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10УДК 615.21+612.822
нейропротекция усиливает нейротрофические механизмы, предотвращая повреждение нейронов и нейроглии при гипоксии
МОЗГА
© Николай Васильевич Цыган1, Александр Петрович Трашков2, Андрей Глебович Васильев3, Василий Николаевич Цыган1, Мирослав Михайлович Одинак1
1 ФГБВОУ ВПО «Военно-медицинская академия имени С.М. Кирова» Министерства обороны РФ. 194044, г. Санкт-Петербург, ул. Академика Лебедева, д. 6.
2 ФГБУН «Институт эволюционной физиологии и биохимии им. И.М. Сеченова» РАН. 194223, г. Санкт-Петербург, пр. Тореза, д. 44.
3 ФГБОУ ВО «Санкт-Петербургский государственный педиатрический медицинский университет» Минздрава России. 194100, Санкт-Петербург, ул. Литовская, д. 2.
Контактная информация: Андрей Глебович Васильев — кафедра патофизиологии с курсом иммунопатологии ФГБОУ ВО СПбГПМУ Минздрава России, e-mail: avas7@mail.ru
РЕЗЮМЕ. Исследовали морфологию головного мозга и нейротрофическую активность на модели острой церебральной гипоксии. Острая тромбоэмболия с последующей окклюзией правой сонной артерии воспроизводилась на 150 крысах; впоследствии 75 животных не получали никакого лечения (группа «тромбоэмболия»), а другие 75 животных получали лечение Цитофлавином на протяжении 10 дней (группа «Цитофлавин»). Все животные исследовались неврологически. В лабораторных исследованиях уровни такого биомаркера нейроглии как NSE оказались увеличенными в головном мозге на 1-й день, а в крови на 3-й день исследования в группе «тромбоэмболия» (в группе «цитофлавин» — достоверно ниже). Уровни других биомаркеров нейроглии увеличивались в крови на 1-й день ^АР), на 3-й и 10-й дни опыта ^100р) в группе «тромбоэмболия» (в группе «цитофлавин» — достоверно ниже). В обеих группах концентрации ростовых факторов нервной ткани превышали референсные значения на 3-й день исследования (NGF в мозговом цитолизате) и на 10-й день (VEGF-A в плазме крови и мозговом цитолизате, IGF-1 в плазме крови). В группе «цитофлавин» уровень VEGF-A в мозговом цитолизате был достоверно выше референсных значений на 1-й и 3-й дни с последующим значительным повышением между 3-м и 10-м днями, концентрация IGF-1 в плазме крови на 10-й день была достоверно выше, чем в группе «тромбоэмболия». Таким образом, острая церебральная гипоксия приводит как к немедленному, так и отсроченному повреждению нейронов и нейроглии, а также в основном к отсроченной активации синтеза ростовых факторов нервной ткани. Цитофлавин оказал продолжительный мультимодальный нейропротективный эффект, сопровождавшийся ранней активацией нейротрофических механизмов в ответ на острую церебральную гипоксию.
КЛЮЧЕВЫЕ СЛОВА: острое гипоксическое повреждение головного мозга; факторы роста нервной ткани; нейротрофические механизмы; нейропротекция; цитофлавин; головной мозг.
neuroprotection boosts neurotrophic mechanisms preventing
DAMAGE OF NEuRONS AND NEuROGLIA IN CASE OF CEREBRAL HYPOXIA
© Nikolay V. Tsygan1, Alexandr P. Trashkov2, Andrey G. Vasiliev3, Vasiliy N. Tsygan1, Miroslav M. Odinak1
1 Medicomilitary Academy named after S.M. Kirov. Academician Lebedev St., 6, Saint-Petersburg, Russia, 194044.
2 I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences. Thorez Ave., 44, Saint-Petersburg, Russia, 194223.
3 Saint-Petersburg State Pediatric Medical University. Litovskaya str., 2, Saint-Petersburg, Russia, 194100.
Contact Information: Andrey G. Vasiliev — department of a pathophysiology with a course of immunopathology of Saint-Petersburg State Pediatric Medical University. e-mail: avas7@mail.ru
ABSTRACT. Brain morphology and neurotrophic activity were studied in an acute cerebral hypoxia model. Acute thromboembolism with subsequent occlusion of the right carotid artery was reproduced in 150 rats; afterwards 75 animals did not get any treatment ("Thromboembolism" group), while 75 rats got Cytoflavin treatment for 10 days ("Treatment" group). All animals were subject to neurologic examination. NSE increased in brain on the 1st day and in blood on the 3rd day in "Thromboembolism" group (significantly lower in "Treatment" group). Neuroglia biomarkers' levels increased in blood on the 1st day (GFAP), 3rd and 10th day (S100P) in "Thromboembolism" group (significantly lower in the "Treatment" group). In both groups the concentrations of the nerve growth factors surpassed the reference values on the 3rd day (NGF in brain cytolysate) and on the 10th day (VEGF-A in blood plasma and brain cytolysate, IGF-1 in blood plasma). In the "Treatment" group VEGF-A brain cytolysate level was significantly higher than the reference values on the 1st and 3rd days with subsequent considerable increase between the 3rd and the 10th days, IGF-1 blood plasma concentration on the 10th day was significantly higher than in the "Thromboembolism" group. Thus, acute cerebral hypoxia results in acute and delayed alteration of neurons and neuroglia as well as mostly delayed activation of synthesis of nerve growth factors. Cytoflavin has produced a prolonged multimodal neuroprotection effect accompanied by early activation of neurotrophic mechanisms after acute cerebral hypoxia.
KEY WORDS: acute cerebral hypoxic alteration; nerve growth factors; neurotrophic mechanisms; neuroprotection, cytoflavin; brain.
INTRODUCTION
Nowadays neuroprotection is considered to be based on complex intracellular as well as intercellular mechanisms in neurons, neuroglia, endothelium, etc. One of these universal neuroprotection mechanisms is presented by boosted synthesis and activation of multifunction regulatory growth factors. Considering high degree of differentiation of neurons and neuroglia as well as significance of blood-brain barrier functioning the neurotrophic protection mechanisms involving nerve growth factors are of particular interest (Odinak and Tsygan, 2005).
The activation of the neurotrophic mechanisms has been proven for acute as well as chronic nervous system pathology including hypoxic and ischemic brain damage. Besides neurotrophins (e. g. nerve growth factor, NGF), glial neurotrophic factor family and ciliary neurotrophic factor family, nerve growth factors also comprise other growth factors including vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) (Reichardt, 2001; Gomazkov, 2002; Odinak and Tsygan, 2005).
NGF was the first of growth factors to be discovered back in 1951 (Levi-Montalcini, 1987). NGF is synthesized by astrocytes in central nervous system and possesses classic neurotrophic effects (Furukawa and Furukawa, 1990; Aloe et al., 2012).
VEFG is synthesized in the brain by astrocytes, endothelial cells and to a minor extent by neurons. Outside the central nervous system the sources of VEFG are various cells including macrophages and platelets (Chodobski et al., 2003; Kiselyovaet al., 2009; Bozoyan et al., 2012). VEFG takes part in the development as well as restoration of central nervous system, promotes angiogenesis, stimulatesproliferation and migration of endotheliocytes of blood-brain barrier as well as inhibits these cells' apoptosis (Lafuente et al., 2002; Emanueli et al., 2003; Rosenstein et al., 2003). Intraventricular administration of
recombinant human VEFG has been demonstrated to increase the density of blood vessels in rats (Harrigan et al., 2002). VEFG effects are implemented through four receptor types widely represented in mammalian brain (Yang et al., 2003). Intracellular cascades mediated by VEGF receptors include phospholipase C, diacilglycerol and MAP-kinase (Khaibullina et al., 2004). Endotheliocytes and paravasal cells including macrophages are also known to be important sources of VEGF (Kiselyova et al., 2009).
IGF-1 is synthesized in brain by glial cells, microcirculatory endotheliocytes and neurons (Daftary et al., 2005; Wang et al., 2013). Besides IGF-1 may be transported into the brain by means of transcytosis throughthe endotheliocytes of blood-brain barrier (Duffy et al., 1988). During the nervous system development the highest IGF-1 concentrations have been revealed in the optical tract, thalamus and cerebellum while IGF-1 receptor's — in all parts of central nervous system (Gomazkov, 2002). IGF-1 is in charge of neurons and glia proliferation, differentiation and restoration as well as stimulation of synaptogenesis and myelinization (D'Ercole et al., 2002; Guan et al., 2003) and antiapoptotic effect upon neurons through phosphatidilinositol-3-kinase pathway activation (Zheng et al., 2000; Yamagishi et al., 2003; Wang et al., 2013). IGF-1also influences cognitive functions in adult mammals (Lupien et al., 2003).
Cerebral hypoxia mostly results fromalteration of systemic hemodynamics and cerebral blood-flow autoregulation which is common in clinical and surgical practice (Tsygan et al., 2012). In chronic cerebral hypoperfusion VEFG is mostly synthesized by endotheliocytes of blood-brain barrier (Hai et al., 2003). On the other hand central nervous system hypoxia is accompanied by elevated neuronal expression of VEGF receptors, VEGF mRNAas well as VEGF itself thus producing neuroprotective effect (Yang et al., 2003; Khaibullina et al., 2004). In case of hypoxic and ischemic brain damage IGF-1 protects neurons probably
through the activation of antiapoptotic mechanisms (Guan et al., 2003; Mackay et al., 2003).
Experimental studies of cerebral cytolysate and blood plasma levels of nervous tissue biomarkersin experimental animals yield highly specific data due to absence of accompanying somatic pathology. Neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) and S100P protein biomarkers are used for the assessment of nervous tissue state (Butterworth et al., 1996; Odinak et al., 2011; Svetlov et al., 2012).
A combination of succinic acid, inosine, riboflavin and nicotinamide improves cell metabolism and composes Cytoflavin drug, which is widely used in the treatment of acute and chronic cerebrovascular pathology (Piradov et al., 2006; Rumyantseva et al., 2007; Odinak et al., 2010; Suslina et al., 2010). Neurochemical analysisof Cytoflavin cytoprotective effect in case of cerebral hypoxiais of interest for clinical practiceinneurology, neurosurgery, cardiology and cardiosurgery.
GOAL OF THE STuDY
Assessment of the brain functional state and neurotrophic mechanisms of protection in experimental model of acute cerebral hypoxia.
MATERIALS AND METHODS
The study involved 186 male albino rats of 215-230 g bodyweight. All rats have been quarantined for 14 days prior to the experiment in order to expel animals with somatic or/and infectious pathology.
Acute hypoxia of the brain was reproduced by means of introducing 0.3 ml of 75% homogenized thrombus suspension in physiologic saline solution into the right carotid artery. The thrombus was taken from rat donor blood left without anticoagulants in a glass test tube for 40 minutes at room temperature. The serum was then drained and the thrombus homogenized by hand in a glass homogenizer. The right carotid artery was separated under zoletil narcotization, mobilized and then catheterized, and thrombotic mass was introduced at 1.0 ml/min rate. The carotid artery was then ligated and the skin wound subjected to layer-by-layer closure. This model produces a combination of acute hypoperfusion with cerebral thromboembolism, i. e. key factors that influence the brain state in cardiac pathology (Shevchenko et al., 2006; Bokeria et al., 2008; Tsygan et al., 2012).
The experimental animals were divided by means of randomization into four groups:
• "Control" (n=20) — intact rats;
• "Sham-operated" (n=16) — animals subjected to right carotid artery catheterization with subsequent ligation without introduction of thrombotic mass;
• "Thromboembolism" (n=75) — animals subjected to right carotid artery catheterization with subsequent intra-arterial introduction of thrombotic mass and ligation;
• "Treatment" (n=75) — animals subjected to right carotid artery catheterization with subsequent intra-arterial introduction of thrombotic mass and ligation and then treated with
Cytoflavin infusions ("NTFF Polysun, Ltd", Russian Federation). Cytoflavin solution (0.2 ml of Cytoflavin and 0.1 ml of 0.9% sodium chloride solution) was introduced into caudal vein with the aid of infusion pump at 0.5 ml/min rate once a day for 10 days.
• Functional stateof the nervous system has been assessed by means of neurologic examination, nervous tissue biomarkers' and nerve growthfactors' concentrations determined in rat brain cytolysate.
Neurologic examination of intact rats was conducted on the day of inclusion into the study, in other experimental groups it was carried out directly prior to manipulations and then every day until the experiment termination. Neurologic examination included assessment of the tail tone, truncal ataxia during movement in horizontal plane, extremities lagging during movement in horizontal plane, resistance to alternate extension of contralateral extremities. Each parameter was rated according to three-point scale:
• tail tone: unchanged (2), decreased (1), atony (0);
• truncal ataxia during movement in horizontal plane: absent (2), moderate (1), substantial (0);
• contralateral extremities lagging during movement in horizontal plane: absent in both extremities (2), moderate in one or two extremities (1), substantial in one or two extremities (0);
• resistance to alternate extension of contralateral extremities: present in both extremities (2), moderately decreased in one or two extremities (1), substantially decreased in one or two extremities (0).
Blood samples were taken by means of transcutaneoustapping of the heart under ether narcotization into vacuum "Monovette" systems ("Sarstedt", Germany) with EDTA anticoagulant. Blood was centrifuged at 240 g for 7 min in order to get platelet-rich plasma, which was transferred into plastic tubes for additional centrifugation at 1200 g for 15 min.The resultant platelet-depleted plasma was transferred into cryotubes to be stored at -20 °C for subsequent laboratory studies.
After blood tapping the animals were euthanized by additional inhalation of ethylic ether. Brain tissue samples were taken directly after euthanasia and processed according to A. Scherbakov et al. (2006) for subsequent enzyme-linked immunosorbent assay.
Biological material was sampled from rats of "Thromboembolism" and "Treatment" groups on the 1st (n=15), 3rd (n=15) and 10th (n=15) days while that of "Control" and "Sham-operated" animals — on the 10th day after modeling acute cerebral hypoxia.
The blood plasma and brain cytolysate concentrations of nervous tissue biomarkers (NSE, GFAP, S100P) and growth factors (NGF, VEGF-A, IGF-1) were determined by means of enzyme-linked immunosorbent assaywith "Cusabio" EIA kits (China).
The results of the study were statistically processed according to procedure as follows: variables' values calculation; comparison of empirical distribution of quantitative variables
Table 1
Rat lethality after acute thromboembolism and occlusion in right carotid artery basin
Experimental groups
Observation period «Control» (N=20) «Sham-operated» (N=16) «Thromboembolism» (N=75) «Treatment» (N=75)
1 hour n;%; 95% [CI] - 3; 18.8; [4.1-45.7] 17; 22.7; [13.8-33.8] 16; 21.3; [12.7-32.3]
1 day n;%; 95% [CI] - 1; 6.3; [0.2-30.2] 12; 16.0; [8.6-26.3] 12; 16.0; [8.6-26.3]
>1 day n;%; 95% [CI] - - 1; 1.3; [0.0-7.2] 2; 2.7; [0.3-9.3]
0-10 days n;%; 95% [CI] - 4; 25.0; [7.3-52.4] 30; 40.0; [28.9-52.0] 30; 40.0; [28.9-52.0]
Comments: N — total number of animals per group; n — number of lethal outcomes per group; % — percentage of lethal outcomes per group; CI — confidence interval
with normal theoretical distribution by Shapiro-Wilk Test; assessment of quantitative variables difference significance in independent samples by Mann-Whitney U-test; assessment of significance of qualitative factor influence upon quantitative parameters' dispersion by ANOVA Kruskal-Wallis H-test (in case of more than two groups); assessment ofbond strength and direction between quantitative parameters by non-parametric Kendall correlation coefficient. Multifactor dispersion ANOVA analysis (calculating the sum of squared deviations SS and the level of factor's influence upon the dispersion of the parameter response rate Kfactor) was used to assess the influence of several qualitative factors upon quantitative factors' dispersion. The hypothesis of the origin of groups (formed according to quantitative value out of the same population) was checked using expected and observed rates' contingency tables, Pearson'sChi-square test, maximum likelihood method and in case of instability of the latter — two-sided Fisher exact test. Quantitative values were described with median, lower and upper quartile. While describing qualitative parameters 95% probability confidence interval (CI) was calculated besides the relative rate value. Zero statistical
hypothesis was rejected at confidence level p<0.05. Statistical analysis was carried out using STATISTICA 8.0 software package.
RESULTS AND DISCUSSION
Surgical manipulations on carotid artery, thromboembolism and subsequent occlusion in the carotid artery basin caused acute brain damage. Resultant high lethality in "Thromboembolism" and "Treatment" groups reflects the intensity of the utilized experimental model (table 1).
No statistically significant difference in lethality levels between "Thromboembolism" and "Treatment" groups was revealed during the entire experimental study thus testifying to safety of Cytoflavin.
Neurologic examination of the animals has revealed no signs of impairment of nervous system functions in the "Control" group and has demonstrated focal neurologic symptoms in the rats in other experimental groups (table 2).
The results of neurologic examination together with lethality level analysis confirm the validity of choice and the intensity of used experimental model of hypoxic brain damage. High
Table 2
Results of neurologic examination of rats on the 1st-10th daysafter the acute thromboembolism and occlusion
in right carotid artery basin
Experimental groups Number of animals with signs of nervous system impairment during the observation period
Decreased tail tone Truncal ataxia during movement in horizontal plane Lagging of contralateral extremities Decreased resistance to alternate extension of contralateral extremities
«Sham-operated» (N=12) n;%; 95% [CI] 8; 66.6; [34.9-90.1] 3; 25; [5.4-57.2] 3; 25; [5.4-57.2] 8; 66.6; [34.9-90.1]
«Thrombo-embolism» (N=46) n;%; 95% [CI] 40; 87; [73.7-95.1] 38; 82.6; [68.6-92.2] 36; 78.3; [63.6-89.1] 42; 91.3; [79.2-97.6]
«Treatment» (N=47) n;%; 95% [CI] 37; 78.7; [64.3-89.3] 34; 72.3; [57.4-84.4] 38; 80.9; [66.7-90.9] 41; 87.2; [74.3-95.2]
Comments: N — total number of animals per group; n — number of animals with the symptoms of nervous system impairment per group;% — percentage of animals with the symptoms of nervous system impairment per group; CI — confidence interval.
Pg/g
70605040302010-
■ Thromboembolism □ Treatment - Reference values
I
1
3 10
Days after thromboembolism
Fig. 1.
NSE concentrations in brain cytolysate (the figure reflects medians; the reference values—29.0 [21.0; 39.5] pg/g). Significant difference:! — from reference values, * — from the previous values in the group
Mcg/l 101
a
6-
4
2
Thromboembolism Treatment Reference values
1 ' 3 ' 10
Days after thromboembolism Fig. 2. NSE concentrations in blood plasma (the figure reflects medians; the reference values — 6.0 [5.65; 6.6] mcg/l). Significant difference:! — from reference values, * — from the previous values in the group, ** — "Thromboembolism" group values from "Treatment" group values
Pg/ml s5
30-
25
20-
15-
10-
5
Fig. 3.
■ Thromboembolism □ Treatment - Reference values
1 3 10
Days after thromboembolism
GFAP concentrations in blood plasma (the figure reflects medians; the reference values — 19.0 [14.5; 22.0] pg/ml). Significant difference:! — from reference values, * — from the previous values in the group, ** — "Thromboembolism" group values from "Treatment" group values
Thromboembolism i i Treatment
Pg/g -----Reference values
1,8-1 * ! ** ! ** 1,61,41,21,00,80,60,40,20,0
1 3 10
Days after thromboembolism
Fig. 4. S100p concentrations in blood plasma (the figure reflects medians; the reference values — 0.16 [0.12; 0.18] pg/l). Significant difference:! — from reference values, * — from the previous values in the group, ** — "Thromboembolism" group values from "Treatment" group values
quality of experiments was additionally documented by absence of impairment of nervous system functions in the "Control" group. The lethality levels' assessment as well as neurologic examination results indicate the association between surgical manipulations and cessation of carotid blood flow, which lead to cerebral dysfunction, considerably complicated by artificial thromboembolism.No significant difference of neurologic examination results have been revealed in "Thromboembolism" and "Treatment" groups.
The results of laboratory assessment of nervous tissue biomarkers are presented in figures 1 through 4.
"Control" group has yielded reference values of concentrations of nervous tissue biomarkers comparable to previously published data (in blood: NSE ~ 1.7 mcg/l, GFAP ~ 80 pg/ml, S100P ~ 0.4 pg/l) (Shahsavand et al., 2012; Svetlov et al., 2012).
On the 10th day of the experiment the neuron and neuroglia biomarkers' levels in the rats of "Sham-operated" group did not differ from the reference values thus indicating the absence of delayed cerebral changes after surgical manipulations and ligation of carotid artery without infusion of thrombotic masses.
NSE level in brain cytolysate in the experimental groups ("Thromboembolism" and "Treatment") increased only on the
Q
Q
1st day while NSE blood plasma concentration have been significantly higher than the reference values at all assessed time points. On the 3rd day NSE blood plasma level in "Treatment" group was lower than that in the "Thromboembolism" one. This pattern of NSE concentration changes may reflect an acute or delayed alteration of neurons as well as a decrease of its rate due to treatment with Cytoflavin.
An increase of GFAP blood plasma level on the 1st day was significant in "Thromboembolism" group both in comparison to the reference values and those of the "Treatment" group.
Similar differences were found in S100P blood plasma levels on the 3rd and the 10th day. The peculiarities of pattern of biomarkers' dynamics may be due to disparate involvement of neuroglial cells into pathologic process: GFAP reflects the state of astrocytes (Kwon et al., 2011) whereas S100P — predominantly astrocytes and other neuroglial cells including olygodendrocytes (Odinak et al., 2011). Multifactor dispersion analysis has yielded significant influence of the time upon GFAP blood plasma level changes (Ktime=14.2%, SS=1211, p=0.002; K =79%, SS=6694), thus testifying to the acute
Pg/g
2501 200 150 100500
Thromboembolism Treatment Reference values
! *
* !
Pg/g
Thromboembolism
1 3 10
Days after thromboembolism
Fig. 5. NGF concentrations in brain cytolysate (the figure reflects medians; the reference values — 100.9 [92.65; 112.05] pg/g). Significant difference:! — from reference values, * — from the previous values in the group
1 3 10
Days after thromboembolism
Fig. 6. VEGF-A concentrations in brain cytolysate (the figure reflects medians; the reference values — 20.7 [17.7; 31.35] pg/g). Significant difference:! — from reference values, * — from the previous values in the group
Pg/ml 18161412 10 8 6 4 2 0
Thromboembolism Treatment Reference values
Pg/ml
20-
16-
12-
8-
- 4-
0 1
1 3 10
Days after thromboembolism
Fig. 7. VEGF-A concentrations in blood plasma (the figure reflects medians; the reference values — 3.7 [2.24; 12.82] pg/ml). Significant difference:! — from reference values, * — from the previous values in the group
Thromboembolism Treatment Reference values
* * ! *
1 3 10
Days after thromboembolism
Fig. 8. IGF-1 concentrations in blood plasma (the figure reflects medians; the reference values — 8.0 [7.55; 8.85] mcg/l). Significant difference:! — from reference values, * — from the previous values in the group, ** — "Thromboembolism" group values from "Treatment" groupvalues
*
character of astrocytes' alteration as a result of cerebral perfusion impairment (Fig. 3).
Thus carotid artery perfusion disturbance appears to be accompanied by acute as well as delayed alteration and/or activation of neuroglia. Taking into consideration the following data regarding growth factors synthesis in glia and stable levels of most growth factors on the 1st and 3rd day, increase of glial biomarker's concentration on the 1st and 3rd day may be regarded as a result of neuroglia damage.
Cytoflavin treatment appears to decrease both the acute and delayed alteration of neuroglial cells. The therapy itself as well as its duration produce significant effect on S100P blood plasma level changes (K,h =30.7%, SS=6.82, p<0.001; K
~ o \ therapy ' ' ~ ' time
d th =11.4%, SS=2.53, p=0.002; K =56%, SS=12.4), thus
and therapy ' ' ~ ' error ' "
confirming long-term neuroprotective effect of Cytoflavin upon neuroglial cells.
The results yielded in "Control" group have been used as reference values of growth factors' concentrations. The results of laboratory assessment of nerve growth factors are represented in figures 5 through 8.
The method of the assessment of growth factors' concentrations used in the present study proved to be specific for the nervous tissue. Due to absence of accompanying somatic pathology in the examined animals, the changes of growth factors' blood plasma levels were regarded as a specific reaction towards nervous system alteration. On the 10th day the growth factors' levels in "Sham-operation" group were practically equal to the reference values while those of the experimental groups increased to a maximum thus confirming the association of neurotrophic mechanisms' activation with acute hypoxic brain damage.
Growth factors concentrations' reference values in "Thromboembolism" group were surpassed on the 3rd day (NGF in brain cytolysate) and on the 10th day (VEGF-A in blood plasma and brain cytolysate, IGF-1 in blood plasma). Taking into consideration the growth factors' main sources (NGF — astrocytes, VEGF-A, IGF-1 — astrocytes, endotheliocytes, neurons) the changes of the concentrations may be caused by boosted synthesis of growth factors in astrocytes on the 3rd day and in endotheliocytes and neurons on the10th day. Multifactor dispersion analysis revealed significant influence of time upon changes of the levels of NGF in brain cytolysate (Ktime=34.8%, SS=83565; p=0.027; K =59.355, SS=142278), VEFG-A in
error
brain cytolysate (K =19.4%, SS=163712; p<0.001; K =72.5,
time error
SS=611690), VEFG-A in blood plasma (Ktime=21%, SS=17470; p<0.001; K =78.9, SS=65720), IGF-1 "in blood plasma
error
(Kti =27%, SS=585; p<0.001; K =65, SS=1408), which confirm
time error
predominantly delayed enhancement of growth factors' synthesis. A direct correlation between IGF-1 and VEFG-A blood plasma levels (t=0.52; p<0.001) may reflect the timing of neurotrophic mechanisms' activation.
The increase of growth factors' levels in experimental groups on the 3rd and 10th day was accompanied by a decrease in alteration of neurons (NSE brain cytolysate level decrease on the 3rd day)
as well as that of neuroglia (GFAP blood plasma level decrease on the 10th day). The correlation analysis indicates inverse correlations between the levels of the growth factors and nervous tissue biomarkers (VEFG-A in blood plasma and GFAP: t=-0.23, p<0.001; IGF-1 and GFAP: t=-0.14, p=0.03; IGF-1 and NSE in brain cytolysate: t=-0.18, p=0.008) thus confirming decrease of nervous tissue damage accompanied by activation of cerebral neurotrophic protection mechanisms in acute cerebral perfusion disturbance.
The patterns of growth factors' changes in the experimental groups proved to be similar at all tested points of timeline, the medians of growth factors' levels were higher in "Treatment" group. VEGF-A brain cytolysate level was higher in "Treatment" group significantly surpassing the reference values on the 1st and the 3rd day with subsequent considerable increase during the period between the 3rd and the 10th day. Isolated increase of VEGF-A brain cytolysate level may indicate an increase of this growth factor synthesis in astrocytes due to neuroprotective effect of Cytoflavin. According to J. V. Lafuente et al. (2002) the increase of blood vessels permeability caused by VEGF-Amay contribute to brain edema development; however its laboratory signs (judging by nervous tissue biomarkers levels) as well as neurological ones were absent in experimental animals during the entire period of the observation.
Significant decrease of IGF-1 blood plasma concentration (compared to reference values) was revealed on the 1st and 3rd day in "Thromboembolism" group unlike the "Treatment" one, which in the absence of considerable decrease of NGF and VEGF-A levels may indicate insufficient neuronal activation and/or endothelial dysfunction in the absence of pharmacological neuroprotection.
Significant difference between the levels of growth factors in the experimental groups animals was revealed for IGF-1 blood plasma levels on the 10th day only and was higher in "Treatment" group thus suggesting activation of neurotrophic mechanisms in endotheliocytes influenced by Cytoflavin treatment. IGF-1 protects neurons from apoptosis, activates phosphatidylinositol-3-kinase pathway boosting anti-apoptotic Bcl-2 superfamily proteins' (Bcl-2 and Bcl-xL) effects (Guanet al., 2003; Mackayet al., 2003; Chereshnev et al., 2011; Wang et al., 2013). Thus drug-induced improvement of IGF-1 synthesis appears to decrease programmed cell death in the nervous tissue in case of cerebral blood flow insufficiency.
CONCLUSION
Acute cerebral hypoxia is accompanied by acute and delayed alteration of neurons and neuroglia as well as predominantly delayed boost of neurotrophic brain protection mechanisms. Cytoflavin treatment after acute cerebral hypoxia results in prolonged multimodal neuroprotective effect and improvement of neurotrophic protection from the first days of the therapy. The results of the study compared to published data indicate that endogenous and pharmacological neuroprotection in cerebral hypoxia may involve early activation of neurotrophic mechanisms
that prevent delayed alteration and may inhibit programmed cell
death of neurons and neuroglia.
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