This work was supported by grants from ANR (Programme Blanc Neurosciences), FRC (F&#233;d&#233;ration pour la Recherche sur le Cerveau), City of Paris (Programme Emergence), INSERM and La Piti&#233; Salp&#234;tri&#232;re Hospital (Translational research contract) to N.R., from Neurop&#244;le de Recherche Francilien (NeRF) to E.D., from the Universit&#233; Pierre et Marie Curie UPMC (Programme Convergence) and from the Institut du Cerveau et e la Moelle epiniere (Paris) to G.H.&#160;

NOTE: The protocol here described follows the guidelines of the French &#8220;Comit&#233; Consultatif National d&#8217;Ethique&#8221; and is declared as a collection activity by INSERM.
1. Resection of Human Epileptic Tissue
<ol>
	<li>Patients
	<ol>
		<li>Obtain brain tissue from patients suffering from intractable epilepsy. Obtain a written informed consent before surgery.</li>
		<li>For all operated epileptic patients perform an extensive presurgical workup including neurological examination, neuropsychological assessment, surface EEG, and neuroimaging (MRI and sometimes 18FDG-PET scan; Figure 1A), followed by post-surgical histological examination of resected tissue (Figure 1B). NOTE: Surgery is indicated when the diverse explorations localize similarly the seizure onset zone and when the benefit of its removal exceeds the surgical risks.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Maintain antiepileptic drugs pre-operatively. Perform surgery under general anesthesia: induction with inhaled sevoflurane (6% delivered ratio and 100% inhaled fraction of&#160;O2), intra venous sufentanyl (0.3 &#181;g/kg) and antracurium (0.5 mg/kg), followed by maintenance with intra venous sufentanyl (0.5 &#181;g/kg/hr) and inhaled sevoflurane (1 MAC).</li>
		<li>Use a neuronavigation system to allow a precise localization of the lesioned cortex (Figure 1C). After skin incision, perform a burr hole in the bone&#160;followed by a craniotomy with a high spill drill. Gently elevate the bone flap, and open the dura with scissors (Figure 1D).</li>
		<li>Use intra-operative ultra sound to facilitate the identification of the abnormal cortex, and microsurgical instruments and an ultrasonic aspirator under the operating microscope.</li>
		<li>Coagulate and cut the pio-arachnoidal plane according to the&#160;sulcal limits. Use subpial dissection to release the cortex from the pia around the lesioned area.</li>
		<li>Cut the white matter under the abnormal cortex to perform one piece &#8220;en bloc&#8221; resection, sparing as much as possible the vasculature. Avoid traumatic manipulation of the cortex.</li>
		<li>Cut a 1 cm thick slice out of the resected tissue along a direction orthogonal to the pial surface, including the sulcus bottom and transitional cortex.</li>
	</ol>
	</li>
</ol>
2. Artificial Cerebrospinal Fluid (ACSF) for Slice Preparation, Incubation and Recording
<ol>
	<li>ACSF for slice preparation
	<ol>
		<li>Prepare 1 L of sucrose-based ACSF7,8, containing (in mM): 250 sucrose, 3 KCl, 25 NaHCO3, 10 D-Glucose, 1 CaCl2, 10 MgCl2; dissolve these salts in deionized water and oxygenate the solution with carbogen for at least 10 min.</li>
		<li>Put ~700 ml of sucrose ACSF at -80 &#176;C for 50-60 min (or -150 &#176;C for 15-20 min). Keep the rest of the solution under oxygenation in ice. NOTE: The ACSF for slice preparation can be stored at 4 &#176;C; in this case, add CaCl2 and MgCl2 the day of the experiment, before cooling it.</li>
	</ol>
	</li>
	<li>ACSF for slice incubation and recording
	<ol>
		<li>Prepare at least 3 L of recording ACSF7,8, containing (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 10 D-Glucose, 1.6 CaCl2, 1.3 MgCl2; dissolve these salts in deionized water and oxygenate the solution with carbogen. Before adding MgCl2, keep some ACSF to perform recordings in 0 Mg2+ ACSF. NOTE: The incubation/recording ACSF can be stored at 4 &#176;C; in this case, add CaCl2 and MgCl2 the day of the experiment.</li>
	</ol>
	</li>
</ol>
3. Equipment for Slice Incubation and Recording
<ol>
	<li>Interface chamber for slice incubation
	<ol>
		<li>Use a brain slice chamber to maintain living slices ex vivo in interface mode. NOTE: The chamber is composed of a lower section, which contains the heating and the sensor elements and a ceramic bubbler and is filled with distilled water, and an upper part, where the slices rest on a sheet of lens tissue in the flat area in the center of the chamber (Figure 2A-B).</li>
		<li>Use two separate fine PTFE tubes, through which the preoxygenated ACSF enters the chamber. NOTE: The tubes spiral in the heated water and reach the upper part of the chamber; then the solution exits by means of capillary action with lens tissue, that conveys the solution into exit wells.</li>
		<li>Connect the PTFE tubes of the interface chamber to a peristaltic pump, to allow continuous perfusion of the slices with the preoxygenated medium.</li>
		<li>Maintain the high oxygen tension by oxygenating with carbogen (95% O2 and 5% CO2) through the ceramic bubbler. NOTE: This warmed and moistened gas mixture reaches the slices in the upper part of the chamber through appropriate holes.</li>
		<li>Maintain the temperature in the upper part of the chamber by connecting the chamber to a temperature controller, through a special double-ended cable, having a connector for the chamber heating element and an external temperature sensor at one end. Place the sensor near the slices to check the temperature throughout the incubation (Figure 2C).</li>
	</ol>
	</li>
	<li>Multi-electrode array recordings
	<ol>
		<li>Use planar multi-electrode arrays with 120 titanium nitride electrodes (30 &#181;m diameter) insulated with silicon nitride and internal reference electrode, arranged in a 12 x 12 matrix with 200 &#181;m center-to-center spacing. NOTE: Other configurations with different electrode diameter and spacing are possible. In particular, the MEA chip can be widened to sample larger slices of human tissues.</li>
		<li>Acquire electrical signals at 10 kHz using a MEA2100-120 system (pre- and filter amplifier with integrated data acquisition and analog-digital converter, 16 bit data resolution, input voltage range and bandwidth adjustable via software) through a dedicated software (MC_Rack). NOTE: The MEA2100 headstage is endowed of a metal plate for fixing of heatable perfusion element through magnetic holders, to continuously perfuse the slices throughout the recording session.</li>
		<li>Hold the tissue down on the MEA by a small homemade platinum anchor, which ensures a good electrical coupling between the slice and the electrodes.</li>
	</ol>
	</li>
</ol>
4. Preparation of Interface Chamber and Tools for Dissection and Slicing
<ol>
	<li>Interface chamber
	<ol>
		<li>Fill the lower section of the interface chamber with distilled water. Ensure that the level of water completely immerses the heating element and is 2 to 4 mm below the junction between the lower and upper section of the chamber. Check this level daily before switching on the heating, in order to avoid damage of the heating element.</li>
		<li>Connect the chamber to a carbogen source, endowed with a secondary flow regulator for fine adjustments of gas pressure.</li>
		<li>Cut a sheet of lens tissue in order to fit the flat area of the slice chamber, and position it close to input solution tubes, to let any bubble escape from the input line before reaching the slices.</li>
		<li>Cut two pieces of stranded cotton thread as long as the piece of lens tissue, and position them along the main sides of the flat area of the chamber. NOTE: This helps to slightly raise the perfusion solution level in the chamber.</li>
		<li>Set the flow rate of the peristaltic pump at 1 ml/min and activate the pump.</li>
		<li>Start oxygenation of the water in the bottom part of the chamber.</li>
		<li>Set the temperature of the temperature controller at 37 &#176;C and turn it on. Cover the upper part of the interface chamber with a piece of parafilm and wait at least 30 min for the temperature to increase and stabilize.</li>
	</ol>
	</li>
	<li>Tools for dissection and slicing
	<ol>
		<li>Prepare a dissection kit consisting of two fine forceps, a spatula, a small spoon and a blade; two glass petri dishes, two brushes as well as cyanoacrylate glue.</li>
		<li>Set the parameters of the vibratome: thickness, 400 &#181;m; frequency, 70 - 90 Hz; amplitude, 0.9 - 1 mm, speed: 0.1 mm/sec.</li>
	</ol>
	</li>
</ol>
5. Human Cortical Slice Preparation
<ol>
	<li>Remove the sucrose-based ACSF from -80 &#176;C freezer and mix it in order to obtain a sort of slush. Oxygenate it with carbogen, put ~250 ml in a glass bottle and keep it in a Dewar bottle filled with ice, while going to the surgery room of the hospital to get the tissue. In the meantime, keep the rest at -20 &#176;C. 
	NOTE: During the surgery, the neurosurgeon dissects a small block of cortical tissue. Immediately place the sample in ice-cold sucrose ACSF for transfer to the laboratory.</li>
	<li>Keep the time needed for the transfer from the hospital to the laboratory to a maximum of 30 min. In the laboratory, take the sucrose-based oxygenated ACSF out of -20 &#176;C freezer, mix it to have a slush, fill a petri dish and the vibratome buffer tray and start oxygenating both with carbogen. Carefully take out the piece of human tissue from the bottle with a spoon and put it in the petri dish (Figure 2D).</li>
	<li>Using the forceps, carefully remove blood clots, vessels and meninges to avoid resistance during slicing. Remove the meninges, consisting in pia-matter, starting from the sides of the tissue block, paying attention not to damage the cortical surface. With a blade, cut a side of the tissue to obtain a flat surface, which will be glued on the vibratome specimen plate. NOTE: The meninges, consisting in pia-matter, are removed starting from the sides of the tissue block, paying attention not to damage the cortical surface.</li>
	<li>If the block of tissue is bigger than the MEA chamber, cut a piece of appropriate dimensions before gluing it on the specimen plate (Figure 2E).</li>
	<li>Glue the tissue in order to obtain transverse cortical slices, put it into the buffer tray and cut 400 &#181;m thick slices at low speed (0.1 mm/sec) (Figure 2F).</li>
	<li>Cut pieces of lens tissue with slice dimensions; with a spatula and a brush, transfer the slices on these lens tissues and incubate them in the interface chamber at 37 &#176;C for at least 1 hr before starting recordings (Figure 2G-I). Continuously store the slices in the same conditions until use. NOTE: Placing slices on lens paper is an important step to facilitate both the perfusion of bottom side of slices and the removal of slices from the interface chamber before recording.
	<ol>
		<li>While transferring the slices on the lens tissue, manage them very carefully, avoiding to touch the cortical layer area with the brush.</li>
		<li>Prepare the slices one at a time and immediately transfer each of them to the interface chamber, in order to ensure proper oxygen supply and temperature; slice the block of resected tissue as quickly as possible.</li>
	</ol>
	</li>
</ol>
6. Preparation of MEA Setup for Recordings
<ol>
	<li>Plug the perfusion system to a peristaltic pump and the MEA system to a computer. Oxygenate the recording ACSF. Place an empty MEA chip inside the amplifier.</li>
	<li>Set the temperature controller for heating cannula element to reach 37 &#176;C in the MEA chamber and start the perfusion at 5-6 ml/min. NOTE: It is usually necessary to set the temperature 3-4 &#176;C higher than the one desired, depending on the room temperature and the flow rate.
	<ol>
		<li>Check the temperature in the MEA chamber with a fine thermometer, placing it the closest possible to the center, without touching the electrode area.</li>
	</ol>
	</li>
	<li>Start recording software and check that all the MEA electrodes are well connected and that there is no noise due to perfusion.</li>
	<li>Start MEA_Monitor to check the functioning of the camera table.</li>
</ol>
7. Transfer of a Slice From Interface Chamber to MEA Chip for Recording
<ol>
	<li>Pour some warmed recording ACSF in a glass Petri dish; with a forceps, take a slice from the interface chamber, by grabbing it through the lens tissue, and put it in the petri dish. Carefully detach the slice from the tissue. If the level of the solution in the interface chamber is carefully adjusted during incubation time, the slice will detach from the lens tissue only by submerging it in the solution; otherwise, use a small brush to facilitate the detachment.</li>
	<li>Fill a MEA chip with some ACSF and with a spatula, transfer the slice into it. With a plastic Pasteur pipette, gently remove the solution to make the slice adhere on the electrode area and keep it in place with a platinum anchor. Add 1 ml of recording ACSF, place the MEA chip into the amplifier and start perfusion at 5-6 ml/min (Figure 2J-L).</li>
	<li>Check the slice position on the MEA recording area by using MEA_Monitor, adjust it if necessary in order to record from the cortical layers and take a picture. Start the recording.</li>
</ol>

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Sponsorship by Multichannel Systems was provided for the production of the video and open access publication.

Pharmacoresistant epilepsy is a rare condition, which can be explored in human tissue in vitro. This allows studying epileptic human cortex, which displays specific defects that are only partially reproduced in animal models. The method here described allows preparing and recording human postoperative tissues ex vivo with preserved cellular viability and networks so that they spontaneously produce epileptic activities. Retaining similar activities than those recorded in vivo is crucial to study mechanisms of genesis of pathological activities. Further, such methods allow exploring human tissues and avoid non perfect animal models of the disease. However, studying human tissue requires synchronization between neurosurgeons and the experimental laboratory. Tissue transportation requires specific care not to be traumatic. In addition, both the quantity of specimen and tissue is limited. Finally, access to proper control tissues is the main concern. With this preparation, the post operative tissues spontaneously produce interictal-like discharges in normal ACSF7,8. Ictal-like events can also be elicited in modified, proconvulsive ACSF so that the mechanisms of seizure initiation and transition from the interictal state to seizures can be investigated.
Human tissue can be kept viable for up to 10 hr in interface conditions. Slices were considered viable when multi-unit activity or field potentials were observed spontaneously and when such activities were evoked by increasing excitability through extracellular potassium increases and/or magnesium decrease. Although variability in activity occurs, likely reflecting differences in pathologies and cortical areas, we explored epileptogenic characteristics of a tissue only in healthy slices showing spontaneous interictal and evoked ictal discharges. In order to preserve tissue vitality and activity, an interface chamber is used to store the slices at 36-37 &#176;C for recovery before recording with the MEA system. Indeed, several groups have clearly demonstrated the advantages of interface based storage system compared to standard beaker storage and the importance of temperature for the preservation of network activity such as spontaneous sharp wave-ripples or cholinergic-induced oscillations9,10. Interface storage of slices has been previously used to record epileptic activity from human hippocampal and subicular slices1,11. With the current MEA technique, after the recovery period from slicing in interface conditions, the network activity is recorded in submerged conditions, in presence of high flow rate (5-6 ml/min) at 37 &#176;C, with the MEA system. The reduced diameter (1.8 cm) of MEA chip, which delimits a small volume chamber (&#60;1.5 ml), together with the increased flow rate, enhances oxygen supply of the slice, which has been demonstrated to be a critical factor for spontaneous and pharmacologically-induced network activities9,10. Further, the reduced amount of circulating ACSF facilitates pharmacological testing.
The slicing procedure is however a traumatism for the tissues12. Both neuronal architecture and chloride homeostasis appear to be perturbed at the tissue surface (50 &#181;m). The origin of the activities recorded by MEA chips, which sample the tissue mostly superficially without deep penetration, may arise from traumatized areas. However, our data show that the extracellular field potentials detected locally are recorded in most MEA electrodes and previous work show that they are integrated over a 100 to 200 &#181;m distance from the recording site13, suggesting that seizures recorded in our preparation are unlikely to be produced by traumatized areas. Further, in studies performed with tungsten electrodes enabling deep penetration, the epileptic activities recorded in human tissues are similar to the ones observed in epileptic patients1,7,8.
Another limit of ex vivo tissue recording is the disruption of the connections between the different brain areas, thus restricting dynamic neuromodulations. This may explain why in such tissue no ictal-like event is spontaneously recorded, but requires to be triggered by ionic manipulation or pharmacological stimulation enhancing excitability. Accordingly, in this protocol, seizure-like events are induced by combining a change in extracellular K+ from 3 to 6 mM and a reduction of external Mg2+ from 1.3 mM to Mg2+-free ACSF, in order to increase tissue excitability and remove Mg2+-dependent NMDA receptor block. Indeed, it has previously been demonstrated that epileptiform activity induced in human neocortical and hippocampal slices using Mg2+-free ACSF resembles the electrographic seizures recorded in vivo14. Besides, it has been shown that epileptiform discharges obtained in temporal lobe slices become resistant to clinically used anticonvulsants after prolonged exposure to Mg2+-free ACSF15,16, thus providing a model for investigating pharmacoresistant seizure-like events in vitro.
MEAs allow recording of both, field potentials and multi-unit activities consisting in neuronal action potentials ex vivo. Thus, MEAs are a powerful electrophysiological tool compared to EEGs, which explore field potentials generated by synchronous activities of neuronal ensembles in vivo, but do not give access to single neuron behaviors17. Although more recent microelectrodes can record in vivo multi-unit activities consisting in neuronal action potentials, they are invasive, so their use is mostly restricted to research purpose during intracranial recordings. In particular, MEAs recordings represent a technique of choice to study the spatio-temporal patterns of epileptic events, the mechanisms controlling seizure onset and propagation and the action of classic and new antiepileptic drugs. Remarkably, to unravel the cell types and the signaling basis of epileptic discharges, spike sorting techniques and pharmacological testing should be combined with MEA techniques. Although MEAs can give access to individual spikes, they do not provide information about synaptic and biophysical properties. In the future, other techniques should be coupled to MEA recordings in order to better sample cellular behavior, network activities and synaptic signaling. For instance, fluorescence imaging to unravel neurons or glial cells behavior, as well as ion dynamics, may be combined with MEAs recordings. Further post hoc histological analysis can also reveal specific alterations of cell types, proteins or receptors so that the location of the epileptic activities can be correlated with specific rearrangements of the nervous structure. The MEAs system can also be embedded in a patch-clamp set-up to correlate single cells or conductances with population activities. In the future, optogenetic tools could also be used, provided that human slices can be cultured in the long-term, as performed for organotypic slices, so that transfection or infection of specific cell types can be performed.

Epilepsy is a chronic disorder in which epileptic seizures, which are intermittent patterned discharges lasting several seconds to tens of seconds on electroencephalographic (EEG) recordings associated with clinical manifestations, interrupt an interictal state, characterized by the presence of synchronous neuronal discharges lasting tens of milliseconds and called interictal events1. It affects approximately 1% world&#8217;s population and although seizures are controlled in the majority of the patients, approximately one third of people with epilepsy do not show adequate response to antiepileptic drugs2. In this condition, called pharmacoresistant epilepsy and whose mechanisms still have to be clearly identified, the surgical resection of the specific part of the brain identified as the seizure-onset zone remains the only alternative treatment giving a positive outcome to patients. Thus, the resected specimens from surgery yield the opportunity to study the mechanisms of interictal discharges and seizure generation and propagation, as well as pharmacoresistance on viable human central nervous tissue ex vivo.
Multi-electrode arrays (MEAs), consisting of an arrangement of spatially distributed microelectrodes, allow the simultaneous stimulation and recording of electrophysiological activity from several sites of the tissue, thus providing an excellent approach to study the spatio-temporal patterns of spontaneous and evoked activity. This technique, first applied to monitor the developmental changes of neuronal cell culture activity3 and then adapted for acute and organotypic brain and spinal cord slices4&#8211;6, is presently considered a valuable electrophysiological tool.
The current protocol describes how to prepare human cortical slices from surgically resected tissue and to record with MEAs reliable ex vivo interictal and seizure-like events from these slices. This technique thus provides a way to address the basic mechanisms underlying epileptic activities initiation, propagation and effects of antiepileptic drugs at both the cellular and network levels. All the procedures used to obtain, prepare, maintain and record human slices are here described in detail.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:279px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Brain Slice Chamber-2: Interface</td>
			<td style="width:201px;">AutoMate Scientific, Inc.</td>
			<td style="width:119px;">S-BSC2</td>
			<td style="width:279px;">It requires separate temperature controller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">2-channel Temperature Controller</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">TC02</td>
			<td style="width:279px;">It allows to plug in and use 2 interface chambers.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Special cable for connection of TC01 to S-BSC2 with external reference PT100</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">CA3</td>
			<td style="width:279px;">The reference PT100 is placed near the slices&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">6pin plug-connector with PT100</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">TS-PT100</td>
			<td style="width:279px;">External reference for CA3 cable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">MEA workstation for recording data from 120-electrode MEAs</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MEA2100-120</td>
			<td style="width:279px;">It includes MEA2100-120 headstage and MEA2100 interface board. www.multichannelsystems.com</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Microelectrode array for MEA2100-120</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">120MEA200/30</td>
			<td style="width:279px;">Electrode spacing: 200 &#181;m; electrode diameter: 30 &#181;m; glass ring: 6 mm high. Different configurations (spacing, diameter, ring) possible.&#160;</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">Video Microscope Table</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MEA-VMT-1</td>
			<td style="width:279px;">Table with a video microscope underneath to image the electrode field of the MEAs in an amplifier placed on top of the table and transfer the image to a computer.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Perfusion cannula</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">PH01</td>
			<td style="width:279px;">Heatable perfusion cannula with temperature sensor; temperature can be programmed with TC02 controller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">MC_Rack</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td></td>
			<td style="width:279px;">Software for data acquisition and recordings</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Magnetic Perfusion Holder</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MPH</td>
			<td style="width:279px;">Magnetic perfusion holder for PH01 element to fix the perfusion cannula and connect the perfusion system to the amplifier&#39;s ground</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Neuroexplorer</td>
			<td style="width:201px;">Nex Technologies</td>
			<td></td>
			<td style="width:279px;">Software for data analysis; info@neuroexplorer.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Peristaltic pump (drive unit)</td>
			<td style="width:201px;">Gilson</td>
			<td style="width:119px;">F155001</td>
			<td style="width:279px;">0,01 to 48 rp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Peristaltic pump (pump head)</td>
			<td style="width:201px;">Gilson</td>
			<td style="width:119px;">F117800</td>
			<td style="width:279px;">R2 two channel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ultrasonic aspirator</td>
			<td style="width:201px;">Integra Life sciences, USA</td>
			<td style="width:119px;">Cusa Excel +</td>
			<td style="width:279px;">It allows blunt subpial dissection of the cortex</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Neuronavigation</td>
			<td style="width:201px;">Isis Solutions, France</td>
			<td style="width:119px;">Surgiscope</td>
			<td style="width:279px;">It allows real time identification of the brain structure on the preoperative MRI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Vibratome HM 650 V</td>
			<td style="width:201px;">Microm</td>
			<td></td>
			<td style="width:279px;">Block slicing into 400 &#956;m thick slices</td>
		</tr>
	</tbody>
</table>

multi-electrode array recordings of human epileptic postoperative cortical tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

The recording of human cortical slice activity starts while perfusing the tissue with normal recording ACSF (as previously described)7,8. In this condition, when the tissue is well preserved during surgery and later on during tissue transfer from the hospital and slice preparation, one can observe spontaneous activity, in the form of small interictal-like events, detected from small clusters of MEA electrodes. Interictal-like discharges consisted in a field potential, usually biphasic, comprising an initial sharp negative deflection, reaching tens of microvolts, followed by a longer opposite wave lasting several tens of milliseconds. Fast multi-unit activities are usually embedded in the field potential mainly in the initial part. A representative example of interictal-like activity is illustrated in Figure 3Aii, where the trace recorded by a MEA electrode is shown.
To record epileptic seizures from in vitro human cortical slices, it is necessary to perfuse them with an &#8220;epileptogenic&#8221; ACSF, in which Mg2+ is absent and K+ is raised to 6 mM, to boost tissue excitability1 (the increase of K+ from 3 to 6 mM alone is not enough to induce seizures; data not shown). Once the perfusion with 0 Mg2+ 6 mM K+ ACSF has started, the activity of cortical slices gradually increases and the first seizure appears within 15 to 20 min (15.4 &#177; 1.7 min, n = 10, from 5 patients; Figure 3 Ai). We evoked ictal-like activity, as shown in Figure 3, in 75% of patients tested and in 66.7% of all recorded slices.
Epileptic activities are recorded from adjacent electrodes of the MEA chip, and comparison of the onset dynamics of field potential events allows studying their site of genesis and propagation. A representative seizure recorded from a slice of human cerebral cortex with the MEA system is shown in Figure 3B (a representative trace of a seizure recorded by a single MEA electrode is shown at higher temporal resolution in Figure 3 Aiii). Note that the seizure is recorded from almost all the electrodes of the MEA chip, thus indicating that it is able to propagate to a large cortical area. Furthermore, looking at the single traces recorded from each electrode, it is possible to observe the progressive propagation of the seizure across the tissue: indeed, it starts first in the cortical area covering the right half of the electrode array, and it gradually spreads to the area covering the left half (i. e., compare traces from electrode L6 and B6; Figure 3B).
<img alt="Figure 1" fo:content-width="3in" src="/files/ftp_upload/51870/51870fig1highres.jpg" width="300" /> 
Figure 1: A cortical dysplasia and its surgical removal. A Taylor type focal cortical dysplasia (white arrow) embedded in a left frontal lobe sulcus is visualized on the preoperative MRI (A), and later confirmed by histological examination (B) MAP kinase labeling; scale bar: 200 &#956;m) showing a cortical dyslamination, abnormal neurons and a track on neurons with an incomplete migration in the lower white matter. During surgery, the dysplasia is localized according to the MRI data with a neuronavigation system (C). Visualization of the gyrus containing the dysplasia during surgery (D).
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/51870/51870fig2highres.jpg" width="400" /> 
Figure 2: Equipment and procedure for human cortical slice preparation. An interface chamber is used to maintain human cortical slices ex vivo (A); the slices rest on a sheet of lens tissue in the flat area in the upper part of the chamber (B), where a temperature sensor (C, left), connected to a temperature controller (C, right, up) through a double-ended cable (C, right, down), is placed to maintain 37 &#176;C throughout slice incubation time. Once obtained, place the resected block of tissue in a petri dish filled with ice-cold sucrose-based ACSF (D) and remove blood clots, vessels and meninges, to facilitate the slicing. If the tissue block is bigger than MEA chambers, isolate a piece of appropriate dimensions with a blade (E) and glue it on the vibratome specimen plate (F). Cut 400 &#181;m thick slices and with the help of a spatula (G) transfer them on pieces of lens tissue (H) and incubate them in the interface chamber (I). Before recording, remove the lens tissue by submerging the slice in a petri dish filled with ACSF and transfer it to an ACSF filled MEA chip with a spatula (J). Remove the solution to let the slice adhere on the MEA (K) and keep it in position by using a platinum anchor. Place the MEA in the amplifier (L), start perfusion and recording.
<img alt="Figure 3" fo:content-width="3in" src="/files/ftp_upload/51870/51870fig3highres.jpg" width="300" /> 
Figure 3: MEA recordings of epileptic seizures in human cortical slices. (A) A representative trace of human cortical slice activity recorded by a MEA electrode is shown in panel i. In presence of normal ACSF, it is possible to observe interictal-like spontaneous activity (small grey rectangle, zoomed in panel ii); then, when the perfusion with 0 Mg2+ 6 mM K+ ACSF has started, the first seizure appears after a ~15 min delay and is followed by other events at 2-3 min interval. Panel iii shows the seizure in the big grey rectangle in panel i with a zoomed scale. The blue trace illustrates a zoom of the activity, as indicated by the blue line and arrow. Panel iv) shows the data illustrated in panel iii) high pass filtered at 250 Hz, which reveal multi-unit activity when zoomed (blue trace). (B) Representative recording of a seizure in presence of 0 Mg2+ 6 mM K+ ACSF from all MEA electrodes. Each square represents an electrode of 12 x 12 MEA array and shows an 80 sec time window; the dotted line indicates the position of the cortical surface relatively to MEA array.

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue at JoVE.com

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

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20.5K Views.  CNRS UMR 7241, INSERM U1050, Collège de France. We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Video: Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

A Postoperative Evaluation Guideline for Computer-Assisted Reconstruction of the Mandible

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging modalities, mandibular defect classification, and evaluation methodologies between studies. This guideline uses a step-by-step approach guiding the process of imaging, classification of mandibular defects and volume assessment of three-dimensional (3D) models, after which a legitimized quantitative accuracy evaluation method can be performed between the postoperative clinical situation and the preoperative virtual plan. The condyles and the vertical and horizontal corners of the mandible are used as bony landmarks to define virtual lines in the computer-assisted surgery (CAS) software. Between these lines the axial, coronal, and both sagittal mandibular angles are calculated on both pre- and postoperative 3D models of the (neo)mandible and subsequently the deviations are calculated. By superimposing the postoperative 3D model to the preoperative virtually planned 3D model, which is fixed to the XYZ axis, the deviation between pre- and postoperative virtually planned dental implant positions can be calculated. This protocol continues and specifies an earlier publication of this evaluation guideline.

Here, we propose a practical, feasible and reproducible evaluation guideline for computer-assisted reconstruction of the mandible in order to create uniformity between studies regarding postoperative accuracy evaluation. This protocol continues and specifies an earlier publication of this evaluation guideline.

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging ...