Effect of enzymatic hyaluronan depletion on the structure and function of the brain’s extracellular space

Abstract

Hyaluronic acid (HA) is a large, abundant and unique matrix molecule situated in the brain’s extracellular space (ECS). In this project, I aimed to study the role of HA in the ECS and hypothesized that HA plays a significant role in: (1) diffusion-based transport through the ECS, and (2) maintaining ECS structural properties like volume and ultrastructural geometry. HA was cleaved and depleted by treating the brain tissue with hyaluronidase (Hyal) enzyme that was highly selective for HA. For treatment of acute brain slices with this expensive enzyme, we designed BubbleDrive, a 3D printed incubation chamber, that reduced the overall cost of the project by successfully maintaining the health of slices in a very low volume of incubation solution. Health of the BubbleDrive-incubated acute brain slices was validated through quantification of neuronal, glial and ECS-biophysical functions, and comparison with slices that were incubated in a conventional incubation chamber. Using immunohistochemistry, I confirmed that Hyal treatment depleted HA in the ECS. To quantify the transport through ECS in control and Hyal treated tissue, I used diffusion studies with ECS probe molecules (0.5-12 nm in diameter) that were excellent size-surrogates for many physiologically important molecules that are transported through the ECS, like neuroactive substances, metabolic and therapeutic proteins. I found that the extracellular diffusion of molecules was up to 25% more hindered in the absence of HA in the somatosensory cortex, both in acute brain slices and in vivo. Interestingly, the HA-depleted ECS was 50% larger in that same region. This means that the molecules were getting more hindered even when the space available for their diffusion was increasing. This seemingly counterintuitive result can be explained by formation and/or enlargement of residual wide spaces at junctions of cellular components, called dead-space microdomains. When molecules enter such microdomains, they become transiently trapped within, increasing their dwell-time, which delays their diffusive spread within the tissue. ECS ultrastructure analysis using electron microscopy showed a higher number of ECS expansions that can potentially act as dead-space microdomains in the Hyal treated tissue as compared to the control tissue. In addition, HA-depleted tissue had significantly more astrocyte-associated expansions, suggesting that some of these expansions could have formed because of astrocyte remodeling. Together, these results lead me to infer that: (1) HA promotes the extracellular diffusion of molecules with 0.5 -12 nm diameter, and (2) Presence of HA affects the ECS volume and ECS ultrastructure, possibly through astrocyte remodeling. I also found that HA depletion led to a partial loss of other matrix molecules, confirming its scaffold-like function within the extracellular matrix meshwork. These results signify HA’s role in maintaining ECS structure and function to support cellular communication, toxic metabolite clearance and drug delivery, all of which depend on diffusion-based transport through ECS. These results are also relevant in pathologies that involve increased cleavage of HA, like tumor tissue and neuroinflammation, and treatment strategies that involve complete HA synthesis inhibition, like 4-methylumbelliferone administration.