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

Abstract

Source: Xiaoguang, W., et al. Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein. 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's disease.

Protocol

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

1. Surgical Procedure

Note: 20 male Sprague-Dawley rats were microinjected with composited amyloid beta protein (Aβ) into the lateral ventricle and anterodorsal thalamic nucleus and designated as the composited Aβ-treated group. Another 20 rats were subjected to the same operation but received 0.1% dimethyl sulfoxide (DMSO) saline microinjection and were designated as the sham-operated group.

1. Anesthetize the rats with 100% isoflurane by inhalation, then restrain them on a brain stereotaxic apparatus.
2. Shave the fur on the vertex of the head with surgical scissors and disinfect with iodophor. Then, cover a disposable surgical towel on the head.
3. Make an incision on the head skin along the median longitudinal calvaria with surgical bistouries and scissors.
4. Separate the subcutaneous tissue and fascia, wipe the skull calvarium with sterile dry cotton to stop the bleeding, and mark the bregma with a marker pen.
5. Refer to the Rat Brain Stereotaxic Map considering the bregma as the point of origin, mark the three points, the anterodorsal thalamic nucleus (ad) (posterior (P): 2.0 mm to the bregma; lateral (L): 1.4 mm to the midline) for injecting recombinant human transforming growth factor-β1 (RHTGF-β1) and fixing one screw, the lateral ventricle (LV) area (posterior (P): 0.8 mm to the bregma; lateral (L): 2.0 mm to the midline) for injecting amyloid beta protein 25-35 (Aβ25-35) and aluminum trichloride (AlCl₃), and the second screw fixing place (Front (F): 2.0 mm to the bregma; lateral (L): 1.5 mm to the midline).
6. Gently drill three 1 mm diameter holes with a flexible bone drill designated at the above three points of the skull (1.5. step). Do not screw deeper than necessary to avoid stabbing the brain tissue.
7. Stop the bleeding and clean the skull surface repeatedly with sterile dry cotton.
8. Insert a needle linked to the micro-injection pump to the brain at 4.6 mm depth and gently inject 1 μL RHTGF-β1 (10 ng) into the ad area. Stay the needle for 2 min after injection and slowly pull out the needle.
9. Fix the two screws into the skull, designated in the points of the ad, and the second screw fixing place of the skull (which was designated at 1.5. step) with a small screwdriver. Do not screw deeper than necessary to avoid stabbing the brain tissue.
10. Assemble the cannula implantation system and insert the dummy cannula into the guide cannula after disinfection by high pressure.
11. Insert the stainless-steel tubing guide cannula to the brain at 4.6 mm into the LV area through the skull hole with the help of the cannula holder of the rats' stereotaxic apparatus.
12. Mix the denture base material with denture base water at a ratio of 1.5 g per 1 mL, put the paste to cover the guide cannula plastic pedestal, and two screws to immobilize the guide cannula and cover the whole skin incision to avoid skin infection.
13. On day 2 of the operation, the rats were anesthetized with isoflurane inhalation using the small animal anesthesia machine. Draw out the dummy cannula, insert the internal cannula into the guide cannula, and screw the fixing screw to immobilize the internal cannula.
14. Set the polyethylene pipe that links the microinjection pump to the internal cannula and regulate the injection speed to 1 μL/min. Microinject the Aβ25-35 or AlCl₃ to the LV.
15. Microinject 4 μg (1 μL) Aβ25-35 daily for 14 days in the morning and 3 μL AlCl₃ (1%) daily for 5 days in the afternoon under isoflurane anesthesia.
16. Wait 5 min after finishing the injection, gently draw out the internal cannula, and insert the dummy cannula again into the guide cannula.  
17. On day 15, post-surgery (which corresponds to the last injection day of Aβ25-35), dismantle the cannula implantation system. Gently remove the denture base material solid with surgical scissors and forceps, unscrew the two screws, pull out the guide cannula, and disinfect the wound with betadine. Fill in the hole of the skull with bone cement and suture the skin using a simple interrupted suture method.
18. Perform the same operation with the sham-operated group and microinject 0.1% DMSO saline.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sprague-Dawley rat	Beijing Vital River Laboratory Animal Technology Co., Ltd, China	SCXK(Jing) 2012-0001	300–350 g
Movable small animal anesthesia	RWD Life Science Co., Ltd. China	R580
Brain stereotaxic apparatus	RWD Life Science Co., Ltd. China	68001
Flexible bone drill	Shanghai Soft Long Technology Development Co., Ltd. China	BW-sD908
Transmission electron microscope	Japan Co., Ltd. Japan	JEM-1400
Two channel microinjection pump	RWD Life Science Co., Ltd. China	RWD202
EM microtome	Hitachi Co., Ltd. China	H-7650
Dummy cannula	RWD Life Science Co., Ltd. China	62001	0.D.0.64×I.D.0.0.45mm/M3.5 http://www.rwdls.com/English/Product/3985102014.html
Guide cannula	RWD Life Science Co., Ltd. China	62101	0.D.0.40mm/M3.5
Internal cannula	RWD Life Science Co., Ltd. China	62201	0.D.0.41×I.D.0.25mm/M3.5
Tighten the nut	RWD Life Science Co., Ltd. China	62501	0.D.5.5mm/L7.5mm/M3.5
Fixing screw	RWD Life Science Co., Ltd. China	62514	M1.2×L2.0mm(100BAO)
The screwdriver	RWD Life Science Co., Ltd. China	62999	45*1mm
PE Tubing	RWD Life Science Co., Ltd. China	62302
Amyloid beta 25-35	Sigma Aldrich Co. USA	SCP0002-5MG
Recombinant human transforming growth factor-β1	PeproTech Inc. USA	100-21
Aluminium trichloride	Tianjin Kemiou Chemical Reagent Co., Ltd. China	3011080
Zinc phosphate dental cement	Dental Material of Factory Shanghai Medical Instruments Co., Ltd. China	201311