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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. Preparation of the Rat
NOTE: We performed the experiments in male rats to avoid any hormonal changes.
<ol>
	<li>Anesthetize the animal using a mix of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg) injected intramuscularly; with this dose, the rat should be deeply anesthetized for around 1 h.</li>
	<li>Pinch the rat&#39;s toe; absence of a withdrawal reflex indicates that the animal is fully unconscious. If the rat responds to the pinch, give supplementary anesthesia at one third of the initial dose.</li>
	<li>Shave the scalp and disinfect the surgical area with povidone-iodine.</li>
	<li>Place the animal on a heating pad to maintain a temperature of 38 &#176;C and stabilize the animal&#39;s head in a stereotaxic frame using two ear bars and a bite bar. Use caution to avoid piercing the tympanic membrane with the ear bars.</li>
	<li>Protect the eyes by applying a drop of ophthalmic gel or serum saline to each eye.</li>
</ol>
2. Location of the AC in the Temporal Bone of the Rat
<ol>
	<li>Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the surface of the cranium.</li>
	<li>Use a sterile cotton tip to gently remove any blood covering the surface of the skull to visualize bregma, lambda, and interaural 0 according to the Paxinos and Watson atlas of the rat brain.</li>
	<li>Make an incision in the temporal muscle near its dorsal insertion on the skull with a scalpel. Pull the muscle out using a needle and suture material and fix the suture material to the stereotactic frame; this will expose the temporal bone. If bleeding occurs, rinse with cold sterile saline.</li>
	<li>Place a sterile straight needle in the stereotactic micromanipulator, making sure that it is fully secured.</li>
	<li>Slowly lower the needle until it is right above the surface of the skull, so that the tip of the needle is set to interaural 0. Set this point as zero and determine coordinates from this point.</li>
	<li>Depending on the brain area of interest, vary the stereotactic coordinates. Determine these coordinates by utilizing the Paxinos and Watson atlas of the rat brain. Once the coordinates are determined, move the needle to match those coordinates.</li>
	<li>Target the AC using the coordinates of the following four points: A: anteroposterior (A/P) = -5.8 mm, mediolateral (M/L) = +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. Lower the needle to right above the temporal bone to visualize each of these four points. Using a marker pen, mark the points on the temporal bone and connect them in order to draw a rectangle; the rectangle will serve as a guide to open of a window in the bone (Figure 1).</li>
</ol>
3. Surgical Exposure of the AC
<ol>
	<li>Open the window using an electric drill and a small drill bit (0.6 mm &#216;). Drill the perimeter of the rectangle at 8,000 rpm until the bone gives away. Cool the drilling surface by rinsing with cold sterile saline to prevent damage to subcortical structures. When the bone gives way, a drop in resistance can be detected. Be careful to not drill the brain.</li>
	<li>When the borders are loose, pull up the covering bone with fine forceps and store it in cold sterile saline.</li>
</ol>
4. Ablation of the AC
<ol>
	<li>Using a surgical microscope (10X), gently cut the meninges with a microsurgical knife and remove them using two fine-pointed forceps. If bleeding occurs, rinse with cold sterile saline.</li>
	<li>Gently aspirate the AC using a surgical suction device (pressure -0.24 bar) coupled to a sterile 20 G blunt tip needle. This point is critical and needs to be performed very carefully: aspirate only the six cortical layers and not the underlying white matter.</li>
	<li>Aspirate until the perforating arteries stop bleeding.</li>
	<li>When the aspiration is finished, cover the injured area with the extracted bone and apply an absorbable hemostatic gauze.</li>
	<li>Let the temporal muscle recover its original position, and then suture the skin using wound clips (9 mm). Apply antibiotic ointment. Continue to apply ointment to the wound twice daily for three days. 
	NOTE: Each application consists of a thin layer applied on the wound.</li>
	<li>Inject buprenorphine subcutaneously in the back of the rat (0.05 mg/kg) as an analgesic 1 h after the surgery, and then every 8 h during 72 h.</li>
	<li>Keep the animal on the heating pad until it wakes up and return it to its housing cage to recover.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Ins.</td>
			<td>900</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>WILD M650 Heerbrugg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>DAGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental micromotor</td>
			<td>W&#38;H elco</td>
			<td>5118</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond burr</td>
			<td>B Braun</td>
			<td>GD021R</td>
			<td>0.6 mm</td>
		</tr>
		<tr>
			<td>Surgical suction device</td>
			<td>Atmos</td>
			<td>Atmoforte E2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td></td>
			<td>30 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td></td>
			<td>5 mg/kg</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>SM-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lawton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Meda</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile saline serum</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20G sterile needle</td>
			<td>Terumo Neolus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture material</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Ointment</td>
			<td>Quadriderm (Betametasona, Gentamicina, Clotrimazol) - Schering-Plough</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>dimeda</td>
			<td>10.331.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical needles</td>
			<td>World Precision Instruments</td>
			<td>501940</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior UK</td>
			<td>Buprex</td>
			<td>0.01-0.05 mg/kg</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>dimeda</td>
			<td>08.120.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical knife</td>
			<td>MSP</td>
			<td>7503</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable hemostatic gauze</td>
			<td>Surgicel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Reflex 9</td>
			<td></td>
			<td>9 mm</td>
		</tr>
	</tbody>
</table>

a stereotactically guided ablation technique for auditory cortex removal in rats

Source: Lamas, V., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56429">Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates a stereotactically-guided surgical procedure for ablating the auditory cortex in a rat. The steps include identifying anatomical landmarks in an anesthetized rat secured in a stereotactic frame, creating a window on the skull above the auditory cortex, aspirating the cortical layers, and providing post-operative care to assess the impact of the ablation on sound processing.

<img alt="Figure 1" src="/files/ftp_upload/23206/23206_Figure1.jpg" /> 
Figure 1: Images of the rat temporal bone at three different surgical steps.&#160;(A) Transfer of the stereotaxic coordinates of the AC to the temporal bone. The coordinates of the four points are: A: A/P= -5.8 mm, M/L= +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. (B) The coordinates are used ...

A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats

Source: Lamas, V., et al. Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain.&#160;J. Vis. Exp. ...

Take an anesthetized rat secured in a stereotactic frame.
Shave and disinfect the scalp, then make an incision to expose the skull.
Identify the anatomical references the bregma, the lambda, and the interaural line to locate the auditory cortex, the brain region that processes sound signals.
Incise and retract the temporal muscle, revealing the temporal bone.
Position a needle at the interaural line&#39;s midpoint. Using this landmark, determine the coordinates above the auditory cortex.
Drill the marked region and remove the bone to expose the brain.
Cut through the meninges, the layers covering the brain.
Perform ablation by aspirating the cortical layers while preserving the white matter. Auditory cortex removal disrupts the sound signal processing ability.
Replace the excised bone, apply gauze to control bleeding, and suture the skin.
Apply antibiotic ointment to prevent infection and administer subcutaneous analgesics to alleviate pain.
Allow the rat to recover to assess the impact of ablation.

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Universidad San Sebastian

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JoVE Encyclopedia of Experiments

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Neuropathology

Neurodegenerative Diseases

41 Views. Source: Lamas, V., et al. Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain. J. Vis. Exp. (2017). This video demonstrates a stereotactically-guided surgical procedure for ablating the auditory cortex in a rat. The steps include identifying anatomical landmarks in an anesthetized rat secured in a stereotactic frame, creating a window on the skull above the auditory cortex, aspirating the cortical layers, and providing post-operative car...

Video: A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats - Concept

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

JoVE Journal

Developmental Biology

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats

Transkrypcja

A Stereotactically-Guided Ablation Technique for Auditory Cortex Removal in Rats

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Laser-Induced Brain Injury in the Motor Cortex of Rats

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice