Background

The majority of cholinergic afferents in cortical and limbic structures involved in cognition originate from long-range projection neurons located in the basal forebrain (Hampel et al., 2018). Basal forebrain cholinergic neurons (BFCNs) are highly vulnerable in Alzheimer’s disease (AD) and other neurodegenerative diseases, including vascular dementia, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, and Parkinson’s disease, and contribute significantly to cognitive decline (Cykowski et al., 2014; Grothe et al., 2014; Hampel et al., 2018; Ray et al., 2018; Convery et al., 2020), validating BFCNs as an important therapeutic target. Although current clinically approved cholinergic agents for the treatment of AD have shown only modest benefits on cognition, recent studies suggest that they may also modify the course of disease with continued use (Dubois et al., 2015; Cavedo et al., 2016 and 2017). It is possible that the modest impact on cognition may reflect the drug dose that can be achieved; higher doses, which could be of greater benefit, cannot be tolerated due to peripheral side effects (Birks and Harvey, 2018). Other putative cholinergic agents, such as trophic factors and related gene therapies, which have the potential to simulate cholinergic function and prevent cholinergic degeneration, do not cross the blood-brain barrier (BBB) (Chen and Mobley, 2019; Xhima and Aubert, 2021). Thus, a major obstacle for the development of cholinergic-targeted agents, and brain-targeted therapeutics in general, is not only the production of biologically active compounds but a way to deliver them safely and efficiently to the brain.

Approximately 98% of small molecules and nearly 100% of large molecule drugs, including recombinant proteins, antibodies, and gene-related therapeutics, are unable to cross the BBB when administered systemically (Pardridge et al., 2020). Current approaches that accomplish direct drug delivery involve highly invasive intracranial procedures with multiple injections to reach the basal forebrain; these procedures carry substantial risk for surgical complications, and control of drug distribution can be difficult because drug concentration decreases exponentially from the injection site (Honig et al., 2018; Castle et al., 2020; Pardridge et al., 2020). Other approaches include: (1) intra-arterial injection of hyperosmotic solutions, such as mannitol, to increase BBB permeability, coupled with peripheral drug delivery; (2) systemic administration of therapeutics linked to protein vector delivery systems that can cross the BBB; (3) intraocular delivery; and (4) intranasal administration. However, even if therapeutics reach the brain in adequate concentrations with these methods, drug biodistribution will be non-targeted and may cause indiscriminate actions on all cholinergic synapses in the brain rather than BFCNs.

Transcranial focused ultrasound (FUS) in combination with intravenously injected microbubbles (i.e., an air/perfluorocarbon gas core stabilized by a phospholipid/protein/polymer shell) can transiently enhance BBB permeability in the targeted volume (Hynynen et al., 2001). FUS offers a minimally invasive avenue to deliver a variety of therapeutic agents to the brain with the advantages of being able to temporarily permeabilize the BBB in specific brain regions without widespread drug exposure elsewhere in the brain and to potentially reduce the peripheral dose required to achieve the desired bioeffects in the brain (Meng et al., 2020). Additionally, FUS targeting systems can be co-registered to the spatial coordinates of MRI scanners, allowing for highly precise BBB modulation in specific neuroanatomical locations, such as the basal forebrain (Xhima et al., 2020). Gadolinium-based MRI contrast agents, excluded by the intact BBB, offer the added advantage of confirming the site of therapeutic delivery following FUS exposure. In a recent study, we established the feasibility of MRI-guided FUS (MRIgFUS)-induced BBB modulation in the basal forebrain for drug delivery in an AD mouse model (Xhima et al., 2020). Here, we present a detailed protocol for using MRIgFUS to non-invasively and locally deliver intravenously administered therapeutic agents to the murine basal forebrain for preclinical drug development.