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© V. lefremova
UDC 616. 831. -005. 1-085:615. 361 V. lefremova
Comparative Analysis of the Dynamics of Structural Changes that Develop in Mammalian Cerebrocortex under Ischemic and Hemorrhagic Stroke Modeling
ESC «Institute of Biology» Taras Shevchenko National University (Kyiv)
The research conducted as a comprehensive part of research work Department of Histology and Embryology at O. O. Bogomolets National Medical University: «Organs of the nervous, immune and digestive systems in experimental damage», № state registration 0112U001413.
Introduction. Acute ischemic attack is an important medical and social problem, due to their high share in the structure of morbidity and mortality, high levels of temporary and primary disability. According to WHO, the incidence of stroke in the world is about 200 cases per 100 thousand population each year. Number of ischemic strokes in the past 10 years in Ukraine has steadily increased and raised by 1. 5 times [1]. A significant number of patients after stroke are disabled, and 25-30% is observed severe manifestations of neurological deficit. Recurrent stroke within the first year (after the primary) develops only in 2% of patients, but in the next 5 years increases to 25-30%, with most patients (60-70%) are disabled [2].
Thus, the study of acute cerebral circulatory insufficiency of various etiologies, and in particular the dynamics of its development is extremely urgent problem. Over the past two decades has significantly increased the number of new data on the study of cerebrovascular disease [3]. At the same time, the study dedicated to the structural and functional basis of individual cell of the brain in the dynamics represented by single works [4,5].
The aim of the study was comparative analysis of the dynamics of structural changes that develop in mammalian cerebrocortex under ischemic and hemorrhagic stroke modeling.
Object and methods of research. Experiments conducted on rats, 40 females (average weight 214,1±10,2 g), which were divided into 4 groups. The first group consisted of intact animals (n = 10), the second pseudooperated (n = 10), third - animals with ischemic stroke (n = 10), fourth - rats with hemorrhagic stroke (n = 10). Pseudooperated animals exercised operational access to the brain and the skull hole in the right parietal bone of rats according to the diameter of the cannula. However, unlike the operated animals, they did not commit the introduction of the cannula with mandren-knife in brain tissue. Sewn tightly wound polyamide filaments 2 USP (Olympus, Ukraine) and treated with 5% alcoholic solution of iodine.
Model of ischemic stroke was unilateral chronic occlusion of the right common carotid artery. Model of acute hemorrhagic stroke was in the reproduction of the local post traumatic hematoma in the region of the internal capsule (capsula interna, CI) the right hemisphere of the brain of rats [6].
Modeling of intracerebral hematoma with autoblood in anesthetized animals (1% solution of thiopental sodium, 60 mg / kg, ip) was performed by mechanical tissue destruction of the CI (L = 3,5-4,0; H = 6,0 ; AP = 0,6-1,0) [9]. In the zone of the CI with using stereotaxic instrument (STM-3, Russia) and prepared mandren-knife was carried out 4-6 rotating movements declined mandren-knife and following injection into the zone of destruction 0. 15-0. 2 ml of animal’s autoblood. After surgery and a set of consecutive stroke modulating wound manipulation in the region of the cranial sewn tightly polyamide filaments 2 USP (Olympus, Ukraine), this area treated with 5% alcoholic solution of iodine [6].
After 7 and 15 days after modeling of local acute ischemic and hemorrhagic stroke in the area of CI rats were injected with lethal dose of thiopental sodium (1% solution, 200 mg / kg intraperitoneally) and carried out by sampling the brain for subsequent histological examination. After standard preparation techniques of brain slices treated with thickness of 10 microns, stained with standard solutions of hematoxylin and eosin.
Morphometric studies were performed using a microscope Olympus BX51 (Japan). Quantitative analysis of structural changes occurring in the somatosensory-cerebral cortex consisted in measuring the average area of nuclei bodies of neurons, the total number of neurons per unit area (1mm2) and the number of dead neurons. The reliability of the difference between groups of animals was evaluated using the Student t-test and Wilcox-on, programs in MS Excel 2007 and Statistica 6. 0. Reliable results were considered such that the significance level was equal to or more than 95% (P<. 05).
Results and discussion. During the experiment frontal sections of rat’s brain were investigated using modern histological and statistical methods. According to the generally accepted idea stroke lasts more than 24 hours and is accompanied by neurological symptoms [7,8]. Significant structural abnormality changes develop in the brain in the form of cerebral infarction, hemorrhage in brain tissue, ventricles or subarachnoid space. In our study of frontal sections of brain from
Table 1
Dynamics of the neurodegenerative process of the cerebral cortex
№ Group Ipsilateral hemisphere Contralateral hemisphere
Total number of neurons, cells/ mm2 Amount of dead neurons, % Total number of neurons, cells/ mm2 Amount of dead neurons, %
1 Intact 693,5±10,6 1,8±0,1 693,5±10,6 1,8±0,1
2 Pseudo operated 654,8±5,0 a 5,0±0,2 a 686,4±9,7 2,7±0,1 a
3 Ischemic stroke, 7th day 608,9±3,1 a,b 8,1±0,2 a,b,c 622,6±2,4 a,b 6,8±0,2 a,b
4 Ischemic stroke, 15th day 600,3±3,4 a,b 7,4±0,3 a,b 614,7±2,1 a,b 5,3±0,2 a,b,c
5 Hemorrhagic stroke, 7th day 603,9±4,2 a,b 10,9±0,4 a,b 611,1±2,5 a,b 7,8±0,2 a,b
6 Hemorrhagic stroke, 15th day 582,3±5,9 a,b 7,5±0,3 a 608,6±3,6 a,b 4,7±0,2 a,b,
Note: a - significantly relative to intact animals (p<0,05); b - significantly relative to pseudooperated animals (p<0,05); c - significantly relative to previous period of stroke (p<0,05).
experimental rats we found out executed intracerebral hematoma in the region of the right CI according to stereotaxic coordinates. Penetration of the blood in the lateral ventricles is not observed. In a detailed morphological study of sensory-motor areas of the cerebral cortex of the brain revealed significant irregularities, especially in the area of I ocalization of hematomas. In tissue around intracerebral hematoma been registered pattern forming glial scar (gliosis), there was a considerable infiltration area of hemorrhagic stroke modeling by leukocytes.
Comparing the morphological changes in the cerebral hemispheres of rats after 7 days after ischemic stroke modeling should emphasize on the acute necrotic processes in ipsilateral hemisphere, while in the
contralateral hemisphere, these processes are less observed. During morphometric studies we found out a significant increase in medium-sized neurons, which is caused by cells swelling. The average area of neurons in the contralateral and ipsilateralniy hemisphere amounted to 317,4±7,9 mkm2 and 341,1±13,6 mkm2, that is significantly more than in intact animals and pseudooperated. The average number of neurons in ipsilateral hemisphere decreased to 608,9±3,1 cells/mm2, and contralateral -622,6±2,4 cells/mm2 (Table 1).
In ipsilateral hemisphere hemorrhagic stroke in most neurons in a state of acute edema, swelling of cell bodies and nu clei (Fig. 1b). Registered substantial number of neurons with deformed nuclei with signs of degradation (autolysis). There acute swelling observed especiall around the apical and basal dendrites. Glial cells are also characterized by significant structural changes. In the cerebral cortex there is a significant amount of glial cells with deformed nuclei.
The average area of neurons in the contralateral and ipsilateral hemisphere amounted to 433,6±16,0 mkm2 and 397,0±10,6 mkm2 that is significantly greater than in ischemic stroke group. The average number of neurons in ipsilateral hemisphere decreased to 603,9±4,2 cells/mm2, and contralateral - 611, 1 ±2,5 cells/mm2 that is not very different from animals with ischemic stroke.
After 15 days of ischemic stroke modeling morphological changes in the cerebral cortex of rats characterized by exacerbation of nervous tissue edema and death of nerve cells (Fig. 1a). During this period, a significant difference between ipsi-and contralateral hemispheres was not observed. In both hemispheres of the brain recorded significant edema swelling and a large number
Fig. 1 Cerebral cortex of rat’s brain under acute stroke modeling a - ischemic stroke, b - hemorrhagic stroke. 7 day after stroke modeling (stained by toluidine blue, X 200). Increased neurodegeneration and edema of brain tissue.
Table 2
Changes in morphometric parameters of neurons in V layer of the cerebral cortex
№ Group Ipsilateral hemisphere Contralateral hemisphere
Area of neuron Area of nucleus Area of neuron Area of nucleus
1 Intact 284,1+8,0 153,5+5,9 284,1+8,0 153,5+5,9
2 Pseudo operated 296,3+8,5 197,1+7,1 a 292,2+9,7 180,2+6,5 a
3 Ischemic stroke, 7th day 317,4+7,9 a,b 198,6+9,5 a 341,1+13,6 a,b 166,9+5,2 a,b
4 Ischemic stroke, 15th day 333,9+14,0 a,b 199,9 + 13,0 a 295,1+6,6 a,b 150,0+5,8 b
5 Hemorrhagic stroke, 7th day 433,6+16,0 a,b 225,0+10,0 a,b 397,0+10,6 a,b 204,3+7,7 a,b
6 Hemorrhagic stroke, 15th day 379,8+13,1 a,b 210,0+12,4 a 343,8+14,5 a,b 163,8+7,9 b
Note: a- significantly relative to intact animals (p<0,05); b- significantly relative to pseudooperated animals (p<0,05).
of degenerated cells. Nerve cells have hypertrophied swollen appearance; there are a significant number of cells with signs of autolysis and degradation of neurite.
After 15 days of hemorrhagic stroke modeling in ipsilateral hemisphere recorded a significant number of degenerated neurons and in some parts of the cortex there are areas of brain tissue necrosis. The average area of neurons in the contralateral and ipsilater-alniy hemisphere amounted to 379,8±13,1 mkm2 and
343,8±14,5 mkm2 (Table 2). The death of neurons significantly reflected in terms of density of cells in the neocortex. In particular, the average number of neurons in the ipsilateral hemisphere was 582,3±5,9 cells/mm2, and contralateral - 608,6±3,6 cells/mm2 that is significantly less than in 7 days after modeling and group of animals with ischemic stroke.
Conclusions. Our study showed that 7-15 day period of experimental stroke developing pathologies changes that are morphologically different in ischemic and hemorrhagic stroke. Special attention should be paid to irregularities in the contralateral hemisphere, as in the works of other such violations do not pay enough attention. In our study we found out significant morphological changes in con-traletral hemisphere, characterized damage of neurons, loss and swelling of brain tissue and the simultaneous activation of pyramidal neurons. These changes indicate the generalized nature of the pathological process during the stroke and compensatory activation of contralateral hemisphere of the brain due to acute cerebrovascular accident; and there is extremely important for understanding the pathogenesis of this disease.
From our point of view, undoubtedly, that further research in this area with a detailed analysis of risk factors for primary and secondary stroke could help predict the future episodes of stroke and improving scheme of treatment.
References
1. Vylenskyy B. S. Insult: prevention, diagnosis and treatment. 2nd ed., Ext. / B. S. Vylenskyy. - S. - Pb.: Folyant, 2002. - 397 p.
2. Vylenskyy B. S. Insult / B. S. Vylenskyy. - St. Pb.: Med. inform. agenstvo, 1995. - 288 p.
3. Polishchuk M. E. Intra ventricular hemorrhage in the structure of hemorrhagic strokes / M. E. Polishchuk, V. V. Danatsko, I. S. Zozulia [et al.] // Problems of ecological and medical genetics and clinical immunology. - 2002. - Issue 1 (40). - P. 69-74.
4. Krayneva V. A. Neuroprotective properties of fobazol under re-modeling of hemorrhagic stroke in older rats / V. A. Krayneva, S. B. Seredenin // BEBiM. - 2010. -149 (2). - P. 165-168.
5. Zozulia I. S. Treatment of occlusive-stenotic processes internal carotid artery / I. S. Zozulia // Proceedings of employees KMAPE them. PLShupyk. - 2004. - Book 1, № 13. - S. 194-197.
6. Makarenko A. The method of modeling the local hemorrhage in various structures of the brain in experimental animals / A. Makarenko, N. S. Kositsyn, N. V. Pasikova [et al.] // ZHVND. - 2002. - Vol. 52 (6). - P. 765-768.
7. Romodanov A. P. Cerebral hemorrhagic stroke / A. P. Romodanov, G. A. Pedachenko. - K.: Health, 1971. - 228 p.
8. Leker R. R. NAP, a Femtomolar-Acting Peptide, Protects the Brain Against Ischemic Injury by Reducing Apoptotic Death / R. R. Leker, A. Teichner, N. Grigoriadis [et al.] // Stroke. - 2002. - Vol. 33(4). - P. 1085-1092.
9. Paxinos G. The Rat Brainin Stereotaxic Coordinates. 2nd ed. / G. Paxinos, C. Watson. - SanDiego : Acad. Press, 1986. - 920 p.
УДК 616. 831. -005. 1-085:615. 361
ПОРУШЕННЯ КОРИ ВЕЛИКОГО МОЗКУ ЩУРІВ ЗА МОДЕЛЮВАННЯ ІШЕМІЧНОГО ТА ГЕМОРАГІЧНОГО ІНСУЛЬТУ
Єфремова В.
Резюме. Досліджено особливості патологічних змін в корі головного мозку щурів при експериментальному геморагічному та ішемічному інсульті. В період 7-15 днів розвитку інсульту патоморфологічні зміни при ішемічному та геморагічному типах патології суттєво відрізняються, реєструються відміни в розвитку набряку тканини мозку та нейродегенеративних процесів.
Ключові слова: геморагічний інсульт, ішемічний інсульт, набряк мозку.
УДК 616. 831. -005. 1-085:615. 361
ИЗМЕНЕНИЯ В КОРЕ БОЛЬШОГО МОЗГА КРЫС ПРИ МОДЕЛИРОВАНИИ ИШЕМИЧЕСКОГО И ГЕМОРРАГИЧЕСКОГО ИНСУЛЬТА
Резюме. Исследованы особенности патологических изменений в коре головного мозга крыс при экспериментальном геморрагическом и ишемическом инсульте. В период 7-15 дней инсульта патоморфологические изменения при ишемическом и геморрагическом типах патологи существенно отличаются, регистрируются отличия в развитии отека ткани мозга и нейродегенеративных процессов.
Ключевые слова: геморрагический инсульт, ишемический инсульт, отек мозга.
UDC 616. 831. -005. 1-085:615. 361
Comparative Analysis of the Dynamics of Structural Changes that Develop in Mammalian Cerebrocor-tex under Ischemic and Hemorrhagic Stroke Modeling
lefremova V.
Summary. The aim of the study was comparative analysis of the dynamics of structural changes that develop in mammalian cerebrocortex under ischemic and hemorrhagic stroke modeling.
Experiments conducted on rats, 40 females (average weight 214,1±10,2 g), which were divided into 4 groups. The first group consisted of intact animals (n = 10), the second pseudooperated (n = 10), third - animals with ischemic stroke (n = 10), fourth - rats with hemorrhagic stroke (n = 10). Pseudooperated animals exercised operational access to the brain and the skull hole in the right parietal bone of rats according to the diameter of the cannula. However, unlike the operated animals, they did not commit the introduction of the cannula with mandren-knife in brain tissue.
Model of ischemic stroke was unilateral chronic occlusion of the right common carotid artery. Model of acute hemorrhagic stroke was in the reproduction of the local post traumatic hematoma in the region of the internal capsule (capsula interna, CI) the right hemisphere of the brain of rats [6].
Modeling of intracerebral hematoma with autoblood in anesthetized animals (1% solution of thiopental sodium, 60 mg/kg, ip) was performed by mechanical tissue destruction of the CI [9]. In the zone of the CI with using stereotaxic instrument (STM-3, Russia) and prepared mandren-knife was carried out 4-6 rotating movements declined mandren-knife and following injection into the zone of destruction 0. 15-0. 2 ml of animal’s autoblood.
After 7 and 15 days after modeling of local acute ischemic and hemorrhagic stroke in the area of CI rats carried out by sampling the brain for subsequent histological examination.
Morphometric studies consisted in measuring the average area of nuclei bodies of neurons, the total number of neurons per unit area (1mm2) and the number of dead neurons. Comparing the morphological changes in the cerebral hemispheres of rats after 7 days after ischemic stroke modeling should emphasize on the acute necrotic processes in ipsilateral hemisphere, while in the contralateral hemisphere, these processes are less observed. The average area of neurons in the contralateral and ipsilateralniy hemisphere amounted to 317,4±7,9 mkm2 and 341,1±13,6 mkm2, that is significantly more than in intact animals and pseudooperated. The average number of neurons in ipsilateral hemisphere decreased to 608,9±3,1 cells/mm2, and contralateral - 622,6±2,4 cells/mm2
After 15 days of ischemic stroke modeling morphological changes in the cerebral cortex of rats characterized by exacerbation of nervous tissue edema and death of nerve cells. During this period, a significant difference between ipsi- and contralateral hemispheres was not observed. In both hemispheres of the brain recorded significant edema swelling and a large number of degenerated cells. Nerve cells have hypertrophied swollen appearance; there are a significant number of cells with signs of autolysis and degradation of neurite. The average area of neurons in the contralateral and ipsilateralniy hemisphere amounted to 379,8±13,1 mkm2 and 343,8±14,5 mkm2. The death of neurons significantly reflected in terms of density of cells in the neocortex. In particular, the average number of neurons in the ipsilateral hemisphere was 582,3±5,9 cells/mm2, and contralateral - 608,6±3,6 cells/mm2 that is significantly less than in 7 days after modeling and group of animals with ischemic stroke.
Study showed that 7-15 day period of experimental stroke developing pathologies changes that are morphologically different in ischemic and hemorrhagic stroke. Special attention should be paid to irregularities in the contralateral hemisphere, as in the works of other such violations do not pay enough attention. In our research we found out significant morphological changes in contraletral hemisphere, characterized damage of neurons, loss and swelling of brain tissue and the simultaneous activation of pyramidal neurons.
Key words: hemorrhagic and ischemic stroke, swelling of brain tissue, neurodegenerative processes.
Рецензент - проф. брошенко Г. А.
Стаття над1йшла 23. 05. 2013 р.
