DOI 10.1007/s10517-017-3737-7 Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  163,  No.  1,  May,  2017  PHARMACOLOGY AND TOXICOLOGY

57

Dihydroquercetin Improves Microvascularization and Microcirculation in the Brain Cortex of SHR Rats during the Development of Arterial Hypertension

M. B. Plotnikov, O. I. Aliev, A. V. Sidekhmenova, A. Yu. Shamanaev, A. M. Anishchenko, T. I. Fomina, G. A. Chernysheva, V. I. Smol’yakova, and A. M. Arkhipov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 163, No. 1, pp. 69-72, January, 2017 Original article submitted June 28, 2016 The effects of dihydroquercetin (50 mg/kg intragastrically daily for 6 weeks) on the density of capillary network (mean number of capillaries per mm2), mean capillary diameter, structure of capillary network, capillary diameter distribution (<3, 3-5, 5-7, and 7-9 µ), and local cerebral blood flow (by laser Doppler) in the visual cortex were studied in SHR rats during the development of arterial hypertension (from the 6th to the 12th week of life). Normally, the systolic and diastolic BP progressively increased in SHR rats during this period. Dihydroquercetin did not affect the development of arterial hypertension. At the same time, the drug significantly increased the mean diameter of capillaries (by 11%), capillary network density (by 23%), and in the percentage of capillaries with a diameter of 3-9  µ (passable for erythrocytes; by 42%). Positive effects of dihydroquercetin on the structure of microcirculatory bed improved microcirculation: local cerebral blood flow in the visual cortex of SHR rats was significantly higher (by 36%) than in rats receiving no flavonoid and close to the value in Wistar-Kyoto rats. Dihydroquercetin improved microvascularization and microcirculation in the cerebral cortex of SHR rats during the formation of arterial hypertension. Key Words: dihydroquercetin; arterial hypertension; SHR rats; microvascularization; local cerebral blood flow Microcirculatory disorders in arterial hypertension (AH) are in the focus of research [8,9,11]. Microcirculatory disorders in AH (microcirculation disease [9]) are associated with reduction of microvessel density due to loss of terminal arterioles, small venules, and capillaries (microvascular rarefaction), which is considered as a general characteristic of hypertensive syndrome [12,14]. Microvascular rarefaction leads to a further increase in total peripheral resistance, impairs delivery of nutrients and oxygen, and consequently contributes to damage of organs [9]. Microcirculatory disorders in the brain tissue are particularly hazardous Laboratory of Circulation Pharmacology, E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine, Tomsk Research Medical Center, Tomsk, Russia. Address for correspondence: [email protected]. M. B. Plotnikov

in AH, as brain involvement is the most dangerous and often fatal complication of AH [10]. However, it remains unclear whether microvascularization of the brain tissue can serve as the target for drug therapy. Flavonoid dihydroquercetin (DHQ) characterized by antioxidant, anti-inflammatory, neuroprotective, and other activities [6] proved to be effective in combined therapy for AH [2,5]. We studied the effects of DHQ on microvascularization and microcirculation in the cerebral cortex of SHR rats during the development of AH.

MATERIALS AND METHODS Experiments were carried out on SPF animals: normotensive Wistar-Kyoto (WKY) rats (n=10) and SHR rats

0007­-4888/16/16310057 © 2017 Springer Science+Business Media New York

58

Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  163,  No.  1,  May,  2017  PHARMACOLOGY AND TOXICOLOGY

(n=20) from the vivarium of M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Organic Biochemistry (Pushchino; animal health certificate of September 04, 2014). The dynamics of BP, morphological changes in the heart and vessels, and secondary changes in the target organs of SHR rats corresponded to the pathogenesis of essential hypertension and its complications in humans [3]; due to this, SHR line was acknowledged as the most adequate model of essential hypertension [15]. The animals were kept in vivarium of E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine under conditions of partialbarrier system at 20-25oC and 50±20% humidity (1215 air changes per hour) and 12/12 h day/night regimen. The animals were kept and cared in accordance with the rules established in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986). The research protocol was approved by the Laboratory Animal Care and Use Committee of the E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine (Protocol No. 72052014). The rats were used in the experiment after the age of 6 weeks. SHR rats were randomized into 2 equal groups, control and experimental. Experimental animals received DHQ in a daily dose of 50 mg/kg intragastrically in 1% starch gel for 6 weeks. Control SHR and WKY rats received starch gel. The animals received the last doses 3 h before BP measurements, which was registered with a NIBP200A automated noninvasive tail blood pressure monitoring system for use on small lab animals (BIOPAC System Inc.). The data were recorded and computer-processed using AcqKnowledge 4.2 software. Local cerebral blood flow was evaluated by laser Doppler flowmetry using an MP150 instrument

for electrophysiological studies (BIOPAC Systems, Inc.), LDF100C modulus, and fiberoptic needle pickup TSD144 (25 mm long, 1 mm in diameter) as follows. The rats were narcotized with sodium thiopental (80 mg/kg) directly after BP measurements and their heads were fixed in stereotaxis. The skin under the parietal bone was removed, and a hole was drilled with a 1.5 mm bore in the skull bone above the primary visual cortex (7.5 mm caudally from bregma and 2.5 mm to the right from the sagittal suture) without impairing the dura mater. The pickup for measurements of the local blood flow was placed onto the dura mater through the hole. The data were processed using AcqKnowledge 4.2 software. After measurements of local cerebral blood flow the animals were sacrificed in a CO2 box. The microcirculatory bed of the cerebral cortex was examined under a microscope as follows. The brain was removed and fixed in 10% neutral formalin. After fixation, a fragment of the occipital cortex was dehydrated in ascending alcohols, embedded in paraffin, and frontal sections (5 µ) were sliced. Deparaffinated sections were stained with hematoxylin and eosin. A series of 25 visual fields in the cortical zone were photographed at ×400 in histological preparations from each animal under an Axio Lab.A1 microscope (Carl Zeiss) with Axiocam ERc 5s. The diameters of all capillaries in the images were measured using ImageJ software, and the density of the capillary network (mean number of capillaries per mm2), mean diameter of capillaries, and structure of capillary network were evaluated. For quantitative evaluation of the capillary network structure, the capillaries were distributed into 4 groups by diameters: <3, 3-5, 5-7, and 7-9 µ. The results were statistically processed using Statistica 6.0 software. The data were presented as the means±standard errors in the means (M±m). The sig-

TABLE 1. Dynamics of Systolic (SBP) and Diastolic (DBP) BP in WKY and SHR Rats Age of rats

WKY rats (n=10)

SHR rats (control; n=10)

SHR rats (DHQ; n=10)

SBP, mm Hg

125±3

138±8

135±5

DBP, mm Hg

97±4

111±8

112±9

Week 6 (before DHQ course)

Week 9 (middle of DHQ course) SBP, mm Hg

128±11

166±4*

155±6*

DBP, mm Hg

90±9

120±4*

112±4*

SBP, mm Hg

124±4

205±6*

204±4*

DBP, mm Hg

91±6

154±6*

154±3*

Week 12 (directly after DHQ course)

Note. *p<0.05 in comparison with WKY rats.

59

M. B. Plotnikov, O. I. Aliev, et al.

Fig. 1. Effects of DHQ (50 mg/kg once a day for 6 weeks) on numbers of capillaries of various diameters in brain cortex of SHR rats. p<0.05 in comparison with *WKY rats, +control SHR rats.

nificance of differences between the groups was evaluated by nonparametric Kruskal—Wallis and Mann— Whitney tests.

RESULTS BP of WKY rats was stable from week 6 to week 12 of life, while in SHR rats, systolic and diastolic BP progressively increased during this period (Table 1). Systolic BP of control SHR rats was by 30 and 60% higher than in normotensive rats after 3 and 6 weeks of the experiment (weeks 9 and 12 of life, respectively). This was in line with the results of other authors [15]. Despite the observed trend to less pronounced increase in BP in the experimental rats by the middle of DHQ course, systolic and diastolic BP in the control and experimental groups differed negligibly after complete course (Table 1). Control SHR rats exhibited a significant decrease in the mean diameter of capillaries (by 27%), mean density of capillary network (by 26%), number of 3-5-µ capillaries (by 38%), 5-7-µ (by 66%) and 7-9-µ capillaries (2-fold) in comparison with the parameters of WKY rats, while the relative number of small capillaries (<3 µ) was higher by 78% than in WKY rats (Table 2, Fig. 1). The rarefaction phenomenon detected

in control SHR rats was in good agreement with the previous data [7]. Small capillaries (<3 µ) are impassable for erythrocytes [4], and the formation of this “pathological” structure of the capillary bed can manifest in microcirculatory disorders, in our study by 29% reduction of the local cerebral blood flow in the visual cortex of control SHR rats (Table 2). These microvascularization and microcirculation disorders can lead to stable hypoxia of brain tissue, detected in rats with AH [1]. The course of DHQ promoted an increase in the mean capillary diameter (by 11%), capillary network density (by 23%), and share of capillaries passable for erythrocytes (3-9 µ; by 42%) (Table 2, Fig. 1). Positive effect of DHQ on the microcirculatory bed structure in the cerebral visual cortex of SHR rats led to improvement of microcirculation: local cerebral blood flow was significantly (36%) higher than in control and did not differ from that of WKY rats. It seems that the rheological effects of DHQ, namely, improvement of the erythrocyte deformability and reduction of their aggregation [13], also contributed to improvement of microcirculation of the brain tissue of rats with AH. Hence, a course of DHQ improved the structure of the microcirculatory bed and the microcirculation in the cerebral cortex of SHR rats during the formation of AH. The study was supported by Russian Science Foundation (project No. 14-25-00017).

REFERENCES 1. Bachmanov AA, Maslennikova LS, Markova LA. Changes in oxygen metabolism after active avoidance training in spontaneously hypertensive and normotensive rats. Ross. Fiziol. Zh. 1991;77(12):35-40. Russian. 2. Britov AN, Aparina TV. The role of “Kapilar” (dihydroquercetin) in the correction of hemodynamic and metabolic disorders in patients with atherosclerosis and arterial hypertension. Regionar. Krovoobr. Mikrotsirk. 2006;5(2):46-53. Russian. 3. Zhuravlev DA. Models of arterial hypertension. Spontaneously hypertensive rats. Arterial. Gipertenz. 2009;15(6):721-722. Russian. 4. Novitskii VV, Rezavtseva NV, Stepovaya EA. Physiology and Pathophysiology of Erythrocyte. Tomsk, 2004. Russian.

TABLE 2. Effects of DHQ (50 mg/kg Daily for 6 Weeks) on Mean Diameter of Capillaries, Mean Capillary Density, and Local Cerebral Blood flow in Cerebral Cortex of SHR Rats Parameter

WKY (n=10)

SHR (control; n=10)

SHR (DHQ; n=10)

Mean capillary diameter, µ

4.8±0.1

3.5±0.1*

3.9±0.1*+

Mean density of capillaries, number/mm2

156±10

115±8*

142±6+

2629±358

1865±229*

2644±306+

Local cerebral blood flow, arb. units

Note. p<0.05 in comparison with *WKY rats, +control SHR rats.

60

Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  163,  No.  1,  May,  2017  PHARMACOLOGY AND TOXICOLOGY

5. Plotnikov DM, Aliev OI, Maslov MYu, Tyukavkina NA, Plotnikov MB. The effect of antioxidative complex on hemorheology and lipid peroxidation in patients with arterial hypertention. Tromboz, Gemostaz, Reologiya. 2001;(3):44-47. Russian. 6. Plotnikov MB, Tyukavkina NA, Plotnikova TM. DiquertinBased Drugs. Tomsk, 2005. Russian. 7. Plotnikov MB, Aliev OI, Anishchenko AM, Sidekhmenova AB, Shamanaev AYu, Fomina TI. Parameters of cerebral cortex capillary network in SHR rats during the development of arterial hypertension and stable high blood pressure. Ross. Fiziol. Zh. 2016;102(5):558-566. Russian. 8. Tokhomirova IA, Murav’ev AV, Petrochenko EP, Mikhailova SG. Microcirculation and Blood Rheology in Circulatory Disorders. Yaroslavl, 2011. Russian. 9. Feihl F, Liaudet L, Waeber B, Levy BI. Hypertension: a disease of the microcirculation? Hypertension. 2006;48(6):1012-1017. 10. Hadaegh F, Mohebi R, Khalili D, Hasheminia M, Sheikholeslami F, Azizi F. High normal blood pressure is an independent

risk factor for cardiovascular disease among middle-aged but not in elderly populations: 9-year results of a population-based study. J. Hum. Hypertens. 2013;27(1):18-23. 11. Jung F, Pindur G, Ohlmann P, Spitzer G, Sternitzky R, Franke RP, Leithäuser B, Wolf S, Park JW. Microcirculation in hypertensive patients. Biorheology. 2013;50(5-6):241-255. 12. Murfee WL, Schmid-Schönbein GW. Structure of microvascular networks in genetic hypertension. Methods Enzymol. 2008;444:271-284. 13. Plotnikov MB, Aliev OI, Maslov MJ, Vasiliev AS, Tjukavkina NA. Correction of the high blood viscosity syndrome by a mixture of diquertin and ascorbic acid in vitro and in vivo. Phytother. Res. 2003;17(3):276-278. 14. Tsioufis C, Dimitriadis K, Katsiki N, Tousoulis D. Microcirculation in Hypertension: An Update on Clinical Significance and Therapy. Curr. Vasc. Pharmacol. 2015;13(3):413-417. 15. Zicha J, Kunes J. Ontogenetic aspects of hypertension development: analysis in the rat. Physiol. Rev. 1991;79(4):1227-1282.