Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

The PING method is designed to produce non-invasive, targeted neuronal lesions. The method derives from a strong and growing foundation of research in the field of focused ultrasound^3,4,5,6,7. The ability to provide focal access to specific areas of the brain parenchyma via transient opening of the BBB has created an avenue for delivering a wide variety of agents that would normally not gain access to the brain. This opportunity is largely being advanced for the central delivery of therapeutic agents that possess poor BBB permeability. The PING method leverages the same opportunity in a different manner by delivering a neurotoxin, with the ultimate goal of destroying circuits that contribute to neurological dysfunction. Aberrant neural circuitry represents an effective target for surgical intervention to attenuate the impact of certain neurological disorders^2,15. The long-term goal for the PING method is to augment the currently available approaches for disconnecting circuitry contributing to neural dysfunction.

The design of a comprehensive study using the PING method would incorporate multiple control groups. These groups would include: (a) an untreated group [No FUS/No QA]; (b) a group controlling the direct effects of FUS [FUS/No QA]; (c) a group assessing the direct effects of systemic neurotoxin in the absence of FUS [No FUS/QA]. Outcomes in these groups would be compared with the primary PING group [FUS/QA].

The PING method requires basic skills for the handling of small animals. These include the induction and maintenance of anesthesia, placement of a tail vein line, intravenous administration of multiple agents in the setting of an MRI unit, intravenous infusion of a drug, and animal care during the operative and postoperative periods. It also requires the ability to perform intracardial perfusion, tissue sectioning, histochemical staining, and microscopic analyses. Institutional training and approval from an Animal Care and Use Committee, or equivalent agency, is necessary for several of the steps in the protocol.

The initial protocol for the PING procedure utilized repetitive intraperitoneal injection of QA over multiple days^8,9. This approach is still viable and effective. However, the current protocol modified the route and rate of QA administration. In particular, QA was administered intravenously during a 1 h post-sonication period of infusion. Again, either administration protocol is effective. The 4–5 day survival period utilized in the current protocol was adopted to optimize the use of the Fluoro-Jade method for detecting degenerating neurons. If longer survival periods are required, then alternative tissue staining methods would be indicated. One such approach would be to use a neuron-specific antibody (e.g., anti-NeuN), to demonstrate immunohistochemically where surviving neurons remained after a lengthier post-sonication survival period. A variety of outcomes, in addition to the current structural analyses, would also be useful. For instance, post-PING assessments of electrophysiological and behavioral outcomes would significantly advance the characterization of the impact of the PING method.

One potential complication of FUS procedures is that temperatures could be elevated in the vicinity of the skull, particularly when targeting cortical areas located near the skull. This complication currently restricts the range of sites (treatment envelope) amenable to thermal lesioning using high intensity FUS (HIFU). In the context of PING, thermal increases near the skull may also represent a limitation of the technique. However, the low intensity of sonication used with PING reduces the risk of thermal injury, and should expand the treatment envelope as compared to HIFU.

The PING method possesses several features that are both infrastructure-intensive and training-intensive. MRI-compatible FUS equipment and an MRI facility equipped for animal research are required. This represents a considerable front-end investment, in order to provide the necessary research infrastructure. However, it is encouraging to note that the number of sites equipped with FUS technology is rapidly expanding for both research and clinical endeavors. Thus, as the number of available sites for performing such work increases, the opportunities for on-site and/or collaborative investigation will grow. In terms of training, any investigator seeking to undertake FUS studies would benefit from training by a research group experienced in FUS experimentation. For instance, the MRgFUS targeting software, while relatively straightforward, is more easily acquired when advised by an experienced investigator.

Multiple, alternative surgical modalities exist for the removal of disturbed neuronal circuitry. These include resective surgery, stereotactic laser ablation, radiosurgery, high-intensity focused ultrasound, and radiofrequency treatment. Each of these approaches is effective and can be of considerable therapeutic value for certain medically intractable disorders. However, these surgical modalities possess one or more specific limitations: (a) invasive procedure, (b) pannecrotic impact on target tissue, (c) damage to adjacent, non-target tissue (e.g., axons of passage), and (d) delay in achieving effective outcomes. Significant advantages of the PING method are that it: (a) is non-invasive, (b) is not pannecrotic, (c) limits effects on adjacent non-target tissue, and (d) should provide rapid outcomes.

There are several potential future directions for the PING method, including both preclinical and clinical applications. With respect to animal experimentation, the method provides a means for producing focal neuronal lesions in preclinical models of neurological disease. For instance, we have recently used this approach to test the effect of PING in a rodent model of limbic epilepsy¹⁹. Treatment with PING reduced the frequency of chronic, spontaneous seizures in a pilocarpine model of limbic epilepsy. This study provided the first, preclinical proof of concept for the utility of PING in treating a neurological disorder.

Quinolinic acid was selected for the PING procedure for two primary reasons. First, existing literature¹¹ indicates that neuronal cell body lesions can be produced while sparing other non-target tissue, such as axons of passage. Second, although QA is a neurotoxic when delivered directly to the brain, it is well tolerated when administered systemically because of its limited permeability through the BBB. The other toxins that are well tolerated peripherally, such as monosodium glutamate (MSG), could be considered as alternatives for QA. A key advantage of MSG would be that there exists a substantial literature regarding the use of this compound by humans. An important goal for future research will be to define the specificity of injury produced by QA or other systemically administered neurotoxins in terms of potential cell-type specificity. Another potential preclinical application for this general approach would be to administer a peripherally tolerated compound that is toxic to other cell types in the brain parenchyma, such as glial cells. For instance, peripheral injection of a toxin that affects oligodendroglia could allow focal demyelination in an area of white matter. This would allow targeted demyelination of part of a targeted pathway. Moreover, the approach might conceivably allow assessments of serial demyelination of the same site, which is a hallmark of relapsing-remitting multiple sclerosis.

With respect to the possible future applications of PING in humans, neurological disorders in which disturbed neural circuitries are a target could be amenable to treatment with PING. Based on our initial preclinical findings¹⁹, Drug Resistant Epilepsy (DRE) is a promising example of a disorder that might benefit from PING. Surgical treatment of DRE can be highly beneficial, but remains one of the most underutilized treatments that is actually effective for a major neurological disorder. An advantage of PING in this setting is that the volume of the target area can be conformal and enlarged by utilizing serial sonications at multiple sites. This would allow more precise targeting of irregularly shaped and irregularly sized targets in the brain parenchyma. The translational process for PING would necessarily involve testing for the safety of systemic administration of QA in non-human primates, and ultimately in humans. Kynurenine metabolites are typically cleared from the blood through the kidneys and eliminated via urination^20,21; however, the fate of injected QA has not been assessed in this model. Nonetheless, it is important to note in this context that high levels of systemically administered QA are well tolerated in rodents. Importantly, the use of PING would not preclude Standard of Care treatment. Were single or multiple PING treatments to prove ineffective in a given patient, then other procedures, such as resective or ablative surgery, would remain viable options. It is also important to recognize that the clinical environment into which PING would emerge is ideally poised for translation and implementation^{22,23,24,25,26}. Structural and functional imaging modalities are rapidly becoming more refined, which will better allow for the identification of appropriate target(s) for precise interventions. Also, there is no need for the design, construction, or approval of a new medical device for PING. MR-guided, high-intensity FUS is already an established and approved modality for multiple indications, including neurological applications. And, as mentioned earlier, recent years have witnessed a proliferation of sites at which FUS is already in clinical use. Together, these advantages suggest a promising course of development for PING for multiple applications.