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J Thorac Cardiovasc Surg 2003;125:678-685
© 2003 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

Preconditioned somatothermal stimulation on median nerve territory increases myocardial heat shock protein 70 and protects rat hearts against ischemia-reperfusion injury

From the Institute of Traditional Medicine, School of Medicine, National Yang-Ming University^a; the Division of General Surgery, Department of Surgery, Veterans General Hospital, Taipei; Taiwan, Republic of China^b; the Department of Family Physicians, Mackay Memorial Hospital^c; the Department of Anesthesia, School of Medicine, National Yang-Ming University^d; the Department of Surgery, School of Medicine, National Yang-Ming University^e; the Institute of Chinese Medical Science, China Medical College,^f Taichung, Taiwan, Republic of China.

This work was supported by grants from National Science Council, Republic of China (NSC 89-2314-B-010-014 and NSC 90-2320-B-010-054).

Received for publication Jan 4, 2002. Revisions requested April 16, 2002; revisions received June 12, 2002. Accepted for publication July 15, 2002. Address for reprints: Jen-Hwey Chiu, MD, Institute of Traditional Medicine, National Yang-Ming University, No 155, Section 2, Li-Nong St, Peitou, Taipei, 112, Taiwan (E-mail: chiujh{at}mailsrv.ym.edu.tw).

	Abstract

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Objective: This study was designed to test the hypotheses that local somatothermal stimulation on the left median nerve territory increases myocardial heat shock protein 70 and that preconditioning of rats with local somatothermal stimulation protects the hearts against subsequent ischemia-reperfusion injury.
Methods: Local somatothermal stimulation was brought about by means of application of a heating rod over and above the left median nerve territory (1.5 cm proximal to the palm crease) in male Sprague-Dawley rats. After rats were treated with local somatothermal stimulation, the gene expression of heat shock protein 70 in regional muscle, heart, and liver was assessed by means of Western blotting and reverse transcription—polymerase chain reaction. In addition, durations of arrhythmia, mortality rates, and mitochondrial functions were compared between groups preconditioned with or without local somatothermal stimulation followed by subsequent myocardial ischemia-reperfusion injury.
Results: The results showed that the gene expression of heat shock protein 70 was upregulated in the muscle beneath the area of local somatothermal stimulation, as well as in the heart, although not in the liver. When animals were preconditioned with local somatothermal stimulation on the left median nerve territory followed by subsequent ischemia-reperfusion injury of the heart, there were significant decreases of creatine kinase level from the heart, duration of arrhythmia, mortality rate, and improved mitochondrial respiratory function compared with that seen in those without local somatothermal stimulation preconditioning.
Conclusion: We conclude that local heat stress preconditioning on the left median nerve territory has a potential cardioprotective effect against subsequent ischemia-reperfusion injury.

	Introduction

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View larger version (111K): [in this window] [in a new window]   Chen, Wu, Lay, Chiu, Wang, Lin, Tsou (left to right).

Previous studies suggest that an inverse correlation exists between expression of Hsp70 induced by means of ischemic or thermal preconditioning and injury of many organs, such as the kidney, heart, and small intestine. ⁵ By using transgenic mice, ^6,7 protein delivery virus vector, ⁸ and gene-transfected rats ⁹ as working systems, there is evidence that overexpressed Hsp70 plays an important and direct role in the enhancement of myocardial tolerance against ischemia-reperfusion injury. In most animal models, experimental heatstroke preconditioning could be achieved by exposing animals to a high ambient temperature, usually up to 41°C, to induce Hsps in many organs. ¹⁰ However, maneuvers with either tissue or whole-body hyperthermia are cumbersome and impractical in human subjects.

Recent scientific studies suggest that stimulation of the median nerve regulates the functions of the cardiovascular and autonomic systems. For example, median nerve stimulation improves left ventricular function in those patients with coronary heart disease, improves myocardial microcirculation in rabbits, and reverses the reflex-induced myocardial ischemia in cats. ^11-13 Nevertheless, information about how the gene expression of myocardial Hsp70 is affected by median nerve stimulation remains lacking.

Our recent works have shown that local somatothermal stimulation (LSTS), in contrast to whole-body hyperthermia, regulates the activity of the sphincter of Oddi in cats, rabbits, and human subjects, as well as the motility of the internal anal sphincter in rabbits and human subjects. In both circumstances the effects of LSTS are mediated by the activation of heat-sensitive neural release of nitric oxide. ^14,15 Moreover, we also demonstrate that preconditioned LSTS on the right seventh intercostal nerve territory increases hepatic Hsp70 and protects the liver from ischemia-reperfusion injury in rats. ¹⁶ Interestingly, the critical temperatures evoking such visceral responses are around 41°C to 43°C, which are similar to those inducing Hsp expression in many studies. ^10,14-16 In this study 2 major questions are to be answered. First, is the myocardial gene expression of Hsp70 upregulated when local heat stress (LSTS) is applied to left median nerve territory? Second, does preconditioning the animals with LSTS protect the heart against subsequent ischemia-reperfusion injury?

	Methods

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Animals
Male Sprague-Dawley rats weighting 250 to 300 g were obtained from the animal center of the National Science Council, Taiwan, Republic of China. They were fed with standard diet and water ad libitum and treated according to the "Guide for the Care and Use of Laboratory Animals" (National Academy Press, 1996). The studies were approved by the committee of experimental animals of National Yang-Ming University.

Antibody and western blot analysis for Hsp expression
Mouse monoclonal antibody against inducible Hsp70 was purchased commercially (H53220-050, Transduction Laboratory). Tissue homogenates from rat livers, hearts, and the muscle beneath the LSTS site were obtained in the presence of protease inhibitors on ice, and the homogenate protein was assayed by using the method of Bradford. ¹⁷ Fifty micrograms of homogenate was separated on 10% sodium dodecylsulfate—polyacrylamide gel electrophoresis transferred to a nitrocellulose membrane. The membranes were blocked with skim milk and incubated with 10,000-fold diluted monoclonal antibody against Hsp70. After washing and incubation with 5000-fold diluted biotin-conjugated secondary antibody (anti-mouse IgG, horseradish peroxidase, ME5345; Transduction Laboratory), Hsp70 was detected by using enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc) and analyzed with autoradiography. Monoclonal antibody against -tubulin was used as an internal control. The optical density values were analyzed from a computer equipped with image analysis software (PhotoCapt, Vilber Lourmat).

Extraction of RNA and reverse transcription—polymerase chain reaction
Total RNA was isolated from rat organs (muscle, heart, and liver) by using a modified single-step guanidinium thiocyanate method (TRI REAGENT, T-9424; Sigma). ¹⁸ Reverse transcription (RT) was performed with an RT kit. One microgram of rat tissue RNA was used as a template, and RT-generated cDNAs encoding Hsp70 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; internal control) were amplified with polymerase chain reaction (PCR). The primers used in this study are Hsp70 (354 bp; sense, 5-AAC-GTG-CTG-CGG-ATC-ATC-AA-3; antisense, 5-CTG-GAT-GGA-CGT-GTA-GAA-GT-3) and G3PDH (983-bp; sense, 5-TGA-AGG-TCG-GGT-GTG-AAC-GGA-TTT-GGC-3; antisense, 5-CAT-GTA-GGC-CAT-GAG-GTC-CAC-CAC-3). The possible contamination of any PCR component was excluded by performing a PCR reaction with these components in the absence of RT product in each set of experiments (negative control). Twenty microliters of RT-PCR product was checked by using 2% agarose gel electrophoresis containing 0.2 µg/mL ethidium bromide, followed by photography under UV transillumination. Quantification of RNA transcripts was analyzed according to the method described previously with some modification. ¹⁹ In brief, the levels of expression of the transcripts were normalized to that of G3PDH mRNA in the same tissue sample. The appropriate number of PCR cycles and the optimal combination of the amount of first-strand cDNAs were determined for each set of primers to ensure that all PCR reactions were carried out in the linear quantitative phase of the amplification process. The PCR products were size fractionated on agarose gels, visualized by means of ethidium bromide staining, and photographed. The photographs of the ethidium bromide—stained gels were scanned with an imaging densitometer linked to a personal computer equipped with image analysis software. The size and optical density of the bands produced by the PCR products were quantified by using the Kodak digital science 1-dimensional image analysis software (EDAS 120, Eastman Kodak Company). For statistical comparison, more than 3 independent samples in each experiment were performed to obtain the ratio of the density of Hsp70 to G3PDH mRNA levels.

Local heat stress by LSTS
Application of local heat stress compared with whole-body hyperthermia was achieved by means of LSTS on the skin area but no direct contact with the skin surface (Figure 1, A). The effect of LSTS has been reported in our laboratory to induce relaxation of the sphincter of Oddi in cats and rabbits, as well as the internal sphincter in rabbits. ^14-16 In brief, LSTS was achieved by application of a heat generator to and above (0.5 cm) the skin area, and a fluctuating skin temperature curve was obtained by intermittently turning on and off the switch of the heat generator (ie, 4 minutes on and 5 minutes off for 3 courses; Figure 1, B). Usually, it took 27 minutes to complete one cycle of LSTS. LSTS was applied to and above the left median nerve territory to study the myocardial expression of Hsp70, and the gene expression of myocardial Hsp70 was analyzed by means of Western blotting and RT-PCR. The left median nerve territory was located 1.5 cm proximal to left palm crease just above the median nerve.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Experimental setup (A) and skin temperature curve obtained by means of LSTS (B). LSTS was applied above (0.5 cm) and to the skin area 1.5 cm proximal to the left palm crease. The dashed line indicates the assumptive median nerve. LSTS was brought about by means of intermittent turning on and off of the heat generator, as described in the "Methods" section.

Animal model and parameters for ischemia-reperfusion injury of the heart
Ischemia-reperfusion injury in a rat model was made as described previously. ^20,21 In brief, male Sprague-Dawley rats preconditioned with LSTS were anesthetized with urethane (1.25 gm/kg) administered intraperitoneally, and the trachea was cannulated for artificial respiration with a ventilator. Polyethylene catheters were cannulated into the femoral artery to monitor the blood pressure, and a standard lead-1 electrocardiogram (ECG) was recorded by attaching silver electrodes to the extremities of animals and monitored by a polygrapher (RS 2400, Gould). After the chest was opened by means of a left thoracotomy and the fourth and fifth ribs were sectioned, the pericardium was incised, followed by easing the heart out of the chest, with gentle pressure on the rib cage. A 6-0 silk suture was placed under the left main coronary artery, and the heart was repositioned in the chest for 15 minutes. Animals in which the procedure produced arrhythmia or death during the first 5 minutes were excluded from this study. The procedure whereby the silk was fashioned into a snare allowed occlusion of the blood supply to the left coronary artery for 30 minutes. The reperfusion was achieved by releasing the tension applied to the ligature for 10 minutes. Successful ligation of the coronary artery was validated by means of observation of a decrease in arterial pressure and ECG changes (increase in R-wave and ST-segment elevation), which is indicative of ischemia. Reperfusion was confirmed by means of an increase in arterial pressure, ECG changes (decrease of the R wave and normalization of the ST segment), and sudden occurrence of arrhythmia. Before and during the ischemia-reperfusion period, heart rate, blood pressure, and ECG changes were recorded on a computer equipped with an offline data analysis software (AcqKnowlege, Biopac System). Arrhythmia, such as ventricular fibrillation and premature ventricular beat, were evaluated as described previously. ²² Serum creatine phosphokinase and creatine kinase isozyme (MB fraction) levels were assayed by means of electrophoresis to determine the degree of myocardial injury.

Mitochondria preparation and mitochondrial functions
Mitochondria were prepared from the hearts of male rats, as previously described by Vercesi and colleagues. ²³ Sprague-Dawley rats were killed by means of decapitation, and their hearts were quickly excised and thoroughly washed with ice-cold SEH buffer (0.25 mol/L sucrose, 0.5 mmol/L ethyleneglycol-bis-[ß-aminoethylether]-N,N,N,N, tetra-acetic acid, and 3 mmol/L N-2-hydroxyethylpiperazine-N-ethanesulfonic acid, pH 7.2). All the following procedures were performed at 4°C. The hearts were finely minced with a pair of scissors in SEH buffer. The dispersed heart tissue was homogenized with a glass pestle and diluted with SEH and pronase (0.1 mg/mL SEH buffer) solution to 10 mL per gram of heart tissue for 15 minutes. The hearts were washed in 0.25 mol/L sucrose twice. The homogenates were centrifuged at 400g for 5 minutes in the SS34 rotor of an RC-5C Sorvall refrigerated high-speed centrifuge (Du Pont). The supernatant was decanted and centrifuged at 700g for 5 minutes. After recentrifugation of the supernatant at 2500g for 10 minutes, the pellet was resuspended in SEH solution and centrifuged again for 10 minutes at 6000g. The mitochondria-rich red-brown lower layer was suspended in 0.5 mL of SEH solution per gram of heart tissue. Because the respiratory function of mitochondria decreased rapidly, all respiratory experiments were performed with the freshly prepared mitochondria.

Determination of Respiratory Control Ratios
The glutamate and malate solution—supported (Sigma) respiration rates of the mitochondria were determined polarographically in an oxygen monitor equipped with a Clark oxygen electrode (Gilson Medical Electronics). Usually, 0.4 to 0.8 mg of freshly prepared mitochondria was incubated in the reaction chamber containing 1.6 mL of assay mixture before the addition of substrates. The addition of glutamate and malate solution induced slow oxygen uptake (state 4), and 400 nmol of adenosine diphosphate was introduced to the assay system to stimulate respiration to reach an active state (state 3). States 3 and 4 of respiration were defined as described previously. ²⁴ The respiratory control ratio (RCR) was measured as the quotient of the respiration rate of state 3 to that of state 4.

Statistics
Data were expressed as means ± SEM. Significance was determined by using 1-way analysis of variance, followed by the Bonferroni-corrected t test or the Wilcoxon rank sum test for stepwise comparison. For noncontinuous variables, the difference between groups was analyzed by using the Fisher exact test.

	Results

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Dose selection for local heat stress (LSTS)
To determine the optimal cycle of local heat stress or LSTS on myocardial Hsp70 expression, cycle-related experiments were performed. The results showed that there was an increase of myocardial expression of Hsp70 after different cycles of LSTS on the left median nerve territory, with the peak expression at 3 cycles of LSTS (Figure 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Dose response of Hsp70 expression after LSTS on the right lower abdominal wall. The expression of Hsp70 in the muscle beneath the LSTS skin area was analyzed by means of Western blotting, as described in the "Methods" section.

View larger version (21K): [in this window] [in a new window]   Fig. 3. The expression of Hsp70 in the muscle near the left median nerve territory after 3 cycles of LSTS. The expression of Hsp70 in the muscle underlying the left median nerve was analyzed by means of Western blotting, as described in the "Methods" section. The asterisk indicates statistical significance (P < 0.05) between the LSTS(+) group (filled column) and the LSTS(-) group (open column). OD, Optical density.

View larger version (19K): [in this window] [in a new window]   Fig. 4. The expression of Hsp70 in the heart and liver after 3 cycles of LSTS on the left median nerve territory. The expression of Hsp70 in the heart and liver tissues was analyzed by means of Western blotting, as described in the "Methods" section. The asterisk indicates statistical significance (P < .05) between the LSTS(+) group (filled columns) and the LSTS(-) group (open columns). OD, Optical density.

View larger version (31K): [in this window] [in a new window]   Fig. 5. RT-PCR analysis of Hsp70 in the heart (A) and liver and muscle (B) after 3 cycles of LSTS on the left median nerve territory. For the primer sequences of Hsp70, reactions of RT-PCR were described in the "Methods" section. The density of Hsp70 to G3PDH mRNA levels were analyzed by means of computerized densitometry and expressed as the Hsp70/G3PDH ratio (C). The data were reproduced from more than 3 independent experiments. The asterisk indicates statistical significance between the LSTS(+) group (filled columns) and the LSTS(-) group (open columns). H, L, and M indicate heart, liver, and muscle tissues in the LSTS group, respectively; whereas Hc, Lc, and Mc indicate heart, liver, and muscle tissues in the control or LSTS(-) group, respectively. PC, Positive control.

View larger version (55K): [in this window] [in a new window]   Fig. 6. ECG and blood pressure monitoring during ischemia-reperfusion injury in rats preconditioned with (A) and without (B) LSTS on the left median nerve territory. The changes in blood pressure (upper panel) and ECG results (lower panel) in 2 groups receiving ischemia-reperfusion injury was recorded. BP, Blood pressure.

View larger version (34K): [in this window] [in a new window]   Fig. 7. RCRs of heart mitochondria were obtained by means of analysis of 3 independent experiments. The asterisk indicates statistical significance (P < .05) between the LSTS(+) group (filled column) and the LSTS(-) group (open column) during 10 minutes of ischemia and 10 minutes of reperfusion. The normal control group is shown in the shaded column. Isc/Rep, Ischemia and reperfusion.

	Discussion

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Among the systemic consequences of reperfusion, lipid peroxidation is probably the most damaging effect of free radicals. Endogenous free radicals generated during ischemia-reperfusion injury induce damage to the cytoskeleton and disruption of microtubules and result in mitochondrial swelling and loss of mitochondrial functions. ^25,26 Respiratory functions are very sensitive to minor membrane damage. ²⁷ Any interruption of mitochondrial membrane integrity leads to proton leakage, uncoupling of the electron transport chain, oxidative phosphorylation, cellular dysfunction, and death. ²⁸ Although the heat shock or stress response refers to the progress whereby stressful stimuli (eg, hyperthermia) induce a transient resistance of the cells to subsequent noxious stimuli, it remains controversial between the expression of Hsp70 and its cardioprotective function in many studies. For example, some studies have suggested that overexpressed Hsp70 plays a direct role in the enhancement of myocardial tolerance against ischemia-reperfusion injury, ^6-9 whereas others have presented differing opinions. ²⁹ Our results showed that there was an increase of myocardial Hsp70 gene expression and improved myocardial functions in terms of durations of arrhythmia, mortality rates, and mitochondrial functions when animals were preconditioned with LSTS on the left median nerve territory. Because no Hsp70 gene knockout or gene transfer experiments were done in our system to provide direct evidence of the protective role of Hsp70, the direct cause-and-effect relationship requires further elucidation.

There is consensus that it is not feasible to perform whole-body hyperthermia in human subjects, although it might induce Hsp70 expression in the heart. In addition, it is impractical to perform whole-body hyperthermia preconditioning in human subjects. On the contrary, LSTS is an alternative to local heat stress, which can be easily applied on the skin area of the human body. In our system the rate of increase of the temperature varied if the distance between the heating rod and the skin changed. We found that LSTS at a distance of 0.5 cm and a stimulated area of 1 cm (diameter) was the optimal condition to induce myocardial Hsps (data not shown). Our previous works showed that temperatures ranging from 41°C to 43°C are critical points in modulating many visceral functions, which is consistent with previous studies on inducing Hsp expression. ^10,14-16 It is noteworthy that the fluctuating curve of temperature on LSTS (Figure 1, B) is designed to make the temperature increase and decrease from the critical point of 41°C so that the heat-sensitive neural transmission would not be tolerated. In addition, in such mild heat stimulation, no skin damage, such as burn injury or median nerve injury, was noticed.

In contrast to whole-body heat stress (hyperthermia), LSTS is a kind of local heat stress. Although there was an increased expression of Hsp70 in the muscle underlying the LSTS area, this was not the case for hepatic Hsp70 expression when LSTS was applied to the left median nerve territory. Furthermore, our previous study showed that LSTS on the right seventh intercostal nerve territories increases hepatic, but not myocardial, Hsp70 expression. The results also demonstrated that sublethal preconditioning of rats with LSTS on the right seventh intercostal nerve territory increases Hsp70 expression and decreases malondialdehyde formation in subsequent ischemia-reperfusion injury in the liver. ¹⁶ These facts make the possibility that the myocardial responses to LSTS on the left median nerve territory are only reflective of a global whole-body response less likely. On the contrary, the visceral responses evoked by LSTS on skin area are organ specific. With regard to the result that there was more abundant expression of myocardial Hsp70 after 3 cycles than after 4 cycles of LSTS on the left median nerve territory (Figure 2), we attributed this phenomenon to the possibilities of more degradation of Hsp70 or a plateau effect of 4 cycles of LSTS. In addition, the increase of relative abundance of myocardial Hsp70 mRNA after LSTS on the left median nerve territory might be caused by an increase in gene expression, an increase in mRNA stability, or both. Nuclear run-on studies are under investigation.

With regard to the potential of clinical implication of LSTS, we have applied a heat-generating pad instead of a heating rod in animal studies to relax the hypertonic anal sphincter in human subjects. ³⁰ The effect of LSTS is postulated to be through the activation of heat-sensitive neural release of nitric oxide. ¹⁵

In summary, preconditioned LSTS on the left median nerve territory has a beneficial effect in protecting the rat heart against ischemia-reperfusion injury. This is the advantage compared with pretreatment by means of whole-body hyperthermia or brief myocardial ischemia. LSTS is an easily applicable alternative and will bring into perspective the clinical prevention of ischemic heart disease or heart transplantation.

	Acknowledgments

 
We thank Miss Yi-Hong Hsieh for her excellent technical assist.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Bukau B, Horwich AL. The Hsp70 and Hsp60 chaperone machines. Cell. 1998;92:351-66.[Medline]
   
2. Jaattela M. Heat shock proteins as cellular life guards. Ann Med. 1999;31:261-71.[Medline]
   
3. Mosser DD, Caron AW, Bourget L, Denis-Larose C, Massie B. Role of human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol. 1997;17:5317-27.[Abstract]
   
4. Akagawa H, Ishii A, Mizuno S. Suppression of thermotolerance development through cycloheximide-induced negative control of stress protein gene expression. J Biochem. 1998;123:226-32.[Abstract/Free Full Text]
   
5. Hutter MM, Sievers RE, Barbosa V, Wolfe CL. Heat-shock protein induction in rat hearts. A direct correlation between the amount of heat-shock protein induced the degree of myocardial protection. Circulation. 1994;89:355-60.[Abstract/Free Full Text]
   
6. Marber MS, Mestril R, Chi SH, Sayen R, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70KD heat stress protein in a transgenic mouse increase the resistance of the heart to ischemic injury. J Clin Invest. 1995;95:1446-56.
   
7. Plumier JCL, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, et al. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest. 1995;95:1854-60.
   
8. Brar BK, Stephanou A, Wagstaff MJ, Coffin RS, Marber MS, Engelmann G, et al. Heat shock proteins delivered with a virus vector can protect cardiac cells against apoptosis as well as against thermal or hypoxic stress. J Mol Cellular Cardiol. 1999;31:135-46.[Medline]
   
9. Suzuki K, Sawa Y, Kaneda Y, Ichikawa H, Shirakura R, Matsuda H. In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia- reperfusion injury in rat. J Clin Invest. 1997;99:1645-50.[Medline]
   
10. Lin MT. Heatshock-induced cerebral ischemia and neuronal damage: involvement of cytokines and monoamines. Ann N Y Acad Sci. 1997;813:572-80.[Medline]
    
11. Chao DM, Shen LL, Tjen-A-Looi S, Pitsillides KF, Li P, Longhurst JC. Naloxone reverse inhibitory effect of electroacupuncture on sympathetic cardiovascular reflex response. Am J Physiol. 1999;276:2127-34.
    
12. Cao Q, Lin J, Chen S, Han Z. Effect of electroacupuncture at neiguan on myocardial microcirculation in rabbits with acute myocardial ischemia. J Tradit Chin Med. 1998;18:134-9.[Medline]
    
13. Li P, Pitsillides KF, Rendig SV, Pan HL, Longhurst JC. Reversal of reflex-induced myocardial ischemia by medial nerve stimulation, a feline model of electroacupuncture. Circulation. 1998;97:1186-94.[Abstract/Free Full Text]
    
14. Chiu JH, Lui WY, Chen YL, Hong CY. Local somatothermal stimulation inhibits sphincter of Oddi motility in cats, rabbits, and humans through the neural release of nitric oxide. Life Sci. 1998;63:413-28.[Medline]
    
15. Jiang JK, Chiu JH, Lin JK. Local thermal stimulation inhibits the motility of internal anal sphincter through nitrergic neural release of nitric oxide. Dis Colon Rectum. 2000;43:381-8.[Medline]
    
16. Lin YH, Chiu JH, Tung HH, Tsou MT, Lui WY, Wu CW. Preconditioned somatothermal stimulation on right 7th intercostal nerve territory increases hepatic heat shock protein 70 and protects the liver from ischemia-reperfusion injury in rats. J Surg Res. 2001;99:328-34.[Medline]
    
17. Bradford M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Chem. 1976;72:248-54.
    
18. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-9.[Medline]
    
19. Wasser S, Ho JMS, Ang HK, Tan CEL. Salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats. J Hepatol. 1998;29:760-71.[Medline]
    
20. Tsai SK, Huang SS, Hong CY. Myocardial protective effect of honokiol: an active component in Magnolia officinalis. Planta Med. 1996;62:503-6.[Medline]
    
21. Hong CY, Huang SS, Tsai SK. Magnolol reduces infarct size and suppresses ventricular arrhythmia in rats subjected to coronary ligation. Clin Exp Pharmacol. 1996;23:660-4.
    
22. Heimburger RF. Injection into pericardial sac and ligation of coronary artery of rat. Arch Surg. 1946;52:677-89.[Abstract/Free Full Text]
    
23. Vercesi A, Reynafarje B, Lehninger AL. Stoichiometry of H⁺ ejection and Ca²⁺ uptake couples to electron transport in rat heart mitochondria. J Biol Chem. 1978;253:6379-85.[Free Full Text]
    
24. Chance B, William GR. The respiratory chain and oxidative phosphorylation. Adv Enzymol. 1956;17:65.
    
25. Patriarca EJ, Kobayashi GS, Maresca B. Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum.Biochem Cell Biol. 1992;70:207-14.[Medline]
    
26. Patriarca EJ, Maresca B. Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae. Exp Cell Res. 1990;190:57-64.[Medline]
    
27. Gronzalez-Flecha B, Cutrin JC, Boveris A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J Clin Invest. 1993;91:456-64.
    
28. Grace PA. Ischemia-reperfusion injury. Br J Surg. 1994;81:637-47.[Medline]
    
29. Su CY, Chong KY, Chen J, Ryter S, Khardori R, Lai CC. A physiologically relevant hyperthermia selectively activates constitutive hsp70 in H9c2 cardiac myoblasts and confers oxidative protection. J Mol Cell Cardiol. 1999;31:845-55.[Medline]
    
30. Local thermal stimulation relaxes hypertonic anal sphincter: evidence of somatoanal reflex. Dis Colon Rectum 1999;42:1152-9.[Medline]
    

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chiu, J.-H. Articles by Wu, C.-W. Search for Related Content PubMed PubMed Citation Articles by Chiu, J.-H. Articles by Wu, C.-W. Related Collections Myocardial protection