Protocols in Current Issue
Protocols in Past Issues
0 Q&A 476 Views Jan 20, 2024

The blood–brain barrier (BBB) is a major obstacle to the diagnostics and treatment of many central nervous system (CNS) diseases. A prime example of this challenge is seen in glioblastoma (GBM), the most aggressive and malignant primary brain tumor. The BBB in brain tumors, or the blood–brain–tumor barrier (BBTB), prevents the efficient delivery of most therapeutics to brain tumors. Current strategies to overcome the BBB for therapeutic delivery, such as using hyperosmotic agents (mannitol), have impeded progress in clinical translation limited by the lack of spatial resolution, high incidences of complications, and potential for toxicity. Focused ultrasound combined with intravenously administered microbubbles enables the transient disruption of the BBB and has progressed to early-phase clinical trials. However, the poor survival with currently approved treatments for GBM highlights the compelling need to develop and validate treatment strategies as well as the screening for more potent anticancer drugs. In this protocol, we introduce an optical method to open the BBTB (OptoBBTB) for therapeutic delivery via ultrashort pulse laser stimulation of vascular targeting plasmonic gold nanoparticles (AuNPs). Specifically, the protocol includes the synthesis and characterization of vascular-targeting AuNPs and a detailed procedure of optoBBTB. We also report the downstream characterization of the drug delivery and tumor treatment efficacy after BBB modulation. Compared with other barrier modulation methods, our optical approach has advantages in high spatial resolution and minimally invasive access to tissues. Overall, optoBBTB allows for the delivery of a variety of therapeutics into the brain and will accelerate drug delivery and screening for CNS disease treatment.

Key features

• Pulsed laser excitation of vascular-targeting gold nanoparticles non-invasively and reversibly modulates the blood–brain barrier permeability.

• OptoBBTB enhances drug delivery in clinically relevant glioblastoma models.

• OptoBBTB has the potential for drug screening and evaluation for superficial brain tumor treatment.

Graphical overview

0 Q&A 449 Views Feb 20, 2023

The zebrafish retina is a canonical vertebrate retina. Since the past few years, with the continually growing genetic toolbox and imaging techniques, zebrafish plays a crucial role in retinal research. This protocol describes a method to quantitatively evaluate the expression of Arrestin3a (Arr3a) and G-protein receptor kinase7a (Grk7a) in the adult zebrafish retina at protein levels by infrared fluorescence western blot. Our protocol can be easily adapted to measure protein levels in additional zebrafish tissues.

0 Q&A 1640 Views Mar 5, 2022

Optogenetics has the potential to transform the study of the peripheral nervous system (PNS), but the complex anatomy of the PNS poses unique challenges for the focused delivery of light to specific tissues. This protocol describes the fabrication of a wireless telemetry system for studying peripheral sensory pathways. Unlike existing wireless approaches, the low-power wireless telemetry offers organ specificity via a sandwiched pre-curved tether, and enables high-throughput analysis of behavioral experiments with a channel isolation strategy. We describe the technical procedures for the construction of these devices, the wireless power transmission (TX) system with antenna coils, and their implementation for in vivo experimental applications. In total, the timeline of the procedure, including device fabrication, implantation, and preparation to begin in vivo experimentation can be completed in ~2-4 weeks. Implementation of these devices allows for chronic (>1 month) wireless optogenetic manipulation of peripheral neural pathways in freely behaving animals navigating homecage environments (up to 8).

0 Q&A 3536 Views Oct 20, 2019
Appetite is tightly linked to the sensory experience of feeding, including the smell, taste, and sight of food. Sensory perception can affect the palatability of food, modulating appetite beyond homeostatic requirements. Hypothalamic neurons that govern feeding are responsive to sensory cues associated with food, including food odors. However, the circuit mechanisms by which sensory information is processed and relayed to feeding nodes to affect feeding behavior is not well understood. Recent work has identified a population of excitatory basal forebrain neurons that modulate potent appetite suppression, as well as respond to food-associated and innately aversive odorants. To investigate this circuitry, we stereotaxically targeted virus expressing Cre-dependent channelrhodopsin to the basal forebrain and implanted fiber optic cannulas over the injection site. Mice were allowed to recover and underwent training to form a passive association of chow with a unique monomolecular odorant. After training, mice were fasted overnight, and were then presented with both the food-associated odor as well as a similar, novel odor in zones of an arena with and without photostimulation. To evaluate whether stimulation of this circuitry influenced sensory modulation of feeding behavior, video recording and behavioral tracking analysis were used to compare time spent investigating either odor. Thus, this protocol provides a useful paradigm to assay the contribution of different circuits in appetitive and aversive behaviors.

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