Vasodilatation Function of Cerebral Vessels at Arterial Hypertension in OXYS Rats
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- Agafonova, I.G., Kotel’nikov, V.N. & Eichhoff, U. Appl Magn Reson (2014) 45: 527. doi:10.1007/s00723-014-0538-2
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OXYS rats represent a selection strain of laboratory animals, which are characterized by the accelerated senescence. Substantial morphologic changes of cerebral vessels were revealed in the senescence-accelerated OXYS rats by the noninvasive MRI diagnostics using the induced arterial hypertension and the author’s original methods. These changes appeared as vascular lesions with a thickening of the intimae and the increasing of the signal intensity from blood in cerebral vessels. Cerebral arterial hypertension in normotensive Wistar rats developed as result of intraperitoneal injections of hydrocortisone acetate and diet enriched by sodium ions. The pathology of cerebral vessels in OXYS rats began its development much earlier and was based on spontaneous hypertension in connection with initially elevated blood pressure as well as the genotype of the animals. Four different inductors (two vasodilators and two vasoconstrictors) were used in the study of the endothelium-independent and endothelium-dependent vasodilatation and vasoconstrictions. We have compared previously unknown effects of these inductors on cerebral vessels in senescence-accelerated OXYS rats and those in normal Wistar rats. The response of the arteries to the action of inductors showed changes in the status of anterior, middle, and posterior cerebral arteries of circle Willis. Thus, we have indicated the changes in compensatory and adaptive characters of arteries in hypertensive OXYS rats in comparison with hypertensive Wistar rats. Reduced vasodilator response in the middle cerebral arteries suggests an elevated risk for development of arterial hypertension in these rats.
Cerebrovascular brain anomalies can be a reason of various serious disorders in elderly people [1, 2]. In recent years, the abundance of cerebrovascular diseases in combination with arterial hypertension was increased in spite of a great attention of medicine to this problem [3, 4]. Functional stability of the cerebral circulation system depends on the reactivity of cerebral vessels. The reactivity index of vessels indicates their ability to response on the influence of different inductors and other drugs regulating blood pressure. Studies with experimental animals which have disorders of cerebral vessels substantially extend the spectrum of therapeutic interventions directed to a secondary prophylaxis of vascular catastrophes. Therefore, they are considered as a pathway to further development of new compounds, which may be useful to prevent and/or discharge the cerebrovascular diseases [5, 6].
The endothelium plays an important biological role in different metabolic and regulatory vital processes. The concept about the endothelium as a target point for the prophylaxis and the treatment of vascular diseases deserves an intensive attention in modern medicine and must be experimentally verified . Endothelium dysfunction is the misbalance between the production of the vasodilatory factors of cerebral vessels such as nitric oxide, prostacycline, plasminogen on the one hand, and vasoconstriction factors such as prothrombotin, endotelin, superoxide anion, thromboxane A2 on the other hand .
Nitric oxide (NO) is the most important bioactive substance produced by the endothelium. The functions of endothelium-relaxing factors (NO, prostacycline I2) consist in regularly reproducing and keeping the cerebral arteries in the condition of dilatation in normotensive animals [9, 10]. The function of endothelium-relaxing factors in hypertensive animals is suppressed. The muscles of the vascular system of hypertensive animals do not respond to relaxing factors .
The response of the endothelium in 1-year-old OXYS rat strain, which is characterized by the senescence acceleration, on different vasodilatation and vasoconstriction factors was not yet studied. Angiographic methods may reveal the stricture of cerebral vessels, which appeared at arterial hypertension. As shown before, OXYS rats exhibit early changes in emotional and cognitive spheres, as well as age––dependent chronic pathologies . Such processes are typical of elderly patients and animals . Brain development of OXYS rats is connected with the delayed formation of the vascular arteries: delayed proliferation of capillaries through the membrane, the changes in energy metabolism within early postnatal period, typical of adaptation to tissue hypoxia [14–16].
We have studied the changes in brain tissue of this type of experimental animals. The first diffusion changes of OXYS rat brain was shown at age of 6 months. Further we have noted deep changes in brain tissue of OXYS rats at the age of 12 months . It was preliminarily indicated that the induction of arterial hypertension by acetylcholine in OXYS rats leads to some changes of cerebral vessels. In the present paper, we report the dilation and constriction properties of vasomotor functions of cerebral arteries of experimental animals during induced arterial hypertension. We compared two groups of rats (Wistar and OXYS), namely normotensive Wistar rats and accelerated senescence OXYS rats. The experimental arterial hypertension was induced in both these groups.
2 Materials and Methods
2.1 Animals, Treatment, and Blood Pressure Measurements
The male OXYS (n = 36) and Wistar (n = 36) rats were obtained from the Breeding Experimental Animal Laboratory of the Institute of Cytology (Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia). The rats were housed two animals per cage (57 × 36 × 20 cm), kept under standard laboratory conditions (at 22 ± 2 °C, 60 % relative humidity and natural light), and provided with a standard rodent feed, PK-120-1 (Laboratorsnab Ltd., Russia) and water ad libitum . Experiments were carried out in accordance with international guidelines (Council of the European Communities Directive 86/609/EES). Wistar rat strain was used as control animals.
One group of animals of age 6 months (control) received a standard diet and another group of animals received drinking water that contained 1 % NaCl plus a special semisynthetic diet with added electrolyte content according to our original model of hypertension . Electrolyte imbalance was aggravated by an acute deficit of potassium and magnesium salts in the diet (<50 g/kg) and by an excess of sodium salts, thus violating the ratio of K+/Na+. It is well known that excess glucocorticoids have hypertensive effect mediated by the suppression of expression of the endothelial nitric oxide synthase (eNOS). To achieve a steady high blood pressure, the animals were injected intramuscularly with 1.5 mg of hydrocortisone per kilogram of body weight per day in addition to the hypertensive diet. The blood pressure was measured using the tail-cuff method pneumatic transducer using ML U/4c501 (MedLab, China) during experiment. The blood pressure equal to or greater than 140/90 mmHg was considered as hypertensive factor for rats .
2.2 Magnetic Resonance Imaging
The animals were anesthetized by intraperitoneal injection of Xylazine (SPORA, PRAHA, Abbott Laboratories) before scanning in a concentration of 1 mg/mL and Sevoflurane in a concentration of 2 mg/mL. Magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) were performed on a «PharmaScan US 70/16» Tomograph (Bruker, Germany) designed for experimental studies at 7.0 T magnetic field strength and 300 MHz proton frequency and equipped with a BGA 09P gradient coil (Gradient strength 100 A (125,000 Hz/cm); Max linear Slew rate (5,622 T/m/s) .
Angiography was performed using the time-of flight-angiography (TOF-MRA) with the following parameters: TR/TE-50.0/5.6 ms; pulse angle-25.0; field of view (FOV)-3.0/3.0/3.0 cm; effective slice thickness-30 mm; slide overlap-30.0 mm; matrix-256/256/64; single scan, scanning time of 14 min. The cerebral vessels were reconstructed using the maximum intensity projection algorithm (MIP) in the region of interest (ROI) to reveal vasomotor reaction. We studied the changes of cerebral vessels after 3D-MIP reconstruction.
The visualization of brain tissue changes T1- and T2-weighted images have been obtained in frontal, sagittal, and horizontal planes using pulse sequence RARE_8 (Rapid Acquisition with Relaxation Enhancement), MSME (Multi Slice of Multi Echo), GEFI (Gradient Echo Fast Imaging). Adequate evaluation of brain tissue changes was based on the signal decrease in T1-weighted and signal enhancement in T2-weighted images. The parameters of T1 were TR/TE-1,500/12 ms; FOV-4.0 × 4.0 cm; effective slice thickness-1 mm; matrix-256/192; four scans, scanning time of 12 min. The parameters used for T2 were TR/TE-4,200/41.0 ms; FOV-4.0 × 4.0 cm; effective slice thickness-1 mm; matrix-256/192; four scans, scanning time of 16 min [19, 20].
2.3 Determination of Vasodilator Response
Acetylcholine (AcCh, Novartis Animal Health Inc. Switzerland) was intravenously injected daily in a dose 7 mg/kg to control the effect of endothelium-dependent dilatations (EDVD) of cerebral vessels. Glyceryl trinitrate (GTN, Nycomed, Austria) in a dose 0.15 mg/kg was added sublingually to control the effect of endothelium-independent dilatations (ENVD) of cerebral vessels (one dose/another day) .
2.4 Determination of Vasoconstriction Response
N (ω)-Nitro-L-arginine methyl ester (L-NAME, Santa Cruz Biotechnology, Inc.) in a dose of 20 mg/Kg was intravenously injected for control of the effect of endothelium-dependent vasoconstriction (EDVC). Noradrenalin (Bitartrate, USA) in a dose 0.04 mg/Kg was injected intraperitoneally for control of the effect of endothelium-independent vasoconstriction (ENVC, one dose/another day).
2.5 Morphological Measurements
The diameter was determined along the boundary between the middle and adventitial layers of the lateral and the medial wall of the artery. The size change, as percentage of maximal response, was calculated as (Dd − Db)/Db × 100, where Dd is the measured diameter for inductor and Db is the baseline diameter before a drug intervention was started. The effect of inductor was calculated as the ratio of artery diameter change in experiment in comparison with control. These measurements were presented as a percent in relation to the initial diameter accepted for 100 % .
2.6 Data Analysis
The estimation of vasomotor functions of cerebral arteries of Wistar and OXYS rats. The response of the endothelium to injection of inductors
Middle diameter of arteries, (mm)
Percentage in relation to control
The initial diameter of arteries
0.87 ± 0.03
1.05 ± 0.05
22.83 ± 1.66
1.21 ± 0.07
31.92 ± 2.43
0.71 ± 0.04
−11.16 ± 0.94
0.61 ± 0.01
−28.71 ± 1.04
The initial diameter of arteries
0.77 ± 0.03
0.90 ± 0.05
11.31 ± 0.91
0.93 ± 0.04
29.42 ± 0.71
0.55 ± 0.03
−26.31 ± 0.97
0.58 ± 0.01
−29.83 ± 1.95
The initial diameter of arteries
0.79 ± 0.04
0.89 ± 0.07
11.2 ± 0.02
0.86 ± 0.04
6.2 ± 1.1
0.68 ± 0.02
−10.12 ± 0.15
0.64 ± 0.03
−14.45 ± 0.26
The initial diameter of arteries
0.63 ± 0.06
0.54 ± 0.08
2.7 ± 0.32
0.71 ± 0.04
4.50 ± 0.15
0.64 ± 0.03
−3.98 ± 0.46
0.52 ± 0.05
−8.67 ± 0.12
3.1 Blood Pressure
The average systolic blood pressure of intact Wistar and OXYS rats was previously measured as 110.5 ± 6.3 and 118.2 ± 4.1 mmHg, respectively (P > 0.05) and the diastolic blood pressure as 75.3 ± 3.6 and 78.2 ± 5.2 mmHg, respectively. The average systolic blood pressure in Wistar and OXYS rats exposed to the action of hypertensive factors was 156.2 ± 9.6 and 168.2 ± 6.3 mmHg, respectively (P > 0.05), and the diastolic blood pressure was of 94.6 ± 5.2 and 102.7 ± 4.9 mmHg, respectively (P > 0.05) .
3.2 The Endothelial Dysfunctions of Cerebral Vessels After Induction of Arterial Hypertension
The sizes of the cerebral arteries in response to injection of inductors were changed in the range of 24 % in normotensive Wistar rats, while these changes ranged within 12–16 % in hypertensive Wistar rats. The size of the cerebral arteries in response to injection of inductors changed in range: 19 % for normotensive OXYS rats and 8–10 % in hypertensive OXYS rats. The diameter of cerebral arteries was measured using two points determined by MRI cursor: at the adventitia/media interface of the arterial lateral walls and at the media/adventitia interface of the medial walls.
Dynamic monitoring of cerebral vessels architecture for both animal strains (Wistar and OXYS) was carried out weekly using the time-of-flight-angiography (TOF-MRA). The endothelial mechanism of cerebral arterials of intact Wistar rats was changed after induction of experimental arterial hypertension. The hypertension was caused by intraperitoneal injections of hydrocortisone acetate and diet enriched by sodium ions, which damaged the humoral mechanism of the animals. We have carried out the morphometry of cerebral vessels of all animals before and after induction of arterial hypertension.
We evaluated the changes of endothelium of rat brain vessels during the experiment after treatment of endothelial inductors. The increase of EDVD up to 10 % and of ENVD up to 15 % was considered being within the framework of norm. Besides, we calculated the index of vasodilatation (IVD). IVD is ratio of the value within EDVD and ENVD (normal index is 1.5–1.9). Moreover, we have calculated the index of vasoconstriction (IVC). IVC is ratio of the value within EDVC and ENVC (normal index is 1.5–2.0) . The estimation of thickness of arterial wall (intimae/media) up to 1.0–1.4 mm was considered as being within the framework of norm. If the thickness of arterial wall increased up to 1.4 mm it was estimated as the formation of an atherosclerotic plaque . We studied the changes of anterior, middle, and posterior vessels of Willis circle. In present paper we showed the changes of middle cerebral arteries only. The reaction of middle cerebral vessels to various inductors is summarized in Table 1.
The reaction of the endothelium of middle cerebral arteries of normotensive Wistar rats to all inductors showed the adequate changes. The reaction of endothelium of middle cerebral arteries to acetylcholine of hypertensive Wistar was two times lower in comparison with normotensive Wistar rats; it was expressed as (22.83 ± 1.66 %, control) and (11.31 ± 0.91 %, experiment, P < 0.001). The reaction of endothelium of middle cerebral arteries to nitroglycerine was slightly changed. The reaction of endothelium of middle cerebral arteries to L-NAME was measured as (−11.16 ± 0.94 %, control) and (−26.31 ± 0.97 %, experiment). Thus, EDVC of hypertensive Wistar rats was reduced approximately twice. Responses of arteries to noradrenaline in both of groups were measured to be of (−28.71 ± 1.04 %, normotensive), and (−29.83 ± 1.95 %, hypertensive). Therefore, the reaction to this inductor was insignificant.
The reaction of endothelium of middle cerebral arteries of hypertensive OXYS rats to acetylcholine was decreased about four times in comparison with normotensive OXYS rats; it was expressed as (2.7 ± 0.32 % hypertensive) and (11.2 ± 0.02 % normotensive), respectively. The reaction of endothelium of middle cerebral arteries to nitroglycerine showed insignificant dilatation of cerebral vessels; it was expressed as (4.50 ± 0.15 %, hypertensive) and (6.2 ± 1.1 %, normotensive, P < 0.05), respectively. Probably, the dilatation of middle cerebral arteries of hypertensive OXYS rats was suppressed in the both groups, treated with acetylcholine and nitroglycerine. The reaction of endothelium of middle cerebral arteries of hypertensive OXYS rats to L-NAME was about three times lower in comparison with normotensive OXYS rats. It was expressed as (−3.98 ± 0.46 %) and (−10.12 ± 0.15 %), respectively. The reaction of endothelium of middle cerebral arteries to noradrenaline of hypertensive OXYS rats was (−8.67 ± 0.12 %) in comparison with normotensive OXYS rats (−14.45 ± 0.26 %). Thus, the reaction of endothelium to inductors of “normotensive” OXYS rats was reduced and the reaction of hypertensive OXYS rats was completely suppressed.
Further, we have compared the reaction of endothelium in two strains of rats. The reaction of endothelium of middle arteries to acetylcholine of normotensive OXYS rats was expressed as (11.2 ± 0.02 %) in comparison with Wistar rats (22.83 ± 1.66 %). The reaction of endothelium of middle cerebral arteries to nitroglycerine of normotensive OXYS was reduced about four times in comparison with normotensive Wistar rats (6.2 ± 1.1 and 31.92 ± 2.43 %, respectively). The reaction of endothelium of middle cerebral arteries to L-NAME of normotensive OXYS rats was of (−10.12 ± 0.15 %) in comparison with normotensive Wistar rats (−11.16 ± 0.94 %). The reaction of endothelium of middle arteries to noradrenaline of normotensive OXYS rats was of (−14.45 ± 0.26 %), in comparison with normotensive Wistar rats (−28.71 ± 1.04 %). Thus, the reaction of endothelium to inductors for normotensive OXYS rat strain was decreased in comparison with normotensive Wistar rat strain.
The reaction of endothelium of middle cerebral arteries to acetylcholine of hypertensive OXYS rats was expressed as (2.7 ± 0.32 %) in comparison with hypertensive Wistar rats; it was expressed as (11.31 ± 0.91 %). The reaction of endothelium of middle cerebral arteries to nitroglycerine for hypertensive OXYS rats was expressed as (4.50 ± 0.15 %) in comparison with hypertensive Wistar rats (29.42 ± 0.71 %). The reaction of endothelium of middle cerebral arteries to L-NAME for hypertensive OXYS rats was expressed as (−3.98 ± 0.46 %) in comparison with hypertensive Wistar rats (−26.31 ± 0.97 %). The reaction of endothelium of middle cerebral arteries to noradrenaline in hypertensive OXYS rats was of (−8.67 ± 0.12 %) when compared with hypertensive Wistar rats (−29.83 ± 1.95 %). Therefore, the mechanism of dilatation and constriction of hypertensive OXYS was suppressed in comparison to hypertensive Wistar rats.
It was established that the thickening of the endothelial walls during arterial hypertension is characteristic of arteries with size of 200–300 micrometers. This effect has been estimated in both rat strains. Hypertrophy of the walls and the accumulation of elastic fibers were observed in the middle and small (anterior) cerebral arteries in both of rat strains.
The reaction of endothelium of middle cerebral arteries to acetylcholine injection in hypertensive Wistar when compared with that of normotensive Wistar was lowered about two times. The reaction of endothelium of middle cerebral arteries to nitroglycerine showed no changes. Probably, the internal surface of endothelial walls were partly damaged and reacted to inductors of ENVD only. The reaction of endothelium of middle cerebral arteries of hypertensive Wistar to L-NAME was lowered by a factor of roughly two. The reaction of endothelium of middle cerebral arteries to noradrenaline showed no changes. Thus, it was confirmed that the arterial hypertension was really induced in Wistar rats. Our data concerning Wistar rats agreed with the data of other authors [23–25].
Our results showed the slow changes in cerebral endothelial reactivity of OXYS rats to inductors within 6 months of age. Probably, the ischemic pathology started to develop into cerebral vessels of “normotensive” OXYS during this age. That is why the reaction of endothelium of middle cerebral arteries to all inductors of normotensive OXYS was lowered. Additional induction of arterial hypertension in OXYS rats decreased the answer of the endothelium against inductors more significantly. We suggest that the arterial hypertension began its development in OXYS rats spontaneously. However, in the first 6 months adaptive and protective mechanisms were initiated in their blood vessels. The vasodilatory and constrictory mechanisms were connected with low content of nitric oxide in the blood and in the wall of the endothelium.
We consider that induced arterial hypertension in rats is a model closely related to human essential hypertension [26, 27]. The decreasing of endothelium relaxation factors and as a consequence of the disorder of the ability of the arteries to increase of blood flow occurs in hypertensive rats. The disorder of the physiological balance of vasomotor function leads to the changes of constrictory function. So, the decreasing ability of the arteries to produce the nitric oxide enhances the constrictory mechanism [28, 29]. The damage of the system of L-arginine––NO causes also the dysfunction of endothelium [30–32]. Probably the damage of NO production was primary while the increase of constrictor function was secondary event.
Thus, the endothelial dysfunction of the cerebral blood vessels of hypertensive animals was verified by us. The development of arterial hypertension is closely connected with the constantly increased arterial pressure. Statistical analysis of vasomotor effect of cerebral arteries and parameters of circulatory homeostasis revealed the correlation between the endothelium-dependent vasodilatation of cerebral arteries and blood pressure.
This work was supported by the President of the Russian Federation (grant no. 06 – III – A – 05 - 464). We are very grateful to the Head of the Sector of Genomic and Postgenomic Pharmacology, Professor Kolosova N.G. from the Breeding Experimental Animal Laboratory of the Institute of Cytology (Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia) for providing the animals and effective cooperation.