The aim of this procedure is to image the intact brain tissue interface around chronically implanted micro devices in rodent brain tissue. This is accomplished by first utilizing quick cell silicone elastomer and a two part dental acrylic to cover the craniotomy and form a skull cap around the brain implanted device. After perfusing the animal with fixative and post-mortem tissue processing, the brain implanted portion of the device is separated from the head cap by burning away the dental acrylic and cutting through the quick cell.
Next, the brain is removed and sliced with a viome to capture the implant in a tissue section. The final step is to prepare the tissue for fluorescent imaging by washing labeling, and then clearing the tissue section in optical clearing solution. Ultimately, confocal microscopy is used to show the intact biological response that the penetrating brain implant produce in surrounding tissue at the subcellular scale.
The main advantage of this technique over existing methods like explaning, the device, is that the technique avoids disrupting the tissue surrounding and adherent to the implanted device so that the interfacing tissue can be examined intact. This method relies on advanced immunohistochemistry and microscopy techniques and results in confocal microscopy data that describes in detail the region surrounding a brain implanted device. This method can help answer key questions in the neuroprosthetics field, such as the degree to which local cell death glial scar formation and blood vessel reorganization truly impact the lifetime of brain implanted micro electrode devices.
Though this method can provide insight into the chronic neural interface of brain implanted micro electrode arrays. The techniques involved in this method can also be applied to other areas of neuroscience research, including implanted cannulas or fiber optics In a fume hood, begin the procedure by carefully removing the skin and other tissues around the acrylic skull cap, using forceps and small scissors while wearing the heat insensitive gloves. Use a soldiering iron to produce a window in the dent acrylic and expose an area of underlying quick seal.
The device should be visible encased in the clear quick seal. Next, under a surgical microscope, cut into the silicon elastomer with a pair of curved micro scissors. Then remove small pieces of quick seal with tweezers and micro scissors down to the position where the device enters the brain.
Continue cutting along the surface of the craniotomy to separate the device components attaching to polysilicon and the skull from the components implanted in the brain. Be careful when cuttings through the exposed device components. Avoid pushing or dragging the implants in the fixed tissue.
Then remove the bone around the head cap with care using the raw shes after that. Use a spatula to separate the brain with the implanted device from the skull. Now place the brain in a glass Petri dish filled with HBHS.
Use the brain block and to razor blades to section the brain and create a flat plane closely matching the angle of the implanted devices. After that, press two razor blades through the brain tissue exposing the flat surface that will be mounted on the Vibram. Next, place some ice under the Vibram platform.
Then briefly dry the tissue surface on a paper towel. After that, orient the sample such that the widest dimension is parallel to the movement direction of the vibrate tone blade to help avoid the blade potentially knocking the tissue over immediately glue the flat plane to the stage with super glue. After the glue sets a chilled PBS to the vibrato dish around the tissue.
Now cut the tissue slices between 250 to 500 microns using the maximum vibration rate and a slow blade progression rate with a blade angle of 10 degrees from horizontal. Then collect slices carefully with a brush and scoop spatula and store an HBHS in a 24 well plate at four degrees Celsius while collecting slices, keep a record of the thickness and location of each slice as they're removed to the 24 well plate. Once the Insitu device is visible, collect a tissue slice around 500 microns thick.
In order to capture the device in a single tissue slice to collect a thinner tissue section, 100 micron slices may be carefully collected to closely approach the device. At which point a slice less than 500 microns thick can be collected with the device inside. Begin tissue processing by washing the slices three times at five minutes per wash in HBHS.
Then incubate them in sodium borah hydride for 15 minutes. Each side of the slice slowly flip the slice using a small paint brush and avoid touching any areas around the implanted device. A stainless steel or Teflon coated micro spoon may also be used along with the small paintbrush.
Now wash the slices three times at five minutes per wash in wash solution at room temperature. Block them in wash solution for two hours at room temperature while flipping the slices. After one hour after two hours, incubate the slices in primary antibody for approximately 48 hours at four degrees Celsius.
Next, perform six quick three minute washes with wash solution. Then perform six one hour washes in wash solution. After that, incubate them in secondary antibody for approximately 48 hours at four degrees Celsius.
Again, perform six quick three minute washes followed by six one hour washes in wash solution. Subsequently, remove the wash solution and add the glycerol based U2 scale clearing solution. Cover the plate with foil and store at four degrees Celsius.
Allow the thick slices to clear approximately one week or very thick tissue sections for two to three weeks of four degrees Celsius. After the clearance, mount the tissue sections on slides using clearing solution as mounting media. Seal all the slides with clear nail polish and store them at four degrees Celsius away from light, using longer working distance microscope objectives.
Begin imaging with a laser scanning confocal microscope or two photon microscope. We will demonstrate with a XS L SM seven 10 Carl Xi send software and a computer controlled XY translating stage to image A 3D panorama around an implant to correct the refractive index of the clearing solution. In this demonstration, we enter a refractive index value of 1.4 in the Leica Zen microscope software for the U2 clearing solution.
This setting subtly adjusts Z step intervals to account for refractive index mismatch in the tissue near the device. Roughly assess the appropriate Z axis window and data collection settings in the zdi dimension for each fluorescent channel using low laser power, about 0.5 to 5%and large Z step sizes. Then use the Zen panorama software to set the objective at the XY center of the eventual panorama and to determine the number of collection positions required for the x and y axis.
To cover your region of interest, reassess and finalize the Z axis collection window by quickly checking the first and last frames of the stack. Next, manually set the detector sensitivity and laser power to ramp appropriately with the increased depth. The aim is typically to maintain approximately the same image intensity regardless of imaging depth into the tissue slice, and also to avoid high background noise.
Collect each fluorescent image data series if necessary to avoid overlapping signals, run laser lines sequentially. Change the software settings to collect the data or the transmittance data and use a longer more penetrative wavelength laser, such as 6 33 nanometers to capture these channels. Then determine the appropriate laser power and detect a sensitivity for reflectance and transmittance collection and repeat the same collection series around the implanted device.
Finally, export the data for later processing quantification and analysis. Imaging through either side of the slide can allow you to assess the interface around a device as shown here where microglia along the silicon backing of an MEA are visible from one side and microglia along electrode sites and traces are visible on the other side. Once mounted tissue can be imaged using an XY translation stage, overviews of the fluorescent labels across the entire tissue slice can be generated at the desired resolution.
Here, cell nuclei stained with hooks 3, 3, 3 4 2 and monocytes microglia labeled with 3 2 9. Anti IBA one was simultaneously imaged on separate channels. Transmission light and reflectance were also collected showing the location of the four week implant with respect to hooks and IVA one data, the overlay of all channels, but transmission is shown here.
Detailed examination of the morphologically preserved tissue interface around implanted micro devices can be collected under high magnification for analysis of the local tissue response, the reflectance and transmittance allow the localization of the device while fluorescent antibody or chemical labels allow detailed imaging of tissue components along the intact tissue interface. While attempting this procedure, it's important to remember to really take time with the careful separation of the implanted devices through the head cap and plan for this step to take upwards of 30 minutes. After watching this video, you should have a good understanding of how to collect and process brain slices containing implanted devices.
You should also be able to label and collect histological data describing the neurobiology surrounding these devices.
