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Study of Microcirculation of the Ocular Ciliary Body in Experimental Kidney Disease S. A. Obrubov, A. O. Ivanova, S. O. Klyuchnikov*, A. A. Dreval, and O. A. Boginskaya Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 4, pp. 435-438, April, 2014 Original article submitted June 20, 2013 We studied disorders in ciliary body microcirculation in experimental chronic glomerulonephritis with tubulointerstitial nephritis and evaluated the hemodynamic effects of low-frequency pulsed electromagnetic field in this pathology. Laser Doppler flowmetry demonstrated vasospasm with reduced nutrient blood flow in the ciliary body of animals with experimental chronic glomerulonephritis with tubulointerstitial nephritis. The exposure to low-frequency pulsed electromagnetic field using developed technology will lead to significant reduction of the vascular tone and improve arterial blood supply to the microcirculatory bed. Key Words: microcirculation; laser Doppler flowmetry; chronic glomerulonephritis with secondary tubulointerstitial nephritis; pulsed low-frequency electromagnetic field An important role in the pathogenesis of glomerulonephritis along with renal impairment is played by extrarenal factors, for instance, pathological changes in small vessels and capillaries. Microcirculatory disturbances are involved in the pathogenesis of ophthalmic manifestations of nephrology pathology [2]. The similarity of renal glomeruli and ocular vascular tract (complex of choriocapillaris, Bruch’s membrane, and retinal pigment epithelium) consists in abundant vascularization, high volume flow rate per weight unit, and the presence of IgG Fc-receptors and receptors for complement C3 fragments on renal glomerular cells and endothelial cells (quoted by [2]). The studies of microcirculation of the eye during glomerulonephritis are scarce and inconsistent. We propose a method for the treatment of progressive myopia combined with extraocular pathology (inflammatory diseases of kidneys) in children [4], which consists in the use of frequency pulsed electromagnetic field (PEMF). Pathogenetic effect of PEMF N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow; *Center for Physical Therapy and Sports Medicine, Federal Medical-Biological Agency, Moscow, Russia. Address for correspondence: obrubovs@yandex. ru. S. A. Obrubov
is determined by its regulatory effect on microcirculation [4], which makes relevant the study of the microcirculation of the ciliar body in experimental renal pathology. We analyzed disturbances of microhemodynamics of the ciliar body in experimental chronic glomerulonephritis with tubulointerstitial nephritis and evaluation of the hemodynamic effects of PEMF in this pathology.
MATERIALS AND METHODS The study was conducted on 10 male Chinchilla rabbits weighing 2.0-2.5 kg (vivarium of the Research Institute for Fundamental and Applied Biomedical Research). In the experimental group (n=5; 10 eyes), chronic inflammatory pathology (chronic glomerulonephritis with secondary tubulointerstitial nephritis) was modeled [3]. Control group comprised 5 intact rabbits (10 eyes). For modeling renal pathology in experimental animals, normal horse serum (BioloT) heated to 37oC was injected into the ear vein in a dose of 12-15 ml/ kg body weight. For desensitization and prevention of allergic shock, the same serum was injected in a dose
0007-4888/14/15740440 © 2014 Springer Science+Business Media New York
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of 1 ml/kg 24 h prior to serum administration (preimmunization). Starting from experimental day 28, the heads of experimental animals were exposed to PEMF generated by an INFITA device for 10 days; the emitter was located at a distance of 20 to 30 cm from the eyes. Field strength in the affected area was 1-2 mV/cm2, field exposure at the same frequency 9 min. Pulse repetition rate was changed daily [4]. Microcirculation in the ciliary body of the eye was evaluated by direct-contact laser Doppler flowmetry (LDF) using LAKK-02 analyzer (LAZMA, Biofizika) with λ=0.63 μ He-Ne transmitter. Probed tissue volume was up to 1 mm3. The main indicators of ocular hemodynamics were evaluated: microcirculation perfusion index, standard deviation of pulse amplitude fluctuations, and coefficient of variation [1]. A wavelet transform of LDF blood flow signals was used in calculations after preliminary correction of the segments of LDF signals. The amplitudes of the oscillations related to endothelial, neurogenic, and myogenic activity, respiration rate, and HR in the range of 50-180 oscillations per minute were evaluated quantitatively. The neurogenic and myogenic tone and bypass index were also calculated [1]. In experimental animals, the parameters of microcirculation in the ciliary body were recorded before and on days 5 and 10 after the PEMF exposure. In
control animals, the microcirculation parameters in the ocular body were recorded only once. Microhemodynamics of the ciliary body in animals was examined under general anesthesia. Double anesthesia was used. Rometar (2% solution xylazine hydrochloride) was injected intramuscularly for premedication. In 10-15 min, Zoletil 50 in a dose of 6.6 mg/kg body weight was administered intravenously in marginal ear vein. For LDF recording of the ciliary body, the sensor was positioned 2–3 mm from the limbus in the upper part of the eyeball (projection area of the ciliary body) at the maximum eye opening. Statistical analysis was performed using Statistica 6.0 software.
RESULTS In experimental group, microcirculation tended to decrease (p>0.05) and the significant reduction in the standard deviation of pulse amplitude fluctuations indicating a decrease in blood flow modulation were revealed before PEMF exposure (Table 1). Significant decrease in correlation coefficient in the experimental group before PEMF exposure reflected the deterioration of microcirculation [1]. Analysis of amplitude and frequency spectrum of perfusion oscillations conducted using wavelet transform of blood flow signals showed a significant decrease in endothelial, neurogenic, and myogenic
TABLE 1. Parameters of Basal Blood Flow and Amplitude-Frequency Spectrum in the Microvasculature of the Ciliary Body in Experimental Animals before and after PEMF Exposure Experimental group Indicator
Control group before the exposure
day 5 after exposure
day 10 after exposure
M, PU
21.64±0.67
20.49±0.86
21.41±0.69
25.52±1.53*+о
σ, PU
1.19±0.08
0.64±0.03*
1.35±0.02+
2.29±0.17*+о
Cv, %
5.49±0.30
3.12±0.13*
6.31±0.13*+
8.97±0.43*+о
+
0.61±0.01*+о
AE, PU
0.36±0.02
0.17±0.02*
0.53±0.01*
AN, PU
0.53±0.05
0.26±0.02*
0.58±0.02+
1.13±0.09*+о
AM, PU
0.61±0.04
0.30±0.02*
0.68±0.02*+
1.36±0.17+о
AR, PU
0.59±0.05
0.29±0.01*
0.60±0.01+
0.61±0.16+
+
0.65±0.07*+о
AC, PU
0.38±0.04
0.21±0.01*
0.43±0.01
NT
2.24±0.17
2.48±0.11
2.32±0.02+
2.01±0.02*+о
MT
1.96±0.05
2.15±0.03*
1.98±0.01+
1.68±0.13*+о
BI
0.87±0.06
0.87±0.05
0.85±0.01
0.84±0.07
Note. M: microcirculation index; σ: standard deviation of pulse amplitude fluctuations, Cv: variation coefficient; AE: amplitude of endothelial activity; AN: amplitude of neurogenic activity, AM: amplitude of myogenic activity; AR: amplitude of respiratory oscillations, AC: amplitude of heart rhythm within the range of 50-180 oscillations per minute, NT: neurogenic tone, MT: miogenic tone, BI: bypass index, PU: perfusion units. p<0.05 in comparison with *the control group, +values before the exposure, оvalues on day 5 after the exposure.
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activities as well as amplitude of respiration and heart rate oscillations before PEMF exposure (Table 1). The amplitude of endothelial activity is determined by the function of the endothelium. Vasodilator NO release and decrease in amplitude indicate increased endothelium-dependent component of the tone in animals with simulated pathology [1]. The lowering of the amplitude of neurogenic oscillations (p<0.05 in comparison with the control group) indicates an increase in arteriolar tone determined by neurogenic activity (neurogenic tone) [1]. However, in this study the increase in neurogenic tone in the experimental group was insignificant in comparison with the controls. Significantly reduced amplitude of miogenic oscillations in experimental animals before PEMF exposure attests to increased myogenic tone (p<0.05 in comparison with the control group). The amplitude of respiratory oscillations was also significantly reduced in animals with modeled chronic pathology, which probably was indicative of elevated microcirculatory pressure. Significant decrease in the amplitude of HR oscillations in the experimental group in comparison with the baseline indicated reduction of the pulse wave [1]. The analysis of bypass index calculated as the ratio of myogenic to neurogenic component revealed no changes in this indicator in the experimental group of animals in comparison with the control group. PEMF exposure had a positive effect on hemodynamics in the experimental group. Thus, the analysis of the performance of basal blood flow revealed enhanced indicator of microcirculation (perfusion; significant on day 10 post exposure), significant increase in the standard deviation of pulse amplitude fluctuations and amplitude correlation coefficient (on days 5 and 10 post exposure) reflecting microcirculation improvement [1]. Analysis of perfusion oscillation amplitude and frequency spectrum showed significantly elevated amplitudes of endothelial, neurogenic, and myogenic domains as well as respiration rate and heart rate os-
cillations on days 5 and 10 after PEMF exposure. The increase in these parameters was more pronounced on day 10 after exposure and attested to significant reduction of the endothelium-dependent component of the tone, neurogenic tone, and myogenic tone, reduced microcirculatory pressure, and enhanced pulse wave in animals with modeled pathology compared to the corresponding values before PEMF exposure. Simultaneous increase in pulse wave and perfusion means enhanced arterial blood supply to microcirculatory bed [1]. Analysis of bypass index revealed no significant changes in its level, but its value remained <1 and demonstrated a tendency to decrease, which attests to an increase in blood supply of ocular tissues associated with shunt spasm [1]. Thus, vasospasm with reduced nutrient blood flow was revealed in the ciliary body of animals in experimental chronic glomerulonephritis with tubulointerstitial nephritis. The exposure to PEMF generated by INFITA device using the developed technology resulted in a significant decrease in vascular tone with increased arterial flow in the microcirculatory bed. LDF provided an accurate assessment of the changes in microhemodynamics of the ciliary body of rabbit eyes and hemodynamic effects of PEMF application during experimental chronic glomerulonephritis with tubulointerstitial nephritis.
REFERENCES 1. Laser Doppler Flowmetry of Microcirculation. Guide for Physicians [in Russian], Eds. A. I. Krupatkin, et al., Moscow (2005). 2. S. A. Obrubov, A. O. Ivanova, and M. B. Sagalovich, Ros. Pediatr. Oftalmol., No. 3, 48-54 (2009). 3. E. P. Proskurneva, N. V. Trishkina, E. S. Leonova, and V. A. Varshavskii, Arkhiv Patol., 53, No. 2, 46-53 (1991). 4. S. A. Obrubov, M. Yu. Demidova, Yu. G. Bespalyuk, et al., Patent RF No. 2371146, The Method for the Treatment of Child’s Progressive Myopia Combined with Extraocular Pathology as Inflammatory Diseases of Kidneys, Byul., No. 30 from October 27, 2009.
