(483g) Invited Talk: Treatment of Traumatic Brain Injury with Microbubbles

Traumatic brain injury (TBI) is a serious public health concern, accounting for hundreds of thousands of morbidities and thousands of fatalities per year in the United States alone. TBI includes injuries suffered on a regular basis by military personnel, athletes in certain sports, victims of domestic violence, and everyday falls, and accidents. Research has shown that the adverse effects of TBI persist for days or months, sometimes even years. Effects can include post-concussion syndrome and cognitive and behavioral deficits. Existing drug candidates under research require high doses to compensate for drug loss due to partial or complete blood brain barrier (BBB) impermeability, which can cause adverse side-effects. No clinically approved drug to directly treat TBI exists in the market, with the inability to deliver a drug into the brain being a major challenge. In this talk, we will discuss our recent and continuing work on using a novel drug delivery platform wherein the noble gases xenon or argon are delivered individually or combinatorially to the brain after a moderate brain injury.

Therapeutic gases like xenon (Xe) and argon (Ar) have been shown to be an effective treatment for arresting the progression of acute TBI. Xenon (Xe) is known to be an antagonist of NMDA and AMPA receptors that are key in excitotoxic signaling following TBI. Argon (Ar) is postulated to upregulate PI3/Akt and ERK1/2 pathways to arrest neuronal degeneration. Importantly, both gases freely diffuse across the blood brain barrier and have excellent safety profiles. The problem remains that while Xe treatment especially has shown great promise in both pre-clinical and clinical settings, delivery is via inhalation. Inhalation is systemic, may cause side effects due to high dosage, and leads to low and uncontrolled gas concentration in the brain.

The most effective method would be to locally deliver high quantities of gas via a standard IV methods, while being able to image the delivery vehicle through a low cost, noninvasive technique like ultrasound. To that end, we use microbubbles (MBs) as a solution to these issues since bubbles have high gas payload while being extremely echogenic under clinical ultrasound. Microbubbles (MBs) are 1-10 µm gas particles, stabilized by a biocompatible phospholipid, polymer, or protein shell. Most commonly, the core is a water-insoluble, inert perfluorocarbon (PFC) gas, while the shell is a phospholipid-lipopolymer mixture which reduces the surface tension at the aqueous/gas interface. MBs are mainly used for contrast-enhanced ultrasound (CEUS) imaging due to their exceptional scattering and non-linear volumetric oscillation properties under clinical ultrasound frequencies. In recent years, MBs have been used to carry and deliver therapeutic gases like oxygen and nitric oxide, or xenon stabilized by perfluorocarbons. Due to the high water-solubility and diffusivity of Xe, Xe is difficult to stabilize in an MB core. To address that, we developed the first examples of stable, echogenic MBs encapsulating pure xenon or argon without any solubilizing agents, through optimization of the phospholipid composition of the microbubble shell. We have demonstrated that a long-chain phospholipid dibehenoylphosphatidylcholine (DBPC, C22:0), can formulate storage-stable sub-10 µm Xe and Ar MBs – these MBs shown consistent echogenicity in vivo in a mouse and a porcine model. This is likely due to the greater intermolecular van der waals cohesive energy for long-chain lipids which provides rigid packing to reduce gas efflux.

Before starting our current lab, we gathered preliminary data that demonstrates neuroprotection by administration of XeMBs in a porcine model of moderate TBI. We showed reduced edema after IV treatment with XeMBs following a controlled cortical impact injury. We also observed the protection of the cerebral vasculature, a therapeutic effect of xenon that hasn’t been explored in detail previously. Fibrinogen staining showed lower protein extravasation outside the cytoplasm in brain tissue, indicating greater vascular integrity post-XeMB treatment. Preliminary trials with an in vitro model of the BBB composed of benD.3 cells showed the positive effect of XeMBs in preserving tight junction protein (ZO-1) expression after excitotoxic shock as an indication of potential BBB protection.

In the recently established Chattaraj Lab, we are conducting studies to explore the therapeutic efficacy of Xe and Ar MBs in detail, including both behavioral and histological studies. In our study, adult Sprague-Dawley rats are subjected to moderate TBI via a fluid percussion injury apparatus, at 1.8-2 atm peak pressure. MBs containing Xe are administered at 1 hr and 24 hr post injury through a tail vein infusion over 5 min. A clinical ultrasound scanner (Siemens S3000 9L4) is held at the carotid artery to first visualize the signal from the MBs under CEUS. Then, the acoustic intensity of the scanner is increased to rupture the bubbles and release the gas near the brain. At 3 days post-injury, animals are tested for memory and anxiety using Novel Object Recognition and Open Field tests. Next, animals are sacrificed, and brain tissue is collected. Tissue sections are stained for reactive microglia (Iba1 protein), reactive astrocytes (GFAP), ZO-1 and Connexin-43 (tight and gap junction proteins to test for the potential efficacy of MBs in treating the BBB). Our preliminary results show significant improvement of memory with treated animals as compared to that of the control (injured, no treatment) animals. Similarly, we notice lower levels of reactive glial cells and higher levels of ZO-1 indicating a distinguishable positive effect of XeMBs for reducing secondary injury progression. We are currently conducting trials on a greater number of animals to increase the statistical significance of our findings. Soon, we will conduct studies with Ar MBs with the same outcome measurements. We also plan to conduct studies in the next few months for longer-term effect of MBs following injury (7, 30 days).

Finally, this talk will also briefly discuss neuroprotective peptides and polymers that our lab is using to treat moderate TBI, both systemically, and through their incorporation into microbubbles for non-invasive localized delivery.