Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Solutions

Phosphate Buffered Saline (PBS) – in g/l; 9 g NaCl, 0.144 g KH₂PO₄, 0.795 g Na₂HPO₄, at pH 7.4

4% Formaldehyde – in ml/l; 202 ml sodium phosphate dibasic solution (0.4 M Na₂HPO₄), 48 ml sodium phosphate monobasic solution (0.4 M NaH₂PO₄), 500 ml 8% formaldehyde solution, 250 ml Milli-Q DDi water, at pH 7.4

HEPES Buffered Hank’s Saline with Sodium Azide (HBHS) – in g/l; 7.5 g NaCl, 0.3 g KCl, 0.06 g KH₂PO₄, 0.13 g Na₂HPO₄, 2 g glucose, 2.4 g HEPES, 0.05 g MgCl₂ 6 parts H₂O, 0.05 g MgSO₄ 7 parts H₂O, 0.165 g CaCl₂, 0.09 g NaN₃ at pH 7.4

Wash Solution (WS) – 1% Vol/Vol, Normal Goat Serum, 0.3% Triton X-100, in HBHS with sodium azide (NaN₃). Refrigerate the WS at 4 °C before use in subsequent steps.

U2 Scale Solution – 4 M urea, 30% glycerol and 0.1% Triton X-100 ¹⁷

PROCEDURE

All experiments were done under the supervision of the Purdue Animal Care and Use Committee and the Laboratory Animal Program at Purdue University.

1. Surgery

1. Various aseptic surgical methods are compatible with the presented histological method. The use of a stereotaxic frame and automated inserters are recommended to improve reproducibility and control of the microelectrode arrays (MEA) implantation angle with respect to stereotaxic planes.

Note: In this demonstration our surgical method is as follows: hold the rodent subject in stereotaxic earbars while under isoflurane anesthetic (between 1-3%) carried by medical grade oxygen. Test for the lack of a toe-pinch reflect in the rat or lack of a tail pinch reflex in the mouse to confirm that the animal is fully anesthetized. Apply eye ointment and clean the surgical site with three alternating washes of Betadine and ethanol. Wash hands, dawn surgical gloves, hairnet, facemask and gown. Inject a bolus of Lidocaine to numb the surgical area, create an incision using scissors or a scalpel, clear periosteum with bone scraper and cotton applicators, and create a craniotomy using a dental drill handpiece and drill bit. Drive a single-shank MEA into the cortex using a micromanipulator. Have a surgical assistant aid in maintaining aseptic surgery conditions and document the progress of the surgery.

2. Following implantation of the MEA into the brain, collect images through a surgical microscope to inform the eventual removal of the skull from around the brain. Implanted portions of the devices will be preserved in situ.

Note: The eventual collection of tissue with in situ devices relies on closely matching the plane of device insertion with the plane of tissue sectioning. Detailed notes about the device insertion plane relative to the brain are thus very important.

3. After device implantation, apply the silicone elastomer Kwik-Sil (WPI), around any exposed MEA shanks or cabling, and allow to cure. A sterilized plastic piece, such as a cut pipette tip, may be used to help contain the silicone elastomer in a small well around the craniotomy while curing.
4. Apply a layer of two-part dental acrylic (“Jet Liquid” and “Jet Powder” from Lang Dental) over the Kwik-Sil and any exposed skull.

Note: In a later step, the headcap will be melted through to allow for the implant to be separated from the skull. UV-curing acrylic should be avoided over the Kwik-Sil and craniotomy, as this very hard acrylic is difficult to remove later. Visually opaque materials around the implant should also be avoided; clear Kwik-Sil provides a view of the implant during subsequent steps of this protocol.

5. Perform post-operative care procedures as per the local protocol of the animal welfare regulations, and return ambulatory subjects to single-housed cage-boxes.

2. Perfusion and Tissue Collection

Note: See Gage et al. for a detailed perfusion procedure walkthrough in the rat animal model¹⁹.

1. Use the anesthesia and perfusion protocol approved by the institution’s animal care and use committee. After deeply anesthetizing the rodent (confirmed by a lack of toe-pinch reflex in rat or tail-pinch reflex in mice) and exposing the heart, make shallow scissor cuts into the left ventricle and right atrium. Insert a blunt needle into the left ventricle and deliver room temp PBS. Deliver a total of ~10 ml PBS at ~5 ml/min in mice and ~200 ml PBS at ~100 ml/min in rats. Look for blood clearance from the liver during.
2. Inject histologically relevant chemical labels transcardially, if so desired, by syringe delivery with care taken to avoid introducing bubbles.

Note: Vascular labels (e.g. DiI) and nucleic acid counterstain dyes (e.g. Hoechst 33342) are examples of chemical labels deliverable during perfusion. When administered properly, these histological markers should label the whole animal²⁰.

3. Deliver buffered, room temperature, 4% formaldehyde transcardially to fix the animal. Avoid septum perforation across the heart, which can be evidenced by lung-nose drainage during perfusion. Look for fixation tremors in the large muscles to indicate complete perfusion. Deliver a total of ~10 ml fixative at ~5 ml/min in mice and ~200 ml fixative at ~100 ml/min in rats.
4. Following perfusion, decapitate the animal, expose part of the brain stem or cerebellum using rongeurs or scissors, and place head in 4% formaldehyde solution for overnight at 4 °C.
5. Wash the head in three changes of PBS with one to four hour intervals between washes.
6. Store fixed heads in HBHS containing sodium azide at 4 °C for days to months.

3. Brain Removal with in situ Devices

Note: Perform this section in a fume hood while wearing appropriate personal protective equipment. Avoid tissue desiccation by returning the fixed head to HBHS solution periodically.

1. Carefully dissect away skin and other tissues from around the acrylic skull cap using forceps and small scissors.
2. While wearing heat-insensitive gloves, use a soldering iron to remove dental acrylic and expose an area of underlying clear Kwik-Sil (Figure 2a).

Note: The goal of this step is to allow micro-scissors an appropriate angle of access to positions where the implanted devices enter the brain, so that brain-implanted components may be separated from components secured to the skull.

3. Under a surgical microscope, cut into the silicone elastomer with curved micro-scissors, removing small pieces of Kwik-Sil with tweezers down to the position where the devices enter the brain.
4. Continue cutting along the surface of the craniotomy with curved micro-scissors to separate device components attached to polysilicon and skull from components implanted in the brain. Again use microscope magnification and great care when cutting through the exposed device components, taking care to avoid pushing or dragging the implants in the fixed tissue.
5. Remove bone around the headcap with care using rongeurs. Use a spatula to separate the brain with implanted devices from the skull. Store the brain in HBHS solution until ready to slice.
6. Place the brain in a glass Petri dish filled with HBHS, and use a razor blade to remove extraneous tissue, such as spinal cord, cerebellum, or olfactory bulb, depending on whether they are relevant to the location of the device implantation. Use a brain block (Ted Pella) and razor blades to section the brain and create a flat plane closely matching the angle of the implanted devices; this is accomplished by placing the brain in an appropriately-sized brain block, orienting the brain such that any visible portion of the implant is parallel with the razor blade guide, and placing a razor blade in a blade guide at least 2 mm from the implant. Press the razor blade through the tissue. Mount this surface to a vibratome platform. Alternatively, a razor blade mounted to a micromanipulator can be used in a similarly controlled fashion as the brain block to create a plane closely matching the direction of the implant.
7. Place ice under the vibratome platform, and, after allowing the tissue surface to briefly dry on a paper towel, adhere the brain to a vibratome using Super Glue. Glue the flat tissue plane created in the previous step is to the stage. With asymmetric tissue dimensions, orient the sample such that the widest dimension is glued down parallel with the movement direction of the vibratome blade to help avoid the blade potentially knocking the tissue over. After the glue sets (~1-2 min), add chilled (~4 °C) PBS to the vibratome dish around the tissue.
8. Collect thick tissue slices between 200-400 μm, including control tissue, using the maximum vibration rate (~100 Hz) and a slow blade progression rate (< 0.2 mm per second), with a blade angle of 10 ° from horizontal. Collect slices carefully with a brush or scoop spatula and store in HBHS in a 24 well plate at 4 °C.
9. Once the in situ device is visible to the unaided eye or a surgical microscope, collect thinner sections to approach the device in slice increments of 100 μm or less.

Note: Typical silicon micro-implants are visible with the unaided eye in formaldehyde-fixed rodent cerebral cortex tissue between 300 and 500 μm from the device surface.

10. With the in situ device now close to the surface of the tissue, collect a >250 μm tissue slice containing the device within. Estimation of the necessary thickness can be aided by referencing previously collected slices.

Note: Larger devices, multishank devices, and angled devices may require thicker tissue slices (>500 μm) to capture the device in one tissue piece; remember that both the penetration of applied labels and the microscopy imaging depth will be limited in the eventual data collection from these very thick slices. If possible, avoid striking the device with the vibratome blade, as this can cause dragging of the device through the tissue and morphological deformation.

4. Tissue Processing and Clearing

1. Wash slices 3x at 5 min per wash in HBHS
2. Incubate in sodium borohydride (5 mg NaBH₄/ 1 ml of HBHS) for 30 min total (15 min each side of the slice). Flip the slices using a stainless steel or Teflon coated micro spoon and small paintbrush (Ted Pella). Slow and thoughtful movements improve the success of each slice flip. Avoid touching any areas around the implanted device with the brush. When possible, adding significantly more solution than required (>2 ml) allows one to manipulate and flip the tissue slices more easily.

Note: This step reduces endogenous autofluorescence; skip this step if fluorescent labels were applied during perfusion or xFP transgenic markers are present.

3. Wash slices 3x at 5 min per wash in Wash Solution at room temperature.

Note: Agitation during wash and incubation steps is optional. The authors speculate that subtle jarring of the stiff implant with respect to the surrounding brain tissue may occur with agitation. However, an orbital mixer moving at a gentle rate (< 60 rpm) may avoid this while increasing the movement of solutions.

4. Block for 2 hr in Wash Solution at room temperature (flip slices after one hour).
5. Incubate in primary antibodies (diluted in Wash Solution) for approximately 48 hr (24 hr on each side of the slice) at 4 °C.
6. Perform 6 quick 3 min washes with Wash Solution.
7. Perform 6, 1 hr washes (3 washes on each side of the tissue slice) in Wash Solution (store at 4 °C if washing overnight).
8. Incubate in secondary antibodies (diluted with Wash Solution) for approximately 48 hr (24 hr on each side) in 4 °C.
9. Perform 6 quick 3 min washes with Wash Solution.
10. Perform 6, 1 hr washes (3 washes on each side) in Wash Solution; store at 4 °C if washing overnight.
11. Remove Wash Solution and add the glycerol-based “U2 – Scale” clearing solution. Allow to clear approximately 1 week for thick slices (250-500 μm) or 2-3 weeks for very thick tissue sections (>500 μm) at 4 °C.
12. Cover plate with foil and store at 4 °C.
13. Mount in slides accommodating thick tissue sections (Figure 2b, 2c) using clearing solution as mounting media and sealing with clear nail polish. Store at 4 °C away from light.

5. Panorama Imaging of Full Tissue and Device Interface

1. Using longer-working distance microscope objectives (> 2 mm), begin imaging with a laser scanning confocal microscope or 2-photon microscope. We will demonstrate with a Zeiss LSM 710, Carl Zeiss “Zen” software (2010), and a computer-controlled XY translating stage to image a 3D panorama around an implant.

Note: Correct for the refractive index of the clearing solution either in software, through a corrective collar on the objective, or in data processing after collection. In this demonstration we enter a refractive index value for the “U2” clearing solution of 1.4 into the Leica Zen microscope software; this setting subtly adjusts z-step intervals to account for refractive index mismatch.

2. In tissue near the device, roughly assess the appropriate z-axis window and data collection settings in the z-dimension for each fluorescent channel using low laser power (~0.5 to 5%) and large z-step sizes (>25 μm).

Note: Include additional space above and below when setting the z-axis when setting up for panoramic data collection, as the tissue may not lie entirely flat on the slide glass (~20 μm each direction).

3. Set the objective at the x-y center of your eventual panorama; using the Zen panorama software, determine the number of collection positions required for each axis (x and y) to cover your region of interest.
4. Reassess and finalize the z-axis collection window.
5. Manually set the detector sensitivity and laser power to ramp appropriately with increased depth; the aim is typically to maintain approximately the same image intensity regardless of imaging depth into the tissue slice, but to also avoid high background noise.
6. Collect each fluorescent image data series. If necessary to avoid overlapping signals, run laser lines sequentially.

Note: If available, set the software to automatically save data to the hard drive during collection.

7. Change the software settings to collect the “reflectance” and “transmittance” data, and use a longer, more penetrative wavelength laser (e.g. 633 nm) to capture these channels. Determine the appropriate laser power and detector sensitivity for “reflectance” and “transmittance” collection and repeat the same collection series around the implanted device.
8. Export the data for later processing, quantification, and analysis.