We appreciate the financial support from the National Natural Science Foundation of China (82130113) and the Key R&#38;D and Transformation Program of the Science &#38; Technology Department of Qinghai Province (2020-SF-C33).

The commercially obtained H9c2 rat cardiomyocytes (see the Table of Materials) were incubated in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in a 5% CO2&#160;humidified atmosphere. The whole-cell patch-clamp technique was then employed to detect the changes in IKv in normal H9c2 cells and 4-AP- or AC-treated cells (Figure 1 and Figure 2).
1. Solution preparation
<ol>
	<li>Prepare the DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin (see Table of Materials).</li>
	<li>Prepare 1.5 M 4-AP solution by adding 14.1165 mg of 4-AP to 100 &#181;L of DMSO solution (see Table of Materials). Dilute 1.5 M 4-AP to 1.5 mM by adding the extracellular solution (step 1.5).</li>
	<li>Prepare 400 mM AC by adding 5 mg of AC to 19.36 &#181;L of DMSO solution (see Table of Materials). 
	NOTE: All the above solutions were stored in a 4 &#176;C refrigerator, and the final concentration of the DMSO must not exceed 0.1% (v/v).</li>
	<li>Prepare 50 mL of intracellular solution by adding 0.4100 g of KCl (110.0 mM), 0.0120 g of MgCl2&#160;(1.2 mM), 0.1270 g of Na2&#160;ATP (5.0 mM), 0.1190 g of HEPES (10.0 mM), and 0.1900 g of EGTA (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials). 
	NOTE: The pH value of the intracellular solution was adjusted to 7.2 using 1 M KOH and was aliquoted into small volumes (1.5-2 mL) and stored at &#8722;20 &#176;C.</li>
	<li>Prepare 50 mL of extracellular solution by adding 0.0185 g of KCl (5.0 mM), 0.3945 g of NaCl (135.0 mM), 0.0203 g of MgCl2&#183;6H2O (2.0 mM), 0.0595 g of HEPES (5.0 mM), and 0.0990 g of D-glucose (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials). 
	NOTE: The extracellular solution must be prepared for immediate use, and its pH value needs to be adjusted to 7.4 by using 1 M NaOH.</li>
</ol>
2. Cell culture
<ol>
	<li>Once the H9c2 culture dish becomes 80% confluent, digest the cells with 0.25% trypsin for 30 s.</li>
	<li>Culture 2 x 105 cells/mL with normal medium or drug-containing medium (25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M AC) in a 35 mm dish carpeted with glass plates for 24 h at 37 &#176;C, with a 5% CO2&#160;atmosphere and 70% to 80% relative humidity (see Table of Materials).</li>
</ol>
3. Fabrication of micropipettes
<ol>
	<li>Turn on the micropipette puller (see Table of Materials), and preheat for 30 min.</li>
	<li>Put a borosilicate glass capillary with a filament (OD: 1.5 mm, ID: 1.10 mm, 10 cm length) on the micropipette puller. Select the program set in step 3.3, and click on Enter on the control panel. Click on the Ramp program in the upper-right corner to determine the &#34;Heat&#34; value of the glass capillary.</li>
	<li>Write the program for pulling the electrode according to the value determined by the &#34;Ramp&#34; test, and use the following steps: &#34;Heat&#34; value = &#34;Ramp&#34; value, Pull = 0, Vel = pulling rod moving speed, Time = 200-250. Click on Pull to start fabricating the pipettes. 
	NOTE: Observe the color of the desiccants in the sealed container of the micropipette puller before using it. If the color changes from blue to pink, the desiccants must be replaced. To prevent the contamination of the electrode tip, try not to touch the middle part of the glass capillary during the pipette preparation. Subsequently, carefully remove the produced pipettes, and place them in a closed and sealed container.</li>
</ol>
4. Instrument setup
<ol>
	<li>Turn on the corresponding instruments in the following order: the digital-analog converter, the signal amplifier, the micromanipulator, the microscope, and the camera (see Table of Materials).</li>
	<li>Open the imaging application, the signal-amplifier software, and the data acquisition software in sequence (see Table of Materials). 
	NOTE: The instrument must be turned on sequentially; otherwise, the &#34;Demo Digitizer&#34; state will appear after opening the software.</li>
</ol>
5. IKv parameter setting
<ol>
	<li>Edit the protocol for recording the IKv following the steps below.
	<ol>
		<li>Click on Edit, and select Edit Protocol in the data acquisition software (see Table of Materials).</li>
		<li>Edit the program in the &#34;Waveform&#34; interface: Epoch A (First level = &#8722;60 mV, Delta level = 0 mV, First duration = 20 ms, and Delta duration = 0 ms); Epoch B (First level = &#8722;40 mV, Delta level = 10 mV, First duration = 150 ms, and Delta duration = 0 ms), and Epoch C (First level = &#8722;60 mV, Delta level = 0 mV, First duration = 30 ms, and Delta duration = 0 ms).</li>
		<li>Then, click on the Mode/Rate interface, and set the &#34;Trial Hierarchy&#34; data: Trial delay = 0 s, Runs = 1, Sweeps = 11, and Sweep duration = 0.22 s22,23. 
		NOTE: Acquire the IKv channel currents by applying 150 ms depolarizing steps from &#8722;40 mV to +60 mV with an increment of 10 mV at a holding potential of &#8722;60 mV under control conditions. The total time of the protocol needs to be greater than the time set in the &#34;Waveform&#34; program.</li>
	</ol>
	</li>
	<li>Edit the P/N Leak Subtraction program: click on Edit Protocol to select the Stimulus button, and set the leak subtraction program in the P/N Leak Subtraction dialog box: number of Subsweeps = 2-8 (4 in this study), Settling time = 100-1,000 ms (200 in this study), &#34;Polarity&#34; = &#34;Opposite to waveform&#34;, holding level = &#8722;80 mV. 
	NOTE: The P/N Leak subtraction must have a holding level lower than the &#34;First holding&#34; (&#8722;60 mV).</li>
</ol>
6. Whole-cell patch-clamp recording of the I Kv in voltage-clamp mode
<ol>
	<li>Establish the data storage path: click on File, and select Set Data File Names in the data acquisition software. Open the established IKv protocol (step 5.1) by selecting Edit and clicking on Open Protocol in the data acquisition software. Finally, click on the Tools option, and select Membrane Test to start running the protocol.</li>
	<li>Add the extracellular solution (step 1.5) to the cell bath on the whole-cell patch-clamp apparatus (see Table of Materials), and place the coverslip upward with the H9c2 cells (cultured in step 2.1) in the bath.</li>
	<li>Fill 30% of the pipette (fabricated in step 3) with the extracellular solution, and install it on the recording electrode holder integrated into the patch-clamp apparatus. Tighten the pipette with the o-ring rubber gasket and plastic washer. Then, drop the pipette into the bath with the micromanipulator. Click on the Pipette offset interface of the signal-amplifier software (see Table of Materials) to maintain the IKv current baseline at 0 pA. 
	NOTE: The intracellular solution (step 1.4) must be in contact with the AgCl/Ag portion of the silver wire, and the silver wire must not be close to the inner wall of the pipette. The resistance of the pipette needs to be 2-6 M&#937;. To avoid the occlusion of the pipette tip, a continuous positive pressure must be delivered to the pipette using a 1.0 mL syringe connected to the recording electrode holder via plastic tubing24.</li>
	<li>Deliver an appropriate positive pressure manually with a 1.0 mL syringe connected to the recording electrode holder through a plastic tube. Move the pipette slightly by manipulating the micromanipulator in three dimensions to contact the cell.</li>
	<li>Once the membrane test square wave drops by 1/3 to 1/2 after the pipette touches the cell membrane, remove the positive pressure, and manually deliver an appropriate negative pressure. Then, click on the Patch interface of the data acquisition software to form the G&#937; seal. Use the &#34;Cp Fast&#34; and &#34;Cp Slow&#34; signal-amplifier software during cell sealing to compensate for the fast and slow capacitance. 
	NOTE: When the membrane test is &#8805;1 G&#937;, a cell-attached configuration is formed between the pipette tip and the cell.</li>
	<li>Apply brief pulses of negative pressure to rupture a patch of the cell membrane. 
	NOTE: A precipitous reduction in membrane resistance characterizes a successful whole-cell recording pattern. If the access resistance (Ra) &#8805;30 M&#937;, the cells must be reselected for steps 6.3-6.6. In order to ensure the accuracy of the recorded IKv data, the negative pressure must be removed after the cell membrane is broken.</li>
	<li>Execute whole-cell membrane capacitance compensation by clicking on the Whole-cell button of the signal-amplifier software. Finally, save and record the data by clicking on the Data recording button.</li>
	<li>Open the saved IKv data with the data analysis software. Save the current-voltage (I&#8722;V) relationships: click on Analyze to select Statistics to do the following: select the Range as Cousors 1,2 for data analysis; click on the Peak Amplitude and Mean buttons, and then click on OK to view the data in the Results page. Finally, copy the column of IKv mean data into the function drawing software (see Table of Materials) for further analysis.</li>
	<li>Save the representative IKv current traces: click on Edit to select Transfer Traces; then, select the Full trace in the Region to transfer; next, click on Select in Trace Selection, and select IN 0 (pA) in Signals; finally, click on OK to view the data in the &#34;Results&#34; page, and copy the total data into the function drawing software to draw the current traces.</li>
	<li>Save the IKv protocol: click on Edit to select Create Stimulus Waveform Signal, and select OK. Then, redo step 6.9 except for selecting A0 # 0 (mA) in &#34;Signals&#34;.</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+kinetics+and+selectivity+of+sodium+channels+in+frog+skeletal+muscle+fibers+treated+with+aconitine.">Modified kinetics and selectivity of sodium channels in frog skeletal muscle fibers treated with aconitine.</a> The Journal of General Physiology. 80 (5), 713-731 (1982).</li><li>Huang, X. Y., Ying, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+specific+protein+1+on+Kv2.1+potassium+channel+in+cardiac+myocytes.">The effect of specific protein 1 on Kv2.1 potassium channel in cardiac myocytes.</a> Journal of Electrocardiology and Circulation. 39 (4), 338-341 (2020).</li><li>Cao, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+patch+clamp+technique.">Development and application of patch clamp technique.</a> Journal of Yuncheng University. 27 (2), 53-55 (2009).</li></ol>

The authors have nothing to disclose.

The patch-clamp electrophysiological technique is mainly used to record and reflect the electrical activity and functional characteristics of ion channels on the cell membrane25. At present, the main recording methods of the patch-clamp technique include single-channel recording and whole-cell recording26. For the whole-cell mode, the glass microelectrode and negative pressure are used to form a high-resistance seal between a small area of the cell membrane and a pipette ti...

Ion channels are special integrated proteins embedded in the lipid bilayer of the cell membrane. In the presence of activators, the centers of such special integrated proteins form highly selective hydrophilic pores, allowing ions of an appropriate size and charge to pass through in a passive transport manner1. Ion channels are the basis of cell excitability and bioelectricity and play a key role in a variety of cellular activities2. The heart supplies blood to other organs through regular contractions resulting from an excitation-contraction-coupled process initiated by action potentials3. Previous studies have confirmed that the generation of action potentials in cardiomyocytes is caused by the change in intracellular ion concentration, and the activation and inactivation of Na+, Ca2+, and K+ ion channels in human cardiomyocytes lead to the formation of action potentials in a certain sequence4,5,6. Disturbed voltage-gated potassium (Kv) channel currents (IKv) could change the normal heart rhythm, leading to arrhythmias, which are one of the leading causes of death. Therefore, recording the IKv is critical for understanding the mechanisms of drugs for treating life-threatening arrhythmias7.
The Kv channel is an important component of the potassium channel. The coordination function of the Kv channel plays an important role in the electrical activity and myocardial contractility of the mammalian heart8,9,10. In cardiomyocytes, the amplitude and duration of action potentials depend on the co-conduction of outward K+ currents by multiple Kv channel subtypes11. The regulation of the Kv channel function is very important for the normal repolarization of the cardiac action potential. Even the slightest change in Kv conductance greatly impacts cardiac repolarization and increases the possibility of arrhythmia12,13.
Representing a fundamental method in cellular electrophysiological research, a high-resistance seal between a small area of the cell membrane and a pipette tip for whole-cell patch-clamp recording can be established by applying a negative pressure. The continuous negative pressure makes the cell membrane come into contact with the pipette tip and stick onto the inner wall of the pipette. The resulting complete electrical circuit allows one to record any single ion channel current across the surface of the cell membrane14. This technique has a very high sensitivity for the cell membrane ion channel current and can be used to detect currents in all ion channels, and the applications are extremely broad15. Moreover, compared with fluorescent labeling and radioactive labeling, patch-clamp has higher authority and accuracy16. At present, the whole-cell patch-clamp technique has been used to detect the traditional Chinese medicine components acting on Kv channel currents17,18,19. For example, Wang et al. used the whole-cell patch-clamp technique and confirmed that the effective component of the lotus seed might achieve the inhibition of the Kv4.3 channel by blocking the activated state channels19. Aconitine (AC) is one of the effective and active ingredients of Aconitum species, such as Aconitum carmichaeli Debx and Aconitum pendulum Busch. Numerous studies have shown that overdoses of AC can cause arrhythmias and even cardiac arrest20. The interaction between AC and voltage-gated ion channels leads to the disruption of intracellular ion homeostasis, which is the key mechanism of cardiotoxicity21. Therefore, in this study, the whole-cell patch-clamp technique is used to determine the effects of AC on the IKv of cardiomyocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma</td>
			<td>MKCJ2184</td>
			<td></td>
		</tr>
		<tr>
			<td>Aconitine</td>
			<td>Chengdu Lemetian Medical Technology Co., Ltd</td>
			<td>DSTDW000602</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Axon Instrument</td>
			<td>MultiClamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Sartorius</td>
			<td>124S-CW</td>
			<td></td>
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		<tr>
			<td>ATP Na2</td>
			<td>Solarbio</td>
			<td>416O022</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length)&#160;</td>
			<td>Sutter Instrument</td>
			<td>163225-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (100 mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>1192022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (35 mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>3012022</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex software</td>
			<td>Molecular Devices, LLC.</td>
			<td>Version 10. 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit software</td>
			<td>Molecular Devices, LLC.</td>
			<td>Version 10. 6. 0. 13</td>
			<td>data acqusition software</td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Rhawn</td>
			<td>RH289133</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Hamamatsu</td>
			<td>C11440</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Axon Instrument</td>
			<td>Axon digidata 1550B</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (1x)</td>
			<td>Gibco</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Biofroxx</td>
			<td>EZ6789D115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>Hunan Fenghui Biotechnology Co., Ltd</td>
			<td>CL0111</td>
			<td></td>
		</tr>
		<tr>
			<td>HCImageLive</td>
			<td>Hamamatsu</td>
			<td>4.5.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sichuan Xilong Scientific Co., Ltd</td>
			<td>2106081</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Xiya Chemical Technology (Shandong) Co., Ltd</td>
			<td>20210221</td>
			<td></td>
		</tr>
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			<td>KCl</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2020082501</td>
			<td></td>
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			<td>KOH</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2020112601</td>
			<td></td>
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			<td>MgCl2</td>
			<td>Tianjin Guangfu Fine Chemical Research Institute</td>
			<td>20160408</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2021020101</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass (20 &#215; 20 mm)</td>
			<td>Jiangsu Citotest Experimental Equipment Co. Ltd</td>
			<td>80340-0630</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q</td>
			<td>Chengdu Bioscience Technology Co., Ltd</td>
			<td>Milli-Q IQ 7005</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700B commander</td>
			<td>Axon Instrument</td>
			<td>MultiClamp commander 2.0</td>
			<td>signal-amplifier software&#160;</td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>17C18B16</td>
			<td></td>
		</tr>
		<tr>
			<td>PH&#160;meter&#160;</td>
			<td>Mettler Toledo</td>
			<td>S201K</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)</td>
			<td>HyClone</td>
			<td>J210045</td>
			<td></td>
		</tr>
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voltage-dependent potassium current recording on h9c2 cardiomyocytes via the whole-cell patch-clamp technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

This protocol allowed the recording of the IKv according to the parameters set in the whole-cell patch-clamp technique. The IKv was triggered by 150 ms of depolarizing pulse stimulus from &#8722;40 to +60 mV at a holding potential of &#8722;60 mV (Figure 3A). The IKv of the H9c2 rat cardiomyocytes first appeared around &#8722;20 mV, and then the amplitude increased with further depolarization. The mean relationship between the IKv and membrane pote...

Watch this Scientific Journal Video about Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique at JoVE.com

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...

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2.6K Views.  Chengdu University of Traditional Chinese Medicine. The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Video: Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes.
Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation HD-tDCS

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...