Windows 10 Product Key Shows Bbbb-bbbb-bbbb-bbbb-bbbb
Pinkie Mclucas
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Ultrasound-induced blood-brain barrier (BBB) opening using microbubbles is a promising technique for local delivery of therapeutic molecules into the brain. The real-time control of the ultrasound dose delivered through the skull is necessary as the range of pressure for efficient and safe BBB opening is very narrow. Passive cavitation detection (PCD) is a method proposed to monitor the microbubble activity during ultrasound exposure. However, there is still no consensus on a reliable safety indicator able to predict potential damage in the brain. Current approaches for the control of the beam intensity based on PCD employ a full-pulse analysis and may suffer from a lack of sensitivity and poor reaction time. To overcome these limitations, we propose an intra-pulse analysis to monitor the evolution of the frequency content during ultrasound bursts. We hypothesized that the destabilization of microbubbles exposed to a critical level of ultrasound would result in the instantaneous generation of subharmonic and ultra-harmonic components. This specific signature was exploited to define a new sensitive indicator of the safety of the ultrasound protocol. The approach was validated in vivo in rats and non-human primates using a retrospective analysis. Our results demonstrate that intra-pulse monitoring was able to exhibit a sudden appearance of ultra-harmonics during the ultrasound excitation pulse. The repeated detection of such a signature within the excitation pulse was highly correlated with the occurrence of side effects such as hemorrhage and edema. Keeping the acoustic pressure at levels where no such sign of microbubble destabilization occurred resulted in safe BBB openings, as shown by MR images and gross pathology. This new indicator should be more sensitive than conventional full-pulse analysis and can be used to distinguish between potentially harmful and safe ultrasound conditions in the brain with very short reaction time.
The blood-brain barrier (BBB) regulates cerebral homeostasis and prevents the passage of xenobiotics and pathogens from the vasculature into the central nervous system (CNS). Due to the selective permeability of the BBB, the majority of drugs cannot reach the brain parenchyma at therapeutic concentrations1. Transcranial focused ultrasound (FUS) can induce cavitation, which may induce transient, localized BBB disruption to allow the passage of molecules into the brain2. Promising results have been shown for the treatment of brain diseases and other conditions using this technique. Nevertheless, fine control of acoustic parameters is crucial to avoid excessive microbubble activity that could result in vascular damage3,4. Thus, work along these lines is necessary for (i) the definition of a reliable monitoring readout during the procedure for safe and efficient ultrasound-assisted BBB disruption; and (ii) a better understanding of biophysical phenomena involved in this process.
Microbubble cavitation can be controlled by tuning ultrasound parameters (e.g., pressure amplitude, frequency, pulse repetition frequency, burst length). Low acoustic pressures induce stable cavitation (alternation of expansion and shrinkage of microbubbles) near the vessel wall that can result in tight junction loosening due to local stress via push-pull mechanisms or microstreaming4,5. On the other hand, higher ultrasound intensities may induce inertial cavitation that leads to violent collapse and fragmentation of microbubbles accompanied by micro-jets and shock waves. It is generally accepted that inertial cavitation should be avoided as it is not required for successful BBB opening and may be associated with the presence of undesirable vessel damage3,4,6. Recording and using cavitation signals during the BBB opening is now widely accepted as a very efficient way to control the procedure in real-time and to ensure repeatable, efficient, and safe delivery to the brain. However, there is still no consensus on the best method to do so.
Commercial ultrasound contrast agents currently available for BBB opening in humans consist of lipid-stabilized microbubbles (e.g., Definity, Lantheus Medical Imaging; SonoVue, Bracco Imaging). The time for microbubble distribution and the intensity of microbubble oscillation induced by ultrasound are directly related to their therapeutic efficacy and safety7. Ultrasound waves set circulating microbubbles into nonlinear oscillations that generate specific harmonic components (e.g., subharmonic, harmonic, ultra-harmonic)8. Conversely, at higher pressures, inertial cavitation collapses microbubbles, generating broadband emissions. The occurrence of subharmonic and ultra-harmonic frequencies is typically interpreted as a threshold for inertial cavitation that is associated with a risky oscillation regime of microbubbles9. These unique acoustic signatures provide monitoring information about microbubble activity through passive cavitation detection (PCD)6,10,11. Feedback-control algorithms based on the detection of inertial cavitation12,13 have yielded safe BBB disruption. However, the tolerable cavitation threshold allowing efficient opening remains challenging to determine. Hence, other approaches based on the monitoring of specific frequency components such as harmonic14, subharmonic15, and ultra-harmonic10,14,16 have also been proposed as indicators of efficient and safe BBB opening.
Some studies reported the difficulty of isolating both sub- and ultra-harmonic from broadband emissions that indicated inertial cavitation13,14 and concluded that these specific signatures were not useful for predicting BBB disruption or damage. However, in all these studies, cavitation was calculated by averaging the frequency response on the total burst length (few ms). As a result, many considerations are necessary for the analysis of the ultrasound-induced microbubble activity and to conclude on its influence on the resulting BBB opening18,19.
In this study, we monitored the evolution of ultra-harmonic frequency components throughout the ultrasound burst during in vivo ultrasound-mediated BBB opening sessions in rats and non-human primates (NHP). We propose a new intra-pulse analysis that allows the detection of these specific frequency components during the ultrasound bursts. By doing so, we enable very short reaction time for future feedback controllers. The acquisition of baseline signals before microbubbles are injected, a step that is both time consuming and sensitive to motion or transducer coupling artifacts, is not needed anymore (12). Intra-pulse processing also eliminates the averaging process over the full-pulse that could decrease the sensitivity to detect sudden changes in subharmonic and ultra-harmonic emissions. Finally, we interpreted the relation of these specific frequency components with the safety and efficacy of BBB opening.
For ethical reasons, macaques were not sacrificed at the end of the BBB opening session, and some of them participated in multiple sessions of BBB opening in different brain locations (i.e., 15 sessions in total).
A traditional ultrasound sequence designed for BBB opening is usually composed of multiple bursts, as shown in Fig. 1A. For each ultrasound burst, the frequency response of the backscattered signal is calculated in real-time, and its frequency content is used to determine the cavitation doses (i.e., stable or inertial) and can be used to adjust the beam intensity. In practice, the frequency response is averaged over the total length of an echo (i.e., the length of the corresponding burst excitation). In this study, we propose a new safety index named intrapulse ultra-harmonic dose (IUD), defined as the relative amplitude in the intrapulse evolution of ultra-harmonic emissions. The raw PCD data is processed in the following way for the calculation of the IUD:
The feedback control described in12 was applied for NHP and rat experiments. Briefly, the sonication started at half the maximum tolerated pressure (i.e., 580 kPa for NHP, 501 kPa for rats) and increased gradually with 9 kPa steps until a defined stable cavitation index was reached or an inertial cavitation event was detected . The stable cavitation dose (SCD) was determined as the sum of the root mean square (RMS) of the harmonic and ultra-harmonic frequency components. The inertial cavitation dose (ICD) was determined as the RMS of the broadband signal, excluding the frequency bandwidths of the SCD.
A total of 15 BBB-opening sessions in macaques have been re-processed or newly performed in this paper. Four of these sessions were executed without feedback control sonication and eleven with the feedback control. Based on MRI follow up, only one session resulted in an evident brain hemorrhage and the second one in a suspicious hemorrhage case.
Another BBB opening session resulted in an intermediate case showing a suspicious hyposignal in the sonicated area on late T2 images. IUD calculation showed an abrupt increase of IUD for a small portion of bursts (8.0% of the total), as illustrated in Fig. 4. Interestingly, multiple IUD events were detected for consecutive bursts. This case illustrates the sensitivity of the index that seems dose-dependent with the severity of bleeding. The results for NHP are summarized in Table 2.
Sudden variation in the frequency content can also be induced by the initial cavitation of bubble clusters confined at the focus44. This initial cavitation activity could result in an uncontrolled expansion of the BBB opening in the treated area. Responses from microbubbles located at different positions in the brain cannot be isolated using single-element PCD due to a lack of spatial resolution. Regardless of the hypothesis used to interpret the data, the safety indicator defined in our study shows that a sudden and repeated variation of ultra-harmonic content during the procedure is deleterious.
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