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, 218 (1), 121-30

A Cranial Window Imaging Method for Monitoring Vascular Growth Around Chronically Implanted micro-ECoG Devices

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A Cranial Window Imaging Method for Monitoring Vascular Growth Around Chronically Implanted micro-ECoG Devices

Amelia A Schendel et al. J Neurosci Methods.

Abstract

Implantable neural micro-electrode arrays have the potential to restore lost sensory or motor function to many different areas of the body. However, the invasiveness of these implants often results in scar tissue formation, which can have detrimental effects on recorded signal quality and longevity. Traditional histological techniques can be employed to study the tissue reaction to implanted micro-electrode arrays, but these techniques require removal of the brain from the skull, often causing damage to the meninges and cortical surface. This is especially unfavorable when studying the tissue response to electrode arrays such as the micro-electrocorticography (micro-ECoG) device, which sits on the surface of the cerebral cortex. In order to better understand the biological changes occurring around these types of devices, a cranial window implantation scheme has been developed, through which the tissue response can be studied in vivo over the entire implantation period. Rats were implanted with epidural micro-ECoG arrays, over which glass coverslips were placed and sealed to the skull, creating cranial windows. Vascular growth around the devices was monitored for one month after implantation. It was found that blood vessels grew through holes in the micro-ECoG substrate, spreading over the top of the device. Micro-hematomas were observed at varying time points after device implantation in every animal, and tissue growth between the micro-ECoG array and the window occurred in several cases. Use of the cranial window imaging technique with these devices enabled the observation of tissue changes that would normally go unnoticed with a standard device implantation scheme.

Keywords: Brain computer interfacing; Cranial window; Micro-electrocorticography; Neural electrode; Tissue response; Vasculature.

Figures

Figure 1
Figure 1. Micro-ECoG device and fabrication process
A.The micro-ECoG device consisted of a Parylene C substrate with 16 Cr/Au/Pt electrode sites. 12 holes through the Parylene substrate are visible between the electrode sites. Scale bar represents 750 μm. B. Micro-ECoG fabrication process. A: 15 μm of Parylene was deposited on a blank Si wafer. B: 1813 photoresist was used to pattern the electrode sites and traces onto the Parylene. C: 10 nm of chrome, 200 nm of gold, and 20 nm of platinum was deposited using an electron beam evaporator. D. Lift off techniques were used to remove the metal that was on top of the photoresist. E. 10 μm of Parylene was deposited onto the sample. F. Photoligthography was used to create an etch mask. G. RIE was used to etch through the 1st 15 μm of Parylene, creating the holes and the outline of the micro-ECoG device. H. A second photoresist etch mask was patterned. I. RIE was used to etch the Parylene off of the electrode sites and finish etching through the holes and the MEA outline. J. The sample was soaked in water and the devices were released from the Si wafer.
Figure 2
Figure 2. Implantation scheme
A.Cross-sectional diagram of the window imaging implantation scheme, in which a glass cover slip was affixed over the top of an epidurally implanted micro-ECoG array. B. Top view of the cranial window implant setup. C. Rat implanted with a micro-ECoG array and cranial window.
Figure 3
Figure 3. Example of observed vascular growth
Blood vessels growing over the top of an epidurally implanted micro-ECoG array 30 days after implantation. Image was taken using an upright fluorescent microscope with broad spectrum light and a GFP-2 filter. Rat was injected with 6 mg of FITC-Dextran dissolved in 0.5 ml saline solution. Scale bar represents 750 μm.
Figure 4
Figure 4. The in vivo progression of vascular growth over an epidurally implanted micro-ECoG device
A. 3 days post implantation. B. 5 days post implantation. C. 8 days post implantation. D. 10 days post implantation. E. 15 days post implantation. F. 17 days post implantation. G. 22 days post implantation. H. 24 days post implantation. Scale bars represent 750 μm. At day 5 blood vessels begin to grow through a hole in the upper left corner of the Parylene substrate. Over time, the vessels spread out over the entire top surface of the device. After 24 days, the vasculature seems to have stabilized, no more significant vessel changes occur after this point. Images in A. and E. were taken in bright field because the tail-vein injection of FITC-dextran was unsuccessful on those days.
Figure 5
Figure 5. Micro-hematomas occurring in different animals at different time points after device implantation
A. 2 days post implantation, B. 3 days post implantation, C. 15 days post implantation, D. 20 days post implantation. Scale bars represent 750 μm.
Figure 6
Figure 6. Cranial windows remain clear for varying lengths of time
A and B. Images of the vasculature around a micro-ECoG device 2 months after implantation. C and D. Images of the micro-ECoG device 19 days after implantation. Scale bars represent 750 μm. Note the growth of opaque connective tissue over the majority of the cranial window.
Figure 7
Figure 7. Impedance changes over time
Plot of average change in impedance (from initial impedance measurement after implantation) at 1 kHz (A.) and 90 Hz (B.) for four animals and all viable channels (60 channels) over time. Error bars represent +/- one standard deviation.
Figure 8
Figure 8. Cortical vasculature labeled with DiI in coronal brain sections
A. Region away from implant. B. Region beneath control window. C. Region beneath Micro-ECoG device. Scale bars represent 500 μm.
Figure 9
Figure 9. Vasculature beneath the array
Blood vessels visible beneath the micro-ECoG device two days after implantation. Scale bar represents 750 μm.

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