establishing an alzheimer's disease rat model through an amyloid-beta peptide injection

Establishing an Alzheimer's Disease Rat Model Through an Amyloid-Beta Peptide Injection

On an anesthetized rat, begin by snipping the fur on the vertex of the head with surgical scissors and disinfect with iodophor.
Next, make an incision on the head skin along the median longitudinal calvaria with surgical bistouries and scissors.
Separate the subcutaneous tissue and fascia. Wipe the skull calvarium with 0.3% hydrogen peroxide, and mark the bregma with a marker pen. Next, mark three points: the anterodorsal thalamic nucleus for injecting RHTGF-&#946;1 and fixing one screw, the lateral ventricle or LV area for injecting A&#946;25-35 and AICI3, and lastly the second screw fixing place.
Gently, drill three 1-millimeter diameter holes with a flexible bone drill at the three marked points on the skull. Stop the bleeding and clean the skull surface repeatedly with sterile dry cotton.
Next, insert a needle linked to the microinjection pump to the brain at 4.2 millimeters depth, and gently inject 1 microliter RHTGF-&#946;1 into the ad area. Keep the microinjection two minutes after injection, then slowly pull it out.
Fix the two screws into the skull, designated in the previous marked points with a small screwdriver. Following screw placement, assemble the cannula implantation system by first inserting the dummy cannula into the guide cannula after disinfection with 75% alcohol immersion for 24 hours. Then, insert the stainless steel tubing guide cannula to the brain at 4.6 millimeters into LV area through the skull hole, with the help of a cannula holder on the rat&#39;s stereotaxic apparatus.
Next, mix the denture base material with denture base water, at a ratio of 1.5 grams per 1 milliliters. Put the paste to cover the guide cannula plastic pedestal and two screws for immobilizing the guide cannula. Cover the whole skin incision to avoid skin infection.
The next day, draw out the dummy cannula and insert the internal cannula into the guide cannula. Screw the fixing screw to immobilize the internal cannula.
Set the polyethylene pipe that links the microinjection pump to the internal cannula, and regulate the injection speed to 1 microliter per minute. Microinject the A&#946;25-35 to the LV. Wait five minutes after finishing the injection, and gently draw out the internal cannula. Then, insert the dummy cannula again into the guide cannula.
On day 15, post-surgery, dismantle the cannula implantation system by removing the denture base material solid with surgical scissors and forceps, and disinfecting the wound with iodophor.
Finally, fill in the hole of the skull with bone cement and suture the skin with a simple interrupted suture method.

establishing-an-alzheimer-s-disease-rat-model-through-an-amyloid-beta

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

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. Surgical Procedure
Note: 20 male Sprague-Dawley rats were microinjected with composited amyloid beta protein (A&#946;) into the lateral ventricle and anterodorsal thalamic nucleus and designated as the composited A&#946;-treated group. Another 20 rats were subjected to the same operation but received 0.1% dimethyl sulfoxide (DMSO) saline microinjection and were designated as the sham-operated group.
<ol>
	<li>Anesthetize the rats with 100% isoflurane by inhalation, then restrain them on a brain stereotaxic apparatus.</li>
	<li>Shave the fur on the vertex of the head with surgical scissors and disinfect with iodophor. Then, cover a disposable surgical towel on the head.</li>
	<li>Make an incision on the head skin along the median longitudinal calvaria with surgical bistouries and scissors.</li>
	<li>Separate the subcutaneous tissue and fascia, wipe the skull calvarium with sterile dry cotton to stop the bleeding, and mark the bregma with a marker pen.</li>
	<li>Refer to the Rat Brain Stereotaxic Map considering the bregma as the point of origin, mark the three points, the anterodorsal thalamic nucleus (ad) (posterior (P): 2.0 mm to the bregma; lateral (L): 1.4 mm to the midline) for injecting recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) and fixing one screw, the lateral ventricle (LV) area (posterior (P): 0.8 mm to the bregma; lateral (L): 2.0 mm to the midline) for injecting amyloid beta protein 25-35 (A&#946;25-35) and aluminum trichloride (AlCl3), and the second screw fixing place (Front (F): 2.0 mm to the bregma; lateral (L): 1.5 mm to the midline).</li>
	<li>Gently drill three 1 mm diameter holes with a flexible bone drill designated at the above three points of the skull (1.5. step). Do not screw deeper than necessary to avoid stabbing the brain tissue.</li>
	<li>Stop the bleeding and clean the skull surface repeatedly with sterile dry cotton.</li>
	<li>Insert a needle linked to the micro-injection pump to the brain at 4.6 mm depth and gently inject 1 &#956;L RHTGF-&#946;1 (10 ng) into the ad area. Stay the needle for 2 min after injection and slowly pull out the needle.</li>
	<li>Fix the two screws into the skull, designated in the points of the ad, and the second screw fixing place of the skull (which was designated at 1.5. step) with a small screwdriver. Do not screw deeper than necessary to avoid stabbing the brain tissue.</li>
	<li>Assemble the cannula implantation system and insert the dummy cannula into the guide cannula after disinfection by high pressure.</li>
	<li>Insert the stainless-steel tubing guide cannula to the brain at 4.6 mm into the LV area through the skull hole with the help of the cannula holder of the rats&#39; stereotaxic apparatus.</li>
	<li>Mix the denture base material with denture base water at a ratio of 1.5 g per 1 mL, put the paste to cover the guide cannula plastic pedestal, and two screws to immobilize the guide cannula and cover the whole skin incision to avoid skin infection.</li>
	<li>On day 2 of the operation, the rats were anesthetized with isoflurane inhalation using the small animal anesthesia machine. Draw out the dummy cannula, insert the internal cannula into the guide cannula, and screw the fixing screw to immobilize the internal cannula.</li>
	<li>Set the polyethylene pipe that links the microinjection pump to the internal cannula and regulate the injection speed to 1 &#956;L/min. Microinject the A&#946;25-35 or AlCl3 to the LV.</li>
	<li>Microinject 4 &#956;g (1 &#956;L) A&#946;25-35 daily for 14 days in the morning and 3 &#956;L AlCl3 (1%) daily for 5 days in the afternoon under isoflurane anesthesia.</li>
	<li>Wait 5 min after finishing the injection, gently draw out the internal cannula, and insert the dummy cannula again into the guide cannula.&#160;&#160;</li>
	<li>On day 15, post-surgery (which corresponds to the last injection day of A&#946;25-35), dismantle the cannula implantation system. Gently remove the denture base material solid with surgical scissors and forceps, unscrew the two screws, pull out the guide cannula, and disinfect the wound with betadine. Fill in the hole of the skull with bone cement and suture the skin using a simple interrupted suture method.</li>
	<li>Perform the same operation with the sham-operated group and microinject 0.1% DMSO saline.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sprague-Dawley rat</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd, China</td>
			<td>SCXK(Jing) 2012-0001</td>
			<td>300&#8211;350 g</td>
		</tr>
		<tr>
			<td>Movable small animal anesthesia</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>R580</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain stereotaxic apparatus</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>68001</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible bone drill</td>
			<td>Shanghai Soft Long Technology Development Co., Ltd. China</td>
			<td>BW-sD908</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Japan Co., Ltd. Japan</td>
			<td>JEM-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Two channel microinjection pump</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>RWD202</td>
			<td></td>
		</tr>
		<tr>
			<td>EM microtome</td>
			<td>Hitachi Co., Ltd. China</td>
			<td>H-7650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dummy cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62001</td>
			<td>0.D.0.64&#215;I.D.0.0.45mm/M3.5 
			http://www.rwdls.com/English/Product/3985102014.html</td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62101</td>
			<td>0.D.0.40mm/M3.5</td>
		</tr>
		<tr>
			<td>Internal cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62201</td>
			<td>0.D.0.41&#215;I.D.0.25mm/M3.5</td>
		</tr>
		<tr>
			<td>Tighten the nut</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62501</td>
			<td>0.D.5.5mm/L7.5mm/M3.5</td>
		</tr>
		<tr>
			<td>Fixing screw</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62514</td>
			<td>M1.2&#215;L2.0mm(100BAO)</td>
		</tr>
		<tr>
			<td>The screwdriver</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62999</td>
			<td>45*1mm</td>
		</tr>
		<tr>
			<td>PE Tubing</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62302</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid beta 25-35</td>
			<td>Sigma Aldrich Co. USA</td>
			<td>SCP0002-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human transforming growth factor-&#946;1</td>
			<td>PeproTech Inc. USA</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium trichloride</td>
			<td>Tianjin Kemiou Chemical Reagent Co., Ltd. China</td>
			<td>3011080</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc phosphate dental cement</td>
			<td>Dental Material of Factory Shanghai Medical Instruments Co., Ltd. China</td>
			<td>201311</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Xiaoguang, W., et al. <a href="https://app.jove.com/v/56157">Establishment of a Valuable Mimic of Alzheimer&#39;s Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for injecting amyloid-beta peptides, aluminum chloride, and a neuroinflammatory growth factor, which collectively aids in toxic amyloid plaque formation in the hippocampus region. This causes memory impairment in the rat, characteristic of Alzheimer&#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. ...

Begin with an immobilized, anesthetized rat with its skull exposed.
Locate the bregma, mark three reference points, and carefully drill the holes.&#160; Clean the area.
Through one of the holes, inject a growth factor into the anterodorsal thalamic nucleus near the hippocampus.
Secure two screws in two of the holes, insert a guide cannula into the third hole, and fix them with dental cement. Cover the guide cannula with a dummy cannula.
The next day, insert the internal cannula into the guide cannula and connect it to the injection system.
Regularly inject amyloid-beta peptides and aluminum trichloride into the brain&#39;s lateral ventricle to facilitate their diffusion in the hippocampus.
Later, remove the cannula implantation system. Disinfect the area, fill the holes with bone cement, and suture the incision.
Over time, the amyloid-beta peptides aggregate into toxic plaques.
The injected growth factor and aluminum trichloride also enhance plaque formation around the hippocampal neurons, which are crucial for memory.
This leads to neuronal death, causing memory impairment similar to Alzheimer&#39;s disease.

20 Görüntüleme. Amiloid-Beta Peptit Enjeksiyonu ile Alzheimer Hastalığı Sıçan Modelinin Oluşturulması

Video: Amiloid-Beta Peptit Enjeksiyonu ile Alzheimer Hastalığı Sıçan Modelinin Oluşturulması - Deney

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Fare Hippocampus içine Oligomerik Amiloid-beta Stereotaksik İnfüzyon

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

JoVE Journal

Nörobilim

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Normal Farelerde Amiloid-β Peptitler intraserebroventriküler enjeksiyon Akut Alzheimer gibi Bilişsel Açıkları tetikleyebilecek

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

kantitatif 3D<em&gt; Siliko</emAlzheimer Hastalığının Kemirgen Modelleri Serebral Amiloid-beta Fagositoz&gt; Modelleme (q3DISM)

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Establishing an Alzheimer's Disease Rat Model Through an Amyloid-Beta Peptide Injection

TRANSKRIPT