Good morning and thank you, section moderators, for letting us present our research work. I would like to thank the AANS-CNS Joint Section on Disorders of the Spine and Peripheral Nerves for awarding this work the Kline Award for Peripheral Nerves Research. It is indeed a great honor. Of course, I wish we could have met in person in Orlando. We have nothing to disclose for this presentation. Focus ultrasound combined with circulating microbubbles have been proven to effectively open the blood-brain barrier of the spinal cord barrier transiently and reversibly with continuous ongoing burst tonication. That's for localized delivery of therapeutic agents. It's non-invasive, targeted, and leads to reversible permeability change. Its effect on the blood-nerve barrier, however, has not been investigated. Blood-nerve barrier, or blood-nerve interface, is a physiological boundary between the peripheral nerve axons and the bloodstream that basically prevents the transfer of substances from the plasma to the nerve fibers. In the peripheral nerve, the innermost layer, or the endoneurium, is a highly regulated microenvironment of loose collagen arrays where myelinated and unmyelinated axons and unfenestrated type junctions forming by whoever's soles reside. The microenvironment of the endoneurium is regulated by two blood-nerve interfaces possessing non-fenestrated type junctions. First, the BNB made of the innermost perineural layer, and second, the endoneurial microvessels. Endoneurial microvessels, which are in direct contact with circling blood, are essentially considered the true interface forming the BNB. Several physiological differences are, however, different from the BBB and are beyond the scope of this talk. It's important to understand that in chronic nerve injuries, the BNB is usually intact, limiting the delivery of therapeutic agents into the injury site, and the goal of this study is to investigate the effect of focus ultrasound with microbubbles on the BNB for potential cell-directed treatment, modality, and drug delivery. Extensive work performing targeted ablation and neuromodulation in both the CNS and PNS has been performed. PNS works have concentrated on thermal effects in comparison with spine and brain, but early on, interest in control versatility of ultrasonic energy took place without specifying BBB or BNB targeting. Foreign colleagues are perhaps the first to acknowledge the need for control of reversibility and action of ultrasound on both barriers. However, no works evaluated reversibility of BNB closure by controlled focus ultrasound, nor the use in conjunction with microbubbles. Our interest in focus ultrasound used with microbubbles follows current use in CNS, and we believe it has promising clinical roles in the PNS too. Our group's first aim was to optimize dose response and characteristics for reversible opening of the BNB using both direct vision and MR guidance, and this is the part of the presented work today. For this aim, we used the histological criteria. Evans blue binds to albumin and can leak out of the microvascular into the endoneural environment, and detection is possible due to specific wavelength by direct fluorescence of Evans blue. In addition, H&E staining was used to rule out bleeding and extravasation of red blood cells in the targeted field. We're currently working on short- and long-term behavioral aspects of the treatment along MR-guided planning and targeting, and hypothesize that the sonication effect of focus ultrasound can create a transient and reversible opening of the blood-nerve barrier, which will allow future clinical utilization in both drug and viral vector delivery. Circulating microbubbles have been shown to amplify focus ultrasound cavitation process by encountering acoustic-actuated harmonic extraction and expansion, and thus providing physical stimulation to the endothelial cells, followed by temporal tight junction disruption. Microbubbles are essentially octofluoropropane gas vesicles covered by a lipid coat, which will undergo stable cavitation, and that's for creating a mechanical disruption on the endothelial cells and tight junctions, and for a safe barrier disruption. This point is crucial, as the unstable inertial cavitation will cause harmful tissue damage. Control of these two is through the sonication energy, or peak pressure and amplitude used, and can be done with real-time detectors, as we will show. Focus ultrasound of investigated nerves were compared to both uninjured and crush-injured nerves, and also followed parallel-targeted BBB targeting as a control condition. This work is currently under peer review, and the reviewer's main concern was technical detailed aspects of the work, so I will try to be more concise on that matter today. Two main phases formed the methods, MR-guided focus ultrasound and direct vision ultrasound. In phase one, we used MR-guided focus ultrasound. Static nerve crush injury control group model was used, where we caused at least a Sunderman grade 2 nerve incontinuity injury. A 9.4 Tesla MR custom saddle coil was used in the magnet, and we acquired FATSAT T2 weighted, FATSAT T1 weighted, and gradient echo T1 with and without gadolinium. MR-guided focus ultrasound was used with a 1 MHz system with a passive cavitation detector, where 1 MPa of acoustic pressure was used for 5 minutes. Here we injected Evans blue, along with a constant 10 microliter per kilogram microwavable concentration and latex microbeads. Both histology and MR chemistry were carried at the end. Phase two consisted of direct vision focus ultrasound using a benchtop system. Here we were able to compare the studies to in parallel cranial focus ultrasound BBB investigations, and use those BBB investigations as a baseline treatment protocol. Here the system is in lower amplitude using 476 kHz with a passive cavitation detector, but we used different peak pressures along with different microwavable concentrations. Again, histology and MR chemistry. Here is the focus ultrasound benchtop setup. The static nerve is surgically exposed, and the ultrasound focus is registered at the targeted nerve using the stereotactic system, so sonication can be carried. The animal model in this study is a Taiwan GFPG modified rat. The experiment was carried out with a benchtop focus ultrasound system. During preparation, the static nerve on both legs is surgically exposed for focus ultrasound sonication and control. After moving the transducer to the target, Evans blue dye is injected for BNB disruption indication, and 5 minutes later, microwavable solution is injected, and ultrasound sonication is delivered simultaneously. 15-20 minutes after sonication, compound muscle action potential NEMG is measured on both nerves, and the animal is sacrificed for physiological evaluation. Various peak negative pressure and microwavable doses were used. Phase 1 consisted of MR guided focus ultrasound. Here are real-time in vivo targeting and sonication images of brown mouse right sciatic nerve. First on the left panel, MR targeting rendered T2 weighted planning images of the sciatic nerve. Axial, where you can see the tibia and arrow, the arrowhead corresponds to the iliac crest and coronal section. Backscattering detection of microwavables before injection of the bubbles and after injections shows software baseline scatter at 1.5 MHz, and after injection of the microwavables shows supraharmonic oscillations at 2.25 MHz, which is 1.5 times from the baseline. On the right panel, MR sequences in T1 and T2 fat suppress creating spin echo before and after injection of gadolinium, following sonication of the right sciatic nerve. Here, the white arrow corresponds to the sciatic nerve. Circle is our target point. The white arrowhead is a conscious enhancement following sonication, whereas the empty arrowhead corresponds to hyperintensity signal change of the sonicated nerve. In phase two, we used the Benchtop system for direct vision sonication. The acoustic emissions during sonication were collected with an acoustic detector, and here panel A shows the baseline frequency spectrum before bubble injection, where focal pressure is estimated at 0.3 MPa and ultrasound transmitting frequency at 476 kHz, which is approximately half of the MR guided system shown before. No acoustic emission was detected at ultraharmonic frequency range, which is about 715 kHz, while after microwavable injection, we do see the frequency spectrum with ultraharmonic detection, seen here by the red arrowhead, indicating potential stable cavitation and opening effect. These are phase two direct vision focus ultrasound results of gross microscopically and microscopically fly-1-GFP rat sciatic nerve sections. All nerves were injected with 3% Evans Blue and latex microbreeds prior to harvesting, and first on the left panel, we see a microscopic naïve control sciatic nerve where no injury or Evans Blue leakage is apparent. Next to it, there is apparent Evans Blue leakage in the crushed injury sciatic nerve. We can see extensive Evans Blue leakage, both proximal and distal to the injury cross-site. And if we go to the longitudinal sections of the naïve nerve, obviously there is no Evans Blue leakage. But when we look into the crushed nerve, we do see extensive axonal microarchitectural disruption, vacuolization and edema. When we look at the 1.2 megapascal, which is our highest energy sonication, along with a high concentration of microwaves at 167 micrograms per milliliter, which is approximately four times our ideal dosage, and the sonication pressure is also approximately three times our ideal sonication pressure, we do see, again, significant Evans Blue leakage along axonal architecture, which is disrupted, vacuolization and edema. However, the stained perineurium is intact. And a high permagnification, we do see microbreed presence, which are the gold spheres inside the vessels not leaking to the surrounding endoneurium. And we can see them again inside the epineural vessel with the following Evans Blue externalization around them. On the right panel, these are results of 0.5 megapascal peak pressure with 40 micrograms per milliliter. This is approximately our ideal combination. This is a targeted tibial branch of the sciatic nerve. Here are microscopic Evans Blue leakage apparent in the tibial branch, not extensive as the crush injury and confirmed to a single branch, also under fluorescent microscopy and limited Evans Blue leakage. This is also apparent in the H&E section, which we do not see any red blood cell extravasation, nor do we see any signs that we saw earlier in the higher amplitude, nor is there any vacuolization, edema, or axonal disorganization. And we can see that also in a high permagnification, where fluorescent microscopy shows Evans Blue leakage at the targeted site with preserved axonal architecture and without vacuolization or edema. This was also verified when we were using the CMAP results, and here is a representative one using 1.2 megapascal peak pressure. This is a relatively high sonication energy and intra-animal sonication compared to non-sonicated contralateral leg. Each recording averages three stimulations, and you can see delayed onset latency in the left panel and lower CMAP amplitudes on the right panel in the sonicated targeted group compared to the non-treated control group. In conclusion, the blood nerve barrier disruption can be achieved with focus ultrasound combined with microbials. And based on our results, 0.3 megapascal with 40 microliter per kilogram of microbial dosage is adequate to induce BNB disruption without causing tissue damage. Changes in nerve conduction activity were observed in the sonicated nerve, but more work will be done for further investigation, and the future work includes investigating the MR-guided focus ultrasound sonication to avoid surgery, increasing the number of animals in each group to optimize the focus ultrasound parameters for reversible disruption, and conducting the efficacy study for localized drug delivery into the PNS. We have investigated its potential for BNB opening in a sciatic nerve in vivo rodent study based on our previous knowledge of peripheral nerve injury models, and used both MR-guided focus ultrasound as well as direct vision ultrasound, both coupled with commercial microbubbles, and basically developed a pilot protocol in this study. We also show clear histological and imaging evidence in targeted sites in both lower and upper threshold limits for focus ultrasound sonication parameters and microbubble dosages, allowing us to optimize our treatment Certain limitations still remain. We have achieved our first and partial second aim of the planned work, and we are concentrating on timely reversible disruption, which is the key issue in this model. And again, there are several physiological differences which still remain to be studied and understood, which differentiate and discriminate the BNB from the BBB. I would like to first thank my instructor and supervisor, Dr. Raj Mehta, with whom we hypothesized and executed this work, alongside Sam Picharder from the Experimental Radiology Department at UFC. Chenchen Vink, Sam's research associate, helped us in technical focus ultrasound aspects, along with our lab research associate, Tak Chu. Special thanks again to the generous grant support from the Denise Lajoie Lake Fellowship in Brain Research and the Hotchkiss Brain Institute, awarded to this work and to this audience, and the AANS-CNS Section on Disorders of the Spine and Peripheral Nerves Award. Without it, we would not be able to do this work. Thank you very much again. Finally, I was asked to provide a post-test question, and this is a single-choice answer. The blood nerve barrier is believed to be formed by which of the following? A. Epineural segmental vessels, B. Endoneural microvessels, C. Perineural layer, or D. Schwann cells. Thank you.

Kline Award for Peripheral Nerves Research

focused ultrasound

circulating microbubbles

blood-nerve barrier

localized delivery of therapeutic agents