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8. Hossniann K.A. Pathophysiology and therapy of experimental stroke / K.A. Hossmann // Cellular and Molecular Neurobiology. - 2006. - Vol. 26, Issue 7-8. - P.1057-1083.
9. Mohr J.M. Stroke: pathophysiological diagnosis and management / J.M. Mohr, D.W. Choi, J.C. Grotta. - Philadelphia: Livingstone, 2004. - 1616 p.
10. Morphological changes in capillaries in the ischemic brain in Wistar rats / Y. Taguchi, S. Takashima, E. Sasahara //
Archives of Histology and Cytology. - 2004. - Vol. 67, № 3. - P. 253-261.
11. Wahtgren N. Thrombolisis with alteplase for acute ischemic stroke in the Safe imptementation of Thrombolisis in Stroke- Monitoring Study (SITS-MOST): an observational study // N. Wahtgren, N. Ahmed, A. Davalos // The Lancet Neurology. - 2007. - Vol. 369. - P. 275.
English version: CHRONOLOGY OF DESTRUCTIVE
AND DEGENERATIVE INFLAMATION PROCESSES IN WISTAR RATS
WITH EXPIREMENTAL BRAIN STROKE
Koiesnyk V. V.
SI A. P. Romodanov Institute of Neurosurgery, Kyiv
SI I. I. Mechnikov Institute of Microbiology and Immunology, Kharkiv
Three-month-old intact maie Wistar rats (n= 84) and animals with modeeed ischemic stroke (n= 84), observed during 28 days, served as material for this research. Microscopic examination was carried out in a traditional way Bits of the brain were removed, washed, fixed in 12 % formaldehyde, subjected to postfixation and dehydrated. Sections were contrasted, analyzed under a microscope and photographed with a digital camera "Canon EOS-3000". It was found that a pronounced structural-functional regression of neurocytes and endotheiium of cerebral microvessels took place during days 1-17 of the observation. The above regression was attributed to development of thrombosis, trophic changes, development of destruction and necrosis. Degeneration and development of inflammation were considered characteristic morphological features. Thus, microscopic changes in the cerebral cortex of male Wistar rats were of a phase character; depended upon terms of the début of modeled ischemic emboic stroke; consisted in destruction of endotheliocytes, gia and nucleocytoplasmic components of neurons and formation of inflammatory focuses.
Key words: destruction, degeneration, inflammation, male Wistar rats, modeled ischemic embolic stroke.
This work is a part of the research: "Development of technology for obtaining autocells of various types of biological tissue from the bone marrow stromal cells and their use for the treatment of diseases of various origins using autotransplantation", No. 0106 U003995.
Introduction
From the point of view of domestic and foreign researchers, stroke at the present time is a major issue both of the operative and conservative neurology [2, 4]. WHO statistics indicate the annual incidence of stroke is 16 million people, 5 million of whom die. In Ukraine, these values reach 110-130 thousand people. Despite modern imaging techniques, certain aspects of stroke remain poorly understood. The principles of structural and functional studies of chronology of the stroke process, as well as certain aspects of the pathogenesis, clinical disease and morphogenesis of the delayed period demand further development [8]. Undoubtedly, an auxiliary factor in reaching this goal may become experimental investigations on the chronology of the pathogenesis of ischemic stroke (development and consistency of degenerative and destructive, inflammatory reactions) in which a substantial part is to be played by laboratory animals (linear rats, mice, rabbits, etc.) [6]. The ability to conduct a reliable extrapolation model as well as a relatively short period of the observation of the life cycle of animals contribute to a detailed analysis of the results and their further implementation in clinical practice. Thus, the study of the chronology of degenerative-destructive and inflammatory reactions in rats with experimental stroke appears to be important.
Purpose
To study the chronology of destructive-degenerative and inflammatory processes in Wistar rats with experimental stroke.
Material and methods of research
The material for the study were three-month-old male Wistar rats of the control (n=84) and experimental (modeled cerebral embolic ischemic stroke; n=84) groups. Modeling of a stroke took place in strict accordance with the established pattern-algorithm [1]. The animals were housed in standard metal cages with dry sawdust (pine, birch) litter, under standard conditions of vivarium climate control (t= 18-24° C, humidity 50-70%, luminance 60 lx). Extruded feed and water were given ad libitum 30-32 g, 2 times a day. Rats were taken out of the experiment by an overdose of ketamine anesthesia (according to the European Convention for the Protection of vertebral animals used for experimental purposes, Strasbourg, 1986). Slices of the brain and its vessels (size 0.5 cu. cm) of control and experimental males were researched post mortem. For this purpose, the slices were fixed in 12 % solution of formalin on phosphate buffer (pH=7,0-7,2), with t0=18-200C, dehydrated with alcohols of increasing concentration and embedded in paraffin/celloidin. From the blocks, cuts were made 10-15 microns thick (microtome MK-25, Russia); the latter were dyed with hema-
* To cite this English version: Kolesnyk V. V. Chronology of destructive and degenerative inflamation processes in Wistar rats with expiremental brain stroke - //Problemy ekologii ta medytsyny. - 2013. - Vol 17, № 5-6. - P. 28 -31.
npoSAeMH eKOAoriï Ta MejHUHHH
toxylin and eosin, and impregnated with nitrogen acid silver. Histological studies were performed in the traditional way, on the stage-by-stage basis (on the 1st, 3rd, 7th, 14th, 17 th, 21st and 28th day of the experiment ). For microscopic analysis of the material, light-optical system of Lieca microscope (Germany) was used: x 300; x 600.
Results and discussion
Morphological study of the brain specimens of the rats from the control group demonstrated the integrity of its cyto- and myeloarchitectonics. The microstructure of the cortex and subcortical centers is pronounced, layered structure undamaged. Vessels show no defects, hemorrhage, or paravascular edema. In general, the structure of the examined sample of the specimens met the indicators of gender and age norms for animals of the corresponding laboratory line.
The results of the morphological analysis of sectioned material taken from the animals of the experimental group with modeled ischemic stroke showed the presence of disorders characteristic for ischemic disease. The latter being of phase character were dependent on the nature and timing of the debut of the stroke process, depth and area of damage. Due to the above mentioned, observation was performed sequentially, based on critical periods of development of signs of ischemia and its natural non-medicamentation removal.
In histological specimens obtained from the animals from a group of modelled ischemic stroke, areas of necrosis were observed (the first day of the observation) distributed on the cortex of the cerebral hemispheres. Areas of tissue located next to the injuries contained leukocyte infiltrates and appeared more contrasting. One of the most distinctive diagnostic criteria of ischemic stroke were heart attacks, which in their structure meet the typical focal necroses of the brain. Development of ischemia and the associated disorder trophic processes led to the destruction of the cortex with formation of a network structure of the brain. It remained intact during the first day of the post-stroke period, gradually replaced by fully developed destruction areas in course of further observation. The areas held the remains of the cell pool of the destroyed neurocytes. Their nuclei were characterized by homogeneity and hyperchromy, as well as a tendency to repeat the cell shape (elongated, sharp). Analysis of individual specimens of rat brains, however, did not provide opportunity for histological registration of total necrosis, generalization areas of "softening" foci, and formation of full-scale scar. Against this background, the death of neurocytes cortex (phase of partial necrosis) was discovered by microscopy. Attention was paid to changes in the endothelium of blood vessels, which were related to the endothelium's heterogeneity and its capacity of desquamation under the conditions of postnatal ontogeny [7]. These phenomena provoked further intensive migration of leukocytes, "melting" of necrotized tissue, "softening" of the structures in damaged areas. These processes have added their contribution to the pathogenesis of ischemic disorder that developed against the backdrop of massive embolism. In micrographs, the light gray substance of the emboli, placing themselves along the damaged inner layer of the wall, filled the vessel outlet completely, increased it, and pressed on surrounding structures. Over time, in the surveyed areas defects of wall layers devel-
oped, accompanied by inflammation and swelling. The latter may have contributed to vascular isolation and future development of tissue hypoxia. These facts led to the emergence of additional reasons to strengthen the structural changes in the vascular wall. The latter, of course, contributed to the development of ischemia [10, 11].
On the third day of the experiment, the increasing infiltration of various cellular elements, mainly lymphocytes, a small number of macrophages and eosinophils was detected. Necrotic foci, fully formed, were distributed in nature. Erytrocites were distributed into typical groups according to the intensity of diapedesis into the parabasal space (hemorrhagic component). The first and most typical group consisted of white infarctions distributed as gray and white matter of mainly temporal and parietal areas and external capsule. Microscopically, the tissue formation at the site of the latter's formation appeared crumbly, fragile, friable, with pale, almost transparent coloration (hematoxylin-eosin). The other group consisted of mixed type infarctions, numerically small, diagnosed only on some histological specimens. The so-called red hemorrhagic infarctions were registered in certain areas (close to the microvessels, capillaries and their branches) of gray matter of the brain. They differ by a tendency to generalization and formation of large foci. In the abovementioned areas, diapedesis of erythrocyte cells into the parabasal space, edema, necrobiotic changes in glial components, and ischemia were diagnosed [7]. Endothelial cells were characterized by signs of nuclear hyperchromy, the shift of the nucleus towards one of the poles of the cell, the appearance of hetero-chromatin. Unlike the specimens from the control group, the contacts between cells in the vessels of the animals of the experimental group were attenuated, with cracks formed in the surface laye [5]. The perivascular edema which occurred as a result of hypersensitivity led to certain isolation of individual vessels from surrounding tissue with subsequent development of tissue hypoxia. Thus, the additional reasons were formed that worsened the structure of the vascular wall [10]. In the vicinity of the damaged area, concentration of thrombocyte plates and individual megakaryocytes with signs of sequestration was detected. In the medium to long observation range, notable destructive-degenerative changes in the vessel walls and desquamation of the surface layer cell were registered. In some specimens, angiospasm, stasis and the consequent pronounced perivascular edema were detected. The substance of brain edema in the centers subjected to pronounced necrosis (from local areas with clearly marked boundary to fragments with signs of generalization ). It is obvious that these facts led to the development of the clinical picture hypoxia and marked acidosis in rats. It is possible that the recorded pathogenetic picture of ischemic stroke contributed to the cerebral vascular insufficiency, which is reinforced by blocking of cerebral blood flow [9]. Changes in adhesive properties of erythrocytes and possible changes in the blood coagulation system led to the emergence of microthrombs (Fig. 1) in vessels, contributing to violation of the surrounding tissue's trophics and development of microbiotic processes.
Fig. 1. Red thrombuses of brain microvessels in Wistar rat aged 3 months (the group of animals with simulated ishemic stroke,
the 3rd day of observation). Staining: hematoxylin-eosin.
Magnification: x 300.
One of the most striking examples was cerebral vascular thromboses of the vertebral-basilar basin, parietal front corner, subcortical sites of the right hemisphere. Occlusion of major vessels created conditions for further (day 7 of the observation) tissue damage of the cerebral hemispheres. The above observation period was marked by the appearance of astrocytes and their proliferating forms. A week after the modeled stroke, the formation of a large area of the necrotic focus softening was observed. In the border area of necrosis in the cortex, numerous accumulations of leukocytes (leukocyte shaft) were recorded. In some areas between the necrotic focus and neighboring area, a structured limit started taking shape. Activation of astrocytes was recorded between the site of the necrosis and nervous tissue. The cells were located at some distance from each other, without a clear arrangement. On the verge of tissue relatively intact to ischemic disorders and injuries a large number of vessels of different diameters was noted. Around the latter, there were foci of inflammation — infiltrative events (neutrophils) which proliferated in the zones of localization of almond-shaped and caudate nuclei. In some cases, thromboses and defects in the layers of vessel walls were observed while parabasal hemorrhages (Fig. 2) were not established. The above study period was marked by the beginning of the process of active formation of collagen fibers, which in turn contributed to appearance of small in size gliomezodermal damage zones and conjunctive tissue scars and cysts, subject to the availability of large areas of destruction. Modeled ischemic strokes that were observed in the mentioned period, accompanied by the development of a number of vascular responses. First of all, they were connected with the phenomenon of deformation of cells that form the layers of the vessel wall. On the other hand, the major role was played by changes in the surface architectonics of erythrocytes as cells of a circulating link of the hema-topoiesis system. At the heart of each of the cases lay physicochemical reorganization of membrane lipids, proteins, and the connected electrical properties, as ion penetration of the cell membrane.
Fig. 2. Right cerebral hemisphere in male Wistar rat aged 3 months (the group of animals with simulated ishemic stroke, the 7th day of observation). Red thrombuses of brain microvessels, paravasal hemorrhages. Staining: hematoxylin-eosin.
Magnification: x 300.
On day 14 of the observation, the cerebral cortex's cleaning of the necrotic masses was seen throughout the whole damaged area. During this period, signs of inflammation reactions (Fig. 3) were still observed (infiltration in most cases by neutrophils and macrophages). In the former foci of necrosis, full-sized liquor-glial cysts were observed. Between areas of damaged and intact tissue, formation of glial scar continued. The latter was formed with multiple layers of astrocytes. In the case of appearance of bulk areas of necrosis, when the pathological process concerned not only the cerebral cortex but also subcortical structures, the appearance of a large number of capillaries, conjunctive tissue and glial cells on the border of the damaged area was observed. That is, in terms of mass lesions, mixed glial and conjunctive tissue scar was formed. In the areas neighboring with the ischemic zone, just as in the cases recorded in the early stages, numerically small, diffusely scattered and damaged neurocytes were detected. In the zones located next to the areas of necrosis, damaged cortex structure and, in some cases, lack of the first and second layers were observed. Search for the areas with signs of cell proliferation did not bring any results.
Fig. 3. Right cerebral hemisphere in male Wistar rat aged 3 months (the group of animals with simulated ishemic stroke, the 14th day of observation). The widespread focal points of infiltration. Staining: hematoxylin-eosin. Magnification: x 300.
On day 17 of the observation, changes in the structure of the cerebral hemispheres of rats with modeled ischemic stroke resembled injuries reported in the previ-
npoSAeMH eKOAoriï Ta MejHUHHH
ous study periods (day 14). Microscopically, the last few differed among themselves. Formation of glial scar continued. In the former foci of necrosis, full-sized liquor-glial cysts were observed. Microvessels of the cerebral hemispheres were characterized by a clear structure and absence of gross defects in walls (Fig. 4). The processes of elimination of the cellular detritus continued, gradually attaining their final stage. No facts of cell proliferation were established.
Fig. 4. Cerebral hemisphere in male Wistar rat aged 3 months (the group of animals with simulated ishemic stroke, the 17th day of observation). Lumen of microvessels. Staining: hematoxylin-eosin. Magnification: x 300.
Three weeks later (21st day of the observation), the morphological pattern of changes in the cerebral hemispheres of rats was limited to total distribution and completion of the processes of purification from necrotic masses. During this period, some areas still showed infiltration of the damaged areas of the brain by neutrophils and macrophages; however, these events were isolated in nature. In areas of necrotic foci, liquor-glial cysts were observed. The formation of the latter was in its final phase (their fragments were completely covered with coating formed mostly by glial cells). Between the injured and undamaged tissues of the brain, a glial scar remained. The newly formed scar absorbed astrocytes were located in layers sequentially (three to four layers). As in the previous observation period (day 14) under conditions of large, deep injuries (when destruction concerned subcortical centers), the development of capillaries occurred at the border of the injuries. Next to them, glial and connective tissue cells concentrated, forming a glial and connective tissue scar. At the boundary of the ischemic zone, as in previous periods, diffuse dispersal of damaged forms of neurocytes was observed. In the areas of the cortex close to the former zones of necrosis, certain violations of the latter's layered structure were noted. Microvessels in histological specimens had the appearance of complete structures.
On the fourth week (28th day of the observation) the process of cleaning fabrics from cellular detritus was fully completed. At the local foci of necrosis, glio-conjunctive scars and fully developed liquor cysts were observed. A highlight of the observation period was discovering the
characteristic features of the structure next to the damaged zone areas, where the vast majority of neurons appeared intact. In areas not contiguous with the former zone of necrosis, isolated hyperchromic intact neurons were recorded.
Conclusions: the development of ischemic stroke in linear rats was characteristic of distinct chronobiologism (days 1-28 of the experiment), which was clearly dependent on the timing of the prescription process, accompanied by degeneration of neurocytes, their processes, damage to cytoplasmic elements and the structure of the nucleus. Amid these changes proliferative and infiltrative reactions developed actively.
Prospects for future research are to explore the chro-nobiological patterns of development of experimental cerebral ischemic (embolic) stroke in male Wistar rats and the subsequent implementation of the results into clinical practice.
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Marepian Hagii/iinOB go pegamii 2.12.2013
