preparing wedge brain slices to visualize auditory neurons

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Begin by using a razor blade to cut the skin at the midline of the skull from the nose to the back of the neck. Peel back the skin to expose the skull, and starting at the base of the skull and continuing towards the nose, use small scissors to make an incision in the skull through the midline.
At the lambda suture, make cuts in the skull from the midline laterally towards the ear on both sides to peel back the skull to expose the brain. Starting at the rostral end, use a small lab spatula to gently lift the brain from the skull to allow the optic nerves to be severed.
Continue to gently work the brain backwards, exposing the ventral surface, and use fine forceps to carefully pinch the trigeminal nerves near the ventral surface of the brain stem to cut them. Place the preparation in a glass Petri dish filled with cold slicing solution, and place the dish under a dissecting microscope. Trim the facial nerves close to the brain stem to expose the vestibulocochlear nerves.
Use fine forceps to push the tips into the foramina where the vestibulocochlear nerve exits the skull as far as possible. Pinch to sever the nerves on both sides, leaving the nerve root attached to the brain stem.
Remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body. Then, pinch the remaining cranial nerves and connective tissue to free the brain completely from the skull, taking care to preserve the remaining spinal cord, if possible.
To prepare the surface of the brain for fixture to the stage, place the brain ventral side up, and use a blunt tool to gently immobilize the spinal cord to stabilize the brain tissue. Insert open forceps at an approximately 20-degree angle through the brain to the bottom of the dish so that the tips exit the dorsal surface of the brain caudal to the optic chiasm, and use a razor blade to cut along the forceps.
Next, prepare a one cubic centimeter block of 4% agar and spread a small drop of glue into a rectangle on the stage. Use forceps to carefully lift the brain and gently dab the excess liquid with the edge of a paper towel. Then, place the blocked surface onto the glue so that the ventral surface will be facing the direction of the blade during slicing, and push the agar block gently against the dorsal surface as a support.
To acquire wedge slices with the cochlear nerve root on the thick side and the medial, olivocochlear neurons, and the medial nucleus of the trapezoid body on the thin side, place the magnetic disk with the attached brain onto the stage holder, and place the holder in the slicing chamber of a vibratome with the ventral surface of the brain oriented toward the blade. Fill the chamber with ice-cold slicing solution and lower the blade into the carbogen-bubbled solution.
Cut slices caudal to the region of interest to make sure the slices are symmetrical. Then, shift the stage approximately 15 degrees to one side. Continue slicing carefully until the auditory nerve root is close to the surface on one side and the facial nerve can be seen at the surface of the other side of the slice.
Shift the stage 15 degrees back to the original position, and move the blade away from the tissue. Spin the stage base 90 degrees so that the lateral edge of the thin side is facing the blade, and lower the blade several hundred microns before slowly bringing the blade close to the edge of the tissue. With the blade retracted slightly, lower the blade to the desired thickness of the thin edge of the slice.
Move the blade back from the tissue and spin the stage base back so that the ventral surface faces the stage base. Make a cut to designate the rostral surface of the wedge slice, and transfer the slice to a one-square-centimeter piece of interface paper. caudal surface down.
The facial nerve should be visible on both hemispheres of the slice on the rostral surface. Then, move the slice to a 35-degree Celsius incubation chamber to recover for 30 minutes.

preparing-wedge-brain-slices-to-visualize-auditory-neurons

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain removal with intact auditory nerve root for stimulation
NOTE: Mice for these experiments were obtained by crossing ChAT-IRES-Cre transgenic mice on a C57BL/6J background with tdTomato reporter mice (Ai14). Mice used for histology and electrophysiology were post-hearing onset (P14-P23), which is around P12 in mice. Neurons expressing tdTomato in the ventral nucleus of the trapezoid body (VNTB) have been previously characterized as medial olivocochlear (MOC) neurons in this mouse line.
<ol>
	<li>Euthanize (e.g., CO2&#160;asphyxiation) and decapitate the animal using approved institutional procedures.</li>
	<li>Using a razor blade, cut the skin at the midline of the skull from the nose to the back of the neck. Peel back the skin to expose the skull.</li>
	<li>Using small scissors, make an incision in the skull through the midline, starting at the base (caudal end near the spinal cord) of the skull and continuing towards the nose.</li>
	<li>At the lambda suture, make cuts in the skull from the midline, lateral toward the ear on both sides. Peel back the skull to expose the brain.</li>
	<li>Starting at the rostral end, gently lift the brain away from the skull with a small lab spatula or blunt forceps. Cut the optic nerve and continue to gently work the brain backward, exposing the ventral surface.</li>
	<li>Cut the trigeminal nerves by pinching them with fine forceps near the ventral surface of the brainstem.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Do this carefully, as the vestibulocochlear nerve lies just below this and needs to be intact for eventual stimulation.</li>
	<li>Place the preparation in a glass Petri dish filled with cold slicing solution. Place the dish under a dissecting microscope. Gently bubble with carbogen.</li>
	<li>Trim the facial nerve close to the brainstem and expose the vestibulocochlear nerve.</li>
	<li>Using fine forceps, push the tips into the foramina where the vestibulocochlear nerve exits the skull as far as possible and pinch the nerve to sever it, leaving the nerve root attached to the brainstem. Repeat this on the other side.</li>
	<li>Once both nerve roots are free, remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body.</li>
	<li>Free the brain completely from the skull by pinching the remaining cranial nerves and connective tissue taking care to preserve the remaining spinal cord if possible.</li>
</ol>
2. Block and mount brain on stage (magnetic disc)
<ol>
	<li>Prepare the surface of the brain to fix to the stage by blocking the brain at the level of the optic chiasm.
	<ol>
		<li>With the ventral surface up, stabilize the brain using a blunt tool to gently immobilize the spinal cord so that the brain does not tilt during the following step.</li>
		<li>At the level of the optic chiasm, use open forceps to create the plane for blocking the brain by inserting through the brain down to the bottom of the dish. Insert the forceps at an angle of approximately 20&#730; from vertical so that the tips exit the dorsal surface of the brain caudal to the optic chiasm.</li>
		<li>Cut along the forceps using the razor blade.</li>
	</ol>
	</li>
	<li>Glue the brain to the surface of the stage.
	<ol>
		<li>Prepare a small block (~1 cm3) of 4% agar for supporting the brain.</li>
		<li>Place a small drop of glue on the stage and spread it into a rectangle so both the brain and agar block can be glued down.</li>
		<li>Using forceps, carefully lift the brain and gently dab the excess liquid using the edge of a paper towel. Place the blocked surface onto the glue, the ventral surface will be towards the blade during slicing.</li>
		<li>Push the agar block gently against the dorsal surface of the brain to support it during slicing and to ensure proper brain positioning (i.e., angle).</li>
	</ol>
	</li>
</ol>
3. Slice brain to create wedge slice
NOTE: Prepare a brain slice using a vibratome that has the cochlear nerve root on the thick side and medial olivocochlear (MOC) neurons and the medial nucleus of the trapezoid body (MNTB) on the thin side.
<ol>
	<li>Place the magnetic disc with the attached brain onto the stage base and place it in the slicing chamber with the ventral surface of the brain oriented towards the blade.</li>
	<li>Fill the chamber with ice cold slicing solution and bubble with carbogen.</li>
	<li>Lower the blade into the solution and cut slices caudal to the region of interest to make sure the slices are symmetrical. If the slices appear asymmetrical, tilt the stage slightly to obtain symmetry.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Blade speeds between 0.05-0.10 mm/s were effective for cutting healthy slices and may vary depending on animal age and brain region.</li>
	<li>Once the slices are symmetrical, shift the stage ~15&#730; (corresponding to approximately 3 concentric rings on the stage base) to one side.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Shift the stage away from the auditory nerve root that you want to preserve in the slice.</li>
	<li>Continue slicing carefully until the auditory nerve root is close to the surface on one side, and the facial nerve can be seen at the surface of the other side.</li>
	<li>Shift the stage back 15&#730; to the original position.</li>
	<li>Move the blade away from the tissue and spin the stage base 90&#176; so that the lateral edge of the thin side is facing the blade. Lower the blade several hundred microns and then slowly bring the blade close to the edge of the tissue. Repeat this until the blade touches the lateral edge. Lower the blade to the desired thickness of the thin edge of the slice, here an additional two hundred microns.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The resulting slice is ideally ~300 mm thick at the level of the ventral nucleus of the trapezoid body (VNTB) on the side where patch clamping will take place.</li>
	<li>Move the blade back away from the tissue and spin the stage base back so that the ventral surface is facing it.</li>
	<li>Make the cut that designates the rostral surface of the wedge slice. Transfer the slice to a piece of interface paper (1 cm2) caudal surface down. Move the slice to the incubation chamber or other suitable incubation apparatus for recovery (30 min at 35 &#176;C). 
	NOTE: The facial nerve should be visible on both hemispheres of the slice on the rostral surface (see&#160;Figure 1B).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Experimental Preparations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, powder</td>
			<td>Fisher Scientific</td>
			<td>BP1423500</td>
			<td>4% agar block used to stabilize brain tissue during vibratome sectioning</td>
		</tr>
		<tr>
			<td>AlexaFluor Hydrazide 488</td>
			<td>Invitrogen</td>
			<td>A10436</td>
			<td>Fluorophore used in internal solution to confirm successful MOC neuron patch</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Geneses Scientific (Intramalls)</td>
			<td>AV114</td>
			<td>Weighing chemicals</td>
		</tr>
		<tr>
			<td>Double edged razor blade</td>
			<td>Ted Pella</td>
			<td>121-6</td>
			<td>Vibratome cutting blade</td>
		</tr>
		<tr>
			<td>Kynurenic acid (5g)</td>
			<td>Sigma Aldrich</td>
			<td>K3375-5G</td>
			<td>Slicing ACSF additive used to reduce neuron activity during dissection and slicing in order to improve tissue health for patch clamping</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Fisher Scientific (Intramalls)</td>
			<td>13-620-451</td>
			<td>Solution pH tester</td>
		</tr>
		<tr>
			<td>Plastic petri dishes 100mm dia X 20mm</td>
			<td>Fisher Scientific (Intramalls)</td>
			<td>12-556-002</td>
			<td>4% Agar Prep</td>
		</tr>
		<tr>
			<td>Stirring Hotplate</td>
			<td>Fisher Scientific (Intramalls)</td>
			<td>11-500-150</td>
			<td>Heating for 4% Agar preparation</td>
		</tr>
		<tr>
			<td>Dissection and Slicing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Sigma Aldrich</td>
			<td>B4261-250MG</td>
			<td>Chemical used for axonal tracing (conjugated to Streptavidin 488)</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Amscope</td>
			<td>SM-1BN</td>
			<td>For precision dissection during brain removal</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Fine forceps dissection tool</td>
		</tr>
		<tr>
			<td>Economy tweezers #3</td>
			<td>WPI</td>
			<td>501976</td>
			<td>Forceps dissection tool</td>
		</tr>
		<tr>
			<td>Glass Petri Dish 150mm dia x 15mm H</td>
			<td>Fisher Scientific (Intramalls)</td>
			<td>08-747E</td>
			<td>Dissection dish</td>
		</tr>
		<tr>
			<td>Interface paper (203 X 254mm PCTE Membrane 10um)</td>
			<td>Thomas Scientific</td>
			<td>1220823</td>
			<td>Slice incubation/biocytin application</td>
		</tr>
		<tr>
			<td>Leica VT1200S Vibratome</td>
			<td>Leica</td>
			<td>1491200S001</td>
			<td>Vibratome for wedge slice sectioning</td>
		</tr>
		<tr>
			<td>Mayo scissors</td>
			<td>Roboz</td>
			<td>RS-6872</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Single-edged carbon steel blades</td>
			<td>Fisher Scientific (Intramalls)</td>
			<td>12-640</td>
			<td>Razor blade for dissection</td>
		</tr>
		<tr>
			<td>Specimen disc, orienting</td>
			<td>Leica</td>
			<td>14048142068</td>
			<td>Specialized vibratome stage for reproducible tilting</td>
		</tr>
		<tr>
			<td>Spoonula</td>
			<td>FisherSci</td>
			<td>14-375-10</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Newegg</td>
			<td>15187</td>
			<td>Used for glueing tissue to vibratome stage</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td>Dissection tool</td>
		</tr>
	</tbody>
</table>

Source: Fischl, M. J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

<img alt="Figure 1" src="/files/ftp_upload/22369/22369_Figure1.jpg" /> 
Figure 1: Wedge slice schematic and example image.&#160;(A) Schematic of the medial olivocochlear feedback circuit. Blue arrows indicate the afferent ascending pathway to MOC neurons, and black arrows indicate the descending feedback pathway from MOC neurons to the base of outer hair cells (OHC). (B) Brightfield image of a wedge slice with labels of the auditory ...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...

Begin with a mouse brain and dissect it horizontally below the posterior part to preserve auditory neurons crucial for sound processing.
Mount the posterior section on a magnetic disc. Place an agar block to stabilize the tissue during slicing.
Transfer the section to the stage of a slicing chamber of a vibratome equipped with a blade.
Introduce a cold buffer and bubble carbogen gas that provides oxygen and preserves neuron integrity.
Make an even horizontal cut to remove the upper portion of the brain section.
Then, tilt the stage and continue cutting the remaining section until the auditory nerve root is visible.
Realign the stage. Next, rotate it and adjust the blade position for the desired thickness of the thin edge of the slice.
Move the blade away and realign the stage to its earlier position. Now, slice the brain into a wedge shape, thicker on one side and thinner on the other side.
The thicker side includes the auditory nerve root, while the thinner side features interconnected auditory neurons.

23 次观看. 准备楔形脑切片以可视化听觉神经元

视频: 准备楔形脑切片以可视化听觉神经元 - 实验

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

生理条件下猪心的切片和培育

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

科研

JoVE Journal

医学

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

生物学

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

成績單

Preparing Wedge Brain Slices to Visualize Auditory Neurons