Ultrasound Bioeffects Research Articles

Exploration of Ultrasound‐Sensitive Biomaterials in Cancer Theranostics

AbstractCancer is a serious health problem to be solved. With the development of biomaterials, enhanced diagnostic and controllable therapeutic strategies for cancer have emerged, revealing encouraging results with a promising future. Due to its outstanding tissue penetration and controllability, along with a series of bioeffects on tissues, ultrasound (US), as a common medical technology, has been regarded as an effective tool against cancer, and its use has promoted the exploration of US‐sensitive biomaterials. In this review, the bioeffects of US are first introduced and then the main US‐sensitive biomaterials (containing cavitation nuclei, sonosensitizers, and US‐responsive macromolecular systems) used to treat cancer, followed by a summary of several applications based on US‐sensitive biomaterials, from diagnosis to therapy. Finally, the emerging challenges in this field that need to be overcome are highlighted. This review reports the progress of US‐sensitive biomaterials, providing new possibilities in cancer theranostics and thus benefiting patients.

Safety of Ultrasound Exposure: Awareness of Ultrasound Bioeffects among Saudi Sonographers.

Ultrasound tends to produce biological effects on body tissues. Therefore, the application of ultrasound safety rules is needed in all ultrasound examinations to avoid bioeffects that may occur because of a high thermal index (TI) and mechanical index (MI). This study aimed to explore the Saudi sonographers' awareness of ultrasound safety and bioeffects. A descriptive cross-sectional study was conducted, and the data was collected using an online survey google forms questionnaire disseminated through social media (Telegram, WhatsApp, and Twitter) as an electronic link. A total of 70 participants responded to the survey directly, and then, the data was analyzed using SPSS (Statistical Package for Social Science). Regarding the awareness of TI and MI, 64.3% of the participants were aware of TI and 65.7% of MI as safety ultrasound indices; moreover, 74.3% of the participants were aware of the ALARA principle "As Low as Reasonably Achievable." Participants' knowledge concerning more heat production in pulsed Doppler ultrasound modes was poor (31.5%). Most of the sonographers, i.e., 77.1% were aware of the relationship between the risk of activation and MI, and 58.6% of the participants were acquainted with the current FDA output limits. In comparison, 58.6% of the participants were aware of the bioeffects of ultrasound contrast agents. There was no significant correlation between the educational level and the degree of awareness of ultrasound bioeffects and safety (P-value> 0.05). In this study, it was found that sonographers were aware of the theoretical principles of ultrasound safety indices and the ALARA principle, although they had poor knowledge of ultrasound bioeffects in Doppler mode.

Best Practice Recommendations for the Safe use of Lung Ultrasound.

Die Sicherheit des Ultraschalls ist bei der Untersuchung der Lunge von besonderer Bedeutung, da spezifische Wechselwirkungsmechanismen an der alveolaren Luft-Gewebegrenze auftreten. Lungengewebe ist dabei deutlich empfindlicher als solides Gewebe gegenüber mechanischen Kräften. Die ursächlichen biologischen Effekte basierend auf Totalreflektion von Schallwellen sind zudem nur unzureichend untersucht. Andererseits ist der klinische Nutzen des Lungenultraschalls beträchtlich und hat Aufgrund der Pandemiesituation erheblichen Stellenwert dazugewonnen. Dabei erweist sich dieser bisweilen dem anderer radiologischer Bildgebungsverfahren ebenbürtig. Deshalb widmet sich diese Arbeit, basierend auf derzeit verfügbaren Literaturquellen, dem Einfluss von Schalleffekten auf das Lungenparenchym und evaluiert bestehende Empfehlungen zur Schalldruckreduzierung bei der Durchführung der Lungensonographie. Es wird ein Vorgehen empfohlen, um klinisch relevante Bilder zu erhalten und gleichzeitig die Ultraschallsicherheit zu gewährleisten. Ein besonderes Augenmerk liegt auf der Sicherheit neuer Ultraschall Modalitäten, welche zum Zeitpunkt früherer Empfehlungen noch nicht berücksichtigt waren. Abschließend werden notwendige Forschungs- und Ausbildungsschritte empfohlen, um Wissenslücken auf dem Gebiet des Lungenultraschalls in Zukunft schließen zu können. Diese Empfehlungen für die Praxis wurden von ECMUS, dem Sicherheitskommittee der EFSUMB, unter Mitwirkung von international Experten auf dem Gebiet der Lungensonographie und biologischer Ultraschallwechselwirkung erstellt. The safety of ultrasound is of particular importance when examining the lungs, due to specific bioeffects occurring at the alveolar air-tissue interface. Lung is significantly more sensitive than solid tissue to mechanical stress. The causal biological effects due to the total reflection of sound waves have also not been investigated comprehensively. On the other hand, the clinical benefit of lung ultrasound is outstanding. It has gained considerable importance during the pandemic situation, showing comparable diagnostic value with other radiological imaging modalities. Therefore, based on currently available literature, this work was dedicated determining possible ultrasound effects on the lung parenchyma and evaluating existing recommendations for acoustic output power limits when performing lung sonography. This work recommends a stepwise approach to obtain clinically relevant images while ensuring lung ultrasound safety. A special focus was set on the safety of new ultrasound modalities, which had not yet been introduced at the time of previous recommendations. Finally, necessary research and training steps are recommended in order to close knowledge gaps in the field of lung ultrasound safety in the future. These recommendations for practice were prepared by ECMUS, the safety committee of the EFSUMB, with participation of international experts in the field of lung sonography and ultrasound bioeffects.

The vulnerable air-tissue interface: Summary of ultrasound related bioeffects on lung and their implications for the use of different imaging modes and applications beyond

Introduction: Lung tissue is sensitive to mechanical stress. Animal studies showed induction of pulmonary capillary hemorrhage (PCH) in lung in the diagnostic regime at intensities. Therefore, this work summarizes novel findings regarding the interaction of ultrasound at the alveolar epithelium-gas interface. Method: Literature research related to ultrasound bio-effects on lung was carried out. Furthermore, recent studies reporting thresholds of PCH induction depending on imaging mode and duration will be discussed. Results: Ventilated lung tissue is more vulnerable to mechanical forces than solid tissue. From peak negative pressure below 1.5 MPa the likelihood of PCH induction increases with exposure time. Such PCH is asymptomatic but can falsify the diagnostic outcome. The causes remain still unknown but is not related to cavitation or thermal effects. The extent of PCH in lung is depending of physiological beside acoustic parameters. The extend of PCH is, therefore, not entirely predictable. In addition, observational studies on humans could not confirm the findings as investigated on pathological level on animal models. Conclusion: The vulnerability of lung should become aware in the lung ultrasound community. Current recommendations for output limitations given by AIUM and EFSUMB should be followed and only increased for diagnostics needs.

From sonar to medical ultrasound—The impact of Paul Langevin

The technical developments and discoveries of Paul Langevin figuratively and literally powered an incredible spectrum of scientific achievement. The quartz transducer technology of his 1918 patent (Procédé et appareils d'émission et de réception des ondes élastiques sous-marines à l'aide des propriétés piézo-électriques du quartz. – French Patent No. 505,703) enabled a level of acoustic output power generally unavailable at ultrasonic frequencies along with an increase in detection sensitivity. While there were disputes as to who should have been included on this patent, its effect on the field of ultrasonics is clear. The impetus for this work was largely high-power sonar for military application but his reported observations of effects on fish and even humans ushered in a new era of ultrasound bioeffects investigations including those of the famous Loomis Laboratory and numerous others. This is arguably the genesis of the rich tradition of research in ultrasound bioeffects and helped drive the pursuit of diagnostic and therapeutic applications of ultrasound including work by such famous researchers as the Fry Brothers. This presentation will review the evolution of bioeffects from the Langevin’s work and its impact on the field of medical ultrasound.

Perspective on ultrasound bioeffects and possible implications for continuous post-dive monitoring safety.

Ultrasound monitoring, both in the form of Doppler and 2D echocardiography, has been used post-dive to detect decompression bubbles circulating in the bloodstream. With large variability in both bubble time course and loads, it has been hypothesised that shorter periods between imaging, or even continuous imaging, could provide more accurate post-dive assessments. However, while considering applications of ultrasound imaging post-decompression, it may also be prudent to consider the possibility of ultrasound-induced bioeffects. Clinical ultrasound studies using microbubble contrast agents have shown bioeffect generation with acoustic powers much lower than those used in post-dive monitoring. However, to date no studies have specifically investigated potential bioeffect generation from continuous post-dive echocardiography. This review discusses what can be drawn from the current ultrasound and diving literature on the safety of bubble sonication and highlights areas where more studies are needed. An overview of the ultrasound-bubble mechanisms that lead to bioeffects and analyses of ultrasound contrast agent studies on bioeffect generation in the pulmonary and cardiovascular systems are provided to illustrate how bubbles under ultrasound can cause damage within the body. Along with clinical ultrasound studies, studies investigating the effects of decompression bubbles under ultrasound are analysed and open questions regarding continuous post-dive monitoring safety are discussed.

Establishing C. elegans as a Model for Studying the Bioeffects of Therapeutic Ultrasound

Ultrasound is widely used in diagnostic and therapeutic medical procedures and it is becoming an important tool in biomedical research. During exposure, as an ultrasound beam interacts with the tissues in its path, changes known as “bioeffects” can result. Animal studies have suggested that these changes can alter survival, movement, reproduction, development and learning in various species. Additional studies in animals could provide valuable information about the mechanisms of therapeutic ultrasound and may contribute to the development of additional exciting laboratory techniques. Therefore, we developed methods for exposing C. elegans nematode worms to ultrasound and observed that they exhibited exposure-dependent reductions in movement, fecundity and survival. These effects were prevented by polyvinyl alcohol, which suggested that cavitation was the main mechanism of damage. This work provides a foundation for capitalizing on the advantages of C. elegans as a model to thoroughly characterize ultrasound's bioeffects at the cellular and molecular levels.

Far field during sonication experiments in vitro – Is it really far enough?

In many in vitro experiments studying ultrasound bioeffects the sonicated samples are placed to far field with intention of exposing them to as uniform ultrasound field as possible. The main aim of this work is to assess whether the sonicated samples really experience what they are believed to. Also we would like to suggest basic rules for construction of sonication vessels. We used 3.5 MHz and 7 MHz ultrasound transducers for measurements. We measured ultrasound field inside and behind common culture plates and special 3D printed plates placed to last axial maximum in water sonication tank with use of a needle hydrophone. Our results show that even though the sonication vessels with sonicated samples are placed into far field, the sonicated samples are actually exposed to some kind of a near field pattern which develops due to the interaction between ultrasound and well of culture plate. The variability of local acoustic intensity can reach up to several hundreds of percent. Our results are also supported by theoretical calculation and software for simulation of ultrasound fields. Even though the sonicated samples may have actually been exposed to some kind of near field pattern in many past studies, the whole phenomenon of creation of near field pattern can be controlled to some extent for future studies. Thus, we suggest that the sonication vessel should always be designed for particular ultrasound transducer.

Behavioral and Functional Assessment of Ultrasound Neuromodulation on Caenorhabditis Elegans.

Ultrasound brain stimulation is a promising modality for probing brain function and treating brain diseases. However, its mechanism is as yet unclear, and in vivo effects are not well-understood. Here, we present a top-down strategy for assessing ultrasound bioeffects in vivo, using Caenorhabditis elegans. Behavioral and functional changes of single worms and of large populations upon ultrasound stimulation were studied. Worms were observed to significantly increase their average speed upon ultrasound stimulation, adapting to it upon continued treatment. Worms also generated more reversal turns when ultrasound was ON, and within a minute post-stimulation, they performed significantly more reversal and omega turns than prior to ultrasound. In addition, in vivo calcium imaging showed that the neural activity in the worms' heads and tails was increased significantly by ultrasound stimulation. In all, we conclude that ultrasound can directly activate the neurons of worms in vivo, in both of their major neuronal ganglia, and modify their behavior.

Synchronous temperature variation monitoring during ultrasound imaging and/or treatment pulse application: a phantom study.

Ultrasound attenuation through soft tissues can produce an acoustic radiation force (ARF) and heating. The ARF-induced displacements and temperature evaluations can reveal tissue properties and provide insights into focused ultrasound (FUS) bio-effects. In this study, we describe an interleaving pulse sequence tested in a tissue-mimicking phantom that alternates FUS and plane-wave imaging pulses at a 1 kHz frame rate. The FUS is amplitude modulated, enabling the simultaneous evaluation of tissue-mimicking phantom displacement using harmonic motion imaging (HMI) and temperature rise using thermal strain imaging (TSI). The parameters were varied with a spatial peak temporal average acoustic intensity (I spta ) ranging from 1.5 to 311 W.cm-2, mechanical index (MI) from 0.43 to 4.0, and total energy (E) from 0.24 to 83 J.cm-2. The HMI and TSI processing could estimate displacement and temperature independently for temperatures below 1.80°C and displacements up to ~117 μm (I spta <311 W.cm-2, MI<4.0, and E<83 J.cm-2) indicated by a steady-state tissue-mimicking phantom displacement throughout the sonication and a comparable temperature estimation with simulations in the absence of tissue-mimicking phantom motion. The TSI estimations presented a mean error of ±0.03°C versus thermocouple estimations with a mean error of ±0.24°C. The results presented herein indicate that HMI can operate at diagnostic-temperature levels (i.e., <1°C) even when exceeding diagnostic acoustic intensity levels (720 mW.cm-2 < I spta < 207 W.cm-2). In addition, the combined HMI and TSI can potentially be used for simultaneous evaluation of safety during tissue elasticity imaging as well as FUS mechanism involved in novel ultrasound applications such as ultrasound neuromodulation and tumor ablation.

Non-thermal effects of ultrasound are selectively induced during a critical window of collagen self-assembly

Type I collagen is a self-assembling fibrillar protein widely used in tissue engineering. An established paradigm utilizes ultrasound exposure during the fluid-to-gel phase transition of neutralized collagen solutions, producing site-specific changes in collagen fiber organization via both thermal and non-thermal acoustic mechanisms. In the present study, we investigated the temporal dependence of ultrasound bioeffects with respect to the progression of collagen assembly. Collagen solutions (0.8 mg/ml) were exposed to ultrasound (CW, 7.8 or 8.8 MHz, 0–10 W/cm2) for 5 min during three distinct stages of collagen self-assembly, which were monitored via both temperature and optical turbidity measurements. Assembly rate was manipulated by adjusting the collagen pH and the temperature at which acoustic exposures were performed. The results identify a critical window during which ultrasound exposure produces radially aligned collagen fibers via a non-thermal acoustic mechanism. This window coincides with a stage of collagen polymerization during which nanofibrils associate laterally into microfibril bundles, and a simultaneous increase in the acoustic absorption coefficient. These findings raise the possibility that ultrasound exposures of self-assembling collagen biomaterials in a point-of-care clinical setting can be timed to preferentially induce either thermal or non-thermal bioeffects, thereby enhancing the efficacy of therapeutic ultrasound for regenerative medicine applications.

Ultrasound for the treatment of acute kidney injury and other inflammatory conditions: a promising path toward noninvasive neuroimmune regulation.

Acute kidney injury (AKI) is an important clinical disorder with high prevalence, serious consequences, and limited therapeutic options. Modulation of neuroimmune interaction by nonpharmacological methods is emerging as a novel strategy for treating inflammatory diseases, including AKI. Recently, pulsed ultrasound (US) treatment was shown to protect from AKI by stimulating the cholinergic anti-inflammatory pathway. Because of the relatively simple, portable, and noninvasive nature of US procedures, US stimulation may be a valuable therapeutic option for treating inflammatory conditions. This review discusses potential impacts of US bioeffects on the nervous system and how this may generate feedback onto the immune system. We also discuss recent evidence supporting the use of US as a means to treat AKI and other inflammatory diseases.

How to Interpret the Ultrasound Output Display Standard for Diagnostic Ultrasound Devices: Version 3.

How to Interpret the Ultrasound Output Display Standard for Diagnostic Ultrasound Devices: Version 3.

Biomedical Ultrasound Bioeffects: Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array (Adv. Sci. 17/2019)

Biomedical Ultrasound Bioeffects: Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array (Adv. Sci. 17/2019)

Hepatocyte Injury Induced by Contrast-Enhanced Diagnostic Ultrasound.

Contrast-enhanced diagnostic ultrasound (US) has a potential to induce localized biological effects. The potential for contrast-enhanced diagnostic US bioeffects in liver were researched, with guidance from a report by Yang et al (Ultrasonics 2012; 52:1065-1071). Contact and standoff scanning was performed for 10 minutes with a diagnostic US phased array at 1.6 MHz during bolus injection or infusion of a contrast agent at a high dose. The impact of the imaging on rat liver was investigated by measuring enzyme release, microvascular leakage, and staining of injured hepatocytes. The results showed liver enzyme release at 30 minutes, indicating liver injury, and elevated extraction of Evans blue dye, indicating microvascular leakage. In addition, Evans blue and trypan blue vital-staining methods revealed scattered stained cells within the US scan plane. For the Evans blue method, fluorescent cell counts in frozen sections were greatest for standoff exposure with contrast infusion. The count decreased strongly with depth for bolus injection, which was probably reflective of the high attenuation noted for this agent delivery method. The results qualitatively confirmed the report by Yang et al and additionally showed hepatocyte vital staining. Research is needed to determine the threshold for the effects and the contrast agent dose response.

Hunt Magic: A lifetime of fruitful collaboration

The 1989–1990 F.V. Hunt Fellowship allowed me to spend one year at the University of Rochester in Edwin Carstensen’s lab to investigate the physical mechanisms of shock wave lithotripsy (ref Boston). Coming from the lab of Bob Apfel at Yale, where I had worked on nonlinear propagation and measurement of B/A, at U. Rochester I applied these concepts to shock wave propagation and measurement in lithotripsy. I also investigated the role of inertial cavitation in both lithotripsy and blood clot dissolution, developing collaborations with Mort Miller’s biophysics lab and clinicians at the Strong Memorial Hospital. The fruitful collaborations I developed at U. Rochester during my Hunt year continued for years thereafter, including several summers spent in Rochester until marriage and family intervened. I can credit the awarding of my NSF Presidential Faculty Fellowship and decades of ultrasound bioeffects work to my Hunt Fellowship year, and I relish ongoing collaborations that enrich my teaching and research a...

The role of cavitation in vascular occlusion

Ed Carstensen’s papers on the effects of ultrasound on plants were amongst the first that attracted me to study ultrasound bio-effects. It was little surprise to me that, when I first started looking at HIFU, his name was on many of the seminal papers in this area. And now, his papers on cavitation and vasculature inform our current research. Ed’s influence on bio-effects research stretches far beyond the borders of the USA Vascular occlusion has the potential to treat life-threatening conditions including twin-twin transfusion syndrome (occurring in fetuses that share a placenta). Sheep placental vessels have been used to develop HIFU treatment guidance, delivery (1.66MHz) and monitoring. Ultrasound imaging guidance/flow monitoring, with simultaneous acoustic cavitation detection in 82 targets in 14 pregnant sheep resulted in flow occlusion in 90%. Adverse events included skin erythema (18/82) and burns (10/82), but all resolved over 21 days. Analysis of acoustic cavitation required low pass (software) filtering and careful choice of broadband integration range (0.1 to 1.1 MHz). 9% of successful treatments displayed detectable broadband, 32% with half-harmonic and 1% with drive-voltage fluctuations. In conclusion, treatment is possible, side effects are manageable, and acoustic cavitation appears not to be essential for successful occlusion.

How to Keep Ultrasound Safe in Obstetrics? What have We Learned?

If asked, the vast majority of end users (and patients) will respond that ultrasound is not X-rays and is completely safe. In reality, there is a marked lack of knowledge on effects of ultrasound in tissues being examined (bioeffects) among the majority of these end-users. Being a form of energy, ultrasound has effects in all insonated tissues (bioeffects), secondary to two main mechanisms: thermal and non-thermal (or mechanical). On-screen indicators of these potential bioeffects are the Thermal and Mechanical indices (TI and MI). Biological effects have been reported in animals, mostly for exposure exceeding those usually applied in humans. In humans, autism, abnormal hearing, vision or language development, intrauterine growth restriction, childhood cancer and increase in non-right handedness have been associated with ultrasound but no cause-effect has been proven by science-based analysis (except, possibly for a slight increase in non-right handedness, specifically in males). Yet, essentially all epidemiological studies are based on information obtained with pre-1992 machines, when acoustic output of instruments was allowed to be increased, for fetal use, by a factor of almost 8. Increasingly, ultrasound is performed early in gestation, a time when the fetus is known to be particularly sensitive to external influences. Most examinations are performed with B-mode, but some studies employ Doppler technology, known to expose the fetus to much higher levels of acoustic energy, hence requiring special precautions. There should always be a medical indication and operators should follow the rule of the shortest time possible, at the lowest possible output, compatible with an adequate diagnosis (ALARA). Keeping the TI and MI below 1 will, almost certainly, not cause deleterious effects to the fetus. Education of the operators is vital to maintain the unblemished record of ultrasound in obstetrics. WFUMB. WFUMB Clinical Safety Statement for Diagnostic Ultrasound. Available from: http://www.wfumb.org/about/statements.aspx. Abramowicz JS, Sheiner E:Ultrasound Bioeffects and Safety: what the practitioner should know. In Fleischer a, Abramowicz JS, Gonsalves L, Manning F, Monteagudo A, Timor I, Toy E (eds): Sonography in Obstetrics and Gynecology-Principles and Practice. 8th edition, 2018.

National survey of Australian sonographer knowledge and behaviour surrounding the ALARA principles when conducting the 11-14-week obstetric screening ultrasound.

To identify gaps in Australian sonographer's knowledge and application of as low as reasonably achievable (ALARA) principles during first trimester imaging; Identify relationships between demographic variables and knowledge or application of the Output Display Standard (ODS) value thermal index (TI) and compare Australian sonographers to their international peers. Australian Sonographer Accreditation Registered ( ASAR)-registered sonographers completed a voluntary questionnaire over September 2015 after institutional ethics approval (RDSE-48-15). Seventeen questions detailed their demographics (5); knowledge of ultrasound bioeffects terminology and ALARA principles (9); behaviour surrounding ALARA and first trimester imaging (2) and continuing bioeffects education (1). Exclusion criteria was non-ASAR status. Descriptive (mode frequency) and inferential statistics (Fisher exact test) were used. Significance level was 95%. Ninety-five valid surveys were collected. Ninety-nine per cent knew the meaning of ALARA, 93.55% correctly defined 'TI' and 85.39% knew where to find the TI value via the ODS. Half never monitor the ODS. No correlation (P=0.094) was found between experience and ODS monitoring. No statistical difference (P=0.189) existed between obstetric and non-obstetric sonographers who knew the meaning of TIB (87.18% vs. 76.92%) or those who correctly identified it as the setting for 11-14-week examinations (30.77% vs. 30.77%). When using Doppler during obstetric examinations, no difference existed (P=0.293), between obstetric and non-obstetric sonographers' knowledge of ASUM/WFUMB guidelines for maximum TI (47.37% vs. 46.15%). Despite poor ODS usage, Australian sonographers outperform their international peers for ALARA literacy, and show better application of ALARA principles in the first trimester. No relationships were found between demographic variables and knowledge or conscious monitoring of ODS.

Do Anesthetic Techniques Influence the Threshold for Glomerular Capillary Hemorrhage Induced in Rats by Contrast-Enhanced Diagnostic Ultrasound?

Glomerular capillary hemorrhage can be induced by ultrasonic cavitation during contrast-enhanced diagnostic ultrasound (US) exposure, an important nonthermal US bioeffect. Recent studies of pulmonary US exposure have shown that thresholds for another nonthermal bioeffect of US, pulmonary capillary hemorrhage, is strongly influenced by whether xylazine is included in the specific anesthetic technique. The objective of this study was to determine the influence of xylazine on contrast-enhanced diagnostic US-induced glomerular capillary hemorrhage. In this study, anesthesia with ketamine only was compared to ketamine plus xylazine for induction of glomerular capillary hemorrhage in rats by 1.6-MHz intermittent diagnostic US with a microsphere contrast agent (similar to Definity; Lantheus Medical Imaging, Inc, North Billerica, MA). Glomerular capillary hemorrhage was measured as a percentage of glomeruli with hemorrhage found in histologic sections for groups of rats scanned at different peak rarefactional pressure amplitudes. There was a significant difference between the magnitude of the glomerular capillary hemorrhage between the anesthetics at 2.3 MPa, with 45.6% hemorrhage for ketamine only, increasing to 63.2% hemorrhage for ketamine plus xylazine (P < .001). However, the thresholds for the two anesthetic methods were virtually identical at 1.0 MPa, based on linear regression of the exposure response data. Thresholds for contrast-enhanced diagnostic US-induced injury of the microvasculature appear to be minimally affected by anesthetic methods.