Simulation studies promote technological development of radiofrequency phased array hyperthermia

A treatment planning program package for radiofrequency hyperthermia has been developed. It consists of software modules for processing three-dimensional computerized tomography (CT) data sets, manual segmentation, generation of tetrahedral grids, numerical calculation and optimisation of three-dimensional Ε field distributions using a volume surface integral equation algorithm as well as temperature distributions using an adaptive multilevel finite-elements code, and graphical tools for simultaneous representation of CT data and simulation results. Heat treatments are limited by hot spots in healthy tissues caused by Ε field maxima at electrical interfaces (bone/muscle). In order to reduce or avoid hot spots suitable objective functions are derived from power deposition patterns and temperature distributions, and are utilised to optimise antenna parameters (phases, amplitudes). The simulation and optimisation tools have been applied to estimate the improvements that could be reached by upgrades of the clinically used SIGMA-60 applicator (consisting of a single ring of four antenna pairs). The investigated upgrades are increased number of antennas and channels (triple-ring of 3 × 8 antennas and variation of antenna inclination. Significant improvement of index temperatures (1–2°C) is achieved by upgrading the single ring to a triple ring with free phase selection for every antenna or antenna pair. Antenna amplitudes and inclinations proved as less important parameters.     

Improving locoregional hyperthermia delivery using the 3-D controlled AMC-8 phased array hyperthermia system: A preclinical study

Background: The aim of this study is preclinical evaluation of our newly developed regional hyperthermia system providing 3-D SAR control: the AMC-8 phased array consisting of two rings, each with four 70 MHz waveguides. It was designed to achieve higher tumour temperatures and improve the clinical effectiveness of locoregional hyperthermia.

Methods: The performance of the AMC-8 system was evaluated with simulations and measurements aiming at heating a centrally located target region in rectangular (30 × 30 × 110 cm) and elliptical (36 × 24 × 80 cm) homogeneous tissue equivalent phantoms. Three properties were evaluated and compared to its predecessor, the 2-D AMC-4 single ring four waveguide array: (1) spatial control and (2) size of the SAR focus, (3) the ratio between maximum SAR outside the target region and SAR in the focus. Distance and phase difference between the two rings were varied.

Results: (1) Phase steering provides 3-D SAR control for the AMC-8 system. (2) The SAR focus is more elongated compared to the AMC-4 system, yielding a lower SAR level in the focus when using the same total power. This is counter-balanced by (3) a superficial SAR deposition which is half of that in the AMC-4 system, yielding a more favourable ratio between normal tissue and target SAR and allowing higher total power and up to 30% more SAR in the focus for 3 cm ring distance.

Conclusion: The AMC-8 system is capable of 3-D SAR control and its SAR distribution is more favourable than for the 2-D AMC-4 system. This result promises improvement in clinical tumour temperatures.     

Physical predictors of adequate hyperthermia with the annular phased array

One hundred and fifty-two hyperthermia sessions in 36 consecutive patients treated with the BSD 1000 annular phased array system (APAS) are reviewed with regard to physical predictors of quality of hyperthermia. Although central tumour temperatures exceeding 42°C were momentarily obtained in 62% of the sessions, it frequently proved difficult to maintain the patient at temperature for prolonged periods of time. The time to reach target temperature was negatively associated with quality of hyperthermia. Thus, of 25 sessions which required over 25 min to attain a temperature of 42°C, only one was adequate (defined as central tumour temperature ± 42°C maintained for at least 30 min) as opposed to 28/69 adequate sessions when 42 °C was reached in less than 25 min. Physical parameters measured in the first 3 min of the session found to be associated with adequate hyperthermia include an initial rate of temperature rise at the tumour site exceeding 0.4°C/min achieved with a net forward power less than 1500 W. Only three of 57 sessions not meeting these criteria were adequate. Treatment policy recommendations and recommendations for future research are made.     

Clinical integration of software tool VEDO for adaptive and quantitative application of phased array hyperthermia in the head and neck

Abstract

Background and purpose: In Rotterdam, patient-specific hyperthermia (HT) treatment planning (HTP) is applied for all deep head and neck (H&N) HT treatments. In this paper we introduce VEDO (the Visualisation Tool for Electromagnetic Dosimetry and Optimisation), the software tool required, and demonstrate its value for HTP-guided online complaint-adaptive (CA) steering based on specific absorption rate (SAR) optimisation during a H&N HT treatment.

Materials and methods: VEDO integrates CA steering, visualisation of the SAR patterns and mean tumour SAR (SAR_target) optimisation in a single screen. The pre-calculated electromagnetic fields are loaded into VEDO. During treatment, VEDO shows the SAR pattern, overlaid on the patients’ CT-scan, corresponding to the actually applied power settings and it can (re-)optimise the SAR pattern to minimise SAR at regions where the patient senses discomfort while maintaining a high SAR_target.

Results: The potential of the quantitative SAR steering approach using VEDO is demonstrated by analysis of the first treatment in which VEDO was used for two patients using the HYPERcollar.

These cases show that VEDO allows response to power-related complaints of the patient and to quantify the change in absolute SAR: increasing either SAR_target from 96 to 178?W/kg (case 1); or show that the first SAR distribution was already optimum (case 2).

Conclusion: This analysis shows that VEDO facilitates a quantitative treatment strategy allowing standardised application of HT by technicians of different HT centres, which will potentially lead to improved treatment quality and the possibility of tracking the effectiveness of different treatment strategies.     

Optimum excitation of phases and amplitudes in a phased array hyperthermia system

In order to achieve a desired temperature distribution inside and outside of malignant tissues, optimization techniques could be used in phased array hyperthermia systems to control effectively the amplitude and phase of the radiating elements. The optimization of a four-element phased array hyperthermia system operating at 432 MHz is examined theoretically. The proposed technique is based on a detailed physical model of the tissue medium to be heated in terms of both electromagnetic and thermal properties. A penalty function technique using a Newton method is applied to determine the optimum phases and amplitudes of the array. Several array geometrics have been studied and numerical results are presented.     

Design and characterisation of a phased antenna array for intact breast hyperthermia

Purpose: Currently available hyperthermia technology is not well suited to treating cancer malignancies in the intact breast. This study investigates a microwave applicator incorporating multiple patch antennas, with the goal of facilitating controllable power deposition profiles for treating lesions at diverse locations within the intact breast.

Materials and methods: A 3D-computational model was implemented to assess power deposition profiles with 915?MHz applicators incorporating a hemispheric groundplane and configurations of 2, 4, 8, 12, 16 and 20 antennas. Hemispheric breast models of 90?mm and 150?mm diameter were considered, where cuboid target volumes of 10?mm edge length (1?cm³) and 30?mm edge length (27?cm³) were positioned at the centre of the breast, and also located 15?mm from the chest wall. The average power absorption (αPA) ratio expressed as the ratio of the PA in the target volume and in the full breast was evaluated. A 4-antenna proof-of-concept array was fabricated and experimentally evaluated.

Results: Computational models identified an optimal inter-antenna spacing of 22.5° along the applicator circumference. Applicators with 8 and 12 antennas excited with constant phase presented the highest αPA at centrally located and deep-seated targets, respectively. Experimental measurements with a 4-antenna proof-of-concept array illustrated the potential for electrically steering power deposition profiles by adjusting the relative phase of the signal at antenna inputs.

Conclusions: Computational models and experimental results suggest that the proposed applicator may have potential for delivering conformal thermal therapy in the intact breast.     

Strategies for optimized application of annular-phased-array systems in clinical hyperthermia

A theoretical framework is presented for optimized heating of deep-seated tumours by phase and amplitude steering. The optimization problem for a specific tumour and perfusion case results in a functional dependency between power-level and maximum obtainable therapeutic efficiency. Different optimization criteria and strategies are outlined, which cause an increase of power or thermal dose in the tumour. Three tumour models (central pelvic tumour, eccentric abdominal tumour with or without necrosis) are analysed in detail. The simulation studies predict that appreciable parts of these tumours (50–100%) can be heated efficiently (42.5–43°C) within the range of available and clinically tolerated power levels (1–5 kW/m), if tumour perfusion is less than 20–25 ml/100 g min. Some improvements are obtained by increasing the number of independent channels (from four to eight) and by the application of time-dependent (complementary) power-deposition patterns.     

A practical system for clinical radiofrequency hyperthermia

This paper describes an apparatus for inducing local hyperthermis by passing high-frequency electrical currents through tissues between electrodes placed against the skin of the patient. The electrodes use a temperature-controlled saline solution contained by a thin rubber membrane to make contact. The resistivity of the saline solution is matched to that of body tissues. This yields a smooth transition from electrode to tissue, thereby greatly reducing the possibility of producing the skin burns which frequently appear along the edges of metallic electrodes. Use of the thin rubber membrane allows easy molding of a given set of electrodes to complex body contours for many different patients. The equipment has proven capable in clinical tests of heating bulky tumors in the head and neck and extremities without significant skin toxicity. Excessive beating of the subcutaneous fat, however, restricts the application of this heating method to tumors located in areas of the body with sparse adipose tissue.     

Benefits of superficial hyperthermia treatment planning: Five case studies

Purpose: To demonstrate the benefits of treatment planning in superficial hyperthermia.

Materials and methods: Five patient cases are presented, in which treatment planning was applied to troubleshoot treatment-limiting hotspots, to select the optimum applicator type and orientation, to assess the risk associated with metallic implants, to assess the feasibility of heating a deeper seated tumour, and to analyse the effective SAR coverage resulting from arrays of multiple incoherent applicators. FDTD simulation tools were used to investigate treatment options, either based on segmented or simplified anatomies.

Results: The background, approach and model implementation are presented per case. SAR cross-sections, profiles and isosurfaces are visualized to predict the effective SAR coverage of the target and the location of the maximum power absorption. In addition, the followed treatment strategy and the implications for the clinical treatment are given: for example, higher temperatures, relief of treatment limiting hot-spots or increased power input.

Conclusions: Treatment planning in superficial hyperthermia can be applied to improve clinical routine. Its application supports the selection of the optimum technique in non-standard cases, leading to direct benefits for the patient. In addition, treatment planning has shown to be an excellent tool for education and training for hyperthermia technicians and physicians.     

Evaluation of a dual-arm Archimedean spiral array for microwave hyperthermia

Purpose: This effort describes a third-party performance evaluation of a novel, commercial, dual-armed Archimedean spiral array hyperthermia applicator. The applicator is analysed for its ability to couple efficiently into muscle equivalent phantom loads, operate over a broad bandwidth to help accommodate variable tissue properties and generate predictable and repeatable SAR contours that are adaptable to clinically probable disease shapes.

Materials and methods: Characterization of the applicator includes E-field and return-loss measurements in liquid muscle tissue-equivalent phantom, as well as comparison of ‘treatment-planning’ simulations of several possible array SAR patterns with measured SAR from non-coherently driven spiral array antennae.

Results: The applicator demonstrates a reasonably low return loss over a large bandwidth and the ability to generate a very uniform heating pattern. Ability to adjust SAR contours spatially to fit specific shapes is also demonstrated.

Conclusions: This device should prove a welcome addition to a currently limited set of superficial heating applicators to provide controllable heating of superficial tissue disease.     

The HYPERcollar: A novel applicator for hyperthermia in the head and neck

The purpose of this work was to define all features, and show the potential, of the novel HYPERcollar applicator system for hyperthermia treatments in the head and neck region. The HYPERcollar applicator consists of (1) an antenna ring, (2) a waterbolus system and (3) a positioning system. The specific absorption rate (SAR) profile of this applicator was investigated by performing infra-red measurements in a cylindrical phantom. Mandatory patient-specific treatment planning was performed as an object lesson to a patient with a laryngeal tumour and an artificial lymph node metastasis. Comfort tests with healthy volunteers have revealed that the applicator provides sufficient comfort to maintain in treatment position for an hour: the standard hyperthermia treatment duration in our centre. By phantom measurements, we established that a central focus in the neck can be obtained, with 50% iso-SAR lengths of 3.5?cm in transversal directions (x/y) and 9–11?cm in the axial direction (z). Using treatment planning by detailed electromagnetic simulations, we showed that the SAR pattern can be optimised to enable simultaneous encompassing of a primary laryngeal tumour and a lymph node metastasis at the 25% iso-SAR level. This study shows that the applicator enables a good control, and sufficient possibilities for optimisation, of the SAR pattern. In an ongoing clinical feasibility study, we will investigate the possibilities of heating various target regions in the neck with this apparatus.     

Conformal microwave array (CMA) applicators for hyperthermia of diffuse chest wall recurrence

Purpose: This article summarises the evolution of microwave array applicators for heating large area chest wall disease as an adjuvant to external beam radiation, systemic chemotherapy, and potentially simultaneous brachytherapy.

Methods: Current devices used for thermotherapy of chest wall recurrence are reviewed. The largest conformal array applicator to date is evaluated in four studies: (1) ability to conform to the torso is demonstrated with a CT scan of a torso phantom and MR scan of the conformal water bolus component on a mastectomy patient; (2) specific absorption rate (SAR) and temperature distributions are calculated with electromagnetic and thermal simulation software for a mastectomy patient; (3) SAR patterns are measured with a scanning SAR probe in liquid muscle phantom for a buried coplanar waveguide CMA; and (4) heating patterns and patient tolerance of CMA applicators are characterised in a clinical pilot study with 13 patients.

Results: CT and MR scans demonstrate excellent conformity of CMA applicators to contoured anatomy. Simulations demonstrate effective control of heating over contoured anatomy. Measurements confirm effective coverage of large treatment areas with no gaps. In 42 hyperthermia treatments, CMA applicators provided well-tolerated effective heating of up to 500 cm² regions, achieving target temperatures of T_min = 41.4 ± 0.7°C, T₉₀ = 42.1 ± 0.6°C, T_ave = 42.8 ± 0.6°C, and T_max = 44.3 ± 0.8°C as measured in an average of 90 points per treatment.

Conclusion: The CMA applicator is an effective thermal therapy device for heating large-area superficial disease such as diffuse chest wall recurrence. It is able to cover over three times the treatment area of conventional hyperthermia devices while conforming to typical body contours.     

Performance of a multi-sector ultrasound hyperthermia applicator and control system: in vivo studies

A multi-element ultrasound hyperthermia system (Labthermics Technologies, Inc., Sonotherm 1000) was utilized to deliver energy to two relatively well-defined regions (3 × 8 × 3 × 8 cm) on the lateral aspect of the right rear leg of 22 dogs in 1, 2 and 3 weekly treatments to a target temperature of 42–43°C, at a depth of 1 cm. The total treatment time was 30 min. One area heated on each dog was adjacent to the proximal femur and the other was 2–3 cm away from the distal aspect of the same bone. The system performed in an acceptably controlled manner and was able to deliver energy to a well-defined site. The mean temperature achieved, and rate of temperature rise in the heated regions, was independent of the region being treated. Little difference (0·12°C) was noted between the mean temperatures of the heated regions, and the temperature bounds (42–43°C) were exceeded by a small amount and for a relatively short period of time (out of range RMSE=0·6°C). Unheated regions showed much lower mean temperatures than did heated regions located 3 · 8 cm away, indicating that the energy was delivered in a well-defined manner. Superficial thermal injuries occurred in three of 22 dogs in the area heated 2–3 cm away from bone, and 11 of 22 dogs exhibited injuries in the tissue heated immediately adjacent to the femur.     

Planning,optimisation and evaluation of hyperthermia treatments

Background: Hyperthermia treatment planning using dedicated simulations of power and temperature distributions is very useful to assist in hyperthermia applications. This paper describes an advanced treatment planning software package for a wide variety of applications.

Methods: The in-house developed C++?software package Plan2Heat runs on a Linux operating system. Modules are available to perform electric field and temperature calculations for many heating techniques. The package also contains optimisation routines, post-treatment evaluation tools and a sophisticated thermal model enabling to account for 3D vasculature based on an angiogram or generated artificially using a vessel generation algorithm. The use of the software is illustrated by a simulation of a locoregional hyperthermia treatment for a pancreatic cancer patient and a spherical tumour model heated by interstitial hyperthermia, with detailed 3D vasculature included.

Results: The module-based set-up makes the software flexible and easy to use. The first example demonstrates that treatment planning can help to focus the heating to the tumour. After optimisation, the simulated absorbed power in the tumour increased with 50%. The second example demonstrates the impact of accurately modelling discrete vasculature. Blood at body core temperature entering the heated volume causes relatively cold tracks in the heated volume, where the temperature remains below 40?°C.

Conclusions: A flexible software package for hyperthermia treatment planning has been developed, which can be very useful in many hyperthermia applications. The object-oriented structure of the source code allows relatively easy extension of the software package with additional tools when necessary for future applications.     

Feasibility of on-line temperature-based hyperthermia treatment planning to improve tumour temperatures during locoregional hyperthermia

Background: The effectiveness of hyperthermia is strongly dependent on the achieved tumour temperatures. Phased-array systems allow flexible power steering to realise good tumour heating while avoiding excessive heating in normal tissue, but the limited quantitative accuracy of pre-treatment planning complicates realising optimal tumour heating. On-line hyperthermia treatment planning could help to improve the heating quality. This paper demonstrates the feasibility of using on-line temperature-based treatment planning to improve the heating quality during hyperthermia in three patients.

Methods: Hyperthermia treatment planning was performed using the Plan2Heat software package combined with a dedicated graphical user interface for on-line application. Electric fields were pre-calculated to allow instant update and visualisation of the predicted temperature distribution for user-selected phase-amplitude settings during treatment. On-line treatment planning using manual variation of system settings for the AMC-8 hyperthermia system was applied in one patient with a deep-seated pelvic melanoma metastasis and two cervical cancer patients. For a clinically relevant improvement the increase in average target temperature should be at least 0.2?°C.

Results: With the assistance of on-line treatment planning a substantial improvement in tumour temperatures was realised for all three patients. In the melanoma patient, the average measured target temperature increased from 38.30?°C to 39.15?°C (i.e.?+0.85?°C). In the cervical cancer patients, the average measured target temperature increased from 41.30?°C to 42.05?°C (i.e.?+0.75?°C) and from 41.70?°C to 42.80?°C (i.e.?+1.1?°C), respectively.

Conclusion: On-line temperature-based treatment planning is clinically feasible to improve tumour temperatures. A next, worthwhile step is automatic optimisation for a larger number of patients.     

Intravesical radiofrequency induced hyperthermia enhances mitomycin C accumulation in tumour tissue

Introduction: Non-muscle invasive bladder cancer (NMIBC) is a highly recurrent disease with potential progression to muscle invasive disease despite the standard bladder instillations with mitomycin C (MMC) or Bacille Calmette–Guérin immunotherapy. Therefore, alternatives such as radiofrequency-induced chemohyperthermia (RF-CHT) with MMC are being investigated. The mechanism explaining the efficacy of RF-CHT is only partly understood. We examined whether RF-CHT results in higher MMC tissue concentrations as compared to cold MMC instillation.

Patients and methods: Prior to a planned transurethral resection of bladder tumour (TURBT), patients with stage Ta NMIBC were allocated to either (1) cold MMC instillation or (2) RF-CHT. After MMC instillation, three biopsies were taken of both normal and tumour tissue. Biopsies were snap-frozen and MMC tissue concentrations were analysed using ultra-performance liquid chromatography.

Results: Eleven patients were included of which six received RF-CHT. Ten patients had TaG2-LG/HG papillary tumours at pathology. One patient in the RF-CHT group appeared to be free of malignancy and was excluded from the analysis as no tumour biopsies were available. The median MMC concentration in tumour tissue was higher in the RF-CHT group (median 665.00?ng/g vs. 63.75?ng/g, U?=?51.0, p?=?0.018). Moreover, in both techniques the MMC concentration was lower in normal tissue compared to tumour tissue. Tissue MMC concentration measurements varied substantially within, and between, different patients from the same group.

Conclusion: Intravesical RF-CHT results in higher tumour MMC concentrations vs. cold MMC instillation which contributes to its superior efficacy.     

Radiofrequency hyperthermia: the design of coil transducers for local heating

Simple and complex coils have been designed and built for use as radiofrequency transducers for local and regional hyperthermia. The heating from such coils is produced by currents generated in the dielectric by the electromagnetic field radiated from the coil elements. The heating rate profile, as a function of phantom depth, is dependent upon the size and geometry of the coil, its proximity to the dielectric, and the transducer current. Since the fields of simple pancake coils are inhomogeneous, either complex geometries or some method of smoothing is required. A simple solution is to move the coil over the region to be heated or to use multiple applicators. However, many satisfactory heating patterns have been derived for stationary coils designed to heat well-defined subcutaneous volumes to a depth of 3-5 cm. Thermometry studies in static saline-gelatin phantoms demonstrate extensive heating ranges and flexibility since the coils couple with the dielectric through air.     

3D radiobiological evaluation of combined radiotherapy and hyperthermia treatments

Purpose: Currently, clinical decisions regarding thermoradiotherapy treatments are based on clinical experience. Quantification of the radiosensitising effect of hyperthermia allows comparison of different treatment strategies, and can support clinical decision-making regarding the optimal treatment. The software presented here enables biological evaluation of thermoradiotherapy plans through calculation of equivalent 3D dose distributions.

Methods: Our in-house developed software (X-Term) uses an extended version of the linear-quadratic model to calculate equivalent radiation dose, i.e. the radiation dose yielding the same effect as the thermoradiotherapy treatment. Separate sets of model parameters can be assigned to each delineated structure, allowing tissue specific modelling of hyperthermic radiosensitisation. After calculation, the equivalent radiation dose can be evaluated according to conventional radiotherapy planning criteria. The procedure is illustrated using two realistic examples. First, for a previously irradiated patient, normal tissue dose for a radiotherapy and thermoradiotherapy plan (with equal predicted tumour control) is compared. Second, tumour control probability (TCP) is assessed for two (otherwise identical) thermoradiotherapy schedules with different time intervals between radiotherapy and hyperthermia.

Results: The examples demonstrate that our software can be used for individualised treatment decisions (first example) and treatment optimisation (second example) in thermoradiotherapy. In the first example, clinically acceptable doses to the bowel were exceeded for the conventional plan, and a substantial reduction of this excess was predicted for the thermoradiotherapy plan. In the second example, the thermoradiotherapy schedule with long time interval was shown to result in a substantially lower TCP.

Conclusions: Using biological modelling, our software can facilitate the evaluation of thermoradiotherapy plans and support individualised treatment decisions.     

Locoregional hyperthermia of deep-seated tumours applied with capacitive and radiative systems: a simulation study

Background: Locoregional hyperthermia is applied to deep-seated tumours in the pelvic region. Two very different heating techniques are often applied: capacitive and radiative heating. In this paper, numerical simulations are applied to compare the performance of both techniques in heating of deep-seated tumours.

Methods: Phantom simulations were performed for small (30?×?20?×?50?cm³) and large (45?×?30?×?50?cm³), homogeneous fatless and inhomogeneous fat-muscle, tissue-equivalent phantoms with a central or eccentric target region. Radiative heating was simulated with the 70?MHz AMC-4 system and capacitive heating was simulated at 13.56?MHz. Simulations were performed for small fatless, small (i.e. fat layer typically <2?cm) and large (i.e. fat layer typically >3?cm) patients with cervix, prostate, bladder and rectum cancer. Temperature distributions were simulated using constant hyperthermic-level perfusion values with tissue constraints of 44?°C and compared for both heating techniques.

Results: For the small homogeneous phantom, similar target heating was predicted with radiative and capacitive heating. For the large homogeneous phantom, most effective target heating was predicted with capacitive heating. For inhomogeneous phantoms, hot spots in the fat layer limit adequate capacitive heating, and simulated target temperatures with radiative heating were 2–4?°C higher. Patient simulations predicted therapeutic target temperatures with capacitive heating for fatless patients, but radiative heating was more robust for all tumour sites and patient sizes, yielding target temperatures 1–3?°C higher than those predicted for capacitive heating.

Conclusion: Generally, radiative locoregional heating yields more favourable simulated temperature distributions for deep-seated pelvic tumours, compared with capacitive heating. Therapeutic temperatures are predicted for capacitive heating in patients with (almost) no fat.     

A multi-element ultrasonic hyperthermia applicator with independent element control

Acoustic field patterns from a planar multi-element ultrasonic applicator were determined experimentally and compared with theory. Measurements were obtained from square arrays of 4 and 16 elements. The acoustic fields produced by various configurations of individual square elements (3 × 6 cm × 3–6 cm) driven at 1 MHz were measured in water. Transverse and axial scans paths were used to characterize the acoustic beam for different aperture sizes and individual element excitations. Unequal power excitation of adjacent elements produced multiple peaked acoustic intensity patterns. While a simple theoretical model was not able to account for all the experimentally determined transverse and axial field patterns, a model including mechanical damping improved the agreement between theory and experiment. However, less ripple in the axial pattern was measured than predicted by either theoretical model. The ability of the applicator to generate acoustic field patterns suitable for local tissue heating was demonstrated by an experimental study in dog thigh muscle.