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. Catheter Preparation before Stereotaxic Surgery
<ol>
	<li>Cut PE50 catheters (6 cm in length) that will be used to connect the cannula with osmotic pumps (see Figure 1). Fill PE50 catheters with artificial cerebrospinal fluid (aCSF) and seal both ends of the catheters. Keep the catheter at 4 &#176;C until used.</li>
</ol>
2. Cannula Implantation by Stereotaxy
<ol>
	<li>Perform the surgery in sterile conditions. Sterilize all the surgical instruments and materials by autoclaving. Clean the stereotaxic apparatus and the working area thoroughly and disinfect with a 70% ethanol solution. Wear a surgical mask, hair bonnet, and sterile gloves.</li>
	<li>Anesthetize rats by injecting intraperitoneally (i.p.) a solution of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). Confirm anesthesia by checking movement after a gentle toe pinch.</li>
	<li>Inject a non-steroidal anti-inflammatory drug (Meloxicam; 1 mg/kg) subcutaneously (s.c.) to the anesthetized animal at least 30 min before surgery.</li>
	<li>Shave the head of the animal using clippers and disinfect the skin three times with a solution of chlorhexidine gluconate 2% and isopropyl alcohol 2%. Apply veterinary ophthalmic ointment on the eyes to prevent dryness while under anesthesia.</li>
	<li>Place the animal on a stereotaxic frame with the ear bars. Fix one cannula on the holder arm of the stereotaxic frame. Inject (s.c.) a local anesthetic agent (bupivacaine (1.5 mg/kg) and lidocaine (1.5 mg/kg) on the top of the head. Anesthesia is maintained during the whole procedure by placing a nose cone delivering 3% isoflurane. The entire surgical field should be draped off, but it was not done here to demonstrate the technique better.</li>
	<li>Make an incision of 3 cm on the top of the head with a scalpel. Install 4 clamps around the incision to leave the skull clear. Using a round-tip scissor, make a pocket (2 x 2 cm2) under the skin between the shoulder blades of the animal.</li>
	<li>Scrape the periosteum of the skull with a blade. Apply a gauze pad on the skull if it is bleeding.</li>
	<li>Verify that the skull is flat and well aligned on the stereotaxic frame. To ensure the skull is flat, check the height coordinates at the bregma and lambda. Take the bregma coordinates that will be used as the reference point.</li>
	<li>Starting from the bregma, calculate the coordinates of the two guide cannula that will be implanted bilaterally in the hippocampus (anteroposterior: - 0.42 cm; mediolateral: &#177; 0.30 cm; dorsoventral: - 0.28 cm according to the Paxinos and Watson rat brain atlas). Indicate the position of the cannula with a marker.</li>
	<li>Drill a hole (0.5 mm) in the skull at the implantation point of both cannulas. Drill two other holes approximately 5 mm above and below those points to insert screws that will solidify the cannula when applying dental cement.</li>
	<li>Cut one end of the catheter previously filled with aCSF with a scalpel and insert it in the angle arm of the cannula. Fix the first cannula with dental cement. Avoid putting dental cement around the position on the second cannula.</li>
	<li>Let the dental cement dry for 2 - 3 min, then remove the holder arm from the first cannula, and fix the second cannula in it. Repeat steps 2.11 and 2.12. Put a dummy cannula on both guide cannulas. Ensure that the dummy and guide cannula are the same length to prevent tissue infiltration and blocking of the cannula.</li>
	<li>Insert the free end of both catheters in the pocket previously made between the shoulder blades of the animal. Remove the clamps and stitch the skin with a suture thread 4-0. 
	NOTE: The top guide cannula must be accessible for upcoming A&#946;o infusions.</li>
	<li>Remove the animal from the stereotaxic frame and put it back in its cage. During post-surgical recovery, place the cage on a heating water blanket until the animal wakes up. The cage should be partially on the pad so the rat can escape the heat if needed. Monitor the animal constantly until it regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>Return rats to the animal facility to recover from the surgery for 10 days under close monitoring. 
	NOTE: Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Meloxicam (1 mg/kg) is given once per day for two days following surgery to treat pain.</li>
</ol>
3. Osmotic Pump Installation
<ol>
	<li>One day before installation, fill osmotic pumps with 6E10 antibody (1 mg/ml; 100 &#181;l) and control IgG1 antibody (1 mg/ml; 100 &#181;l) according to the manufacturer&#39;s instructions under sterile conditions. Keep the pumps in sterile distilled water at 37 &#176;C overnight to activate pumps.</li>
	<li>Anesthetize rats with isoflurane 3% to install osmotic pumps (0.5 &#181;l/hr for 7 days). Shave between shoulder blades and disinfect the skin with a solution of chlorhexidine gluconate 2% and isopropyl alcohol 2%. Make an incision of 2 cm with a scalpel between the shoulder blades to locate the PE50 catheters connected to the guide cannula.</li>
	<li>Cut the end of the PE50 catheters containing dental cement with a scalpel. Connect the osmotic pumps to the PE50 catheters and add some dental cement at the pump and catheter junction to secure the connection.</li>
	<li>Stitch the skin tightly with a suture thread 4-0. Put the animal back in its cage on a heating water pad. Observe the animal wake up rapidly from an isoflurane anesthesia (about 5 min). 
	NOTE: The surgery takes approximately 5 to 10 min.</li>
</ol>
4. A&#946;o Infusions in Awake and Freely Moving Rats
<ol>
	<li>Prepare the A&#946;o solution (0.2 &#181;g/&#181;l). Allow A&#946;o to aggregate dynamically and spontaneously for 1 hr at room temperature before infusion.</li>
	<li>Install two 10 &#181;l syringes on an infusion pump. Fill the syringes with 5 &#181;l of sterile distilled water. Cut two PE50 catheters to about 60 cm in length with a scalpel. Fill both catheters with sterile distilled water using 1 ml syringes and 21G needles.</li>
	<li>Keep the 1 ml syringes at one end of the catheter and connect the other end to the Hamilton syringes. Remove the 1 ml syringes and check that there are no air bubbles within the catheters.</li>
	<li>Insert the internal cannula at the end of the PE50 catheters. Using sterile distilled water, fill the internal cannula connected to PE50 catheters up to1 &#181;l on Hamilton syringes. Make an air bubble by pulling back the pistons up to 2 &#181;l.</li>
	<li>Mix the A&#946;o solution by pipetting up and down using a tip of minimum adherence. Avoid forming bubbles during mixing. Fill both internal cannulas with 1.5 &#181;l of A&#946;o solution by pulling back the pistons up to 3.5 &#181;l.</li>
	<li>Make lines with a marker before and after the air bubble in both catheters. This serves as a checkpoint for ongoing infusion. For infusion, bring the cage near the infusion pump and remove its cover.</li>
	<li>Place the rat in a snuggle and tightly scrape the arm of the snuggle around its neck to immobilize the head of the rat. Remove the dummy cannula from the two guide cannula. Insert the internal cannula previously prepared in the guide cannula. Verify that they are fully inserted and well-fixed to the base of the guide cannula.</li>
	<li>Release the rat from the snuggle and put it back in its cage to limit contention stress. Monitor closely to ensure the catheters do not twist together and the infusion is done correctly.</li>
	<li>Turn on the infusion pump. Inject 1 &#181;l of A&#946;o solution at a rate of 0.1 &#181;l/min (10 min). During infusion, check that the syringe piston moves from 3.5 &#181;l to 2.5 &#181;l and that the air bubble in both PE50 catheters moves continuously.</li>
	<li>Leave the internal cannula in place for another 5 min after infusion to allow efficient diffusion of A&#946;o solution. Bring the rat back in the snuggle to remove the internal cannula and capped guide cannula to prevent reflux of the injected solution. Use a dummy cannula that stops just before the angle arm of the cannula. Return the animal to its cage.</li>
	<li>Repeat A&#946;o infusion once per day over 6 consecutive days. Euthanize the animal by decapitation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artificial Cerebrospinal Fluid (aCSF)</td>
			<td>Harvard Apparatus</td>
			<td>59-7316</td>
			<td></td>
		</tr>
		<tr>
			<td>PE50 Catheter thin wall</td>
			<td>Plastics one</td>
			<td>C232CT</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride (100 mg/mL)</td>
			<td>Bioniche</td>
			<td>1989529</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylaxine Hydrochloride (100 mg/mL)</td>
			<td>Bimeda</td>
			<td>8XYL004C</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (5 mg/mL)</td>
			<td>Norbrook</td>
			<td>215670I01</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution of chlorhexidine gluconate 2% and isopropyl alcohol 2%</td>
			<td>Carefusion</td>
			<td>260100C</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine Hydrochloride</td>
			<td>Alveda Pharma</td>
			<td>0122AG01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bupivacaine Hydrochloride</td>
			<td>Hospira</td>
			<td>1559</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Baussh and Lomb inc.</td>
			<td>2125706</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting</td>
			<td>51600</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic cannula holder arm</td>
			<td>Harvard Apparatus</td>
			<td>72-4837</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>8050-N/18</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide Cannula</td>
			<td>Plastics one</td>
			<td>326OPG/spc</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Cannula</td>
			<td>Plastics one</td>
			<td>C315I/spc</td>
			<td></td>
		</tr>
		<tr>
			<td>Dummy Cannula</td>
			<td>Plastics one</td>
			<td>C315DC/spc</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture thread coated vicryl rapide 4-0</td>
			<td>Ethicon</td>
			<td>VR2297</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Acrylic Cement</td>
			<td>Harvard Apparatus</td>
			<td>72-6906</td>
			<td></td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>JI Morris Company</td>
			<td>P0090CE125</td>
			<td></td>
		</tr>
		<tr>
			<td>6E10 antibody (mouse IgG1 isotype)</td>
			<td>BioLegend</td>
			<td>803003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 isotype control antibody</td>
			<td>Abcam</td>
			<td>AB18447</td>
			<td></td>
		</tr>
		<tr>
			<td>Alzet osmotic pumps model 1007D</td>
			<td>Durect corporation</td>
			<td>290</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>CA2L9100</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid-beta 1-42</td>
			<td>rPeptide</td>
			<td>A-1163-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (10 &#181;L)</td>
			<td>Fisher Scientique</td>
			<td>14815279</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion Syringe Pump CMA 402</td>
			<td>Harvard Apparatus</td>
			<td>CMA8003110</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 21G</td>
			<td>Terumo</td>
			<td>NN-2125R</td>
			<td></td>
		</tr>
		<tr>
			<td>Snuggle</td>
			<td>Lomir Biomedical</td>
			<td>RTS04</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled infusion of a&beta; peptide-specific antibodies and a&beta; oligomers into the rat hippocampus

Source: Sajadi, A., et al. <a href="https://app.jove.com/v/54215">Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for the continuous infusion of amyloid-beta (A&#946;) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of A&#946; peptide-specific antibodies coupled with the infusion of soluble A&#946; oligomers (A&#946;o) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

<img alt="Figure 1" src="/files/ftp_upload/22127/22127_Figure1.jpg" /> 
Figure 1. Photo of the Animal with Bilateral Cannula During A&#946;o Infusion. A solution of A&#946;o (0.2 &#181;g/&#181;l; 1 &#181;l) was injected in awake and freely moving rats (once a day for 6 days) using PE50 catheters connected to an internal cannula inserted into a guide cannula. Treatment with control (Ctl) IgG1 or 6E10 antibody was infused directly at the site of A&#946...

Controlled Infusion of A&beta; Peptide-Specific Antibodies and A&beta; Oligomers into the Rat Hippocampus

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

Take an awake rat with two guide cannulae implanted bilaterally into the hippocampus.
The cannulae are connected to subcutaneously implanted osmotic pumps containing amyloid-beta or A&#946;-specific and control antibodies, respectively.
A high antibody concentration within the pump induces water diffusion from the surrounding tissue through its outer semipermeable membrane, expanding the osmotic layer.
This expansion compresses the antibody reservoir, facilitating controlled antibody release.
Attach two internal cannulae to sterile-water-filled catheters connected to an infusion pump.
Fill the cannulae with water.
Next, aspirate a small volume of air, creating a bubble.
Now, fill the cannulae with neurotoxic A&#946; oligomers, soluble aggregates of A&#946; peptides.
Insert the internal cannulae into the guide cannulae. Start the pump for a controlled delivery of the oligomers, using the air bubble to prevent backflow and track the infusion rate.
Administer repeated oligomer injections for the required duration.
The model is ready for studying A&#946; oligomer-antibody interactions.

controlled-infusion-peptide-specific-antibodies-oligomers-into-rat

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

96 次观看. 来源:Sajadi， A. 等人 慢性β淀粉样蛋白寡聚物输注动物模型中的神经退行性变被注入渗透泵的抗体处理抵消。J. Vis. Exp. （2016）。 该视频演示了一种将淀粉样蛋白-β （Aβ） 肽特异性抗体连续输注到大鼠海马体中的技术。该研究采用植入双侧套管系统以受控输注 Aβ 肽特异性抗体，同时将可溶性 Aβ 寡聚体 （Aβo） 输注到大鼠海马体中，以研究寡聚体和抗体之间的相互作用。 

视频: 将 Aβ 肽特异性抗体和 Aβ 寡聚体受控输注到大鼠海马体中 - 概念

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

科研

JoVE Journal

神经科学

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Controlled Infusion of Aβ Peptide-Specific Antibodies and Aβ Oligomers into the Rat Hippocampus

成績單

Controlled Infusion of Aβ Peptide-Specific Antibodies and Aβ Oligomers into the Rat Hippocampus