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. Preparation of drugs
<ol>
	<li>Anesthesia with Ketamine/Xylazine 
	NOTE: The dose of ketamine used is 70 mg/kg, and the dose of xylazine is 10 mg/kg.
	<ol>
		<li>To prepare 1 mL of anesthetic using ketamine 100 mg/mL solution and xylazine 20 mg/mL solution, combine 0.35 mL of ketamine solution, 0.25 mL of xylazine solution, and 0.4 mL of 0.9% sterile saline solution. Administer the anesthetic solution at a final volume of 2 mL/kg. 
		NOTE: Ketamine along with xylazine can produce sedation for 60-80 min. If the animal still has reflexes (e.g., hind leg pitching and/or blinking reflex), administer an additional 10% of the individual dose.</li>
	</ol>
	</li>
	<li>Imipramine 
	NOTE: The individual dose of imipramine used is 20 mg/kg.
	<ol>
		<li>To prepare 1 mL of imipramine 20 mg/mL solution, combine 20 mg of imipramine and 1 mL of 0.9% sterile saline solution. Administer the imipramine solution at a final volume of 1 mL/kg.</li>
	</ol>
	</li>
	<li>Meloxicam 
	NOTE: The individual dose of meloxicam used is 1 mg/kg.
	<ol>
		<li>To prepare 1 mL of meloxicam 1 mg/mL solution, combine 0.05 mL of meloxicam 2% and 0.95 mL of 0.9% sterile saline solution. Administer the meloxicam solution at a final volume of 1 mL/kg once a day for two days.</li>
	</ol>
	</li>
	<li>Ascorbic acid 0.1%
	<ol>
		<li>To prepare 1 mL of 0.1% ascorbic acid, combine 1 mg of ascorbic acid and 1 mL of 0.9% sterile saline solution.</li>
	</ol>
	</li>
	<li>6-hydroxydopamine (6-OHDA) 
	NOTE: 6-OHDA is a neurotoxin used to selectively destroy dopaminergic and noradrenergic neurons in the brain. Avoid direct contact with skin and mucous membranes of the eyes, nose, and mouth. When handling 6-OHDA, wear double nitrile gloves, lab coat, disposable gown, eye protection, and surgical mask or face shield. The total infusion volume of the toxin is 4 &#956;L/animal, and the individual amount is 10 &#956;g of 6-OHDA/animal.
	<ol>
		<li>To prepare 1 mL of 6-OHDA at a final concentration of 2.5 mg/mL, mix 2.5 mg of 6-OHDA and 1 mL of 0.9% saline solution containing 0.1% ascorbic acid (described above). 
		&#8203;NOTE: 6-OHDA is light-sensitive and degrades faster when exposed to bright light. It must be properly handled and stored in an environment protected from light. If the color of the solution is reddish, discard it.</li>
	</ol>
	</li>
	<li>Lidocaine hydrochloride (2%)
	<ol>
		<li>Prepare 2% lidocaine solution for local application to the animal. 
		NOTE: The maximum dose that can be applied is 7 mg/kg.</li>
	</ol>
	</li>
	<li>Poly-antibiotic suspension 
	&#8203;NOTE: The poly-antibiotic suspension with streptomycins and penicillins (see the Table of Materials) must be prepared at the time of application with the entire volume of diluent, whose ampoule accompanies the vial with the powder.
	<ol>
		<li>Remove the metallic disc on the rubber stopper. Disinfect the rubber stopper with alcohol.</li>
		<li>Using a syringe with a needle of 23 G, inject the diluent into the vial. Remove the needle and shake the vial vigorously until the suspension is entirely homogenized. Inject a little air into the vial and withdraw the desired volume of suspension.</li>
		<li>Administer a deep intramuscular injection, pulling the plunger before injecting the drug to ensure that no blood vessel is reached. 
		&#8203;NOTE: The final volume of suspension to be applied is 0.5 mL/kg.</li>
	</ol>
	</li>
</ol>
2. Preparation of materials
NOTE: Always follow instructions provided with the material safety data sheet when handling chemicals.
<ol>
	<li>Stereotaxic apparatus
	<ol>
		<li>Place the stereotaxic device on a stable and clean bench with proper illumination to perform the surgery. Disinfect the apparatus with 70% ethanol.</li>
		<li>Check if the ear and incisor bars of the device are correctly aligned. Place a thermal blanket where the animal will be placed during surgery to stay warm during the procedure. Monitor the animal&#39;s temperature with an accurate rectal probe. 
		NOTE: The thermal blanket should be at 37.5 &#176;C so that the animal maintains a body temperature of 37 &#176;C body.</li>
	</ol>
	</li>
	<li>Microinfusion system
	<ol>
		<li>Fill (70-80%) a Hamilton syringe (50 &#956;L or as desired) attached to a medical-grade polyethylene microtubing and a needle with double distilled water (ddH2O) and check for leaks through the system.</li>
		<li>Pull air through the system so that a single air bubble separates the ddH2O in the syringe from the 6-OHDA solution in the microtube. 
		NOTE: This procedure avoids contaminating the Hamilton syringe with 6-OHDA and allows the use of several rats on the same experimental day.</li>
		<li>Position the Hamilton syringe on the infusion pump so that it is firmly attached, and the plunger of the syringe is parallel to the frame that will move to push it. Set the infusion pump to a speed of 0.5 &#956;L/min so that the total application of 4 &#956;L of 6-OHDA lasts for 8 min. Test the infusion system by confirming that there are no leaks, and that the infusion occurs according to the previously set time and volume.</li>
		<li>Attach the needle of infusion attached to the microtube to the apparatus at the end of the stereotaxic arm and check that the needle is positioned at a 180&#176; angle to the surface. Ensure that the needle is straight and not bent. 
		NOTE: Check all the described procedures carefully because if any of the items in the infusion system do not work correctly, it may jeopardize the success of the surgery.</li>
	</ol>
	</li>
	<li>Suture
	<ol>
		<li>Use a sterile nylon non-absorbable suture with a 3/8 circle needle to suture the incision after surgery.</li>
	</ol>
	</li>
	<li>Postsurgical recovery site
	<ol>
		<li>Place a clean and sterilized housing box where animals can be monitored until fully recovered (responsive to touch and manipulation). Put a thermal blanket in the box for thermoregulation. 
		&#8203;NOTE: As thermoregulation is important, include a supplemental heat source to maintain body temperature if necessary.</li>
	</ol>
	</li>
</ol>
3. Surgical procedure
NOTE: In this protocol, adult male Sprague-Dawley rats (200-250 g) were kept under controlled conditions of temperature (22 &#177; 2 &#176;C), air exchange (15-20 exchanges/hour), and light-dark cycles (12 h/12 h), grouped in boxes with 3 or 4 animals, with free access to food and water.
<ol>
	<li>Weigh the animals to monitor weight changes in the days following the surgery. Calculate the dose of drugs to be administered.</li>
	<li>Administer imipramine intraperitoneally 30 min before surgery (~10-15 min before administering anesthesia), using a 27 G needle and a 1 mL syringe. 
	NOTE: The imipramine will block the noradrenaline transporter (NAT) and prevent 6-OHDA uptake by noradrenergic neurons, making the lesion more selective to the dopaminergic neurons40.</li>
	<li>After 10-15 min of the administration of imipramine, administer the intraperitoneal ketamine/xylazine anesthesia using a 27 G needle and a 1 mL syringe. Wait until the animal is completely anesthetized. Verify that the animal is under deep anesthesia when the animal does not respond to hind leg pinching and does not show a blink reflex.</li>
	<li>Shave the rat&#39;s fur in the region of the head where the incision will occur.</li>
	<li>Position the rat in the stereotaxic apparatus.
	<ol>
		<li>Position the head over the incisor bar and fix the bar 3.3 mm below the interaural line.</li>
		<li>Position the ear bars, one side at a time. Position the incisor bar and the ear bars so that the top of the skull is straight and parallel to the surface.</li>
		<li>Adjust the nose clamp and test that the head is firm and does not move to either side.</li>
	</ol>
	</li>
	<li>Apply sterile ophthalmic ointment to the rat eyes to prevent corneas from drying out.</li>
	<li>Apply povidone-iodine to the area to be incised to disinfect the site.</li>
	<li>Apply local lidocaine for analgesia of the incision region; do not exceed 7 mg/kg.</li>
	<li>Administer the meloxicam subcutaneously using a 27 G needle and a 1 mL syringe. 
	NOTE: Meloxicam is a nonsteroidal anti-inflammatory analgesic that will help the animal recover post-surgery.</li>
	<li>Administer the poly-antibiotic suspension intramuscularly using a 23 G needle and a 1 mL syringe. 
	NOTE: The poly-antibiotic suspension is administered as a prophylactic treatment to avoid possible bacterial infections in the post-surgery recovery.</li>
	<li>Check that the animal is in a state of deep anesthesia by checking for blink reflexes or hind limb reflexes by pinching the hind paw with tweezers.</li>
	<li>With a scalpel, make an incision of ~1.5 cm in the region where the microinjection will occur. 
	NOTE: Sterile techniques are applied from this point until the wound closure.</li>
	<li>Clean the skull region with cotton swabs and cotton buds until the Bregma and Lambda can be seen. Mark the Bregma and the Lambda with a sterilized fine pen.</li>
	<li>Check that the dorsal-ventral (DV) coordinates of Bregma and Lambda are similar. If they are different, readjust the rat in the stereotaxic apparatus as the rat&#39;s head is not correctly positioned.</li>
	<li>Note down the anteroposterior (AP) and mediolateral (ML) coordinates of the Bregma.</li>
	<li>Move to the AP and ML coordinates of the right MFB according to 41: AP: -4.3 mm, ML: 1.6 mm from Bregma.</li>
	<li>Mark the region of the trepanation with a sterilized fine pen.</li>
	<li>With a sterilized drill, slowly pierce the animal&#39;s skull, taking care not to injure the dura mater.</li>
	<li>Position the microinjection needle on the dura mater and note the DV coordinates. Take a thin needle and gently rupture the dura mater. Insert the needle to the DV coordinate (8.3 mm ventral) of the MFB, where the microinjection will take place.</li>
	<li>Operate the microinjection pump to release the 6-OHDA solution into the MFB. When the microinjection is finished, check the Hamilton syringe to see if 4 &#956;L of 6-OHDA has been injected. 
	NOTE: The microinjection should last 8 min.</li>
	<li>After administration of the 6-OHDA, wait for 10 min before removing the needle to avoid backflow of the drug. Remove the microinjection needle slowly from the animal&#39;s brain.</li>
	<li>Disinfect the incision region again with povidone-iodine.</li>
	<li>Suture the incision area with ~3-4 surgical knots. 
	NOTE: The knot should not be too strong or too loose.</li>
	<li>Remove the rat from the stereotaxic apparatus and place it in a clean box for recovery on the thermal blanket until the animal has fully recovered from anesthesia. Observe the animal every 15 min until it is fully awake from anesthesia.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-OHDA</td>
			<td>Sigma Aldrich</td>
			<td>H4381</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt&#174;ion=BR&#38;cm_sp=Insite-_-caSrpResults_srpRecs_srpModel 
			_6-ohda-_-srpRecs3-1</td>
		</tr>
		<tr>
			<td>70% Alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>795437</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sial/795437?lang=pt&#174;ion=BR&#38;gclid= 
			Cj0KCQjw4cOEBhDMARIsAA3XD 
			RipyOnxOxkKAm3J1PxvIsvw09 
			_kfaS2jYcD9E5OyuHYr4n89kO 
			6yicaAot6EALw_wcB</td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drill or tap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 50 uL</td>
			<td>Hamilton</td>
			<td>80539</td>
			<td>https://www.hamiltoncompany.com/laboratory-products/syringes/80539</td>
		</tr>
		<tr>
			<td>Imipramine</td>
			<td>Alfa Aeser</td>
			<td>J63723</td>
			<td>https://www.alfa.com/pt/catalog/J63723/</td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>Insight</td>
			<td>EFF-311</td>
			<td>https://insightltda.com.br/produto/eff-311-bomba-de-infusao-2-seringas/</td>
		</tr>
		<tr>
			<td>Ketamine (Dopalen)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/DOPALEN</td>
		</tr>
		<tr>
			<td>Machine for trichotomy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Maxicam 2% Ourofino)</td>
			<td>Ourofino</td>
			<td></td>
			<td>https://terrazoo.com.br/produto/maxicam-injetavel-2-50ml-ouro-fino/</td>
		</tr>
		<tr>
			<td>Metal Disposal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentabiotic</td>
			<td>Zoetis</td>
			<td></td>
			<td>https://www.zoetis.com.br/pentabiotico-veterinario.aspx</td>
		</tr>
		<tr>
			<td>Plastic waste garbage can</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-antibiotic</td>
			<td>Pentabiotic (Wealth)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel and blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scraper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Insight</td>
			<td>EFF-331</td>
			<td>https://insightltda.com.br/produto/eff-331-estereotaxico-1-torre/</td>
		</tr>
		<tr>
			<td>Sterile saline solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Anasedan)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/ANASEDAN</td>
		</tr>
	</tbody>
</table>

generating the 6-hydroxydopamine rat model of parkinson&#39;s disease

Source: Guimar&#227;es, R. P., et al. <a href="https://app.jove.com/v/62923">The 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a procedure to create a rat model of Parkinson&#39;s disease by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the brain. The neurotoxin infusion into the medial forebrain bundle (MFB) specifically targets dopaminergic neurons responsible for motor control, leading to their destruction and replicating Parkinson&#39;s disease symptoms.

Generating the 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease

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

Take a rat and intraperitoneally inject the drug imipramine. Anesthetize the rat, shave its head, and secure it in a stereotaxic frame.&#160;
Administer antibiotics intramuscularly to prevent infections.
Disinfect the head and incise to expose the skull. Identify the bregma and the lambda to locate the medial forebrain bundle or MFB.
The MFB comprises neurons connecting the midbrain to the forebrain, including the nigrostriatal dopaminergic neurons that control motor coordination.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
Drill a hole, position a microsyringe containing 6-hydroxydopamine or 6-OHDA, a neurotoxin, and inject it into the MFB.
Keep the needle in place to prevent neurotoxin backflow, then withdraw it.
Disinfect and suture the incision. Allow the rat to recover.
The previously injected imipramine blocks transporters in non-dopaminergic neurons, ensuring selective 6-OHDA uptake by dopaminergic neurons.
6-OHDA produces reactive oxygen species, causing oxidative damage and neuronal death.
The loss of dopaminergic neurons disrupts motor function, mimicking the motor function loss in Parkinson&#39;s disease. &#160; &#160; &#160; &#160; &#160;

generating-the-6-hydroxydopamine-rat-model-of-parkinson-s-disease

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

92 Views. Source: Guimarães, R. P., et al. The 6-hydroxydopamine Rat Model of Parkinson's Disease. J. Vis. Exp. (2021). This video demonstrates a procedure to create a rat model of Parkinson's disease by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the brain. The neurotoxin infusion into the medial forebrain bundle (MFB) specifically targets dopaminergic neurons responsible for motor control, leading to their destruction and replicating Parkinson's disease symptoms. 

Video: Generating the 6-hydroxydopamine Rat Model of Parkinson's Disease - Concept

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

JoVE Journal

Behavior

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

Generating the 6-hydroxydopamine Rat Model of Parkinson's Disease

Transcript