Hypotension and Bradycardia Produced by Transthoracic Application of Low-Intensity Ultrasound Therapy in Hearts of Healthy Rats - A Preclinical Study

Objective To investigate the cardiovascular effects produced by transthoracic application of low-intensity pulsed ultrasound therapy (LIPUST). Methods Three-month-old male Wistar rats (± 300 g, N=16) were randomly allocated in two groups, namely SHAM (control group, faked procedures) and UST (animals treated with LIPUST). These animals, under anesthesia, were instrumented (femoral artery and vein catheterization) for hemodynamic recordings (mean blood pressure [MBP], heart rate [HR]) and blood biochemical profile (lipids, creatine kinase-myocardial band [CK-MB]). Then, LIPUST (spatial average-temporal average [ISATA] 1-MHz, power 0.1 to 1.2 W/cm2, pulsed 2:8 ms, cycle at 30%, for three minutes) was applied to animals from the UST group, externally to their thorax. SHAM animals were equally manipulated, but without application of ultrasound energy. After the hemodynamic and biochemical measurements, animals were sacrificed, and their hearts were mounted in a Langendorff apparatus for coronary reactivity evaluation. Standard histology techniques were employed to analyze the hearts. Results LIPUST application caused statistically significant reductions in MBP (92±4 vs. 106±1 mmHg) and HR (345±14 vs. 380±17 rpm) when compared with SHAM procedures. UST rats exhibited higher CK-MB levels (318±55 vs. 198±26 U/dL) and lower plasma triglycerides levels (38±7 vs. 70±10 mg/dL) than SHAM animals. Coronary reactivity was not significantly changed by LIPUST. Cardiac histopathology showed an increase in capillary permeability in treated animals when compared with SHAM animals. Conclusion Noninvasive LIPUST induces significant metabolic and hemodynamic changes, including intensity-dependent bradycardia and hypotension, indicating a possible therapeutic effect for cardiac events.

In fact, very few studies have addressed the relationship between US parameters and their biological consequences, including the molecular and cellular mechanisms involved. This preliminary study was therefore designed to further investigate the cardiovascular effects of noninvasive transthoracic lowintensity pulsed ultrasound therapy (LIPUST) in healthy rats.

Animals and Groups
All procedures were performed in accordance with the international guidelines, particularly to those established in the Guide Care and Use of Laboratory Animals (1996) and in the Brazilian Guideline for the Care and Use of Animals for Scientific and Educational Purposes -DBCA, of the National Council for the Control of Animal Experimentation -CONCEA (2013), and approved by Institutional Ethic Committee -CEUA (number# 059/2012 and revised 2018).
Three-month-old male Wistar rats weighing 300 to 350 g, from our breeding stock, were divided into two groups, namely UST (animals treated with LIPUST) and SHAM (control group, faked procedures) (N=8 per group). Throughout the experiment, the animals were individually housed in Perspex ® cages with sawdust bedding material, in a noiseless room, at 22-24 ºC, with lights on from 6:00 h to 18:00 h. Pelleted food and filtered water were freely available. Animals were maintained anesthetized with halothane-3% (vaporized from a Sections of 6 μm thickness were made with a microtome (Spence 820, New York, NY, United States of America) and stained with hematoxylin plus eosin to allow histological analysis. Capillary histomorphometry was performed from pictures captured by a 40x objective; evaluation was made using a computerized imaging processing system (Sigma-Pro™ Image; St. Louis, MO, United States of America). Five pictures from samples from pairs of tissue (i.e., SHAM and UST) were randomly captured under optical microscopy (Olympus™ AX70 Plus, Tokyo, Japan) and were stored for further analysis. Such analysis included a semiquantitative determination of myocardial capillarity and interstitial area, by using digital markers (as color contrasts) as discrimination parameters [9] .

Limitations
In order to minimize the use of experimental animals (N=16), according to the institutional ethic committee, the same hearts (from the UST group) were submitted to the US irradiation at seven different powers (21-minute effective time). This fact could have magnified the observed hemodynamic and cellular effects. Another limitation was the removal of a single blood sample from each animal, to minimize the changes in blood volume. If additional samples could have been taken, perhaps a better idea of the time-effects of biochemical changes could be obtained.

Data Analysis
Data was put into appropriate spreadsheets and analyzed by a Prism-6™ statistical package (GraphPad Software, Inc., San Diego, CA, United States of America). Parametric analyses were Student's t-test (paired and non-paired), analysis of variance (ANOVA) (one-and two-way), and the post hoc Tukey's test, as appropriate. Nonparametric analysis for semiquantitative data was done by the Mann-Whitney U test. Unless otherwise stated, data are presented as means ± standard error. The level of significance for statistical differences was set at P<0.05.

RESULTS
In the in vivo studies, the dose-response curve for application of LIPUST to the heart (UST group) showed a progressive reduction (P<0.05 and P<0.01) in MBP (ΔMBP) from the third US dose ( Figure 3A). The HR curve also showed a reduction (P<0.01) in this parameter in the UST group, which was already observed from the first US dose ( Figure 3B). The body temperature curve showed no significant changes, there was a mean variation of ± 0.6 ºC in the animals from SHAM and UST groups ( Figure 3C).
In comparison, similar curves from SHAM animals did not show any obvious change in the hemodynamic parameters (ΔMBP, HR). Table 1 shows that values of TC and fractions in the UST group were similar to those from the SHAM group. It was also observed that despite the apparent increase in CRP values, there were no significant differences between SHAM and UST animals. However, it was verified that TG decreased (P<0.05) in the UST group and that CK and CK-MB enzymes significantly increased (P<0.01). On the other hand, the in vitro studies on the coronary reactivity did not show any statistically significant difference, regarding the coronary vasodilator effect of adenosine, between total creatine kinase (CK), (f ) CK-myocardial band (CK-MB), and (h) C-reactive protein (CRP). In sequence, Protocol 2 was performed [26] .

Protocol 2 (In Vitro): Isolated Perfused Heart
The animals were sacrificed under halothane (100%) anesthesia, had their thorax opened, and their heart was carefully transferred to a Langendorff perfusion system (Hugo Sachs Electronics, March-Hugstetten, Germany).
The isolated hearts were perfused with a modified Krebs solution (Sigma-Aldrich, St. Louis, MO, United States of America), maintained pH 7.4, and kept at 37 ºC. The solution was equilibrated with a carbogen mixture (O 2 95% + CO 2 5%) (White-Martins, Rio de Janeiro, Brazil). The perfusion flow was maintained at 10 mL/min by a peristaltic pump (4-channel MS-Reglo, Hugo Sachs Electronics).
A small latex balloon filled with saline (10 mmHg pressure) was carefully introduced into the left ventricle. A steel catheter connected the latex balloon to a mechanical transducer (TPS-2 Statham transducer, Incor, São Paulo, Brazil), to allow recording the isovolumetric cardiac contractility. The coronary perfusion pressure (CPP) was continuously recorded by another transducer connected to a data acquisition system Power-Lab™ (ADI Instruments, Bela Vista, NSW, Australia). After 40-minute stabilization, basal CPP was determined and a dose-response curve to bolus injections of adenosine 1, 4, 8, 16, 32, 64, 128, and 256 μg (Sigma-Aldrich, St. Louis, MO, United States of America) was obtained to evaluate the coronary reactivity ( Figure 2).

Histopathology
At the end of the perfusion experiment, the hearts were removed and were fixed for 24 h in 10% formalin solution (pH 7). Thereafter, the myocardial samples were gradually dehydrated with increasing concentrations (70 to 100%) of ethanol, clarified in xylene, and embedded in paraffin blocs, to allow microtomy. SHAM and UST groups ( Figure 4A). In addition, the effects of noninvasive LIPUST on the changes in CPP (ΔCPP) were not statistically different of those from SHAM animals ( Figure 4B).
The morphometric analysis of the hearts from the two groups regarding the capillary density also did not show any obvious difference between SHAM and UST groups ( Figure 5A). Interestingly, however, the hearts treated with LIPUST displayed a greater capillary permeability than SHAM animals and showed small, yet consistent, lipid deposits inside the myocardial cells ( Figure 5B).

DISCUSSION
In the original and present in vivo and in vitro studies, we show some cardiovascular effects elicited by transthoracic LIPUST to the heart (via extracorporeal application) from healthy Wistar rats. In our opinion, the main finding of this study was that LIPUST is able to cause parallel reductions in MBP and HR, without important changes in the coronary function. According to Kuma et al. [14] , application of low-intensity US energy to open thorax of guinea pigs has produced positive inotropic and lusitropic effects. Such findings are similar to our results in rats, in particular with respect to the power-dependency of the systemic hypotension and bradycardia; they also corroborate other studies using distinct parameters of US energy [4,5,14,16,19,22] .
Most of the studies using changes in frequency or time (doses) of US energy, which were interested in verify cardiac effects, were not able to detect systemic hemodynamic implications, as described here; also, most of these studies were done with invasive techniques or using in vitro and in vivo methods. Such invasive procedures may be associated with post-surgery intercurrences as well as a greater probability of infections [14,[22][23][24]26] . In this aspect, the noninvasive LIPUST technique here presented is capable of minimize such adverse effects. However, technical problems persist, since the larger intensities variability and the possibility of activation of multiple biochemical and biomechanical pathways make it difficult to interpret the data, including differing the direct from the indirect effects of the radiation [12] . It deserves mention, however, that some of these mechanisms were already described in the literature, including direct vibration effects (mechanical) on the cellular structure and changes in the turn-over of bioactive substances (biochemical) [7,20,25] .
The observed reduction in HR (Figure 3) found in the present study can be attributed to the direct mechanical activation of a cardiovascular reflex (baroreflex and Bezold-Jarisch reflex),  Similar studies were conducted by Siegel et al. [22] and other authors [1,11,14,15,24,[27][28][29] , showing that the release of NO had an important participation in the capillary permeability effects of US therapy. All together, these data suggest that LIPUST can indeed have good therapeutic potentialities. Additionally, our histopathological results did not shown significant changes in capillarity with LIPUST; however, we were able to identify small intracellular depositions of lipids, that could possibly be due to reesterification of free fatty acids by the myocardial cells ( Figure 5) [21][22][23][24][25]28,31] . This speculation is based on the observation that LIPUST reduced the plasma TG together with an increase in the tissue perfusion and in the cell permeability, situations that theoretically would facilitate the intracellular fat deposition, here found in the morphologic study of the hearts.

CONCLUSION
In summary, the data showed that noninvasive LIPUST, applied to the precordial area of healthy rats, causes powerdependent reductions in both HR an MBP, accompanied by improved myocardial perfusion. Despite its limitations, this study pointed to a good therapeutic potential of noninvasive LIPUST in the primary or secondary treatment of cardiac diseases. To clarify and expand these issues, further studies, especially done in humans and using more sophisticated molecular techniques, will be necessary.

Authors' Roles & Responsibilities
Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
with a subsequent increase in its sensitivity, although the pathways involved cannot be identified in this study. This hypothesis of involvement of cardiovascular reflexes, especially baroreflex and Bezold-Jarisch reflex, was partially supported by the experiments performed by Miwa et al. [17] , who described an increase in sympathetic nerve activity, accompanied by increased norepinephrine release, following application of US energy to subcutaneous adipose tissue in rats. However, one possibility would be that direct stimulation (cumulative tension) of the aortic baroceptors by transthoracic US could trigger the baroceptor reflex, which results in increased parasympathetic tone and reduced sympathetic tone, with consequent bradycardia [17,22,[27][28][29] . Another possibility would be the Bezold-Jarisch reflex, a cardiac inhibitory reflex that results in bradycardia, vasodilation, and hypotension, and originates in cardiac receptors, located mainly in the left ventricular posteroinferior wall, which are activated by mechanical-chemical inputs, leading to increased parasympathetic activity and inhibition of sympathetic activity [17,22,24,[26][27][28][29] . In this study, it was not intended to determine the direct mechanisms responsible for the negative chronotropic effects of US on the heart "in vivo". However, the observation that these chronotropic effects were mainly restricted to the period of application is suggestive of direct chemical-mechanical action of ultrasonic waves in the heart tissue.
In this context, the concomitant participation of the sonomechanical and sonochemical effects (e.g., flow and microflow) of the ultrasonic therapy is well described and evidenced in this study. In addition, they corroborate the observed changes in plasma concentrations of important biochemical markers such as TG, CK, and CK-MB (Table 1), as the literature has shown that therapeutic US is capable of modifying various enzymatic [2,11,13,26] activities and causing release of intracellular molecules, depending on the US energy [6,23,24,26,31] . It is worth mentioning that therapeutic US is capable of activating the release of nitric oxide (NO) and possibly other vasoactive endothelial substances, as well as activating enzymes (metalloproteinases), which could also at least partially explain the hypotensive effects classified and described in this study [1.11.14-16.19,22.24] .
It can also be speculated that both negative chronotropic and systemic hypotensive effects occurred by the piezoelectric action of low intensity US energy in cardiac tissue. The literature states that US waves deform muscle structural proteins, mainly collagen and tropomyosin, producing electrical charges (tension), in this case, in the cardiomyocyte, and consequently providing changes in HR, rhythm, and regularity [11][12][13][14]22,24,25,[27][28][29]31,32] .
It has also been shown that US waves, when focused on peripheral vessels (as a target-tissue), caused activation of tissular plasminogen, which is an important thrombolytic agent [6,15] . We can speculate that the effect of plasminogen contributed to reduce the adherence of lipids to the arterial wall and reduced possible injuries to the coronary endothelium. Another relevant finding of the present study was the augmented capillary permeability observed in the photomicrography of treated hearts ( Figure 5B), further suggesting that the pulsed US therapy acted as a modulator of some physicochemical process involved in cell permeability and perhaps vasomotricity.

Authors' roles & responsibilities
WLSG ANR RC SAG Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; final approval of the version to be published.
Drafting the work or revising it critically for important intellectual content; final approval of the version to be published.
Final approval of the version to be published.
Revising it critically for important intellectual content; final approval of the version to be published.

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