The project was supported by the Hebei Provincial Natural Science Foundation (No. C2009001007, H2014406048), the Hebei Provincial Administration of Traditional Chinese Medicine (No. 05027), and the Key Subject Construction Project of Hebei Provincial College, China.

This procedure was in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China on Oct. 31, 198811. Scientists should follow the guidelines established and approved by their institutional and national animal regulatory organizations.
NOTE: Animal and regents: Four-month-old male Sprague-Dawley rats (300-350 g) were supplied for this experiment. All rats were housed in groups (four or five per cage) at a temperature of 23 &#177; 1 &#176;C with a 12-h light-dark cycle. Food and water were available ad libitum. The rats were acclimatized to the housing conditions for 7 days before the procedure was performed. A&#946;25-35 was dissolved in 1% DMSO saline to 1 mg/mL, and sonicated for 5 min in an ultrasonic oscillator until completely dissolved. AlCl3 and RHTGF-&#946;1 were dissolved in saline at 1% and 0.1 mg/mL, respectively. Congo red, silver nitrate, and other chemicals were of analytical grade and were purchased from ordinary commercial sources.
1. Surgical Procedure
Note: 20 male Sprague-Dawley rats were microinjected with composited 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% DMSO saline microinjection, and designated as the sham-operated group.
<ol>
	<li>Anaesthetize the rats with 100% isoflurane by inhalation and then restrain on a Brain Stereotaxic Apparatus.</li>
	<li>Shave&#160;the fur on the vertex of the head with surgical scissors and disinfect with iodophor.&#160;Then, cover 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 a sterile dry cotton for stopping the bleeding, and mark the bregma with a marker pen.</li>
	<li>Refer to the Rat Brain Stereotaxic Map12; 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 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 A&#946;25-35 and 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 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 needle 2 min after injection and slowly pull out the needle (Supplemental File 1).</li>
	<li>Fix the two screws into the skull, designated in the points of ad and the second screw fixing place of skull (which were 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 (Supplemental File 2), 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 LV area through the skull hole (Supplemental File 3), with the help of cannula holder of rats&#8217; stereotaxic apparatus.</li>
	<li>Mix the denture base material with denture base water at ratio of 1.5 g per 1 mL, put the paste to cover the guide cannula plastic pedestal and two screws for immobilizing the guide cannula and till cover the whole skin incision for avoiding the skin infection.</li>
	<li>On day 2 of the operation, anaesthetize the rats 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 with a simple interrupted suture method.</li>
	<li>Perform the same operation with the sham-operated group and microinject 0.1% DMSO saline. &#160;</li>
	<li>Postoperative nursing
	<ol>
		<li>House 2 rats per cage after operation and provide food for 30 days. 
		Note: 18 rats in the sham-operated group survived (90% success rate of operation), and 19 rats in the composited-treated A&#946; group survived (95% success rate of operation).</li>
	</ol>
	</li>
</ol>
2. Screening for Successful Model Rats and Assessment of Spatial Memory with Morris Water Maze
<ol>
	<li>Morris water maze 
	Note: The Morris water maze was used to assess rat spatial memory13. The Morris water maze is a stainless-steel circular tank with 120 cm diameter and 50 cm depth. The water maze test was performed, based on the &#34;gold standards&#34; paradigm described in Behavioral Neuroscience by J. Nunez14.
	<ol>
		<li>Blacken the pool water with several drops of food coloring.</li>
		<li>Maintain the depth of water at 31.5 cm and the temperature at 23 &#177; 1 &#176;C.</li>
		<li>Set a 1.5 cm circular transparent plexiglass platform below the water surface.</li>
		<li>Maintain that all spatial signals around the water maze are invariable during the water maze tests.</li>
		<li>Divide the pool into 4 equal quadrants by imaginary lines for descriptive data collection.</li>
		<li>Place the hidden platform in the first quadrant (Q1) of water maze.</li>
		<li>Capture the rat swimming behaviors (measured by latency, trajectory, or crossing number) through a video camera, over the water maze linked to a computer-based graphics analytic software.</li>
	</ol>
	</li>
	<li>Screening for successful model rats to the composited A&#946;-treated group
	<ol>
		<li>On day 45 of surgery, perform the Morris water maze training for 4 consecutive days to screen for successful model rats with memory impairment and collect the screening ratio (SR). 
		Note: The SR is defined as the average latency of each composited A&#946;-treated rat and the sham-operated group of rats to find the hidden platform under the water surface on day 4 of water maze training. &#8220;A&#8221; is the average latency of each composited A&#946;-treated rat to find the hidden platform and &#8220;B&#8221; is the average latency of the sham-operated group of rats to find the hidden platform, on day 4 of water maze training, in the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/56157/56157eq1.jpg" /> 
		When SR was larger than 0.2 for one composited A&#946;-treated rat, the rat was regarded as a successful model rat with impaired memory of composited A&#946;-treated rat15. The intraday memory performance was calculated to the data of 2 trials by the average value of rats to find the hidden platform. The process of the Morris water maze test was designed such that the rats were permitted to swim in the water maze tank and search for the hidden platform within 60 s. If a rat did not find out the hidden platform within 60 s, then the rat was put on the platform with hand of experimenter. When a rat reached on the hidden platform (independently or assisted), the rat was let stay there for 20 s. Then, the rat was removed from the tank and allowed a physical recovery for 10 s between the 2 trials.</li>
	</ol>
	</li>
</ol>
3. Neuron examination
<ol>
	<li>On day 86 post surgery, under isoflurane&#160;anesthesia, euthanize the rats by decapitation (Figure 1).</li>
	<li>Put the brain on ice and gently separate the two hemispheres at the raphe. Take the left hemisphere of the optic chiasma and fix in 4% formaldehyde for light microcopy observation of neuron hematoxylin and eosin (HE), Congo red, or silver nitrate stain (see Sections 4-6). Fix the right hemisphere hippocampus CA1 area in 2.5% glutaraldehyde for electron microcopy observation (Section 7).</li>
	<li>Process the brain for the light/electron microcopy sample preparation, as previously described16,17.</li>
</ol>
4. Neuron HE Staining
<ol>
	<li>Deparaffinize each slide (20 min each) with gradient alcohol (100%, 95%, 90%, 80%, and 70% alcohol) to distilled water in a fume hood.</li>
	<li>Stain for 3 min with hematoxylin (0.5% w/v), and then rinse with tap water to remove the unbound dye from slides.</li>
	<li>Rinse with 0.1% hydrochloric acid in alcohol for 1 s to remove the color of unstained nuclei.</li>
	<li>Immerse in 0.5% ammonia solution for 2 min until the background turns light blue.</li>
	<li>Stain for 1 min with 1% eosin.</li>
	<li>Dehydrate for 5 min with gradient alcohol (70%, 80%, 90%, 95%, and 100% alcohol).</li>
	<li>Clear in xylene and mount with resinous mounting medium.</li>
	<li>Observe and count the living neurons of the HE stain per 0.125 mm in the middle CA1 of the hippocampus and per 0.0352 mm2 in the cerebral cortex at a magnification of 400x with an optical microscope by a person blinded to the experimental design.</li>
</ol>
5. Congo Red Staining for Assaying Neuron A&#946; Burden
<ol>
	<li>Deparaffinize each slide (20 min) with gradient alcohol (100%, 95%, 90%, 80%, and 70% alcohol) to distilled water in a fume hood.</li>
	<li>Stain for 20 min with Congo red working solution (0.5 g Congo red, 80 mL methyl alcohol, 20 mL glycerinum).</li>
	<li>Rinse in distilled water for 5 min.</li>
	<li>Differentiate quickly with alkaline 80% alcohol solution (0.2 g alcohol/100 mL potassium hydroxide) for 3 s.</li>
	<li>Rinse twice, each for 5 min with distilled water.</li>
	<li>Counterstain in Gill&#39;s hematoxylin for 3 min.</li>
	<li>Rinse in tap water for 2 min.</li>
	<li>Dip in ammonia water (add a few drops of ammonium hydroxide to tap water and mix well) for 30 s or until the sections turn blue.</li>
	<li>Rinse in tap water for 5 min.</li>
	<li>Dehydrate with gradient alcohol (70%, 80%, 90%, 95%, and 100% alcohol).</li>
	<li>Clear in xylene and mount with resinous mounting medium.</li>
	<li>Observe and count the cells stained with Congo red at a magnification of 400x, with an optical microscope by a person blinded to the experimental design.</li>
</ol>
6. Silver Nitrate Staining for Assaying Neuron NFT Formation
<ol>
	<li>Deparaffinize each slide (20 min) with gradient alcohol (100%, 95%, 90%, 80%, and 70% alcohol) to distilled water in a fume hood.</li>
	<li>Immerse in 20% silver nitrate solution and capping agent for 20 min in the dark.</li>
	<li>Wash in distilled water twice, 5 min for each time.</li>
	<li>Immerse in silver ammonia solution. Drop ammonia solution into 20% silver nitrate solution, and add until the solution goes from turbidity to clarification. Simultaneously, stir the solution with a glass rod for 15 min and then put into the 1% diluted ammonium hydroxide for 2 min.</li>
	<li>Place the slide into the developing working solution for 3-7 min until the black block in the axon can be observed.</li>
	<li>Immerse in the 0.1% diluted ammonia solution for 1 min and then rinse with water for 1 min.</li>
	<li>Dispose with 5% sodium thiosulfate for 2 min and then rinse with water for 5 min.</li>
	<li>Dehydrate with gradient alcohol (70%, 80%, 90%, 95%, and 100% alcohol).</li>
	<li>Clear in xylene and mount with resinous mounting medium.</li>
	<li>Observe and record the cell for silver nitrate stain at a magnification of 400x with an optical microscope by a person blinded to the experimental design.</li>
</ol>
7. Hippocampal Neuron Ultrastructure Measurement
<ol>
	<li>Cut the rat hippocampus CA1 into several cubes (1 x 1 x 1 mm3) and place in 2.5% glutaraldehyde for 2 h at 4 &#176;C.</li>
	<li>Rinse the cubes with PBS three times (pH 7.2, 10 min for each time).</li>
	<li>Fix the cubes in 1% osmic acid for 2 h at 4 &#176;C.</li>
	<li>Rinse the cubes in double distilled water 3x (10 min for each time).</li>
	<li>Dehydrate with gradient alcohol 50%, 70%, and 90% (10 min for each), 100% two times (15 min for each time).</li>
	<li>Replace by propylene oxide two times (15 min for each time), propylene oxide:resin at 1:1 (60 min for each time at room temperature), and propylene oxide:resin 1:4 (60 min for each time at room temperature). Soak in resin (120 min, at room temperature).</li>
	<li>Embed in EPON 812 and polymerize (5 h/35 &#176;C, 5 h/60 &#176;C, 5 h/80 &#176;C).</li>
	<li>Cut the semithin sections (1 &#181;m), stain by methylene blue, and localize under the microscope.</li>
	<li>Stain the ultra-thin sections (50 nm) with uranyl-o-acetate and lead citrate.</li>
	<li>Examine under a JEM-1400 electron microscope and collect images.</li>
</ol>

<ol>
	<li>Robinson, M., Lee, B. Y., Hane, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Progress+in+Alzheimer's+Disease+Research,+Part+2:+Genetics+and+Epidemiology.">Recent Progress in Alzheimer's Disease Research, Part 2: Genetics and Epidemiology.</a> J. Alzheimers. Dis. , (2017).</li><li>Hane, F. T., Lee, B. Y., Leonenko, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Progress+in+Alzheimer's+Disease+Research,+Part+1:+Pathology.">Recent Progress in Alzheimer's Disease Research, Part 1: Pathology.</a> J. Alzheimers Dis. , (2017).</li><li>Perl, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology+of+Alzheimer's+disease.">Neuropathology of Alzheimer's disease.</a> Mt. Sinai. J. Med. 77, 32-42 (2010).</li><li>Wu, X. G., Wang, S. S., Miao, H., Cheng, J. J., Zhang, S. F., Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scutellaria+barbata+flavonoids+alleviate+memory+deficits+and+neuronal+injuries+induced+by+composited+A&#946;+in+rats.">Scutellaria barbata flavonoids alleviate memory deficits and neuronal injuries induced by composited A&#946; in rats.</a> Behav. Brain Funct. 12, 33-43 (2016).</li><li>Guo, K., Wu, X. G., Miao, H., Cheng, J. J., Cui, Y. D., Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+mechanism+of+Scutellaria+bartata+flavonoids+on+apopotosis+of+cortical+neurons+and+cytochondriome+induced+by+composited+A&#946;.">Regulation and mechanism of Scutellaria bartata flavonoids on apopotosis of cortical neurons and cytochondriome induced by composited A&#946;.</a> Chin Hosp Pharm J. 35, 1994-1999 (2015).</li><li>Guo, K., Miao, H., Wang, S. S., Cheng, J. J., Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scutellaria+barbata+flavonoids+inhibits+NFT+aggregation+and+regulatory+mechanism+in+rats+induced+by+composited+A&#946;.">Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited A&#946;.</a> Chin. J. Pathophysio. 32, 2147-2156 (2016).</li><li>Hou, X. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scutellaria+Barbata+flavonoids+inhibit+the+brain's+A&#946;+and+NFT+abnormal+generation+and+affect+the+related+enzymes+expression+in+rats+induced+by+composited+A&#946;.">Scutellaria Barbata flavonoids inhibit the brain's A&#946; and NFT abnormal generation and affect the related enzymes expression in rats induced by composited A&#946;.</a> Chin J New Drugs. , (2017).</li><li>Zhao, H. X., Guo, K., Cui, Y. D., Wu, X. G., Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Scutellaria+barbata+flavonoids+on+abnormal+changes+of+Bcl-2,+Bax,+Bcl-xL+and+Bak+protein+expression+in+mitochondrial+membrane+induced+by+composite+A&#946;25-35.">Effect of Scutellaria barbata flavonoids on abnormal changes of Bcl-2, Bax, Bcl-xL and Bak protein expression in mitochondrial membrane induced by composite A&#946;25-35.</a> Chin J Pathophysiol. 30, 2262-2266 (2014).</li><li>Cheng, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoid+extract+from+Scutellaria+stem+and+leaf+attenuates+composited+A&#946;-+induced+memory+impairment+and+apoptosis+in+rats.">Flavonoid extract from Scutellaria stem and leaf attenuates composited A&#946;- induced memory impairment and apoptosis in rats.</a> Chin.J. New Drugs. 25, 2627-2636 (2016).</li><li>Fang, F., Yan, Y., Feng, Z. H., Liu, X. Q., Wen, M., Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+Alzheimer's+disease+model+induced+multiple+factors.">Study of Alzheimer's disease model induced multiple factors.</a> Chongqing Med. 36, 146-151 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulations+for+the+Administration+of+Affairs+Concerning+Experimental+Animals.">Regulations for the Administration of Affairs Concerning Experimental Animals.</a> The Ministry of Science and Technology of the People's Republic of China. , 10-31 (1988).</li><li>Bao, X. M., Shu, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The stereotaxic atlas of the rat brain. , (1991).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rats.">Developments of a water-maze procedure for studying spatial learning in the rats.</a> J. Neurosic. Methods. 11, 47-60 (1984).</li><li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+Water+Maze+Experiment.">Morris Water Maze Experiment.</a> J. Vis. Exp. (19), e897 (2008).</li><li>Yu, J. C., Liu, C. Z., Zhang, X. Z., Han, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupuncture+improved+cognitive+impairment+caused+by+multi-infarct+dementia+in+rats.">Acupuncture improved cognitive impairment caused by multi-infarct dementia in rats.</a> Physiol. &amp; Behav. 86, 434-441 (2005).</li><li>Shang, Y. Z., Miao, H., Cheng, J. J., Qi, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+amelioration+of+total+flavonoids+from+stems+and+leaves+of+Scutellaria+baicalensis+Georgi+on+cognitive+deficits,+neuronal+damage+and+free+radicals+disorder+induced+by+cerebral+ischemia+in+rats.">Effects of amelioration of total flavonoids from stems and leaves of Scutellaria baicalensis Georgi on cognitive deficits, neuronal damage and free radicals disorder induced by cerebral ischemia in rats.</a> Biol. Pharm. Bull. 29, 805-810 (2006).</li><li>Song, H. R., Cheng, J. J., Miao, H. J., Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scutellaria+flavonoid+supplementation+reverses+ageing-related+cognitive+impairment+and+neuronal+changes+in+aged+rats.">Scutellaria flavonoid supplementation reverses ageing-related cognitive impairment and neuronal changes in aged rats.</a> Brain Inj. 23, 146-153 (2009).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+RAS+inhibitors+Poricoic+Acid+ZG+and+Poricoic+Acid+ZH+attenuate+renal+fibrosis+via+a+Wnt/&#946;-Catenin+patheway+and+targeted+phosphorylation+of+smad3+signaling.">Novel RAS inhibitors Poricoic Acid ZG and Poricoic Acid ZH attenuate renal fibrosis via a Wnt/&#946;-Catenin patheway and targeted phosphorylation of smad3 signaling.</a> J Agric Food Chem. 66, 1828-1842 (2018).</li><li>Pini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+atrophy+in+Alzheimer's+Disease+and+aging.">Brain atrophy in Alzheimer's Disease and aging.</a> Ageing Res. Rev. 30, 25-48 (2016).</li><li>Ubhi, K., Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+recent+advances+and+future+perspectives.">Alzheimer's disease: recent advances and future perspectives.</a> J. Alzheimers Dis. 33, 85-94 (2013).</li><li>Guo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scutellaria barbata flavonoids inhibite NFTs aggregation, tau protein phosphorylation and the regulated mechanism of related enzymes in rats induced by composited A&#946;. , (2016).</li><li>Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Effects and Mechanism of Scutellaria Barbata Flavonoids on Rat's Memory Impairment Induced by Compound A&#946;25-35. , (2013).</li><li>Zussy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+related+markers,+cellular+toxicity+and+behavioral+deficits+induced+six+weeks+after+oligomeric+amyloid-&#946;+peptide+injection+in+rats.">Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-&#946; peptide injection in rats.</a> PLoS One. 8, 1-20 (2013).</li><li>Walton, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aluminum+involvement+in+the+progression+of+Alzheimer's+disease.">Aluminum involvement in the progression of Alzheimer's disease.</a> J. Alzheimers Dis. 35, 7-43 (2013).</li><li>Neils-Strunjas, J., Groves-Wright, K., Mashima, P., Harnish, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysgraphia+in+Alzheimer's+disease:+a+review+for+clinical+and+research+purposes.">Dysgraphia in Alzheimer's disease: a review for clinical and research purposes.</a> J. Speech Lang Hear Res. 49, 1313-1330 (2006).</li><li>Morley, J. E., Farr, S. A., Kumar, V. B., Armbrecht, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SAMP8+mouse:+a+model+to+develop+therapeutic+interventions+for+Alzheimer's+disease.">The SAMP8 mouse: a model to develop therapeutic interventions for Alzheimer's disease.</a> Curr Pharm Des. 18, 1123-1130 (2012).</li><li>Puzzo, D., Gulisano, W., Palmeri, A., Arancio, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+Alzheimer's+disease+drug+discovery.">Rodent models for Alzheimer's disease drug discovery.</a> Expert Opin Drug Discov. 10, 703-711 (2015).</li></ol>

The authors have nothing to disclose.

It is well known that the loss of learning and memory are major clinical symptoms in AD patients2. The procedure described here is an in vivo method to study AD; we have adapted a previously published protocol that tested a medication to alleviate memory deficits and neuronal injuries in a rat model4. Our protocol provides important details to obtain valuable data, as well as a high survival rate of animals that successfully model operation, memory deficits, neuron injuries, A&#946; burden, and NFT deposition, to mimic AD (in the present experiment, the survival rate and successful model rate of operation are more than 90%). These successful model rats were used to measure their spatial memory with the Morris water maze test. The positioning navigation trial found that the composited A&#946; can cause rat memory acquisition impairment; the probe trial found that the composited A&#946; can decrease rat memory retention; and the reversal trial found that the composited A&#946; can result in rat re-learning impairment. These Morris water maze test data show that the composited A&#946; can induce rat spatial memory. Overall, injecting rats intracerebroventricularly with A&#946;25-35 in combination with AlCl3 and TGF-&#946;1 created a feasible and credible in vivo AD-like animal model for the laboratory.
Previous studies have shown that the brain volume in AD patients is 10% less than that of healthy individuals. Various atrophies can be found in the cerebral hemisphere by visual observation. The degree of cortical atrophy is positively related to the memory impairment19. In the histology, the large number of neuron loss and severe morphological pathology directly disturb the memory function in AD patients20. In the present study, light/electron microscopic observation found that the rats microinjected with composited A&#946; displayed dramatic neuropathological changes, including neuron loss, and neuronal and subcellular structure disruption. This result corroborates the rat spatial memory disorder induced by composited A&#946;, and is similar to the state of AD patients.
It is well known that the brain A&#946; burden and NFT aggregation are considered the most important histopathogenic traits in AD. They can destroy the neuronal structure, disturb the neural signaling, disrupt the neuronal function, and result in advanced dementia17. The present animal model found A&#946; burden and NFT aggregation in brain, which agree with the AD patient state. Therefore, the present neuron injuries in rats induced by composited A&#946; can be used as a model to study neuronal pathology and treatment strategy of AD.
The following are examples of screening drug effects in AD rat models: Zhao et al., reported that both flavonoids from Scutellaria stems and leaves (SSF) and Scutellaria barbata (SBF) can attenuate rat memory impairment and apoptosis induced by composited A&#946;8,9. Guo et al., also reported that SBF can inhibit NFT aggregation and tau protein over-phosphorylation at Ser199, Ser214, Ser202, Ser404, and Thr231 side, and decrease GSK-3&#946;, CDK5, and PKA protein and mRNA expression in composited A&#946;-treated rats21. Simultaneously, Shang et al., have also reported that SBF can suppress the astrocyte and microglia proliferation, and lower A&#946;1-40, A&#946;1-42, and &#946;-site APP cleaving enzyme 1 (BACE1) mRNA expression in the brain of composited A&#946; rats22. Based on the above results, our animal model is advantageous over other AD-like model, which involve more neuronal function and structure disorder.
Concerning other AD-like model, single intracerebroventricular injection of A&#946; to rats can cause rat memory deficits, neuron loss, and neurogliocyte proliferation, but may or may not have A&#946; and NFT deposition23. Rats exposed to high dose Al appear to have a high success rate, mimic AD, and a cost-effective animal model, with memory impairment, neuron loss, neurogliocyte proliferation, and senile plaque (SP) and NFT aggregation in the brain. However, the high dose of Al may cause rat liver injuries and anorexia, accompanied with decreased weight24. The aged rat is another AD-like model. The aged rats demonstrate memory deficits, neuronal structure/substructure pathological changes, lipofuscin deposition, but without A&#946; burden and NFT aggregation. Rats of more than 24 months of age are considered aged for this model, and therefore it requires a longer period of feeding and thus the cost is higher17,25. SAMP8 and APP transgenic mice are the closest mimic to AD and they are the most ideal models for investigating AD. But both animal models are higher priced and are limited to use in the laboratory26,27. Compared with the above animal models, our model of composited A&#946;-treated animal model has a lower cost and high performance, making it an ideal tool for studying AD.
In conclusion, intracerebroventricular injection of A&#946;25-35 combined with AlCl3 and TGF-&#946;1 to rats offers a valuable in vivo animal model to better understand the spatial memory impairment, neuronal injuries, A&#946; burden, and NFT deposition underlying AD. This model provides a fast and relatively simple experimental protocol with a high animal survive rate and high model successful rate of operation, as well as a high rate of duplication, which showed to be more economic. The present animal model is an effective model to mimic AD and can further validate itself by being used to mimic various other diseases.

It is well known that AD is a chronic and progressive neurodegenerative disease, with gradual memory loss as the main clinical syndrome. In the general pathology, there is nervous tissue atrophy, neuron and synapse loss, as well as neuronal subcellular structure and function disorders, which are all involved in the development and clinical manifestation of AD1,2. It is reported that when animals were intracerebroventricularly injected with A&#946;, some neurotoxic events occur in the brain involving neuron loss, calcium homeostasis disruption, neuron apoptosis, and reactive oxygen species overproduction3. However, multiple factors are involved in the pathogenesis of AD and thus it is essential to establish a better model of AD.
A detailed protocol is described here for establishing an in vivo mimic AD model through intracerebroventricular injection of A&#946;25-35 and AlCl3, combined with anterodorsal thalamic nucleus injection of RHTGF-&#946;1 to rats. This rat modelhighly mimics human neuronal function and histopathogenesis of AD, including memory impairment, neuron loss and structure damage, apoptosis, intracellular A&#946; burden, and NFT aggregation4,5,6,7,8,9. The AlCl3 prevents the deposited A&#946; from forming soluble A&#946;, and the RHTGF-&#946;1 can promote deposited A&#946; production and facilitate AD occurrence10. This attack from several factors to the neuron is in accordance with the multi-pathogenesis of AD.
The entire experiment spanned 86 days: Figure 1 shows a timeline of the experimental design, with the time point of animal surgery, animal model screening, animal spatial memory test, and sample preparation. On the first day of operation, RHTGF-&#946;1 was microinjected into the anterodorsal thalamic nucleus. On the second day of operation, A&#946;25-35 and AlCl3 were microinjected into the lateral ventricle daily for 14 consecutive days in the morning and 5 consecutive days in the afternoon, respectively. All rats were allowed to recover for 45 days after the operation. The Morris water maze was used to screen for successful model rats with memory impairment and to assess rats&#39; spatial memory. The rats underwent 4 consecutive days of water maze training with 2 trials per day, and on day 4 of training, the rats were evaluated with the Morris water maze performance for memory impairment. All rats continued to be fed 37 days after the animal model screening. The spatial memory of rats was tested in the Morris water maze over 7 consecutive days, from day 79 to day 85 after the operation. All rats were sacrificed by decapitation on day 86 for brain sample preparation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56157/56157fig1.jpg" /> 
Figure 1. Timeline of the experimental design. <a href="//cloudflare.jove.com/files/ftp_upload/56157/56157fig1large.jpg">Please click here to view a larger version of this figure.</a>

<table><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&amp;ndash;350 g</td></tr><tr><td>Morris water maze</td><td>Chinese Academy of Medical Sciences and Peking Union Medical Research Institute, China</td><td>No</td><td></td></tr><tr><td>Movable small animal anesthesi</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&amp;times;I.D.0.0.45mm/M3.5&lt;br/&gt; 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&amp;times;I.D.0.25mm/&lt;img alt=&quot;Special&quot; src=&quot;/files/ftp_upload/56157/special01.jpg&quot;/&gt;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&amp;times;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-&amp;beta;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>Congo red</td><td>Tianjin Kemiou Chemical Reagent Co., Ltd. China</td><td>3010016</td><td></td></tr><tr><td>Silver nitrate</td><td>Sinopharm Chcmical Reagent Co., Ltd. China</td><td>20150720</td><td></td></tr><tr><td>Denture base material</td><td>Shanghai New Century Dental Materials Co., Ltd. China</td><td>20170609</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein at JoVE.c

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

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 ...

establishment-valuable-mimic-alzheimer-s-disease-rat-animal-model

Research

JoVE Journal

Behavior

11.8K Views.  Chengde Medical College. Dit is een protocol om na te bootsen van de ziekte van Alzheimer bij ratten door evaluatie van ruimtelijke geheugen verzwakking, neuronale pathologische veranderingen, neuronale bèta amyloid proteïne (Aβ) lasten en neurofibrillary klitten aggregatie, geïnduceerd door de injectie van Aβ25-35 gecombineerd transformeren met aluminium titaantrichloride en recombinante menselijke groeifactor-β1.

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

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.

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

Neuroscience

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.

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

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Medicine

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Biology

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.

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

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

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

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.

Neuropathology

Neurodegenerative Diseases

Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer's Disease Model

Source: Jean, Y. Y. et al., <a href="https://app.jove.com/v/52805">Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.</a> J. Vis. Exp. (2015)
This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer&#39;s disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer&#39;s disease development.

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

A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling

Source: Van der Perren, A. et al., <a href="https://app.jove.com/v/53670">Development of an Alpha-synuclein Based Rat Model for Parkinson&#39;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the process of injecting a viral vector into the rat substantia nigra to model Parkinson&#39;s disease. It outlines the steps for preparing the rat, microinjecting the viral vector into the substantia nigra, and the recovery procedure.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

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

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