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High thoracic sympathetic block improves coronary microcirculation disturbance in rats with chronic heart failure
Microvascular Research 122 (2019) 94–100
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
High thoracic sympathetic block improves coronary microcirculation disturbance in rats with chronic heart failure
T
⁎
Guifang Suna, , Fengqi Liua, Chunhong Xiub a b
Department of Internal Intensive Medicine, the First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Cardiac Ultrasound, the First Affiliated Hospital of Harbin Medical University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Myocardium Capillary Congestive heart failure Vascular endothelial growth factor Autonomic nerve block
Introduction: Coronary microcirculation disturbance plays an important role in chronic heart failure (CHF). High thoracic sympathetic block (HTSB) is effective to treat CHF, but its impact on coronary microcirculation is unclear. Methods: Forty male Wistar rats were subcutaneously injected with isoproterenol (340 mg/kg) for 2 days. Eight weeks later, 24 surviving rats were randomized to the CHF and HTSB groups and 10 rats were used as the control group. 50 μl of saline and ropivacaine (0.2%) were epidurally infused everyday in the CHF and HTSB group respectively. Four weeks later, echocardiography and pathological and ultrastructural examination, capillary histochemical staining and vascular endothelial growth factor (VEGF) immunohistochemical staining in left ventricular (LV) subendocardial myocardium were performed. Results: Compared with the control group, LV dilation and dysfunction, myocardial focal necrosis, capillary spasm appeared in the CHF group. HTSB ameliorated LV dilation and dysfunction, myocardial necrosis and capillary spasm. Rats in the CHF group had less myocardial capillary density and more VEGF expression than in the control group (1591 ± 99 vs. 1972 ± 118/mm2, 0.62 ± 0.13 vs. 0.33 ± 0.10 optic density, all p < 0.05). Myocardial capillary density (1782 ± 96/mm2) was more and VEGF expression (0.47 ± 0.12 optic density) was less in the HTSB group than in the CHF group (all p < 0.05). Conclusion: HTSB improves coronary microcirculation disturbance in CHF, which may contribute to reversing myocardial remodeling and dysfunction. HTSB stimulates myocardial capillary growth independent of VEGF.
1. Introduction Coronary microcirculation disturbance appears in chronic heart failure (CHF) due to different etiologies such as ischemia (Chen et al., 2015), idiopathic (Laguens et al., 2011; Gil et al., 2015) and hypertrophy (Lazzeroni et al., 2016). The decrease of myocardial microvascular density indicates microvascular rarefaction, which occurs in various types of myocardial remodeling (Laguens et al., 2011; Gil et al., 2015; Huo and Kassab, 2015; Hoenig et al., 2008; Seferović and Paulus, 2015). Myocardial capillaries show decreased lumen diameter and thickened wall in idiopathic dilated cardiomyopathy (Laguens et al., 2011; Gil et al., 2015). Coronary microcirculation disorder as a part of myocardial remodeling, is involved in the progression of myocardial remodeling and dysfunction (Huang et al., 2004), so it is one of the important pathophysiological mechanisms of CHF. The activated sympathetic nerve and increased release of peripheral
circulating catecholamine prevent myocardial microvascular growth (Tomanek, 1989) and perfusion (Head, 1989; Acad and Weiss, 1988). Some methods of sympathetic blockade have been proved to improve myocardial microvascular angiogenesis (Amann et al., 2011; Lu et al., 2017; Torry et al., 1991) or perfusion (Lauten et al., 2011; Skalidis et al., 2007). Low concentrations of local anesthetics introduced via the high thoracic epidural cavity induce high thoracic sympathetic block (HTSB) without affecting sensory and motor nerves. Due to the inhibition of sympathetic drives in the upper thoracic organs including heart and relevant vessels, long-term HTSB is effective to treat CHF (Shuang et al., 2008; Chi et al., 2011; Sun et al., 2017). The effect of HTSB on coronary microcirculation in CHF is unclear. Surviving rats by subcutaneous injection with large doses of isoproterenol exhibit enlarged cardiac chambers and dysfunction, which is a typical animal model of CHF (Feng and Li, 2010). We observed myocardial capillary ultrastructure and density as well as vascular endothelial growth factor
⁎ Corresponding author at: Department of Internal Intensive Medicine, the First Affiliated Hospital of Harbin Medical University, No. 23 Post Street, Harbin 150001, China. E-mail address: [email protected] (G. Sun).
https://doi.org/10.1016/j.mvr.2018.11.013 Received 17 September 2018; Received in revised form 29 November 2018; Accepted 30 November 2018 Available online 01 December 2018 0026-2862/ © 2018 Elsevier Inc. All rights reserved.
Microvascular Research 122 (2019) 94–100
G. Sun et al.
2.6. Griffonia simplicifolia I-B4 (GSI-B4)histochemical staining
(VEGF) expression in rats with CHF treated by HTSB for 4 weeks, in order to evaluate its effect on coronary microcirculation and the role of myocardial VEGF in capillary growth.
GSI-B4 is combined with D‑galactose components in myocardial microvascular endothelial cells to make them positive without dyeing myocardiocytes and other extravascular tissues (Porter et al., 1990; Kerckhoven et al., 2002). Therefore, GSI-B4 is a specific marker for myocardial microvessels. The dewaxed sections were in immersed in 3% methanol hydrogen peroxide to block the activity of endoperoxidase at room temperature for 30 min. After washed by PBS for three times, they were blocked by normal sheep serum for 25 min to reduce background nonspecific staining. After washing, the sections were incubated in 25 μg/ml GSI-B4 (Sigma–Aldrich, St. Louis, MO., USA) in a moist chamber at 4 °C overnight. Then after washing, they were incubated in the 1:200 diluted horseradish peroxidase labeled streptavidin (Beijing Zhongshan Biological Company, China) at 37 °C for 30 min. Finally, GSI-B4 binding was detected by DAB stain and hematoxylin counterstain.
2. Methods 2.1. Animal grouping and model of CHF After approved by the Animal Experimentation Ethic Committees of Harbin Medical University, 50 male Wistar rats (the First Affiliated Hospital of Harbin Medical University), weighing between 250 and 350 g, were included in the study. Forty rats were subcutaneously injected with isoproterenol (Sigma–Aldrich, St. Louis, MO., USA) at a dose of 340 mg/kg for 2 days. The remaining 10 rats were used as the control group. Eight weeks later, 24 rats survived and were randomly divided into the CHF and HTSB groups. They were housed under standard temperature and humidity laboratory conditions with 12 h cycle of day and night and allowed unlimited food and water according to the policy of Good Laboratory Practice.
2.7. VEGF immunohistochemical staining
2.2. HTSB treatment
The procedure was carried out according to the instructions of the kit. The sections were incubated in the 1:100 diluted rabbit-anti-rat VEGF antibody (Wuhan Boster Biological Technology, Co. Ltd., China) in a moist chamber at 4 °C overnight. After washing by PBS, the sections were incubated with horseradish peroxidase labeled goat anti rabbit polymer (Beijing Zhongshan Biological Company, China) at 37 °C for 30 min. VEGF staining was detected by DAB stain and hematoxylin counterstain.
Operations were performed in the HTSB and CHF groups. Rats were anesthetized with pentobarbital sodium (Shanghai Second Biological Chemical Plant, China) at a dose of 40 mg/kg. A finely processed epidural catheter (0.61 mm OD) was inserted at the T4-T5 interspace and advanced to the T1 as described previously (Adolphs et al., 2003). An epidural catheter was introduced through a neck subcutaneous tunnel and protected by a neck splint. Skin temperature at the T1 dermatome was recorded before and 30 min after the first epidural injection of 0.2% ropivacaine (AstraZeneca, England) 50 μl with a microprobe thermometer (World Precision Instruments, UK). Skin temperature elevation of 0.5 °C or more was considered a successful sympathetic blockade. Ropivacaine was epidurally injected at the intervals of 3 h from 8 a.m. to 5 p.m. every day in the HTSB group. Meanwhile, normal saline was given by the same schedule in the CHF group. After completing the experiment, the position of the catheter was verified by autopsy.
2.8. Image analysis Capillary density was the numbers of capillaries in the unit area (mm2) in GSI-B4 slices at high magnification (10 × 40). A capillary was defined as a vessel with a lumen less 8 μm and only one endothelial cell nucleus. VEGF positive intensity was expressed as the average optical density at high magnification (10 × 40). Nine slices were analyzed in each group and the mean values were calculated on five visual fields randomly chosen from each slice. The slices were examined with a computerized pathological image analyzer (Motic Advanced 3.0, Motic China Group Co. LTD., China) by an experienced pathologist blinded to the experiment.
2.3. Echocardiography Four weeks later, rats were examined after anesthesia by a Vevo770 ultrasonic instrument (Visualsonics Inc., Toronto, Canada) with a 10 MHz high-frequency probe. Left ventricular end-systolic diameter (LVDs), end-diastolic diameter (LVDd), ejection fraction (LVEF) and fraction shortening (LVFS) were obtained by M-mode imaging of parasternal short-axis view. Three different cardiac cycles were analyzed and an average value was used for each parameter.
2.9. Statistical analysis Measurement data were expressed as the means ± SD. The significance of the differences between the groups was assessed by oneway analysis of variance with SPSS version 19.0 (SPSS Inc., Chicago, USA). Group t-test was used to compare the differences between the two groups if data was normally distributed. A value of p < 0.05 was defined as significant.
2.4. Myocardial hematoxylin and eosin staining
3. Results
The hearts were excised. Subendocardial myocardium of LV free wall was cut and fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (Beijing Chemical Reagent Company, China) in accordance with normal steps. The slides were observed by a microscope (BX51, Olympus Corporation, Tokyo, Japan).
3.1. Echocardiographic examination Skin temperature of T1 was elevated equal to or > 0.5 °C after starting the first epidural injection in all rats of the HTSB group, which confirmed the effective sympathetic blockade. Compared with the control group, LVDd and LVDs were increased and LVFS and LVEF were decreased in the CHF and HTSB group. LVDd and LVDs were decreased and LVFS and LVEF were increased in the HTSB group as compared with the CHF group. Data are shown in Table 1.
2.5. Myocardial capillary ultrastructural examination LV subendocardial myocardium was chipped into tissue blocks of 1 mm3 on ice and immediately put into a 2.5% glutaraldehyde fixation solution at 4 °C for 2 h. Slices were processed according to the routine methods for transmission electron microscope analysis. Epon embedded sections were stained with uranyl acetate and lead citrate and examined by a JEM-1220 electron microscope (JEOL Co., Japan).
3.2. Myocardial hematoxylin and eosin staining Compared with the control group, focal necrosis appeared in the 95
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Table 1 Echocardiographic results.
Control group (10) CHF group (12) HTSB group (12)
LVDd (mm)
LVDs (mm)
LVFS (%)
LVEF (%)
5.13 ± 0.58 6.81 ± 0.56⁎ 5.96 ± 0.35⁎▲
3.10 ± 0.44 5.27 ± 0.49⁎ 4.31 ± 0.27⁎▲
39.56 ± 6.06 22.56 ± 3.71⁎ 27.67 ± 3.35⁎▲
68.11 ± 6.43 37.22 ± 5.29⁎ 45.5 ± 4.89⁎▲
Data are means ± SD. ⁎ p < 0.05 vs. control group. ▲ p < 0.05 vs. CHF group.
CHF
Control
HTSB
Fig. 1. Myocardial pathology (×400). Compared with the control group, focal necrosis showed irregular deep red in the CHF group. It was reduced in the HTSB group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
blue nucleus, without specific staining as the negative control (Fig. 3). Capillaries were less in the CHF (1591 ± 99/mm2) and HTSB group (1782 ± 96/mm2) than in the control group (1972 ± 118/mm2) (all p < 0.05). They were more in the HTSB group than in the CHF group (p < 0.05).
CHF group, but it was obviously lessened in the HTSB group (Fig. 1). 3.3. Myocardial capillary ultrastructural examination In the CHF group, capillaries exhibited spasm, luminal stenosis or occlusion, irregular vascular wall and endothelial cell nucleus condensation or disintegration. They were relieved in the HTSB group (Fig. 2).
3.5. Myocardial VEGF immunohistochemical staining Brown granules in the cytoplasm of cardiomyocytes were VEGF positive staining (Fig. 4). VEGF staining was stronger in the CHF (0.62 ± 0.13 optic density) and HTSB group (0.47 ± 0.12 optic density) than in the control group (0.33 ± 0.10 optic density) (all
3.4. GSI-B4 histochemical staining Myocardial capillaries appeared small brown rings with only one 96
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CHF
Control
HTSB
Fig. 2. Capillary ultrastructure (×12,000). Capillary spasm, luminal stenosis and disintegration of endothelial cell nucleus appeared in the CHF group. They were relieved in the HTSB group.
microscopic examination of right ventricular biopsies in a patient with idiopathic dilated cardiomyopathy (Toussaint et al., 1987). The decreased myocardial capillary density in the CHF group proved myocardial microvascular rarefaction in dilated cardiomyopathy (Laguens et al., 2011; Gil et al., 2015). It also appears in hypertrophic or restrictive forms of myocardial remodeling (Hoenig et al., 2008; Seferović and Paulus, 2015). The abnormal myocardial capillary morphology and growth in the CHF group prove that coronary microcirculation disturbance exists in CHF and is involved in the progression of it. Isoproterenol directly leads to such changes and further confirms that sympathetic nerve system takes part in coronary circulatory disorder. Four weeks later, HTSB improved LV dilation, dysfunction, myocardial necrosis as well as capillary morphology and growth in rats with CHF. These results are mainly attributed to sympathetic block. HTSB inhibits sympathetic drives in heart and blood vessels of high thoracic organs to reduce myocardial oxygen consumption. HTSB reversed myocardial remodeling and dysfunction in CHF, in accordance with the previous studies (Shuang et al., 2008; Chi et al., 2011; Sun et al., 2017). Although HTSB is effective to treat CHF, its impact on myocardial microcirculation remains unclear. For the first time, we prove that HTSB improves coronary microcirculation disturbance in CHF. Other methods of sympathetic nerve blockade also show the improving effect. Renal sympathectomy prevents the decrease of myocardial microvascular
p < 0.05). It was weaker in the HTSB group than in the CHF group (p < 0.05). 4. Discussion Myocardial β‑adrenergic receptor is excessively activated in rats after subcutaneous injection with large doses of isoproterenol, which leads to myocardial necrosis and acute cardiac dysfunction through intrinsic mechanisms such as intracellular calcium overload, oxidative stress and inflammatory signaling (Willis et al., 2015; Shukla et al., 2015). Eight weeks later, the survival rate of rats subjected to isoproterenol in our research was 60%, which was slightly higher than the previous result (Feng and Li, 2010). Rats in the CHF group showed myocardial necrosis as well as LV dilation and dysfunction. Coronary arteries are not ligatured in rats and the pathological changes are similar to idiopathic dilated cardiomyopathy, so it is a typical animal model of CHF. There is heterogeneity in the distribution of coronary microcirculatory perfusion. Since microcirculatory blood flow is more in the epicardium than in the subendocardial myocardium, the latter is vulnerable to ischemic attack (Hoffman, 1995). Therefore, we focus on the capillaries here. In the CHF group, myocardial capillaries displayed spasm. Myocardial microvascular spasm was confirmed by electron 97
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CHF
Control
HTSB
Fig. 3. GSI-B4 histochemical staining (×400). Myocardial capillaries were stained with small brown rings with only one blue nucleus. Compared with the control group, capillaries were reduced in the CHF and HTSB group. HTSB increased capillaries. Arrows pointed to the capillaries. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
microcirculatory perfusion (Bulte et al., 2017). Since HTSB inhibits cardiac sympathetic drive, it may increase coronary microcirculatory perfusion to stimulate capillary growth. Coronary microcirculation disorder may be the main cause of ischemic injury to myocardium in idiopathic dilated cardiomyopathy. It may result in cardiomyocyte death and fibrosis deposition as well as myocardial dysfunction. HTSB relieves myocardial capillary spasm and rarefaction and reduces ischemia damage, which may help to prevent LV remodeling and dysfunction in rats with CHF. The mechanisms of HTSB in the treatment of CHF are not fully stated. Our study proves that HTSB protects impaired coronary microcirculation in CHF and reveals one of the mechanisms.
angiogenesis in renal failure (Amann et al., 2011) or resistant hypertension (Lu et al., 2017). Partial thoracic sympathectomy stimulates myocardial capillary growth in hypertension (Torry et al., 1991). βadrenergic receptor blockers increase myocardial microcirculatory perfusion in CHF (Lauten et al., 2011; Skalidis et al., 2007). Compared with the control group, myocardial capillary growth was decreased in the CHF group with increased VEGF expression. HTSB promoted myocardial capillary growth in rats with CHF, but myocardial VEGF expression was reduced. It indicates that HTSB stimulates myocardial capillary growth in CHF independent of VEGF, and myocardial VEGF expression is negatively regulated by capillary growth. In contrast, renal sympathectomy restores impaired myocardial capillary growth in renal failure or resistant hypertension just by VEGF regulation (Amann et al., 2011; Lu et al., 2017). Pioglitazone stimulates myocardial capillary growth independent of VEGF expression in streptozotocin-diabetic rats (Ashoff et al., 2012). Thus, it can be seen that myocardial VEGF may be not necessary for microvascular angiogenesis. There are other factors driving it, such as hypoxia (Tomanek et al., 2003), mechanical stretch (Cassino et al., 2012) and blood flow (Lamping et al., 2005). Sympathetic nerve is overactivated in CHF (Triposkiadis et al., 2009), which acts on the myocardial arteriole to reduce coronary
5. Conclusion HTSB improves coronary microcirculation disturbance in CHF, which may contribute to reversing myocardial remodeling and dysfunction. HTSB stimulates myocardial capillary growth independent of VEGF.
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CHF
Control
HTSB
Fig. 4. VEGF immunohistochemical staining (×400). Brown particles in the cytoplasm were VEGF positive staining. Compared to the control group, They were increased in the CHF and HTSB group. HTSB reduced VEGF expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Conflict of interest
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The authors declare that they have no conflict of interest. Funding None. References Acad, B.A., Weiss, H.R., 1988. Chemical sympathectomy and utilization of coronary capillary reserve in rabbits. Microvasc. Res. 36, 250–261. Adolphs, J., Schmid, D.K., Mousa, S.A., Kamin, B., Korsukewitz, I., Habazettl, H., et al., 2003. Thoracic epidural anesthesia attenuates hemorrhage-induced impairment of intestinal perfusion in rats. Anesthesiology 99, 685–692. Amann, K., Odoni, G., Benz, K., Campean, V., Jacobi, J., Hilgers, K.F., et al., 2011. Sympathetic blockade prevents the decrease in cardiac VEGF expression and capillary supply in experimental renal failure. Am. J. Physiol. Ren. Physiol. 300, 105–112. Ashoff, A., Qadri, F., Eggers, R., Jöhren, O., Raasch, W., Dendorfer, A., 2012. Pioglitazone prevents capillary rarefaction in streptozotocin-diabetic rats independently of glucose control and vascular endothelial growth factor expression. J. Vasc. Res. 49, 260–266. Bulte, C.S., Boer, C., Hartemink, K.J., Kamp, O., Heymans, M.W., Loer, S.A., et al., 2017. Myocardial microvascular responsiveness during acute cardiac sympathectomy induced by thoracic epidural anesthesia. J. Cardiothorac. Vasc. Anesth. 31, 134–141. Cassino, T.R., Drowley, L., Okada, M., Beckman, S.A., Keller, B., Tobita, K., et al., 2012. Mechanical loading of stem cells for improvement of transplantation outcome in a
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Kerckhoven, R., Saxena, P.R., Schoemaker, R.G., 2002. Restored capillary density in spared myocardium of infarcted rats improves ischemic tolerance. J. Cardiovasc. Pharmacol. 40, 370–380. Laguens, R., Alvarez, P., Vigliano, C., Cabeza Meckert, P., Favaloro, L., Diez, M., et al., 2011. Coronary microcirculation remodeling in patients with idiopathic dilated cardiomyopathy. Cardiology 119, 191–196. Lamping, K.G., Zheng, W., Xing, D., Christensen, L.P., Martins, J., Tomanek, R.J., 2005. Bradycardia stimulates vascular growth during gradual coronary occlusion. Arterioscler. Thromb. Vasc. Biol. 25, 2122–2127. Lauten, A., Ferrari, M., Goebel, B., Rademacher, W., Schumm, J., Uth, O., et al., 2011. Microvascular tissue perfusion is impaired in acutely decompensated heart failure and improves following standard treatment. Eur. J. Heart Fail. 13, 711–717. Lazzeroni, D., Rimoldi, O., Camici, P.G., 2016. From left ventricular hypertrophy to dysfunction and failure. Circ. J. 80, 555–564. Lu, D., Wang, K., Wang, S., Zhang, B., Liu, Q., Zhang, Q., et al., 2017. Beneficial effects of renal denervation on cardiac angiogenesis in rats with prolonged pressure. Acta Physiol. (Oxf.) 220, 47–57. Porter, G.A., Palade, G.E., Milic, A.J., 1990. Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon exculentum to microvascular endothelium: organ-specific localization and partial glycoprotein characterization. Eur. J. Cell Biol. 51, 85–95. Seferović, P.M., Paulus, W.J., 2015. Clinical diabetic cardiomyopathy: a two-faced disease with restrictive and dilated phenotypes. Eur. Heart J. 36, 1718–1727 (1727a-1727c). Shuang, W., Shiying, F., Fengqi, L., Renhai, Q., Lanfeng, W., Zhuqin, L., et al., 2008. Use of a high thoracic epidural analgesia for treatment of end-stage congestive heart failure secondary to coronary artery disease: effect of HTEA on CHF. Int. J. Cardiol.
100
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
High thoracic sympathetic block improves coronary microcirculation disturbance in rats with chronic heart failure
T
⁎
Guifang Suna, , Fengqi Liua, Chunhong Xiub a b
Department of Internal Intensive Medicine, the First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Cardiac Ultrasound, the First Affiliated Hospital of Harbin Medical University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Myocardium Capillary Congestive heart failure Vascular endothelial growth factor Autonomic nerve block
Introduction: Coronary microcirculation disturbance plays an important role in chronic heart failure (CHF). High thoracic sympathetic block (HTSB) is effective to treat CHF, but its impact on coronary microcirculation is unclear. Methods: Forty male Wistar rats were subcutaneously injected with isoproterenol (340 mg/kg) for 2 days. Eight weeks later, 24 surviving rats were randomized to the CHF and HTSB groups and 10 rats were used as the control group. 50 μl of saline and ropivacaine (0.2%) were epidurally infused everyday in the CHF and HTSB group respectively. Four weeks later, echocardiography and pathological and ultrastructural examination, capillary histochemical staining and vascular endothelial growth factor (VEGF) immunohistochemical staining in left ventricular (LV) subendocardial myocardium were performed. Results: Compared with the control group, LV dilation and dysfunction, myocardial focal necrosis, capillary spasm appeared in the CHF group. HTSB ameliorated LV dilation and dysfunction, myocardial necrosis and capillary spasm. Rats in the CHF group had less myocardial capillary density and more VEGF expression than in the control group (1591 ± 99 vs. 1972 ± 118/mm2, 0.62 ± 0.13 vs. 0.33 ± 0.10 optic density, all p < 0.05). Myocardial capillary density (1782 ± 96/mm2) was more and VEGF expression (0.47 ± 0.12 optic density) was less in the HTSB group than in the CHF group (all p < 0.05). Conclusion: HTSB improves coronary microcirculation disturbance in CHF, which may contribute to reversing myocardial remodeling and dysfunction. HTSB stimulates myocardial capillary growth independent of VEGF.
1. Introduction Coronary microcirculation disturbance appears in chronic heart failure (CHF) due to different etiologies such as ischemia (Chen et al., 2015), idiopathic (Laguens et al., 2011; Gil et al., 2015) and hypertrophy (Lazzeroni et al., 2016). The decrease of myocardial microvascular density indicates microvascular rarefaction, which occurs in various types of myocardial remodeling (Laguens et al., 2011; Gil et al., 2015; Huo and Kassab, 2015; Hoenig et al., 2008; Seferović and Paulus, 2015). Myocardial capillaries show decreased lumen diameter and thickened wall in idiopathic dilated cardiomyopathy (Laguens et al., 2011; Gil et al., 2015). Coronary microcirculation disorder as a part of myocardial remodeling, is involved in the progression of myocardial remodeling and dysfunction (Huang et al., 2004), so it is one of the important pathophysiological mechanisms of CHF. The activated sympathetic nerve and increased release of peripheral
circulating catecholamine prevent myocardial microvascular growth (Tomanek, 1989) and perfusion (Head, 1989; Acad and Weiss, 1988). Some methods of sympathetic blockade have been proved to improve myocardial microvascular angiogenesis (Amann et al., 2011; Lu et al., 2017; Torry et al., 1991) or perfusion (Lauten et al., 2011; Skalidis et al., 2007). Low concentrations of local anesthetics introduced via the high thoracic epidural cavity induce high thoracic sympathetic block (HTSB) without affecting sensory and motor nerves. Due to the inhibition of sympathetic drives in the upper thoracic organs including heart and relevant vessels, long-term HTSB is effective to treat CHF (Shuang et al., 2008; Chi et al., 2011; Sun et al., 2017). The effect of HTSB on coronary microcirculation in CHF is unclear. Surviving rats by subcutaneous injection with large doses of isoproterenol exhibit enlarged cardiac chambers and dysfunction, which is a typical animal model of CHF (Feng and Li, 2010). We observed myocardial capillary ultrastructure and density as well as vascular endothelial growth factor
⁎ Corresponding author at: Department of Internal Intensive Medicine, the First Affiliated Hospital of Harbin Medical University, No. 23 Post Street, Harbin 150001, China. E-mail address: [email protected] (G. Sun).
https://doi.org/10.1016/j.mvr.2018.11.013 Received 17 September 2018; Received in revised form 29 November 2018; Accepted 30 November 2018 Available online 01 December 2018 0026-2862/ © 2018 Elsevier Inc. All rights reserved.
Microvascular Research 122 (2019) 94–100
G. Sun et al.
2.6. Griffonia simplicifolia I-B4 (GSI-B4)histochemical staining
(VEGF) expression in rats with CHF treated by HTSB for 4 weeks, in order to evaluate its effect on coronary microcirculation and the role of myocardial VEGF in capillary growth.
GSI-B4 is combined with D‑galactose components in myocardial microvascular endothelial cells to make them positive without dyeing myocardiocytes and other extravascular tissues (Porter et al., 1990; Kerckhoven et al., 2002). Therefore, GSI-B4 is a specific marker for myocardial microvessels. The dewaxed sections were in immersed in 3% methanol hydrogen peroxide to block the activity of endoperoxidase at room temperature for 30 min. After washed by PBS for three times, they were blocked by normal sheep serum for 25 min to reduce background nonspecific staining. After washing, the sections were incubated in 25 μg/ml GSI-B4 (Sigma–Aldrich, St. Louis, MO., USA) in a moist chamber at 4 °C overnight. Then after washing, they were incubated in the 1:200 diluted horseradish peroxidase labeled streptavidin (Beijing Zhongshan Biological Company, China) at 37 °C for 30 min. Finally, GSI-B4 binding was detected by DAB stain and hematoxylin counterstain.
2. Methods 2.1. Animal grouping and model of CHF After approved by the Animal Experimentation Ethic Committees of Harbin Medical University, 50 male Wistar rats (the First Affiliated Hospital of Harbin Medical University), weighing between 250 and 350 g, were included in the study. Forty rats were subcutaneously injected with isoproterenol (Sigma–Aldrich, St. Louis, MO., USA) at a dose of 340 mg/kg for 2 days. The remaining 10 rats were used as the control group. Eight weeks later, 24 rats survived and were randomly divided into the CHF and HTSB groups. They were housed under standard temperature and humidity laboratory conditions with 12 h cycle of day and night and allowed unlimited food and water according to the policy of Good Laboratory Practice.
2.7. VEGF immunohistochemical staining
2.2. HTSB treatment
The procedure was carried out according to the instructions of the kit. The sections were incubated in the 1:100 diluted rabbit-anti-rat VEGF antibody (Wuhan Boster Biological Technology, Co. Ltd., China) in a moist chamber at 4 °C overnight. After washing by PBS, the sections were incubated with horseradish peroxidase labeled goat anti rabbit polymer (Beijing Zhongshan Biological Company, China) at 37 °C for 30 min. VEGF staining was detected by DAB stain and hematoxylin counterstain.
Operations were performed in the HTSB and CHF groups. Rats were anesthetized with pentobarbital sodium (Shanghai Second Biological Chemical Plant, China) at a dose of 40 mg/kg. A finely processed epidural catheter (0.61 mm OD) was inserted at the T4-T5 interspace and advanced to the T1 as described previously (Adolphs et al., 2003). An epidural catheter was introduced through a neck subcutaneous tunnel and protected by a neck splint. Skin temperature at the T1 dermatome was recorded before and 30 min after the first epidural injection of 0.2% ropivacaine (AstraZeneca, England) 50 μl with a microprobe thermometer (World Precision Instruments, UK). Skin temperature elevation of 0.5 °C or more was considered a successful sympathetic blockade. Ropivacaine was epidurally injected at the intervals of 3 h from 8 a.m. to 5 p.m. every day in the HTSB group. Meanwhile, normal saline was given by the same schedule in the CHF group. After completing the experiment, the position of the catheter was verified by autopsy.
2.8. Image analysis Capillary density was the numbers of capillaries in the unit area (mm2) in GSI-B4 slices at high magnification (10 × 40). A capillary was defined as a vessel with a lumen less 8 μm and only one endothelial cell nucleus. VEGF positive intensity was expressed as the average optical density at high magnification (10 × 40). Nine slices were analyzed in each group and the mean values were calculated on five visual fields randomly chosen from each slice. The slices were examined with a computerized pathological image analyzer (Motic Advanced 3.0, Motic China Group Co. LTD., China) by an experienced pathologist blinded to the experiment.
2.3. Echocardiography Four weeks later, rats were examined after anesthesia by a Vevo770 ultrasonic instrument (Visualsonics Inc., Toronto, Canada) with a 10 MHz high-frequency probe. Left ventricular end-systolic diameter (LVDs), end-diastolic diameter (LVDd), ejection fraction (LVEF) and fraction shortening (LVFS) were obtained by M-mode imaging of parasternal short-axis view. Three different cardiac cycles were analyzed and an average value was used for each parameter.
2.9. Statistical analysis Measurement data were expressed as the means ± SD. The significance of the differences between the groups was assessed by oneway analysis of variance with SPSS version 19.0 (SPSS Inc., Chicago, USA). Group t-test was used to compare the differences between the two groups if data was normally distributed. A value of p < 0.05 was defined as significant.
2.4. Myocardial hematoxylin and eosin staining
3. Results
The hearts were excised. Subendocardial myocardium of LV free wall was cut and fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (Beijing Chemical Reagent Company, China) in accordance with normal steps. The slides were observed by a microscope (BX51, Olympus Corporation, Tokyo, Japan).
3.1. Echocardiographic examination Skin temperature of T1 was elevated equal to or > 0.5 °C after starting the first epidural injection in all rats of the HTSB group, which confirmed the effective sympathetic blockade. Compared with the control group, LVDd and LVDs were increased and LVFS and LVEF were decreased in the CHF and HTSB group. LVDd and LVDs were decreased and LVFS and LVEF were increased in the HTSB group as compared with the CHF group. Data are shown in Table 1.
2.5. Myocardial capillary ultrastructural examination LV subendocardial myocardium was chipped into tissue blocks of 1 mm3 on ice and immediately put into a 2.5% glutaraldehyde fixation solution at 4 °C for 2 h. Slices were processed according to the routine methods for transmission electron microscope analysis. Epon embedded sections were stained with uranyl acetate and lead citrate and examined by a JEM-1220 electron microscope (JEOL Co., Japan).
3.2. Myocardial hematoxylin and eosin staining Compared with the control group, focal necrosis appeared in the 95
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Table 1 Echocardiographic results.
Control group (10) CHF group (12) HTSB group (12)
LVDd (mm)
LVDs (mm)
LVFS (%)
LVEF (%)
5.13 ± 0.58 6.81 ± 0.56⁎ 5.96 ± 0.35⁎▲
3.10 ± 0.44 5.27 ± 0.49⁎ 4.31 ± 0.27⁎▲
39.56 ± 6.06 22.56 ± 3.71⁎ 27.67 ± 3.35⁎▲
68.11 ± 6.43 37.22 ± 5.29⁎ 45.5 ± 4.89⁎▲
Data are means ± SD. ⁎ p < 0.05 vs. control group. ▲ p < 0.05 vs. CHF group.
CHF
Control
HTSB
Fig. 1. Myocardial pathology (×400). Compared with the control group, focal necrosis showed irregular deep red in the CHF group. It was reduced in the HTSB group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
blue nucleus, without specific staining as the negative control (Fig. 3). Capillaries were less in the CHF (1591 ± 99/mm2) and HTSB group (1782 ± 96/mm2) than in the control group (1972 ± 118/mm2) (all p < 0.05). They were more in the HTSB group than in the CHF group (p < 0.05).
CHF group, but it was obviously lessened in the HTSB group (Fig. 1). 3.3. Myocardial capillary ultrastructural examination In the CHF group, capillaries exhibited spasm, luminal stenosis or occlusion, irregular vascular wall and endothelial cell nucleus condensation or disintegration. They were relieved in the HTSB group (Fig. 2).
3.5. Myocardial VEGF immunohistochemical staining Brown granules in the cytoplasm of cardiomyocytes were VEGF positive staining (Fig. 4). VEGF staining was stronger in the CHF (0.62 ± 0.13 optic density) and HTSB group (0.47 ± 0.12 optic density) than in the control group (0.33 ± 0.10 optic density) (all
3.4. GSI-B4 histochemical staining Myocardial capillaries appeared small brown rings with only one 96
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CHF
Control
HTSB
Fig. 2. Capillary ultrastructure (×12,000). Capillary spasm, luminal stenosis and disintegration of endothelial cell nucleus appeared in the CHF group. They were relieved in the HTSB group.
microscopic examination of right ventricular biopsies in a patient with idiopathic dilated cardiomyopathy (Toussaint et al., 1987). The decreased myocardial capillary density in the CHF group proved myocardial microvascular rarefaction in dilated cardiomyopathy (Laguens et al., 2011; Gil et al., 2015). It also appears in hypertrophic or restrictive forms of myocardial remodeling (Hoenig et al., 2008; Seferović and Paulus, 2015). The abnormal myocardial capillary morphology and growth in the CHF group prove that coronary microcirculation disturbance exists in CHF and is involved in the progression of it. Isoproterenol directly leads to such changes and further confirms that sympathetic nerve system takes part in coronary circulatory disorder. Four weeks later, HTSB improved LV dilation, dysfunction, myocardial necrosis as well as capillary morphology and growth in rats with CHF. These results are mainly attributed to sympathetic block. HTSB inhibits sympathetic drives in heart and blood vessels of high thoracic organs to reduce myocardial oxygen consumption. HTSB reversed myocardial remodeling and dysfunction in CHF, in accordance with the previous studies (Shuang et al., 2008; Chi et al., 2011; Sun et al., 2017). Although HTSB is effective to treat CHF, its impact on myocardial microcirculation remains unclear. For the first time, we prove that HTSB improves coronary microcirculation disturbance in CHF. Other methods of sympathetic nerve blockade also show the improving effect. Renal sympathectomy prevents the decrease of myocardial microvascular
p < 0.05). It was weaker in the HTSB group than in the CHF group (p < 0.05). 4. Discussion Myocardial β‑adrenergic receptor is excessively activated in rats after subcutaneous injection with large doses of isoproterenol, which leads to myocardial necrosis and acute cardiac dysfunction through intrinsic mechanisms such as intracellular calcium overload, oxidative stress and inflammatory signaling (Willis et al., 2015; Shukla et al., 2015). Eight weeks later, the survival rate of rats subjected to isoproterenol in our research was 60%, which was slightly higher than the previous result (Feng and Li, 2010). Rats in the CHF group showed myocardial necrosis as well as LV dilation and dysfunction. Coronary arteries are not ligatured in rats and the pathological changes are similar to idiopathic dilated cardiomyopathy, so it is a typical animal model of CHF. There is heterogeneity in the distribution of coronary microcirculatory perfusion. Since microcirculatory blood flow is more in the epicardium than in the subendocardial myocardium, the latter is vulnerable to ischemic attack (Hoffman, 1995). Therefore, we focus on the capillaries here. In the CHF group, myocardial capillaries displayed spasm. Myocardial microvascular spasm was confirmed by electron 97
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CHF
Control
HTSB
Fig. 3. GSI-B4 histochemical staining (×400). Myocardial capillaries were stained with small brown rings with only one blue nucleus. Compared with the control group, capillaries were reduced in the CHF and HTSB group. HTSB increased capillaries. Arrows pointed to the capillaries. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
microcirculatory perfusion (Bulte et al., 2017). Since HTSB inhibits cardiac sympathetic drive, it may increase coronary microcirculatory perfusion to stimulate capillary growth. Coronary microcirculation disorder may be the main cause of ischemic injury to myocardium in idiopathic dilated cardiomyopathy. It may result in cardiomyocyte death and fibrosis deposition as well as myocardial dysfunction. HTSB relieves myocardial capillary spasm and rarefaction and reduces ischemia damage, which may help to prevent LV remodeling and dysfunction in rats with CHF. The mechanisms of HTSB in the treatment of CHF are not fully stated. Our study proves that HTSB protects impaired coronary microcirculation in CHF and reveals one of the mechanisms.
angiogenesis in renal failure (Amann et al., 2011) or resistant hypertension (Lu et al., 2017). Partial thoracic sympathectomy stimulates myocardial capillary growth in hypertension (Torry et al., 1991). βadrenergic receptor blockers increase myocardial microcirculatory perfusion in CHF (Lauten et al., 2011; Skalidis et al., 2007). Compared with the control group, myocardial capillary growth was decreased in the CHF group with increased VEGF expression. HTSB promoted myocardial capillary growth in rats with CHF, but myocardial VEGF expression was reduced. It indicates that HTSB stimulates myocardial capillary growth in CHF independent of VEGF, and myocardial VEGF expression is negatively regulated by capillary growth. In contrast, renal sympathectomy restores impaired myocardial capillary growth in renal failure or resistant hypertension just by VEGF regulation (Amann et al., 2011; Lu et al., 2017). Pioglitazone stimulates myocardial capillary growth independent of VEGF expression in streptozotocin-diabetic rats (Ashoff et al., 2012). Thus, it can be seen that myocardial VEGF may be not necessary for microvascular angiogenesis. There are other factors driving it, such as hypoxia (Tomanek et al., 2003), mechanical stretch (Cassino et al., 2012) and blood flow (Lamping et al., 2005). Sympathetic nerve is overactivated in CHF (Triposkiadis et al., 2009), which acts on the myocardial arteriole to reduce coronary
5. Conclusion HTSB improves coronary microcirculation disturbance in CHF, which may contribute to reversing myocardial remodeling and dysfunction. HTSB stimulates myocardial capillary growth independent of VEGF.
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CHF
Control
HTSB
Fig. 4. VEGF immunohistochemical staining (×400). Brown particles in the cytoplasm were VEGF positive staining. Compared to the control group, They were increased in the CHF and HTSB group. HTSB reduced VEGF expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Conflict of interest
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