DISORDERS OF GLIAL NEOCORTEX HOMEOSTASIS UNDER REPRODUCTION OF ACUTE CEREBRO-VASCULAR PATHOLOGY Makarenko O. M., *Kovtun A. M. Taras Shevchenko National University of Kyiv, Ukraine Hrigoriy Skovoroda State Pedagogical University of Pereyaslav- Khmelnytskiy, Ukraine International Global Summit on Stroke, August, 2015, Birmingham, UK

Development and using of the method of determination of the glial formula and glial index quantitative for studying of the reaction of gliocytes in normal state and in the conditions of CNS pathology and its therapy. Aim of the study:

The current state of a neuroscientific neuroprotection paradigm Glu, Asp NMDA Excitotoxicity INSIG-1 (fragment, protein of erythrocytes) INDIP SREBP-1 Golgi apparatus Excitement Ischemia, cells lesion, death Ca 2+ level

Reactivation of the latent viral infection under acute HS A section of the sensomotor cerebral cortex under acute HS. Encephalomeningitis. Gliosis, tissue edema (Hematoxylin-eosin. 100x).

The system of cytostructural disturbances after modeling acute HS (7-th day) A section of the sensomotor neocortex. Pyramidal neurons of the 3-rd layer of the cerebral cortex and gliocytes. Control. (Hematoxylin- eosin. 400x) 5-th layer of the sensomotor cerebral cortex after acute hemorrhagic stroke modelling. Disturbances of neuroglial interrelations, cell death. Edema. (Hematoxylin-eosin. 400x)

The cellular reaction and structural changes of oligogliocytes after acute insufficiency of brain blood circulation modelling: A section of the sensomotor neocortex of a cat, control. The nuclei of satellites are designated by arrow signs (Hematoxylin-eosin. 400x) A section of the sensomotor cortex of big cerebral hemispheres of a cat, 7-th day of HS modeling. The satellites of pyramidal neurons of the 5th layer are designated by arrow signs. (Hematoxylin-eosin. 400x)

Typical changes of astrocytes after acute insufficiency of brain blood circulation modelling: A section of the cat’s sensomotor neocortex, control. The nuclei and cells of astrocytes are designated by arrow signs (Hematoxylin-eosin. 400x) Reactive astrogliosis of the sensomotor cortex of the cat’s big cerebral hemispheres, 7-th day of HS modeling. Edema. Nuclei of astrocytes are designated by arrow signs (Hematoxylin- eosin. 400x). Reactive astrogliosis of the sensomotor cortex of the cat’s big cerebral hemispheres, 7-th day of HS modeling. Edema. Nuclei of astrocytes are designated by arrow signs (Hematoxylin- eosin. 400x).

A section of the cat’s sensomotor cerebral cortex, control. (Hematoxylin-eosin. 400x). A section of the cat’s cerebral cortex, 7- th day of HS modeling. Microgliocytes are designated by arrow signs (Nissl staining. 400x). Microglia changes after acute insufficiency of brain blood circulation modeling :

Typical changes of ependymocytes after acute insufficiency of brain blood circulation modelling: Lateral ventricle ependymocytes of the cat's brain. Control (Hematoxylin- eosin. 400x). Damaged lateral ventricle the layer of ependymocytes of the cat's brain. Impairment and detachment of the layer of cells into the ventricle cavity. Acute period of HS. (Hematoxylin-eosin. 400x).

Changes of the neuroglial index (NGI) (glial neuron support) of the sensomotor cerebral cortex of cats after acute HS (7-th day) modeling: № Series of investigations NGI (ІІІ-rd layer) NGI (V-th layer) 1Control0,80±0,030,89±0,02 2Acute HS1,22±0,08*1,43±0,09* * – p<0,05 (in comparison with a control group);

Calculation of the glial formula(GF) of cellular brain formations: The glial formula (GF) represents the sum of the part of brain cells of astrocytes, oligogliocytes and microglial cells, and is calculated as follows: GF = A + O + M *; where A – the quantity of astrocytes, or % of the total of gliocytes; O – the quantity of oligogliocytes, or % of the total of gliocytes; and M – the quantity of microglial cells, or their % of the total of gliocytes in the studied field of vision (61,12х10 4 μ m 2 ). GF is a total quantity of gliocytes of the cellular brain formation, which makes 100%. * without ependymocytes.

The dynamics of changes of GF and glial homeostasis of the cerebral cortex of white rats, after primary acute HS (7 th day) modelling and M2 administration Type of gliocytes Intact animals Primary strokePrimary stroke + M 2 Ipsilateral hemisphere Contralateral hemisphere Ipsilateral hemisphere Contralateral hemisphere Astroglia 289,5±6,0128,34±7,0218,67±12,81260,75±3,33165,59±3,67 Oligoglia 880,25±8,08415,75±12,25538,42±13,35210,09±2,49418,67±12,46 Microglia 489,92±6,67587,34±10,78427,0±6,2369,75±3,68462,67±3,04 Σ 1659,671131,431184,09840,591046,43

The dynamics of changes of GF and glial homeostasis of the cerebral cortex of white rats after recurrent HS modelling and M2 administration Type of gliocytes Intact animals Recurrent strokeRecurrent stroke+ M 2 Ipsilateral hemisphere Contralateral hemisphere Ipsilateral hemisphere Contralateral hemisphere Astroglia 289,5±6,0168,17±10,76 447,83±17,38 261,59±5,16272,92±4,85 Oligoglia 880,25±8,08320,67±17,75559,67±20,41351,75±6,38427,75±15,03 Microglia 489,92±6,67974,42±18,83565,5±17,24459,5±9,7570,92±7,08 Σ1659,671463,261573,01072,841271,59

Quantitative changes of GF Type of gliocytes Nor ma Primary HSRecurrent HS IpsCoIps+М 2 Co+М 2 IpsCoIps+М 2 Co+М 2 Astroglia 1 Oligoglia 1 Microglia 1 = == = = = = = = = Conclusions: 1. Аstroprotective and proastroglial effect of М 2 2. There is no any positive effect of M2 in relation to oligoglia. 3. Some partial corrective effect of М 2 on microglia.

The dynamics of changes of gliocytes in the rats cerebral cortex in the conditions of primary stroke modeling (in the limits of the ІІІ-rd and the V-th layers of the ipsilateral hemisphere) Nerve cells ІІІ-rd layer Control III-rd layer HS 7 days III-rd layer HS 7 months V-th layer Control V-th layer HS 7 days V-th layer HS 7 months Astroglia 19,83±0,19%27,57±0,22%*34,21±0,23%*12,33±0,1%22,07±0,16%*28,49±0,16%* Perineuronal satellites 34,34±0,24%19,27±0,17%*21,82±0,18%*38,8±0,19%15,05±0,14%*17,06±0,12%* Microglia45,83±0,31%54,16±0,34%*46,97±0,28%52,8±0,29%62,87±0,27%*54,45±0,24% * – p<0,05 (in comparison with a control group).

The dynamics of changes in the quantity of gliocytes within the III-rd layer of the sensomotor cerebral cortex of white rats when modeling primary hemorrhagic stroke (%) * – p <0,05 (in comparison with a control group); 61,12*10 4 μm 2 - microscopic field area 17

The dynamics of changes in the quantity of gliocytes within the V-th layer of the sensomotor cerebral cortex of white rats when modeling primary hemorrhagic stroke (%) Perineuronal satellites (oligoglia) Microglia * – p <0,05 (in comparison with a control group); 61,12*10 4 μm 2 - microscopic field area

The dynamics of glial formula changes of the cerebral cortex of white rats in the conditions of primary hemorrhagic stroke modeling (in the limits of the ІІІ-rd and the V-th layers of the ipsilateral hemisphere) №Series of investigations GF percentage variant (Σ of A+O+M) 1All layers, intact animals 17,44% + 53,04% + 29,52% = 100% 2ІІІ-rd layer, intact animals 19,83% + 34,34% + 45,83% = 100% 3III-rd layer, 7 days of HS 27,57% + 1 8,27% + 54,16% = 100% 4III-rd layer, 7 months of CIBBC 33,21% + 20,82% + 45,97% = 100% 5V-th layer, intact animals 12,33% + 38,8% + 52,8% = 100% 6V-th layer, 7 days of HS 22,07% + 15,05% + 62,8 8 % = 100% 7V-th layer, 7 months of CIBBC 28,49% + 17,06% + 54,45% = 100%

Calculation of the glial index quantitative (GIQ) of the cellular brain formations The glial index quantitative (GIQ) allows estimating the following types of intercellular ratios (between different types of gliocytes): GIQ 1 = А/М GIQ 2 = О/М GIQ 3 = А/О, where A – the quantity of astrogliocytes, or % of the total of gliocytes; O – the quantity of oligogliocytes, or % of the total of gliocytes, M – the quantity of microgliocytes, or % of the total of gliocytes. GIQ 1,2,3 – a ratio of the total or percent of the corresponding types of gliocytes.

The dynamics of GIQ changes of the cerebral cortex of white rats after primary stroke modelling Index Intact animals Primary strokePrimary stroke + М2 Ipsilateral hemisphere Contralateral hemisphere Ipsilateral hemisphere Contralateral hemisphere GIQ 1 (А/М) 0,59090,21850,5121 0,7052 0,3579 GIQ 2 (О/М) 1,79670,70791,2609 0,56820,9049 GIQ 3 (А/О) 0,32890,30870,4061 1,24120,3955 Σ2,71651,23512,1791 2,51461,6583

The dynamics of GIQ changes of the cerebral cortex of white rats after recurrent stroke modelling Index Intact animals Recurrent strokeRecurrent stroke + М2 Ipsilateral hemisphere Contralateral hemisphere Ipsilateral hemisphere Contralateral hemisphere GIQ 1 (А/М) 0,59090,17260,7919 0,56930,478 GIQ 2 (О/М) 1,79670,84220,9897 0,76550,7492 GIQ 3 (А/О) 0,32890,20490,8002 0,74370,638 Σ2,71651,21972,5818 2,07851,8652

GIQ changes in cellular formations of the rat’s cerebral cortex after HS modeling GIQControl7 days7 months A/М1,0 О/М1,0 A/О1,0 Cerebral cortex (primary stroke, 7 days) =

GIQ changes of the rat’s cerebral cortex in the conditions of primary stroke modeling (in the limits of the ІІІ-rd and the V-th layers of the ipsilateral hemisphere) Index ІІІ-rd layer Control III-rd layer, HS 7 days III-rd layer, HS 7 months V-th layer Control V-th layer, HS 7 days V-th layer, HS 7 months GIQ 1 (А/М) 0,43270,5090,72830,23350,3510,5232 GIQ 2 (О/М) 0,74930,35580,46460,73480,23940,3133 GIQ 3 (А/О) 0,57751,43071,56780,31781,46641,67 Σ1,75952,29552,76071,28612,05682,5065

GIQ changes in cellular formations of the rats cerebral cortex after HS modeling GIQNorma7 days7 months A/MA/M1,0 О/М1,0 A/О1,0 GIQNorma7 days7 months A/М1,0 О/М1,0 A/О1,0 ІІІ-rd layer V-th layer

CHANGES OF THE MORPHOMETRIC INDICATOR (THE DIAMETER OF GLIOCYTES) OF THE SENSOMOTOR CEREBRAL CORTEX IN THE CONDITIONS OF ACUTE PRIMARY HS MODELING (IPSILATERAL HEMISPHERE) *– p<0,05 (in comparison with a control group); the results are presented in μm

CHANGES OF THE MORPHOMETRIC INDICATOR (THE AREA OF GLIOCYTES) OF THE SENSOMOR CEREBRAL CORTEX IN THE CONDITIONS OF ACUTE PRIMARY HS MODELING (IPSILATERAL HEMISPHERE) Astroglia Perineuronal Interfascicular Microglia Choroid Ependymoglia satellites oligodendroglia lamina cells *– p<0,05 (in comparison with a control group). the data are presented in μm

Conclusions: 1. The use of GF and GIQ, developed by us, provides a chance to estimate the features of the disturbances of glial homeostasis of cellular brain formations after modeling AIBBC, CIBBC, the dynamics of the developing processes in gliocytes and after using the specific pharmacotherapy. 2. A decrease in the GIQ oligoglia /microglia is observed in the acute period of hemorrhagic stroke, but the positive dynamics is absent and during the late period. The maximum microglial reaction is recorded on the 7th day of autohemorrhagic stroke modeling. 3. In 7 months after HS modelling (chronic insufficiency of brain blood circulation) a gradual change of the indicators of GF and GIQ of the cerebral cortex is noted. It can testify to the development partial recovery and reparative processes in cellular brain formations after a successfully experienced stroke. 4. According to the resistance degree in the conditions of acute HS gliocytes can be arranged in the following row: astroglia, microglia > oligoglia > choroid epithelium cells > ependymogliocytes.

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