey: Humana Press Inc., 2007, 608 p.
9. Jessica M., Micaela G., Vincenzo M., Cristina C., Saverio N., Roberta M., Miriam S., Francesca B., Federica S., Stefano R., Maria C., Francesca O., Rocco M., Sara P., Ernesto P., Carolina M. And Vincenzo M. The Role of endothelial dysfunction in peripheral blood nerve barrier: molecular mechanisms and pathophysio-logical implications // Int. J. Mol. Sci. 2019, 20, 3022; doi:10.3390/ijms20123022.
CARIES COURSE IN CHILDREN. THE CURRENT STATE OF THE ISSUE
Kotelban A.
PhD, Associate Professor, Chernivtsi, Ukraine, Department of Pediatric Dentistry Bukovynian State Medical University
Moroz P.
PhD, Associate Professor, Chernivtsi, Ukraine, Department of Surgery № 1 Bukovynian State Medical University Zhyrulyk Ju.
Student of stomatological faculty Bukovynian State Medical University
Abstract
Undoubtedly, the most common disease of the oral cavity in childhood is caries. This dental nosology is one of the oldest and most common in the world.
The results of epidemiological studies show that a high prevalence of caries of temporary teeth is observed in children under two years. In different regions of Ukraine, this figure remains consistently high. This indicates the need to start preventive measures from an early age, taking into account the regional characteristics of the formation of dental pathology.
Factors in the development of dental caries are individual. There are more than 100 causes of the risk of dental caries in childhood. They can be of different intensity and nature, there are different options for their interaction.
Keywords: caries, prevalence, dental health, children, S. Mutans.
According to the WHO, the incidence of dental caries in different countries and among different contingents ranges from 80% to 98%. In the last two decades, there has been a trend of increasing incidence among children, especially in economically developed countries, and by 6-7 years, 80-90% of children have caries of varying depth [21]. In Ukraine, these indicators also remain quite high and tend to increase [2,4,6].
Clinical observations indicate the rapid progression of caries, which occurs in the early stages after the eruption of temporary teeth. Thus, in children of the first year of life caries of temporary teeth occurs in approximately 12-15% of cases, in children 3 years of age increases to 60%, and reaching 6 years of age is found in 85% of subjects [7].
In adolescents, the prevalence of caries of permanent teeth is high, in 12-year-olds this figure averages 64.8%, and among 15-year-olds - 76% [4].
In our opinion, children of 5-7 years of age deserve special attention, as this period of a child's life is a turning point due to the significant psychological, physical and mental load on the child's body during preparation and the first year of school. This period coincides with the eruption of the first permanent teeth -
molars, which in turn also has an impact on the growth and development of the body as a whole and on the ecosystem of the oral cavity.
The frequency of dental caries is different in different regions of Ukraine. High levels of dental caries are registered in the studied Lviv, Ivano-Frankivsk, Chernivtsi, Zakarpattia, Chernihiv, Khmelnytsky, Luhansk regions [7]. Regions with an average level of morbidity include Mykolayiv, Kherson, Odesa, Vinny-tsia, Ternopil, Zhytomyr, Zaporizhia regions and the Crimean Autonomous Republic [4, 7]. Low levels of dental caries have been found in Poltava, Sumy, Kyiv, Dnipropetrovsk, Kharkiv, Kirovohrad regions [2, 4, 7]. Particularly relevant medical and social research among the population of regions with a pronounced mi-cronutrient deficiency, which determines the endemic nature of the area. The growth of epidemiological indicators of caries in some regions depends on environmental and geophysical factors, including contamination by radionuclides, heavy metals, pesticides and other xenobiotics; urbanization of the region and the state of reactivity and adaptive-compensatory capabilities of the organism [1].
