生理学前沿 doi: 10.3389/fphys.2016.00211
摘要：已知過去自律神經失衡與心血管血壓異常、組織與器官損傷息息相關，本研究利用自發性高血壓大鼠模式分析經過60天長期攝取克弗爾發酵乳後，探討克弗爾發酵乳對於迷走神經與交感神經對於血壓調節的現象。研究總共分成對照組(Wistar rat)、自發性高血壓大鼠(SHR, Spontaneous Hypertensive Rat)與攝取克弗爾發酵乳自發性高血壓大鼠(SHR Kefir)，分別測量心跳速率、血壓、左心房肥大狀況；同時以神經抑制劑介入自律神經調節進一步探討生理現象。結果顯示SHR Kefir組相較於SHR組能夠有效血壓、心跳速率與左心房肥大的狀況，同時能夠調節迷走神經和交感神經趨於正常。證明長期每天攝取低劑量的克弗爾發酵乳能夠改善因自發性高血壓使自主神經導致心跳速率異常和感壓受體反射敏感度下降。
c. 感壓受體反射敏感度光譜分析：將脈搏間隔以低頻率(0.2-0.8 Hz)和高頻率(0.8-2.8 Hz)光譜分別評估交感神經活性與迷走神經活性，進而計算感壓受體反射敏感度。
Material and Methods
The present study was performed in male 4-month-old SHR and in age-matched Wistar-Kyoto rats obtained from the Vila Velha University animal care and that were maintained in the animal care facility of the Federal University of Espirito Santo, Brazil. The rats were housed in individual acclimatized plastic cages with a controlled temperature (22–23°C), light-dark cycle (12:12-h), and were fed with a standard rat chow and water ad libitum. The study protocols were previously approved by the Institutional Committee on Animal Care (CEUA-UFES, Protocol #040/2014). All experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals as recommended by the National Institutes of Health (NIH).
Kefir Preparation, Identification, and Administration
The identification, preparation and administration of kefir were performed as previously described (Friques et al., 2015). Briefly, kefir was obtained from the fermentation of the grains in whole milk. The kefir beverage was prepared by adding kefir grain to pasteurized whole milk in a ratio of 4% (w/v).
The treatment of the animals was started at the age of 4-month-old and lasted 60 days. One group of animals was treated with kefir (0.3 mL/100 g body weight, by gavage, SHR-kefir) for 60 days. Another group of SHR was administered whole milk (0.3 mL, pH adjusted to 4.5, SHR) for 60 days for use as the hypertensive controls. The rationale for using 0.3 mL/100 g body weight was based on the dose translation from human to animal studies by a simple method using the body surface area normalization and that dose is compatible with that used in human beings. The Wistar rats were administered whole milk for 60 days and were used as normotensive control groups. The reason for treating the animals with kefir for 60 days was based on a previous study from our group (Friques et al., 2015), demonstrating that the treatment for less than 60 days had no effect on cardiovascular parameters in SHR.
Instrumentation for Hemodynamic Measurements
After 60 days of kefir administration, the animals were intraperitoneally anesthetized with a mixture of ketamine and xylazine (91+9.1 mg/kg) and a polyethylene catheter (PE 50) was positioned into the femoral vein for injection of drugs and another into the inferior aorta for measurement of pulsatile pressure, mean BP and HR 48 h later using a data-acquisition system (Biopac Systems, Santa Barbara, CA, USA) in unrestrained animals.
At the end of the evaluation of hemodynamic parameters, the animals were euthanized by an over-dose of thiopental (100 mg/kg) and the hearts were excised. The right and the left ventricles, including the interventricular septum, were dissected from the remaining cardiac tissues. The tibia bone was also dissected and its length was measured. To evaluate the extent of cardiac hypertrophy for each animal the left ventricle weight was normalized by the animal's body weight and tibia length.
Evaluation of the Cardiac Autonomic Tones
Cardiac autonomic tones were estimated through selective pharmacological blockers of the muscarinic receptors (methylatropine) and the β1?adrenergic receptors (atenolol) in conscious animals, based on previous studies from our laboratory and from others (Chapleau and Sabharwal, 2011; Campagnaro et al., 2012). As shown in the scheme in Figure 3 (top), the cardiac parasympathetic tone was estimated by the change in basal HR 15 min after a single injection of methylatropine (1 mg/kg, i.v.), which reaches a plateau effect at approximately this time and lasts for ~30 min. Immediately afterwards, they were injected with atenolol (1 mg/kg, i.v.), which also reached a maximum effect 15 min later, and this value was considered the intrinsic (pacemaker) HR. the next day, the sequence of the injections was the opposite, and the cardiac sympathetic tone was estimated by the change in the basal HR 15 min after atenolol, and the intrinsic HR was estimated 15 min under the double blockade.
Pharmacological Baroreflex Sensitivity
The baroreflex control of arterial pressure was evaluated in conscious animals by measuring the tachycardia and bradycardia in response to an equivalent increase in arterial BP in each of the three groups of conscious animals. Based on previous studies from our laboratory demonstrating that the major sensitivity of the baroreflex is at changes in arterial BP close to the resting values and that changes in BP higher than 40 mmHg could elicit complex humoral mechanisms, we decided to challenge the baroreflex with sudden increase and decrease in BP of ~25 mmHg (Schenberg et al., 1995; Gava et al., 2004). These changes in BP were elicited by a single bolus injection of phenylephrine (1 μg/kg) and sodium nitroprusside (1 μg/kg), respectively.
The relative contribution of the cardiac parasympathetic and sympathetic nerves was assessed by increase or decrease in HR in response to a sudden decrease or increase in BP, under the blockade of cardiac muscarinic receptors with methylatropine (1 mg/kg, i.v.) on day one and the blockade of the cardiac β1-adrenoceptors with atenolol (1 mg/kg, i.v.) on the following day.
An aim of this protocol was to characterize patterns of autonomic control in SHR and normotensive Wistar rats by power spectral analysis of pulse interval (PI) variability and BP variability as previously described (Dantas et al., 2012), Power spectra analysis was performed using a Matlab-customized software validated against the software developed by A. Porta in Italy (Linear Analysis version 8.3, University of Milan, Italy). Pulse intervals from systolic arterial pressure were obtained from 30 min of continuous BP records in conscious animals. A parametric method based on autoregressive model of spectral estimation was performed for systolic arterial BP and PI analysis. The oscillatory components in the rat were quantified as low frequency (LF: 0.2–0.8 Hz) mainly corresponding to sympathetic activity and high frequency (HF: 0.8–2.8 Hz) corresponding to vagal activity (influenced by respiration; Silva et al., 2009; Dantas et al., 2013; Quagliotto et al., 2015).
The estimation of spontaneous baroreflex sensitivity was obtained by measuring oscillations in BP and PI in the LF range using spectral analysis. The baroreceptor sensitivity value was provided by α-LF index, which was calculated as the square root of the ratio between the absolute power of PI-LF/BP-LF, expressed in ms/mmHg. The oscillations in BP and PI needed to be coherent (coherence2), with a coherence higher than 0.5, and a negative phase difference was required between the two variables, as recently reviewed (Chapleau and Sabharwal, 2011).
The values are expressed as means ±SEM. First, a D'Agostino-Pearson omnibus normality test was performed to verify if that the values came from a Gaussian distribution. All data Statistical comparisons between the different groups were performed by a randomized one-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software version 6.07 (GraphPad, Inc., San Diego, CA, USA).
分別將Wistar組、SHR組、SHR Kefir組老鼠經過犧牲之後，同時分別以小鼠體重和脛骨重進行心肌重量校正，結果顯示患有自發性高血壓老鼠相較於一般大鼠有明顯左心房肥大狀況，而同時間攝取60天SHR Kefir組相較於SHR組別無論是經體重或是脛骨重校正後，都有顯著改善左心房肥大的結果。
給予老鼠適度的去氧腎上腺素(phenylephrine)模擬增加血壓後，神經為穩定血壓減緩心搏的心動過緩反射(reflex bradycardia)狀況，來探討自發性高血壓老鼠心動過緩反射敏感性。在本研究發現，經去氧腎上腺素刺激下三組動脈血壓皆上升，然而在心跳速率上SHR組卻無法像對照組Wistar組因血壓上升而產生適度心動過緩反射，導致該組心跳速率僅對照僅為對照組50%降幅；但在SHR Kefir組別能夠些微增加自律神經系統對於心搏過緩反射的敏感性，同時本研究使用迷走神經抑制劑阿托品比較各組後無顯著差異，驗證克弗爾發酵乳可幫助自律神經系統調節血壓。
給予老鼠適度的硝普鈉(sodium-nitroprusside)模擬降低血壓後，神經為穩定血壓增加心搏的心動過速反射(reflex tachycardia)狀況，一樣來探討自發性高血壓老鼠心動過速反射敏感性。在本研究發現，經硝普鈉刺激下三組動脈血壓皆下降，然而在心跳速率上SHR組卻無法像對照組Wistar組因血壓下降而產生適度心動過速反射，導致該組心跳速率無法像對照組有一樣的增幅；而SHR Kefir組別能夠些微增加自律神經系統對於心搏過動反射的敏感性，同時本研究使用β-受體抑制劑阿替洛爾無論是SHR組或是SHR Kefir組都因迷走神經作用有所提升；同時SHR Kefir組改善效果優於SHR組，再次驗證克弗爾發酵乳可幫助自律神經系統調節血壓。
分別以高、低頻率觀察各組心跳、動脈血壓狀況來評估迷走神經與交感神經活性，低頻率光譜分析中發現SHR組別老鼠皆相較於對照組老鼠心跳速率、動脈血壓較高，而攝取克弗爾發酵乳的SHR Kefir組有調節心跳速率與動脈血壓的結果；在以高頻率光譜分析中，三組則在心跳上沒有顯著差異。同時進一步以光譜分析SHR、SHR Kefir組隊於對照組自發性感壓受體反射(Barorelex)相較不敏感。