Brain Mapping Using a Graphene Electrode Array

Podsumowanie

We present a graphene array-based brain mapping procedure to reduce the invasiveness and improve spatiotemporal resolution. Graphene array-based surface electrodes exhibit long-term biocompatibility, mechanical flexibility, and suitability for brain mapping in a convoluted brain. This protocol allows for constructing multiple forms of sensory maps simultaneously and sequentially.

Streszczenie

Cortical maps represent the spatial organization of location-dependent neural responses to sensorimotor stimuli in the cerebral cortex, enabling the prediction of physiologically relevant behaviors. Various methods, such as penetrating electrodes, electroencephalography, positron emission tomography, magnetoencephalography, and functional magnetic resonance imaging, have been used to obtain cortical maps. However, these methods are limited by poor spatiotemporal resolution, low signal-to-noise ratio (SNR), high costs, and non-biocompatibility or cause physical damage to the brain. This study proposes a graphene array-based somatosensory mapping method as a feature of electrocorticography that offers superior biocompatibility, high spatiotemporal resolution, desirable SNR, and minimized tissue damage, overcoming the drawbacks of previous methods. This study demonstrated the feasibility of a graphene electrode array for somatosensory mapping in rats. The presented protocol can be applied not only to the somatosensory cortex but also to other cortices such as the auditory, visual, and motor cortex, providing advanced technology for clinical implementation.

Wprowadzenie

A cortical map is a set of local patches representing response properties to sensorimotor stimuli in the cerebral cortex. They are a spatial formation of neural networks and enable prediction for perception and cognition. Therefore, cortical maps are useful in evaluating neural responses to external stimuli and processing sensorimotor information^1,2,3,4. Invasive and noninvasive methods are available for cortical mapping. One of the most common invasive methods involves the use of intracortical (or penetrating) electrodes for mapping^5,6,7,8.

Assessing the on-demand high-resolution cortical maps using penetrating electrodes has faced several obstacles. The method is too laborious to obtain a decent map and too invasive to implement for clinical use, prohibiting further development. More recent technologies such as electroencephalography (EEG), positron emission tomography (PET), magnetoencephalography (MEG), and functional magnetic resonance imaging (fMRI) have gained popularity because these are less invasive and reproducible. However, given their prohibitive costs and poor resolution, they are used in a limited number of cases^9,10,11. Recently, flexible surface electrodes with superior signal reliability have attracted considerable attention. Graphene-based surface electrodes demonstrate long-term biocompatibility and mechanical flexibility, providing stable recordings in a convoluted brain^{12,13,14,15,16}. Our group has recently developed a graphene-based multichannel array for high-resolution recording and site-specific neurostimulation on the cortical surface. This technology allows us to keep track of the cortical representations of sensory information for an extended period.

This article describes the steps involved in acquiring a brain map of the somatosensory cortex using a 30-channel graphene multielectrode array. To measure brain activity, a graphene electrode array is placed on the subdural area of the cortex, while the forepaw, forelimb, hind paw, hindlimb, trunk, and whiskers are stimulated with a wooden stick. The somatosensory-evoked-potentials (SEPs) are recorded for somatosensory areas. This protocol can also be applied to other brain areas, such as the auditory, visual, and motor cortex.

Protokół

All animal-handling procedures were approved by the Institutional Animal Care and Use Committee of the Incheon National University (INU-ANIM-2017-08).

1. Animal preparation for surgery

NOTE: Use Sprague Dawley Rat (8-10 weeks old) without the sex bias for this experiment.

1. Anesthetize the rat with 90 mg/kg ketamine and 10 mg/kg xylazine cocktail intraperitoneally. To maintain the desired depth of anesthesia throughout the surgery, provide a supplemental 45 mg/kg ketamine and 5 mg/kg xylazine cocktail when the rat shows signs of waking up.
2. Confirm that the rat is under deep anesthesia and regularly check body reflections such as toe pinch, tail pinch, and corneal reflex.
3. Shave the fur between the eyes and the back of the ears using a trimmer.
4. Apply an ophthalmic ointment onto the eyes to prevent them from drying out.

2. Surgery for cortical surface exposure

1. Fix the rat head on the stereotaxic apparatus with a stereotaxic adaptor. To maintain the body temperature of 37 °C during the surgery, place the rat on a temperature-controlled heating pad.
2. Sterilize the shaved area with alternating scrubs of alcohol and povidone-iodine three times.
3. Use forceps to grasp the scalp firmly and inject 0.1 mL of lidocaine (2%) with a syringe directly into the scalp to induce local anesthesia in the surgery area.
4. Make a 2-3 cm long midline incision with a scalpel and pull apart the scalp to expose the skull.
5. Clamp the scalp with mosquito forceps to expose the skull.
6. Scratch the surface of the skull with forceps to remove the periosteum.
7. Blunt dissect the muscles over the occipital skull to expose the cisterna magna above the axis on the top of the spinal cord.
8. Incise the cisterna magna with the blade to drain the cerebrospinal fluid and put a sterile gauze inside the incision of the cisterna magna to absorb the cerebrospinal fluid constantly to prevent brain edema and minimize inflammation.
9. Using a pencil, mark on the skull a rectangular window measuring 3 mm in the anteroposterior axis and 6 mm in the right lateral direction from the bregma of the right hemisphere.
   NOTE: Marking must secure a 1 mm distance from the midline to avoid superior sagittal sinus rupture.
10. Drill the marked area according to the stereotaxic coordinate and remove the skull with a bone rongeur.
11. To remove the dura mater, bend the tip of the 26 G needle to 90°, create a hole in the dura mater, lift the dura mater, insert forceps into that hole, and tear it with forceps.
12. Place saline-wetted gauze on the somatosensory cortex to prevent it from drying out.

3. Preparation of graphene electrode array connected to the recording system

1. Prepare a graphene electrode array with an omnetics connector.
   1. Detach the graphene multielectrode array without causing damage by applying the saline solution.
   2. Remove the outer covering of the reference and ground wires from the connector.
2. Connect the head stage with the graphene electrode array to the connector.
3. Plug the interface cable linked to the head stage into the recording system.
4. Secure the graphene electrode array complex into the stereotaxic arm.
5. To capture neural signals from all channels, position the array on the somatosensory cortex without any bending, following the predetermined stereotaxic coordinates.
6. Place a reference wire underneath the tissue behind the occipital bone and connect the ground wire to the grounded optical table.

4. Physical stimulation and recording SEPs for mapping

1. Open the neural signal recording software.
2. Set the recording software environment: (1) set the sampling rate for SEPs and notch filter (60 or 50 Hz, a frequency of household electrical power) to remove the noise from the power line.
3. For whisker mapping, bend the whisker with a fine stick.
4. Constantly poke the forepaw, forelimb, hind paw, hindlimb, and trunk with a wooden stick for body mapping.
5. Record neural signals in the data acquisition system for the indicated time.

5. Animal euthanasia

1. After all the recording procedures, sacrifice the rats with anesthesia using >5% isoflurane and perform cervical dissection.

6. SEP measurement for cortical mapping

1. Open MATLAB code-named read_Intan_RHS2000_file.m for signal analysis.
   NOTE: read_Intan_RHS2000_file.m can be downloaded from "https://intantech.com/downloads.html?tabSelect=Software”.
2. Click the Run button, select the recording file with the ".rhs" filename extension, and wait for the file to be processed and read.
3. Enter the command "plot (t, amplifier_data("channel number",:))" to create a 2D line plot of the recording data, find the SEPs, and calculate the amplitude of SEPs in all channels.
   NOTE: Enter the channel number at "channel number." For example, "plot(t, amplifier_data(1,:))" creates channel 1's 2D line plot. In addition, when the experimenter calculates the amplitude of the response, choose the response recorded from each channel.
4. Obtain data by coloring the grid with a different hue according to the amplitude of the SEPs.
   NOTE: MATLAB command "imagesc" helps obtain a topographic map more quickly.

Wyniki

This protocol describes how a graphene multichannel array is mounted on the surface of the brain. The somatosensory map was constructed by acquiring neural responses to physical stimuli and calculating the amplitude of the response. Figure 1 shows the schematic of this experiment.

Figure 2A presents the structural characteristics of a graphene electrode array. There are thru-holes of the substrate between the electrodes. These holes h...

Dyskusje

The presented protocol provides an in-depth, step-by-step process that explains how to access and map the somatosensory responses of rats using a graphene electrode array. The protocol-acquired data are SEPs that provide somatosensory information that is synaptically linked to each body part.

Several aspects of this protocol should be considered. When extracting cerebrospinal fluid to prevent brain edema and mitigate inflammation, it is crucial for the experimenter not to damage the brainstem ...

Materiały

Name	Company	Catalog Number	Comments
1mL syringe	KOREAVACCINE CORPORATION		injecting the drug for anesthesia
3mL syringe	KOREAVACCINE CORPORATION		injecting the drug for anesthesia
Bone rongeur	Fine Science Tools	16220-14	remove the skull
connector	Gbrain		Connect graphene electrode to headstage
drill	FALCON tool		grind the skull
drill bits	Osstem implant		grind the skull
Graefe iris forceps slightly curved serrated	vubu	vudu-02-73010	remove the tissue from the skull or hold wiper
graphene multielectrode array	Gbrain		records signals from neuron
isoflurane	Hana Pharm Corporation		sacrifce the subject
ketamine	yuhan corporation		used for anesthesia
lidocaine(2%)	Daihan pharmaceutical		local anesthetic
Matlab R2021b	Mathworks		Data analysis Software
mosquito hemostats	Fine Science Tools	91309-12	fasten the scalp
ointment	Alcon		prevent eye from drying out
povidone	Green Pharmaceutical corporation		disinfect the incision area
RHS 32ch Stim/Record headstage	intan technologies	M4032	connect connector to interface cable and contain intan RHS stim/amplifier chip
RHS 6-ft (1.8m) Stim SPI interface cable	intan technologies	M3206	connect graphene electrode to headstage
RHS Stim/Recording controller software	intan technologies		Data Acquisition Software
RHS stimulation/ Recording controller	intan technologies	M4200
saline	JW Pharmaceutical
scalpel	Hammacher	HSB 805-03
stereotaxic instrument	stoelting		fasten the subject
sterile Hypodermic Needle	KOREAVACCINE CORPORATION		remove the dura mater
Steven Iris Tissue Forceps	KASCO	50-2026	remove the dura mater
surgical blade no.11	FEATHER		inscise the scalp
surgical sicssors	Fine Science Tools	14090-09	inscise the scalp and remove the dura mater
wooden stick			whisker stimulation
xylazine	Bayer Korea		used for anesthesia