Permeabilization of the Blood-Brain Barrier via Mucosal Engrafting: Implications for Drug Delivery to the Brain

Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson’s disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS

Noninvasive optical inhibition with a red-shifted microbial rhodopsin

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.McGovern Institute for Brain Research at MIT (Razin Fellowship)United States. Defense Advanced Research Projects Agency. Living Foundries Program (HR0011-12-C-0068)Harvard-MIT Joint Research Grants Program in Basic NeuroscienceHuman Frontier Science Program (Strasbourg, France)Institution of Engineering and Technology (A. F. Harvey Prize)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) ProgramNew York Stem Cell Foundation (Robertson Investigator Award)National Institutes of Health (U.S.) (New Innovator Award 1DP2OD002002)National Institute of General Medical Sciences (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1RC1MH088182)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (Career Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS0848804)Society for Neuroscience (Research Award for Innovation in Neuroscience)Wallace H. Coulter FoundationNational Institutes of Health (U.S.) (RO1 MH091220-01)Whitehall FoundationEsther A. & Joseph Klingenstein Fund, Inc.JPB FoundationPIIF FundingNational Institute of Mental Health (U.S.) (R01-MH102441-01)National Institutes of Health (U.S.) (DP2-OD-017366-01)Massachusetts Institute of Technology. Simons Center for the Social Brai

Transmucosal marker delivery to striatum over time.

<p>A) Fluorescent microscopic images demonstrating transmucosal rhodamine-dextran delivery to the striatum(bregma 1.18 mm, bar = 0.5 mm, 72 h). Diffusion into the right striatum(ipsilateral to mucosal graft) occurs in a molecular weight dependent fashion while contralateral diffusion to the left striatum is negligible. B) The differential luminosity of the right(ipsilateral) striatum relative to the contralateral striatum following rhodamine-dextran exposure. While detectable striatal diffusion of the 20 and 40 kDa rhodamine-dextran conditions is seen, the 500 kDa rhodamine-dextran solution is associated with negligible striatal delivery. The differential luminosity of the striatum at 72 h following 20 kDa rhodamine-dextran delivery is significantly greater than at 12 h.</p

Murine graft model.

<p><b>A)</b> Sagittal MRI of a patient following endoscopic reconstruction of a skull base defect using a nasal mucosa graft(dotted white line, arrow denotes the proposed transmucosal pathway for HMW agents from the nose into the CNS through the graft). <b>B)</b> Illlustration of the murine graft model with the position of the graft(red circle) relative to the skull. The arrow denotes the equivalent transmucosal pathway to that seen on the MRI(Fig. 1A) utilized in our study. <b>C and D)</b> Cross sectional illustration of the skull base layers prior to and following craniotomy with dural removal and mucosal graft inset, respectively. Note that the dural layer(dura and arachnoid) contains the blood-cerebrospinal fluid barrier which restricts the transport of HMW molecules. <b>E)</b> Hematoxylin and eosin(H&E) section of the intact murine parietal bone with typical appearance of the inner and outer cortical tables with their associated diploic space(D) prior to engrafting(bar = 200 µm). <b>F</b>) H&E section of the mucosal graft implant in direct continuity with underlying brain parenchyma. Note the intact epithelial layer(E) consisting of pseudostratified columnar epithelium.</p

Area and intensity of marker diffusion over time.

<p>A)Fluorescent microscopic images demonstrating an increase in area and intensity of transmucosal rhodamine-dextran delivery over time(bar = 1 mm, bregma −1.06 mm, 40 kDa rhodamine-dextran). B) 3-D map of Fig. 3A quantifying increase in relative pixel luminosity intensity across each cross section over time(bregma −1.06 mm, 40 kDa rhodamine-dextran). C) Percent of the total cross sectional area containing detectable rhodamine fluorescence at bregma −1.06 mm. The overall trend describes an increasing percent area of staining as time increases and molecular weight decreases. Among the 40 kDa conditions, the percent area of rhodamine staining at 72 h is significantly greater than at 12 or 48 h. Among the 20 kDa conditions the percent area of rhodamine staining at 72 h is significantly greater than at 12 h. At 72 h, the percent area subtended by the 20 kDa condition is significantly greater than that of the 40 kDa condition. D) Weighted luminosity of rhodamine staining at bregma −1.06 mm. The overall trend describes an increasing weighted luminosity as time increases and molecular weight decreases. Among the 40 kDa conditions, the weighted luminosity at 72 h is significantly greater than at 12 or 48 h. Among the 20 kDa conditions, the weighted luminosity at 72 h is significantly greater than at 12 h.</p