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>

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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&#8211;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&#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/<img alt="Special" src="/files/ftp_upload/56157/special01.jpg" />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>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>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>

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 ...

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Research

JoVE Journal

Behavior

11.7K Views.  Chengde Medical College. This is a protocol to mimic Alzheimer's Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (Aβ) burden, and neurofibrillary tangles aggregation, induced by the injection of Aβ25-35 combined with aluminum trichloride and recombinant human transforming growth factor-β1.

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

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...