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Ann Thorac Surg 1998;65:1241-1247
© 1998 The Society of Thoracic Surgeons

Cardioprotection by Local Heating: Improved Myocardial Salvage After Ischemia and Reperfusion

Ashok Gowda, MSa, Chun-jie Yang, MDa, Gregory K. Asimakis, PhDb, Johannes Ruef, MDc, Sohi Rastegar, PhDa, Marschall S. Runge, MD, PhDc, Massoud Motamedi, PhDa

a Biomedical Engineering Center, The University of Texas Medical Branch, Galveston, Texas, USA
b Division of Cardiothoracic Surgery, The University of Texas Medical Branch, Galveston, Texas, USA
c Division of Cardiology, The University of Texas Medical Branch, Galveston, Texas, USA

Accepted for publication November 29, 1997.

Address reprint requests to Dr Motamedi, Division of Cardiology, Rt D56, Jennie Sealy Hospital, Rm 625, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX 77555
e-mail: (massoud. motamedi{at}utmb.edu)


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Previous studies have shown that expression of the inducible 70-kD heat-shock protein (HSP72) by whole-body hyperthermia is associated with protection against ischemia-reperfusion injury. To develop techniques for regional elevation of heat-shock proteins that prevent extracardiac sequelae during whole-body hyperthermia, we sought to determine if local heating of the heart in vivo provides protection against ischemia-reperfusion injury in the rat.

Methods. A thermal probe was used to locally heat rat hearts at two adjacent sites on the epicardial surface of the left ventricle. Rats were subjected to either 30 minutes of sham surgery (control; n = 10) or two local applications of the probe at 42.5° to 43.5°C for 15 minutes each (n = 9). After 4 hours, rats were subjected to 30 minutes of regional ischemia followed by 120 minutes of reperfusion. Hearts were removed and area at risk and infarct area were determined.

Results. Localized heat stress resulted in a significant limitation of infarct size in heat-treated animals versus controls (mean ± standard error of the mean infarct area/area at risk = 4.3% ± 0.85 versus 19.2% ± 3.4%; p < 0.005). Western blot experiments confirmed elevated HSP72 expression in left (heated) and right (nonheated) ventricular samples from treated animals (n = 6; left ventricular = 5.5-fold; right ventricular = 3.7-fold) compared with sham-operated controls. Controls treated with the probe at 37°C (n = 4) showed no increases in HSP72.

Conclusions. Local heating of the heart is associated with elevated levels of HSP72 and improved myocardial salvage. The increase in expression of HSP72 is not limited to the heated region, but extends into nonheated regions of the heart as well. This may lead to the development of new techniques that improve methods of myocardial revascularization and heart transplantation procedures.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Cells exposed to hyperthermia or other environmental stresses respond by increased synthesis of heat-shock proteins (HSPs) [1]. Expression of HSPs has been shown to provide protection against subsequent elevated stress conditions [2, 3]. The exact mechanism by which HSPs protect cells is unknown, but it appears that they may prevent cellular denaturation by acting as molecular chaperones, mediating protein conformations and preventing irregular folding interactions caused by stress [4]. Additionally, because this protection is not dependent on the form of the initial stress, a "cross-tolerance" exists by which exposure to one sublethal stress may provide protection from an otherwise lethal dose of an unrelated stress. Providing protection against ischemic stress may ultimately lead to improved myocardial revascularization and heart transplantation procedures.

In recent years there has been significant interest in inducing HSPs in the heart and examining their cardioprotective capabilities. These efforts have led to novel experimental protocols in which different stresses, such as ischemia [5–7], hypoxia [8], mechanical strain [9], hemodynamic overload [10], and hyperthermia [5, 11–19] were used to express HSPs, particularly the inducible form of the 70-kD HSP, HSP72. More recently, studies on transgenic mice that were programmed to overexpress HSP72 in cardiac muscle have shown a direct relation between expression of HSP72 and cardioprotection [20–22].

Previous in vitro and in vivo findings have shown that whole-body hyperthermia induces protection against ischemia-reperfusion (I/R) injury of the heart by reducing the extent of infarction [5, 11, 13, 14, 17]. This protection has been shown to be related to HSP expression and was directly correlated with the amount of HSP72 induced before I/R [12]. Thus, hyperthermia has excellent potential as a means for induction of HSPs while avoiding adverse effects associated with other initial stresses.

In previous hyperthermia studies, HSP expression was achieved by heating buffer solutions of isolated hearts in vitro or by subjecting animals to whole-body hyperthermia 24 hours before I/R. One of the limitations of applying whole-body heat stress is that it may exert negative effects on extracardiac cells such as blood cells. Walker and associates [14] demonstrated these extracardiac effects in experiments in which heat-shocked buffer-perfused hearts and hearts perfused with blood from a non–heat-shocked support animal were able to withstand longer periods of ischemia than animals subjected to whole-body hyperthermia whose hearts were still perfused by heat-shocked blood components.

The present study sought to determine the potential of local heating of the heart in vivo as a way to induce hyperthermia and provide protection against I/R in the rat heart. The success of this method would eliminate limitations of whole-body hyperthermia via direct local heating of the heart. It is possible that this study may lead to the development of novel techniques for upregulating endogenous protective mechanisms for reducing the effects of I/R injury.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Thermal probe
To produce regional elevation of HSP72 in the heart, a thermal probe was constructed from a 6-cm-long stainless steel tube (diameter = 4.0 mm) with a highly conductive synthetic diamond window (surface area = 12.5 mm2) at the distal end and connections for water circulation through the probe at the proximal end. Heated water from a temperature-controlled water bath was circulated through the probe. Localized hyperthermia was achieved through conductive heating from the thermal probe, which was placed directly on the epicardial surface of the heart (Fig 1). The temperature was maintained between 42.5° and 43.5°C at the probe–tissue interface.



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  		Fig 1. A thermal probe with a synthetic diamond window (diameter = 4 mm) located at the distal end was used as a tool for local heating of heart tissue. Heated water from a temperature-controlled water bath was recirculated through the probe to maintain a temperature between 42.5° and 43.5°C at the tip. Treated rats were subjected to two local applications of the probe for 15 minutes each at the points indicated above. (Note: the left anterior descending coronary artery [LAD] is not a surface artery in the rat. It is shown this way for spatial reference only.)

 
Experimental protocol
Thirty-five male Sprague-Dawley rats (300 to 350 g) were divided into three experimental groups (H = 15; C1 = 14; C2 = 6) with protocol end points of either HSP analysis or infarct size assessment (Fig 2). All rats were anesthetized with ketamine (100 mg/kg) and xylazine (40 mg/kg) given intraperitoneally, intubated, and mechanically ventilated with 1% to 2% halothane. Animals were placed on a heating blanket and monitored by a rectal thermometer to maintain normal core body temperature (37° to 38°C). A left thoracotomy through the fifth intercostal space was performed to expose the epicardial surface of the left ventricle. Heat-group animals (H) were treated with local applications of heat at two adjacent sites on the anterior left ventricle wall for 15 minutes each. To confirm elevated myocardial tissue temperature during application of the probe, we constructed a special probe instrumented with three separate thermocouples (Type K, diameter = 0.002; Omega Engineering, Stamford, CT). One thermocouple was secured directly to the probe window to monitor the probe-tissue interface temperature. The second was placed within a needle-tip (23 gauge) located 1 mm directly below the center of the probe to monitor intramyocardial tissue temperature. The third sensor was placed at a lateral distance of 2 mm from the edge of the probe to monitor temperature on the epicardium. All thermocouples were calibrated to a mercury thermometer before temperature measurements. Three additional animals were anesthetized as above and a midline sternotomy was performed to expose the heart. The pericardium was removed and the special probe was applied at two separate sites for 15 minutes each while temperatures were recorded using a PC-based data acquisition system (Omega Engineering). An additional thermocouple was placed in the rectum to monitor core body temperature.



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  		Fig 2. Summary of heat treatment and control protocols. Heat-treated rats (H) were subjected to two 15-minute applications of the thermal probe followed by 4 hours of recovery. Sham-operated controls (C1) underwent 30 minutes of open-chest operation followed by 4 hours of recovery. The heat group and control animals then were subjected to either 30 minutes of left anterior descending artery occlusion followed by 2 hours of reperfusion or Western blot analysis for HSP72 content. An additional control group (C2) was treated with two 15-minute applications of the thermal probe at body temperature (37°C) and then allowed to recover for 4 hours for determination of any HSP72 mechanically induced by application of the probe.

 
In sham-operated control animals (C1; n = 14) there was no intervention, but the chest was left open for 30 minutes. Warm gauze was placed over the thoracotomy to prevent significant heat loss. An additional control group (C2; n = 6) was subjected to two local applications of the thermal probe at 37°C (body temperature) for 15 minutes each to determine if HSP72 expression had been mechanically induced by application of the thermal probe.

The thoracotomy was closed and air was evacuated from the chest using a 20-gauge intravenous catheter connected to a 5-mL syringe. Four hours later the rats were reanesthetized and randomized to undergo either (1) 30 minutes of regional ischemia and 120 minutes of reperfusion or (2) analysis of HSP72 expression by Western blotting. All experiments performed conformed with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Ischemia/reperfusion protocol
A total of 19 rats (H = 9, C1 = 10) were enrolled in the I/R protocol. Animals were mechanically ventilated as above and a midline sternotomy was performed, exposing the entire heart. The left anterior descending coronary artery was isolated approximately 2 to 3 mm from its origin by an RB-2 taper needle with a 6-0 polypropylene stitch suture passed beneath the artery. The suture was then placed within a reversible snare occluder. The snare was tightened, closing the artery and rendering a portion of the left ventricle ischemic. Because the left anterior descending artery in the rat is not a surface artery and therefore not readily visible, occlusion of the artery was confirmed by cyanosis of the area at risk. At 30 minutes the snare was loosened and the artery reperfused. After 120 minutes of reperfusion the animal was sacrificed and its heart excised. The aorta was cannulated and the heart was briefly perfused retrogradely with saline solution to wash away excess blood. The stitch suture surrounding the coronary artery was then retied and 0.8 to 1.0 mL of phthalocyanine blue dye was injected and allowed to perfuse the nonischemic portions of the heart. The heart was sliced transversely into cross-sections 2 mm in thickness. Samples were photographed for measurement of area at risk (area not stained by blue dye) and then incubated in triphenyltetrazolium chloride for 8 minutes at 37°C to delineate infarcted tissue from normal tissue [23]. Samples were fixed in 10% buffered formalin solution for 24 hours and rephotographed for measurement of infarct area (area not stained by triphenyltetrazolium chloride). We have found in our laboratory that infarcts from relatively short periods of ischemia and reperfusion are better delineated after fixing tissue for 24 hours after triphenyltetrazolium chloride staining. Pictures were projected and planimetry was used to determine the area of risk, expressed as a percentage of left ventricle, and the infarct size, expressed as a percentage of area at risk.

Heat-shock protein analysis
A total of 16 rats (H = 6, C1 = 4, C2 = 6) were used for analysis of HSP72 expression. After 4 hours of recovery, hearts from treated and untreated rats were excised, divided along the intraventricular septum into right ventricle (RV) and left ventricle (LV), snap frozen, and stored at -80°C.

Western blot analysis was used to determine the expression of the inducible HSP72 in all myocardial samples. Tissues were weighed and cut into small slices with a razor blade. The slices were thawed in 3 mL/mg of cold lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/mL aprotinin, 1 mmol/L sodium orthovanadate in phosphate-buffered saline solution). Tissues were homogenized with a Polytron Homogenizer (Kinematica AG, Littau, Switzerland) and stored on ice for 30 minutes. After centrifugation at 15,000 g for 20 minutes at 4°C, the supernatant was removed and centrifuged again. Protein concentration of the total cell lysate was determined with a Bradford Assay solution (Bio-Rad protein assay kit; Bio-Rad Laboratories, Inc). Equal amounts of cellular proteins (2 µg) were resolved by electrophoresis on a 0.1% SDS, 12% polyacrylamide gel (SDS-PAGE) under denaturing conditions. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond; Amersham Corp, Arlington Heights, IL). After blocking in 10 mmol/L Tris HCl (pH = 8.0), 150 mmol/L sodium chloride and 5% (weight/volume) nonfat dry milk, the membranes were treated with a primary antibody that recognizes the inducible HSP72 (SPA-810 AP; StressGen, Victoria, BC, Canada) for 90 minutes, followed by incubation with peroxidase-conjugated secondary antibody for 45 minutes (Kirkegaard & Perry Laboratories, Inc, Gaithersburg, MD). The immunocomplexes were detected using a chemoluminescence reagent kit (Amersham Corp). To visualize normalization of loaded proteins, equal amounts of cell lysate (40 µg) were electrophoretically separated and the gel stained with Coumassie brilliant blue, following standard procedure.

Statistics
All values are expressed as mean ± standard error of the mean. Comparisons between groups were assessed for significance by one-way analysis of variance with post hoc analysis using the unpaired Student’s t test for infarct analysis and Bonferroni’s test for HSP72 levels. Statistical significance was accepted at p less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
As was expected, application of the thermal probe resulted in a local increase in tissue temperature at the treated site. Figure 3 shows a typical temperature history for the four sites that were monitored. Probe–tissue interface temperatures remained between 42°C and 43°C while the temperature at 1 mm below the center of the thermal probe was maintained between 41°C and 42°C. There was a minimal increase in temperature (1° to 2°C) on the surface of the heart at a lateral distance of 2 mm from the probe. Body temperature remained stable throughout the applications.



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  		Fig 3. Typical temperature recording during application of the thermal probe for 15 min at 43°C. Thermocouples were located at the probe-tissue interface, axially, 1 mm below the center of the probe in the myocardium, laterally 2 mm from the edge of the probe, and rectally. (Note: the recirculating water bath was shut off and the probe held in place at t = 16 minutes on this figure.)

 
The thermal probe was successfully applied to the LV of heat-treated animals at two adjacent sites for 15 minutes each. On gross pathologic examination, there was no evidence of thermal injury to the epicardium and no complications resulted from application of the thermal probe. All animals recovered successfully from the first surgical procedure and were awake within 30 minutes after closure of the thoracotomy. One H group animal was excluded from the infarct analysis because of coronary artery damage during the I/R protocol, which prevented adequate reperfusion. Two C1 animals and 1 C2 animal died before completion of the infarct analysis protocol during reperfusion and were excluded from further analysis.

Infarct size analysis
There was no significant difference in the area at risk (expressed as a percentage of LV area) as a result of left anterior descending coronary artery occlusion in any of the groups studied (mean ± standard error of the mean: H = 49.5% ± 5.4%, C1 = 51.5% ± 3.5%) (Fig 4A). However, rats treated with two local applications of heat using the conductive thermal probe demonstrated a marked decrease in infarct size. Localized heat stress resulted in a significant (p < 0.005) limitation of infarct size expressed as a percentage of area at risk in heat-treated animals versus controls (H versus C1, 4.26% ± 0.85% versus 19.2% ± 3.4%) (Fig 4B).



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  		Fig 4. Resulting area at risk (AR) (A) and infarct sizes (IA) (B) in heat-treated rats (H; n = 9) and controls (C1; n = 10) after 30 minutes of regional ischemia and 2 hours of reperfusion. (A) There was no difference in area at risk as a percentage of left ventricle (LV) between groups. (B) Compared with controls, heat-treated rats demonstrated a significant (p < 0.005) reduction in infarct size expressed as a percentage of area at risk.

 
Hsp72 analysis
Western blot analysis confirmed increased expression of HSP72 in rats treated with the thermal probe in both RV and LV samples at 4 hours after treatment (H), but the expression of HSP72 in the control groups (C1, C2) was not significantly altered. Figure 5 shows typical immunoblots of RV and LV samples from controls with thoracotomy and 4 hours of recovery (C1), controls with application of the thermal probe at 37°C and 4 hours of recovery (C2), and animals that underwent thoracotomy and local heat application of 42.5° to 43.5°C with 4 hours of recovery (H). The upper panel shows an immunoblot where an antibody specific for the inducible form of HSP70 (HSP72) was used. As a control for equal protein concentrations, the ß-actin band of electrophoresis-separated lysates was stained with Coumassie blue (lower panel). The results indicate that inducible HSP72 is upregulated by local heat application at 4 hours after heating. Densitometric analysis of immunoblots showed a marked difference in the expression of HSP72 between heat-treated animals (H) and sham-operated controls (C1) at 4 hours (Fig 6). There was a 3.7-fold (p < 0.005) and a 5.5-fold (p < 0.001) difference in RV and LV samples, respectively, between heat-treated animals (H) and sham controls (C1). Animals that underwent application of the probe at 37°C (C2) did not show a difference in HSP72 levels compared with sham controls (C1).



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  		Fig 5. Total cell lysates from treated and untreated rat hearts were dissolved on SDS-PAGE and immunoblotted with antibody specific for both HSP72 (top). The B-actin band of electrophoresis-separated lysate was stained with Coumassie blue for confirmation of equal protein concentrations (bottom). Right ventricle (RV) and left ventricle (LV) were analyzed separately. Study groups included controls with thoracotomy and 4 hours of recovery (C1), animals that underwent thoracotomy and application of the probe at 37°C followed by 4 hours of recovery (C2), and animals that underwent thoracotomy and local heat applications followed by 4 hours of recovery (H).

 


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  		Fig 6. Bar graphs showing densitometric analysis of autoradiographs from right ventricle and left ventricle in all groups studied. Local heat application followed by 4 hours of recovery (H; n = 6) resulted in a significant difference (p < 0.001) in myocardial HSP72 content in both right and left ventricular samples when compared with both control groups (C1, n = 4; C2, n = 6).

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Previous studies have shown that whole-body hyperthermia limits myocardial damage during I/R protocols. However, these studies do not address the effect of whole-body hyperthermia on extracardiac cells or whether those cells then contribute positive or negative factors to cardioprotection observed. There is evidence that extracardiac cells may exert a negative effect in in vivo studies that used whole-body hyperthermia for expression of HSPs in the heart. Walker and associates [14] demonstrated the adverse effects of whole-body hyperthermia in an in vivo rabbit model. These effects were shown to be directly related either to heating blood components or to agents released into the blood from other heated tissues, but were eliminated when heat-shocked hearts were perfused with either buffer solution or blood from non–heat-shocked support rabbits. We eliminated extracardiac variables by using a thermal probe applied locally to induce hyperthermia and to subsequently provide protection against 30 minutes of ischemia and 120 minutes of reperfusion in an in vivo rat model.

In animals subjected to ischemia at 4 hours after heating, our data strongly suggest a mechanism related to gene expression and accumulation of HSP72 in the heart. Densitometric analysis of Western blots confirmed elevated levels of HSP72 in rat hearts treated with the thermal probe and 4 hours of recovery. There was a 5.5-fold and 3.7-fold increase in HSP72 expression in LV and RV ventricular samples, respectively, from hearts treated with local heating compared with sham controls at 4 hours. Rats were allowed to recover for 4 hours after heat treatment to allow sufficient time for production of HSPs [16]. There is mounting evidence that the absolute levels of HSP72 in the heart at the time of ischemia may be critical in protecting against ischemia. Donnelly and colleagues [11] demonstrated significant reduction in infarct size after whole-body heat stress, but were unable to reduce infarct size or to significantly increase myocardial HSP72 after a single 20-minute ischemic pretreatment followed by 8 hours of recovery. Marber and colleagues [5] also showed that a greater ischemic-induced elevation of myocardial HSP compared with sham ischemic pretreatment was associated with increased protection in a rabbit model of I/R. Finally, Hutter and coworkers [12] showed a direct correlation between the amount of hyperthermia-induced HSP72 and the level of protection afforded against I/R. Future studies will have to address the effects of rate of heating, exposure time, and increased temperatures on the kinetics of HSP72 production to determine the subsequent time course of protection by HSP72.

Our analysis indicated that HSP72 was elevated in the LV as well as in the untreated area of the RV. This most interesting finding suggests that we were able to induce global myocardial elevation of HSPs in the heart with regional heating of the heart. Przyklenk and associates [24] demonstrated a similar response in a study in which regional ischemic preconditioning protected remote myocardium from a subsequent coronary occlusion. More recently Gho and colleagues [25] showed protection of myocardial tissue by a brief ischemic episode in noncardiac tissue in a rat model. Although it is likely that the mechanism of protection in their studies differs from ours, the underlying cell signaling and cell transport mechanisms may be analogous. Future studies are needed to address this issue on a molecular basis. Whether the proteins are transferred by diffusion through the heart or whether the actual cellular machinery responsible for their production was activated in untreated areas, and whether these remote areas would be protected from I/R by the presence of these proteins, are questions that remain to be answered. Our temperature measurements, illustrated in Figure 3, strongly suggest that it is not possible to generate an increase in temperature and hence elevated expression of HSP72 in RV tissues during application of the probe to the LV. It is possible that the increase in protein content found in the RV could be caused by the activation of genes by mechanical stresses in untreated tissue as a result of changes in contractility in the treated areas (LV). Small changes in myocardial function occurring over a prolonged period of time could be sufficient to cause the observed upregulation of HSP expression. However, because it was not possible to measure the contractility during the recovery period of rats subjected to heat treatment, it is difficult to state whether this mechanism played a role in the current study.

Previous studies have also shown induction of HSPs by mechanical stretch [9], yet this study demonstrated no significant increase in protein synthesis resulting from mechanical forces encountered during application of the thermal probe. Western blots showed no mechanical induction of HSP72 in group C2 (application of thermal probe at 37°C) compared with the sham operation group (C1). Therefore, we can conclude that in heat-treated animals HSPs are expressed as a result of heating and not of mechanical force.

Preconditioning and heat shock have been shown to provide cardioprotection in numerous animal studies. Yet, these techniques have not been accepted in a clinical setting as an adjunct therapy either for myocardial revascularization or for heart transplantation. Clinicians are understandably reluctant to subject their patients to the stresses necessary to elicit these cardioprotective phenomena, fearing that damage to hearts with existing cardiovascular disorders may outweigh any benefits from the treatment. Local hyperthermia may, however, be less traumatic than ischemia, hypoxia, or other pharmacologically induced stresses in these types of patients and may provide a more feasible way for clinical implementation of protective schemes. This technique is also versatile in that many forms of energy, including ultrasound, microwave, radiofrequency, and laser, could be used to induce local hyperthermia and protection during ischemic conditions. Additionally, induction of hyperthermia by local application of energy would be a more practical therapy than whole-body hyperthermia, which would be difficult to realize in a clinical setting.

Study limitations
One of the limitations of our study was an inability to reproducibly apply the thermal probe to the surface of the heart. Transfer of heat to tissue depends on maintaining contact with the tissue (ie, constant heating profile) and on perfusion of the tissue where the probe is applied. Because we applied the probe manually, a degree of difference in temperature distribution and HSP expression can be expected. At steady-state the temperatures were highest directly beneath the probe and decayed exponentially with distance both in the lateral and axial direction from the surface. Therefore, tissue temperature near the endocardial surface did not likely change significantly. Application of other energy sources that allow for deeper, more uniform heating may overcome these problems.

Our study also does not examine the time course of protection or expression of HSP72. Subsequent studies that determine the effects of rate of heating, methods of heating, and amounts of recovery time on expression of HSP72 and protection against I/R injury are needed to address this aspect.

Summary
The results from our study indicate that localized heating of the heart can induce protection in the rat heart from a subsequent ischemic injury 4 hours later. Development of methods to locally induce hyperthermia in the heart may avoid the problems related to heating blood and other vital organs in whole-body hyperthermia models and may allow for a more clinically feasible implementation of cardioprotective schemes.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Mrs Joann Aaron for her editorial assistance in reviewing the manuscript.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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