Name: Chronic Cranial Window Technique for Repeated Cortical Recordings During Anesthesia in Pigs (Video) | JoVE Journal
Uploaded: 2025-06-06T13:45:00.000+00:00
Duration: 7 min 19 s
Description: Learn a chronic cranial window technique for repeated cortical recordings in pigs during anesthesia. This method improves signal stability and reduces device failure risks for neuroscience research.

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The authors would like to thank the animal caretakers and technicians at the laboratory animal facility at Aalborg University Hospital. The Center for Neuroplasticity and Pain (CNAP) is supported by the Danish National Research Foundation (DNRF121). Figure 6 and Figure 8 were created in BioRender.

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</ol>

The authors have no conflicts of interest to disclose.

The significance of the development of this technique lies in removing electrode failure modes, reducing tissue responses, and thereby improving the reliability of invasive cortical recording techniques. No electrode failure modes occurred during the study, and a total of two ECoG arrays were used throughout the study, including 16 animals and 43 recording sessions. This has an additional positive impact on the research economy. Using traditional fully implanted methods12,<sup class="xr...

In general, the purpose of cortical recordings is to extract information from neuronal signaling in the brain. This information can be used in various ways-controlling an external device, communication, disease diagnosis, or rehabilitation1,2,3,4. Each application places unique requirements on the information content and spatial resolution required and the amount of invasiveness that is considered acceptable. Therefore, recording solutions with a range of invasiveness and spatial resolution have been developed since the discovery of the electroencephalogram in 19295.
Generally, these can be divided into electroencephalography (EEG), electrocorticography (ECoG), and intracortical recordings. EEG is a non-invasive recording method that captures neural oscillations and event-related potentials (ERPs) from the entire brain. However, its capability to define the sources of this activity is limited due to its low spatial resolution. ECoG is a more invasive method where electrodes are placed epi- or subdurally, typically covering a smaller portion of the cortex. It has a higher spatial resolution and can record ERP and surface local field potentials (LFP). Therefore, it can localize the source of brain activity more precisely, which makes it helpful, for example, in identifying the origin of focal epilepsy. The intracortical recording is the most invasive recording method and can record spiking activity from individual neurons located superficial or deep inside the brain and LFP from the volume of neurons around the electrodes. These signals have a very high spatial resolution and information content but are produced by a restricted subset of neurons (1-10 neurons per channel)6.
To extract information from the brain for prolonged periods (months-years), the interface must be stable and reliable for the acquired signals to continue to represent the same information during the entire period. EEG recordings require frequent electrode changes, rendering their reliability variable from very low to very high7,8,9,10. ECoG and intracortical methods are, therefore, often selected for prolonged recordings. However, these methods both require that the condition of the recording electrode, as well as the tissue, must remain stable over time. While the electrode usually stays at the same location, the electrode-tissue interface may change due to tissue reactions or electrode failure modes11,12,13,14. Tissue reactions include neuronal death, hemorrhage, biofouling, foreign-body reaction, gliosis, encapsulation, infection, meningitis, and meningeal extrusion15. These reactions compromise the recording capabilities of the electrodes12,13. Common electrode failure modes are delamination or leakage at the insulated parts, electrode surface coating delamination or cracking, wire damage, and electrode dislocation11,12.
To overcome electrode failure modes, we considered the viability of a temporary electrode placement solution that also addresses many of the challenges related to tissue responses, namely neuronal death, foreign-body reaction, gliosis, encapsulation, and meningeal extrusion. Furthermore, consistent electrode placement was a requirement to achieve reliable and reproducible neuronal recordings. Since the electrode was placed epidurally at a few millimeters distance to the nervous tissue, the movement of the electrode should not exceed 1 mm. The cranial window was designed with dimensions to prevent excessive movement between electrode placements. With the development of the cranial window technique, we aim to improve the long-term signal reliability and quality and remove the risk of electrode failure.

<table><tbody><tr><td>Cranial window implantation</td><td></td><td></td><td></td></tr><tr><td>Scalpel</td><td></td><td></td><td>disposable, blade 10</td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Cauterizer</td><td></td><td></td><td></td></tr><tr><td>Periosteum elevator</td><td></td><td></td><td>flat, 10 mm width</td></tr><tr><td>Weitlaner retractor</td><td></td><td></td><td>3x4 prong, blunt, 16.5 cm</td></tr><tr><td>Midas Rex Legend EHS stylus surgical drill system</td><td>Medtronic Powered Surgical Solutions, Fort Worth, USA</td><td></td><td></td></tr><tr><td>Legend Ball Fine drill bits&amp;nbsp;</td><td>Medtronic Powered Surgical Solutions, Fort Worth, USA</td><td>7BA40F-MN and 7BA60F-MN</td><td>MedNext type 4 and 6 mm head diameter</td></tr><tr><td>Sterile cover for the drill</td><td></td><td></td><td></td></tr><tr><td>Syringe</td><td></td><td></td><td>5 ml</td></tr><tr><td>Saline</td><td></td><td></td><td></td></tr><tr><td>Suction</td><td></td><td></td><td></td></tr><tr><td>Ruler</td><td></td><td></td><td></td></tr><tr><td>Cotton buds</td><td></td><td></td><td></td></tr><tr><td>Rongeur</td><td></td><td></td><td>straight jaw, 15 cm, 3 x 20 mm bit size</td></tr><tr><td>2.5 mm hand drill and handle</td><td></td><td></td><td></td></tr><tr><td>Butterfly infusion set</td><td></td><td></td><td></td></tr><tr><td>Six M3 screws in 6-14 mm length</td><td></td><td></td><td></td></tr><tr><td>Screwdriver&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Three sizes of 3D-printed cranial window (4-, 5- and 6-mm depth) and cap</td><td></td><td></td><td></td></tr><tr><td>Deisolated U-shaped connector</td><td></td><td></td><td></td></tr><tr><td>Crocodile connector</td><td></td><td></td><td></td></tr><tr><td>Genta-coll resorbable antibiotic sponge</td><td></td><td></td><td></td></tr><tr><td>VYCRIL resorbable suture&amp;nbsp;</td><td>Ethicon</td><td></td><td>2-0 26 mm round bodied</td></tr><tr><td>Monocryl non-resorbable suture</td><td>Ethicon</td><td></td><td>3-0 24 mm reverse cutting</td></tr><tr><td>Needle holder</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Topical adhesive tissue glue</td><td>Leukosan</td><td></td><td></td></tr><tr><td>Peripheral wire implantation</td><td></td><td></td><td></td></tr><tr><td>Two partially uninsulated Cooner wires</td><td></td><td></td><td></td></tr><tr><td>NOTE: 1-2cm of the wires is uninsulated in the middle and at one of the ends</td><td></td><td></td><td></td></tr><tr><td>Two 23-gauge needles</td><td></td><td></td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Programmable stimulator controlled by a PC running MC_stimulus</td><td>Multichannel Systems, Reutlingen, Germany</td><td>STG4008</td><td></td></tr><tr><td>Two crocodile connectors</td><td></td><td></td><td></td></tr><tr><td>Cortical recordings</td><td></td><td></td><td></td></tr><tr><td>Metal plate for the magnetic micromanipulator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Magnetic micromanipulator stand</td><td></td><td></td><td></td></tr><tr><td>Micromanipulator</td><td></td><td></td><td></td></tr><tr><td>Headstage holder</td><td></td><td></td><td></td></tr><tr><td>32-channel ZIF-clip headstage</td><td>TDT, Alachua, FL, USA</td><td></td><td></td></tr><tr><td>32-channel micro-electrocorticography (&amp;micro;ECoG) array&amp;nbsp;</td><td>Neuronexus, Ann Arbor, USA</td><td>E32-1000-30-200</td><td></td></tr><tr><td>TDT recording equipment including pre-amplifier SI8, data processor RZ2 and workstation WS8&amp;nbsp;</td><td>TDT, Alachua, FL, USA</td><td></td><td></td></tr><tr><td>Anesthesia &amp;amp; Analgesia</td><td></td><td></td><td></td></tr><tr><td>Zoletil 50 Vet</td><td>Virbac, Carros, France</td><td></td><td></td></tr><tr><td>Rompun (Zxylacin)</td><td>Elanco Animal Health Monheim; Germany</td><td></td><td></td></tr><tr><td>Torbugesic (Butorphanol)</td><td>Intervet International, Boxmeer, Netherlands</td><td></td><td>Today a different formulation (dolorex) is used</td></tr><tr><td>Ketaminol</td><td>Intervet International, Boxmeer, Netherlands</td><td></td><td></td></tr><tr><td>Propofol</td><td>Baxter Holding, Utrecht, Netherlands</td><td></td><td></td></tr><tr><td>Fentanyl</td><td>B. Braun, Melsungen, Germany</td><td></td><td></td></tr><tr><td>Metacam</td><td>Borhringer Ingelheim Vetmedica, Ingelheim, Germany</td><td></td><td></td></tr><tr><td>Noromox</td><td>Scanvet Animal Health, Fredensborg, Denmark</td><td></td><td></td></tr><tr><td>Pentobarb</td><td>LeVet, Oudewater, Netherlands</td><td></td><td></td></tr></tbody></table>

chronic cranial window technique for repeated cortical recordings during anesthesia in pigs

Chronic Cranial Window Technique for Repeated Cortical Recordings During Anesthesia in Pigs

Cortical recordings are essential for extracting neuronal signals to inform various applications, including brain-computer interfaces and disease diagnostics. Each application places specific requirements on the recording technique, and invasive solutions are often selected for long-term recordings. However, invasive recording methods are challenged by device failure and adverse tissue responses, which compromise long-term signal quality.
To improve the reliability and quality of chronic cortical recordings while minimizing risks related to device failure and tissue reactions, we developed a cranial window technique. In this protocol, we report methods to implant and access a cranial window in juvenile landrace pigs, which facilitates temporary electrocorticography (ECoG) array placement on the dura mater. We further describe how cortical signals can be recorded using the cranial window technique. Cranial window access can be repeated several times, but a minimum of 2 weeks between implant and access surgeries is advised to facilitate recovery and tissue healing.
The cranial window approach successfully minimized common electrode failure modes and tissue responses, resulting in stable and reliable cortical recordings over time. We recorded event-related potentials (ERPs) from the primary somatosensory cortex as an example. The method provided highly reliable recordings, which also allowed the assessment of the effect of an intervention (high-frequency stimulation) on the ERPs. The absence of significant device failures and the reduced number of electrodes used (two electrodes, 43 recording sessions, 16 animals) suggest an improved research economy. While minor surgical access is required for electrode placement, the method offers advantages such as reduced infection risk and improved animal welfare. 
This study presents a scalable, reliable, and reproducible method for chronic cortical recordings, with potential applications in various fields of neuroscience, including pain research and neurological disease diagnosis. Future adaptations may extend its use to other species and recording modalities, such as intracortical recordings and imaging techniques.

Using the cranial window technique, cortical signals were recorded in 43 sessions in 16 animals. Animals healed appropriately after surgery and were pair-housed throughout the study and monitored daily using the welfare scheme in Supplemental Table S1. All animals received a score 0 at all times, indicating excellent welfare. Figure 7 shows that the windows were indeed placed over the S1 area of the pig cortex. Some scarring was usually observed on the dura in vivo ...

This study presents a scalable, reliable, and reproducible method for repeated chronic cortical recordings in a porcine model. The method has applications in various fields of neuroscience, including pain research and neurological disease diagnostics.

Cortical recordings are essential for extracting neuronal signals to inform various applications, including brain-computer interfaces and disease ...

This research aims to develop a scalable and reliable method for chronic cortical recordings in pigs, addressing challenges of device failure and tissue responses, improving neuroscience, diagnostics and research. To extract information from the brain for prolonged periods, current technologies focus on creating a stable and reliable interface between the electrode and the brain, ensuring consistent signals from the same neural population. Electrocorticography and intracranial recordings are typically used for prolonged brain recordings.These electrodes stay in the same location, but the electrode tissue interface changes due to tissue responses and device failure. To begin, identify the bregma point by drawing straight lines between each ear and the contralateral eye. The bregma point is at the intersection of those lines.Make an incision slightly lateral to the bregma point with a scalpel. Using a cauterizer, continue the incision through the skin. After placing the sterile drape, make the initial incision at the marked side in the dermis with a scalpel.Using a cauterizer, continue the incision through the skin. After placing the retractor, lift the skin using forceps and cut the avascular connective tissue between the periosteum and the subcutus to provide flexibility for suturing later. Place one more retractor to accommodate the additional skin flexibility.Drill a 10 millimeter diameter hole frontal to the sagittal suture line and lateral to the midline suture using a rounded drill bit. Once the dura becomes visible, switch to a smaller drill bit to achieve the maximal width of the hole at the dura level. Clip the edges of the hole using rongeurs.Confirm that the cranial window fits completely into the drilled hole, reaching the bottom without gaps and determine the appropriate depth. While holding the cranial window in place, mark the screw hole positions with a hand drill. Remove the cranial window and drill the marked holes to a depth of at least four millimeters.Then place the cranial window back. After removing the needle from the butterfly infusion set, measure the screw hole depth through the cranial window using the blunt butterfly. Insert and fasten a screw with a matching length into the hole.Place a U-connector under the screw that perforates the skull and touches the dura. Insert two needles perpendicular to the nerve course approximately two centimeters apart. Pierce the skin again, approximately three centimeters away from the insertion point.Pass the Cooner wires through the needles and remove the needles, leaving the wires partially under the skin. After dipping the micro electrocorticography electrode in alcohol, place it in the ZIF-Clip Headstage. Using a micro manipulator and gently guiding it with a cotton bud, bring the micro electrocorticography electrode into place.Connect the ground wires of the electrode to the ground screw using a crocodile connector. Place sterile gauze on the electrode to maintain firm tissue contact. Drip body temperature saline on the gauze to prevent the tissue from drying.Then apply electrical stimulation to the ulnar nerve via the peripheral wire to evoke small movements in the forelimb. Preview spontaneous and evoked brain signals. Place the cranial window cap to close the cranial window, ensuring that it fits precisely in the cranial window and requires no additional fastening.Place an antibiotic pouch on top of the cranial window cap. Close the skin subcutaneously using the buried vertical mattress technique with single resorbable sutures placing them five to 10 millimeters apart. Tie the last three sutures after all the sutures have been placed.Next, place intradermal sutures in a continuous fashion, starting one to two centimeters lateral to the incision site and tunneling under the skin to the dermal layer, but below its surface. Tie a knot on the lateral end to prevent irritation or suture loosening. Then place the continuous suture between the subcutaneous sutures until the other side of the incision is reached.Make a new incision in the dermis with a scalpel slightly lateral to the midline. Using a cauterizer, continue the incision through the skin. Then incise the periosteum so the implant becomes visible.Finally, using gentle circular motions with a cotton bud, remove any connective tissue from the opening until the dura becomes visible. This figure shows the stability and modulation of event related potentials or ERPs recorded at different time points and under varying stimulation conditions. ERPs recorded at three time points showed nearly identical waveforms demonstrating the high reproducibility of the cranial window technique over time.Peak amplitudes and interchannel variability remain stable across sessions indicating consistent signal quality, while a slight delay in the first session likely reflects developmental changes. High frequency stimulation significantly enhanced the N1 peak amplitude confirming ERP sensitivity to neuroplastic changes linked to increased pain sensitivity. This approach reduces risks associated with permanent implants by eliminating device failure and reactions at the electrode.This in turn increases recording fidelity and results in highly reliable recordings. This method also reduces research costs and improves animal welfare. It can further be implemented in other species or to access other brain areas.

chronic-cranial-window-technique-for-repeated-cortical-recordings

Research

JoVE Journal

Neuroscience

This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

A Craniotomy Surgery Procedure for Chronic Brain Imaging

This video demonstrates the measurement of glutamate levels in the brain of an anesthetized neonatal piglet using a microelectrode array (MEA). The procedure involves creating a craniotomy window in the pig skull and inserting the MEA into the brain region of interest. The glutamate-oxidase-coated recording sites of the MEA convert extracellular brain glutamate to hydrogen peroxide, which generates a measurable current. This current is measured and normalized against background noise to...

Measuring Brain Glutamate Levels Under Anesthesia in Neonatal Piglets Using a Microelectrode Array

This video demonstrates the implantation of a soft electrocorticography array and the long-term cortical recordings in a minipig. It outlines the steps involved in surgically implanting the array over the minipig's auditory cortex and recording both baseline and sound stimulus-evoked neuronal action potentials.

Subdural Implantation of a Soft Electrocorticography Array for Cortical Electrophysiology Recording in Minipigs

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges.  An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

This protocol describes performing a craniectomy using a high-speed pneumatic drill on a 3-month-old Danish Landrace pig. The access is made through the frontal bone and reveals the ventral dura mater and underlying cerebral hemispheres. This procedure allows for access to a large portion of the pig brain.

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

This protocol describes a two-phase surgical method to create a large, resealable, dura-sparing cranial window in mice. This technique enables chronic, multi-modal electrophysiological recordings from distributed, deep-brain networks, such as the Default Mode Network, over several weeks.

Surgical Protocol for a Large, Resealable Cranial Window Enabling Longitudinal, Multi-Modal Electrophysiology Recordings of Mouse Default Mode Network

Learn a chronic cranial window technique for repeated cortical recordings in pigs during anesthesia. This method improves signal stability and reduces device failure risks for neuroscience research.

Chronic Cranial Window Technique for Repeated Cortical Recordings During Anesthesia in Pigs

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