Only a small fraction of therapeutic antibodies are taken up by the brain. Using our method, antibodies are delivered into the brain by transiently opening the blood-brain barrier. This technique allows us to deliver antibodies into the mouse brain in a noninvasive manner.
To perform quality control of the micro bubbles using a cell counter, remove the micro bubble solution from the amalgamator and use a 19 gauge needle to pierce the septum of the micro bubble solution vial. Use a one milliliter syringe equipped with a 19 gauge needle to dilute 100 microliters of the micro bubbles in five milliliters of filtered flow solution and pipette 100 microliters of the diluted micro bubble solution in 10 milliliters of filtered flow solution in a cuvette. Lock the cuvette in place on the cell counter platform and make sure that a 30 micron aperture will be used for the sample acquisition.
In the software, load the standard operating method and select edit standard operating method and concentration. Enter 5, 000 times for the dilution and click apply and OK.Click edit info to select a suitable filename. Select preview and verify that the micro bubble sample concentration is less than 10%Select start and OK to begin the sample acquisition.
After the measurement, rinse the aperture of the cell counter with filtered flow solution. Sonicate the cuvette of diluted micro bubble solution for 30 seconds in a water bath. Measure the sonicated micro bubble solution as demonstrated and click edit info to label the data as blank.
After measuring the blank, subtract the final readout from the initial readout to exclude any particles that are not micro bubbles. To label the sections with fluorescent anti-mouse IgG antibody, label one milligram of mouse IgG antibody with Alexa Flour 647 in 0.1 molar sodium bicarbonate buffer according to the manufacturer's instructions for 15 minutes at room temperature. At the end of the incubation, load the fluorescently labeled antibody solution onto a spin column and purify the antibody by centrifugation.
Then use a spectrophotometer to measure the protein concentration. To set up the focused scanning ultrasound system, add a five millimeter space to the water bolus to position the ultrasound focus nine millimeters below the bottom of the water bolus. Fill the water bolus with approximately 300 milliliters of degassed deionized water and place the annular array into the filled water bolus.
Use a dental mirror to check that there are no air bubbles on the surface and launch the application software. In the waveform menu, select set waveform duty cycle and set the pulse repetition frequency to 10 Hertz, the duty cycle to 10%the focus to 80 millimeters, the center frequency to one megahertz, the amplitude to 0.65 megapascals, and the mechanical index to 0.65. Press set to define the waveform and store the waveform in memory.
After selecting the treatment plan, open the scan tab in the motion controller window and enter start, stop, and increment values for motion in the X dimension and start, stop, and increment values for motion in the Y direction. To define the actions for the treatment sites, click event and open the script editing window. Select a list of actions that will be executed in the order selected at each treatment site and set the movement type to raster grid.
In the events tab, select add actions and move the move synchronously start trigger ARB wait and stop trigger ARB actions to the script panel. Then click wait action and select a wait time of 6, 000 milliseconds. To prepare an animal for the analysis, after confirming a lack of response to toe pinch in an anesthetized mouse, use a permanent marker to label the center of the head, then glue plastic wrap to the bottom of a small weigh boat with the bottom cutoff and fill the weigh boat with ultrasound gel.
For focused ultrasound treatment, invert the vial of micro bubbles and gently load one microliter of the solution per gram of the mouse's body weight into a 29 gauge insulin syringe. Add 100 microliters of fluorescently labeled antibody to the syringe and gently invert and roll the syringe between thumb and index finger to mix the antibody and the micro bubbles within the syringe. Carefully and slowly retro-orbitally inject 150 microliters of the micro bubble and antibody solution into the mouse and set a timer for two minutes.
Apply ophthalmic ointment, then place the mouse in the head holder and fix its nose in the holder. Place the small ultrasound gel-filled weigh boat on top of the head and lower the water bolus until it sits on top of the ultrasound gel within the weigh boat. Then use the joystick to visually align the transducer focus within the center of the head.
When the timer goes off, in the motion tab of the software, select reset origin and select a complete scan. When the treatment is complete, apply ophthalmic ointment to the animal's eyes and place the mouse in a warmed recovery chamber. Here, representative results from the culture counter measurements of the size and concentration that can be obtained when the micro bubbles are produced correctly are shown.
Antibody uptake by the brain can be easily visualized in the whole brain or tissue sections using an infrared scanner or fluorescent microscopy of the sections. As demonstrated in these images, representative staining for microglia markers can be used to determine whether microglia become more phagocytic following the delivery of the antibody. It is important to ensure the welfare and safety of the animals especially when performing the retro-orbital injections and applying the transducer above the heads of the mice.
This method allows the application of various scanning patterns to target different regions of the brain, such as the hippocampus, cortex, or individual brain hemispheres. This technique can be used to develop new therapies and drugs that penetrate the blood-brain barrier and to study the mechanism of the blood-brain barrier formation and opening.
