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

Samenvatting

Source: Jean, Y. Y. et al., Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus. 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's disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer's disease development.

Protocol

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

1. Prepare Surgical Instruments and Solutions for Surgery

1. Autoclave all stainless steel surgical instruments.
2. Prepare 70% ethanol by diluting 200 proof absolute ethanol with sterile molecular grade deionized distilled water.
3. Attach the 29 G needle to the Hamilton syringe. Clean the interior of Hamilton syringe and needle by drawing up and ejecting nanopure water repeatedly for 1 min. Repeat this procedure with 70% ethanol.
   1. Remove the plunger and needle from the Hamilton syringe. Air dry the parts in a laminar flow hood O/N.
   2. Irradiate the needle and syringe with ultraviolet light for 30 min before use.
4. Prepare a saline solution by dissolving sodium chloride (NaCl) in molecular-grade water to a final weight-to-volume concentration of 0.9%. Sterilize the solution by filtering it through a 0.2 μm pore filter.

2. Prepare Oligomeric amyloid-beta (Aβ1-42)

1. Monomerize and resuspend recombinant human Aβ1-42 in dimethylsulfoxide (DMSO) to 5 mM exactly as described in the publication of Fa' and others.
2. Dilute 5 mM Aβ1-42 with sterile (1x) phosphate-buffered saline (PBS) to 100 µM on the day before surgery.
3. Mix the solution well by trituration.
4. Incubate Aβ1-42 solution for 12 hr at 4˚C.
   NOTE: Control solutions such as diluting DMSO in PBS or scrambled/reverse Aβ1-42 peptide should be prepared the same way as Aβ1-42.

3. Determine Injection Coordinates

1. Use the adult mouse brain atlas to determine the exact anterior-posterior (AP), medial-lateral (ML), and dorsal-ventral (DV) coordinates for the brain region of interest.
   ​NOTE: To illustrate the technique we picked the dentate gyrus of the hippocampus as the target with the following coordinates from the bregma: AP, -2.00 mm; ML, ±1.3 mm; DV, -2.2 mm. The negative sign preceding the AP value indicates that it is 2.00 mm posterior of the bregma. The ± sign preceding the ML value indicates left and right direction from the center. Lastly, the negative sign preceding the DV indicates it is moving ventrally from the surface of the brain.

4. Stereotaxic Frame Setup

1. Wear a surgical face mask, a clean lab coat, and sterile surgical gloves.
2. Wipe down the stereotaxic instrument and mouse adaptor with 70% ethanol.
3. Lay down a sterile surgical drape on the counter.
4. Place the stereotaxic instrument with a mouse adaptor on top of the sterile surgical drape.
5. Wipe the mouse heating pad with 70% ethanol. Position the heating pad over the stereotaxic frame bed. Use general-purpose laboratory labeling tape to tape down the heating pad over the mouse adaptor bed.
   1. Attach the heating pad to the warm water recirculation pump according to the manufacturer's directions.
   2. Turn on the warm water recirculation pump, set the temperature to 37 °C, and wait until it reaches that temperature.
6. Affix the 50 µl Hamilton syringe with a 29 G needle onto the stereotaxic frame by screwing it onto the motorized vertical injecting shaft according to the manufacturer's directions.
7. Turn on the bead sterilizer and wait until it reaches the manufacturer's preset temperature.
   NOTE: This is used for sterilizing stainless steel instruments between animals. It takes instruments 20 sec of contact with the beads for sterilization.
8. Turn on the hot plate, set it to 42 °C, and place a clean empty cage atop.
9. While the Hamilton syringe is fixed on the stereotaxic frame, use the motorized stereotaxic injector to draw up the Aβ1-42 solution.
   ​NOTE: Draw up more than 4 μl of the solution into the syringe and then use the stereotaxic injector to set the injecting volume. Operate the injector according to the manufacturer's instructions.

5. Animal Preparation

1. Determine the weight of the mouse using the weigh scale.
2. Anesthetize mouse with ketamine/xylazine cocktail at 100 mg/kg ketamine and 10 mg/kg xylazine by injecting intraperitoneally (IP). Monitor the depth of anesthesia by the loss of toe pinch reflex.
3. After the mouse is sedated, take the hair clipper and shave its head to expose the skin over the skull.
4. Place the mouse on top of the heating pad on top of the stereotaxic bed.
5. Use the spatula to open the mouth and place the incisor teeth inside the teeth guard. Secure the nosepiece over the face of the mouse. Clamp it down gently and not too tight.
6. Position the bilateral ear crossbars into the auditory meatus to secure the head. Move each crossbar in until it hits the skull, and then turn the screw to lock it.
   1. To make sure the head is secured use your index finger to gently push down on the head. If the head is properly secured it will not give out or move when pressed.
7. Apply a drop of eye cream or an eye drop to the eyes to keep them moist. Make sure the eyes are moist throughout the surgical procedure.
8. To disinfect the surgical site use sterile cotton swabs to apply betadine solution to the skin over the skull, followed by 70% ethanol. Perform the alternating betadine and 70% ethanol cleaning 2 more times.

6. Surgery and Infusion

1. Use a scalpel to make a 2-3 mm incision in the midline of the scalp, and then use straight fine scissors to extend the incision line to 1.0-1.5 cm to expose the sagittal suture, bregma, and lambda of the skull, landmarks which the stereotaxic coordinates are based on. Use micro clamps to keep the skin apart.
   NOTE: The bregma and lambda positions are explained in the mouse brain atlas "The Mouse Brain in Stereotaxic Coordinates".
2. Take a cotton swab, soak it in sterile saline, and use it to clean the skull. Then, use a clean dry cotton swab to dry the skull.
3. Use a sterile fine point pen to mark a dot on the bregma. Use the stereotaxic micromanipulator to position the Hamilton syringe so that the tip of the needle just touches the dot on the bregma.
   1. Record the DV coordinate.
   2. To check if the AP axis of the skull is level move the Hamilton syringe posteriorly so that the tip of the needle touches the lambda point.
   3. Record the DV position. Make sure the DV coordinates for bregma and lambda are within 0.5 mm.
      NOTE: The goal is to minimize the DV difference between bregma and lambda.
4. Use the micromanipulator to return the Hamilton syringe needle tip back to the bregma dot.
5. Record the starting AP position.
6. Calculate the ending AP position by subtracting 2.00 mm from the starting AP coordinate.
7. Use the micromanipulator to reposition the Hamilton syringe needle to the final AP coordinate.
8. Record the current ML coordinate.
9. Calculate the left and right ending ML coordinates by adding or subtracting 1.3 mm from the starting ML coordinate.
10. Use the micromanipulator to move the Hamilton syringe needle to either the ending ML coordinate.
    1. Touch the needle tip to the surface of the skull on both ending ML coordinates and record the DV coordinates and make sure the values are within 0.5 mm.
       ​NOTE: The goal is to minimize the DV difference between the two ending ML coordinates.
    2. While at an ending coordinate pull the needle up 0.5-1 cm over the skull. Take a sterile fine point pen to mark a dot on the surface of the skull. Repeat this step for the contralateral side of the skull.
11. Swing the Hamilton syringe clear out of the way.
12. Attach 0.8 mm drill head to the drill. Take the drill with both hands, set elbows on the surface of the table for stability, and position the drill head slightly above the sharpie dot on either the left or right ML coordinate. Activate the drill, lower the drill tip onto the skull surface to introduce a hole in the skull. Repeat this step for the contralateral side.
    NOTE: Some bleeding may occur from the newly introduced holes. If there is bleeding then use a clean dry cotton swab to dab the blood.
13. Move the Hamilton syringe back into position over either of the newly introduced ML holes. Lower the Hamilton needle just past the skull. At this point be careful not to puncture the brain. To make sure the needle has passed the skull take your index finger and gently push the needle against the skull to make sure the needle doesn't exit the hole.
14. Record the starting DV coordinate.
15. Calculate the ending DV coordinate by subtracting 2.2 mm from the starting DV coordinate.
16. Lower the needle down to the ending DV coordinate.
17. Activate the stereotaxic injector pump to pump 4 µl of Aβ1-42 into the dentate gyrus at a rate of 0.5 µl/min.
18. When the infusion is complete let the needle remain in place for an additional 1 min to minimize backflow of solution out of the injection site.
19. Move the needle to the other side of the brain and repeat steps 6.13-6.18.

7. Animal Removal and Postoperative Care

1. Unscrew the ear bars and face/nose guard. Remove the mouse from the apparatus.
2. Use a student standard pattern forceps to pull close the scalp and then seal the wound with a suture.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Ketamine HCl (100mg/ml)	Henry Schein Medical	1049007	100 mg ketamine per 1 kg animal
Xylazine (20mg/ml)	Henry Schein Medical	not available	10 mg xylazine per 1 kg animal
Aβ1-42	David Teplow/UCLA	not available	100 μM; This amyloid was used in the paper
Aβ1-42	Bachem	H-1368	Can be used in place of amyloid from the Teplow lab
Aβ1-42	American Peptide	62-0-80B	Can be used in place of amyloid from the Teplow lab
Scrambled Aβ1-42	American Peptide	62-0-46B	Can be used as control peptide for comparing Aβ1-42
GenTeal Lubricant Eye Gel	Novartis	not available	For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores
Refresh Optive Lubricant Eye Drops	Allergan	not available	For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal
Betadine	Stoelting	50998	For sanitizing and disinfecting
Round/Tapered Spatula	VWR	82027-490	For opening animal mouth
Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm)	Fine Science Tools	18050-28	Optional; For keeping scalp skin apart during injection
Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm)	Fine Science Tools	14060-11	For cutting scalp
#3 Scalpel Handle	Fine Science Tools	10003-12
#11 Surgical Blade	Fine Science Tools	10011-00	For making scalp incision
Student Standard Pattern Forcep (Tip Dims. 2.5x1.5mm; Length 11.5cm)	Fine Science Tools	91100-12	For holding scalp closed during suturing
Trimmer Combo Kit	Kent Scientific	CL9990-1201	For shaving hair
T/Pump Warm Water Recirculator	Kent Scientific	TP-700	For warming animal during surgery
Resusable Warmining Pad (5" x 10")	Kent Scientific	TPZ-0510FEA	For attaching it to the T/Pump warm water recirculator to warm the animal during surgery
Cordless Micro Drill	Stoelting	58610	Use 0.8mm steel burrs to drill holes in the skull
Lab Standard Stereotaxic Instrument with Mouse & Neonatal Rat Adaptor	Stoelting	51615
Just for Mouse Stereotaxic Instrument	Stoelting	51730	Can use this in place of Stoelting Cat. #51615
Quintessential Stereotaxic Injector	Stoelting	53311
Dry Glass Bead Sterilizer	Stoelting	50287	For sterilizing stainless steel instruments
Sterile Surgical Drape (18" x 26")	Stoelting	50981
Hamilton Syringe 50 ml, Model 705 RN SYR, NDL	Hamilton Company	7637-01	For brain injection; use different syringes for different solutions
29 Gauge Needle, Small Hub RN NDL	Hamilton Company	7803-06	For attaching to the Hamilton syringe for brain injection
1 ml BD Tuberculin Syringes	VWR	BD309659	For administering anesthesia and saline
30 Gauge Needle (0.5")	VWR	BD305106	For administering anesthesia and saline
Portable Electronic CS Series Scale (Ohaus)	VWR	65500-202	For weighing animals to determine anesthesia dose
Hot plate (Top Plate Dims. 7.25x7.25 in)	VWR	47751-148	For warming animals post-surgery
Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm	Covidien	S1173	For closing wound
Vetbond Tissue Adhesive (3M)	Santa Cruz Biotechnology	sc-361931	Optional: for aiding in wound closure; Use with suture.
Cotton-Tipped Wooden-Shaft Sterile Applicators	Fisher scientific	22-029-488	For cleaning and drying surgical wound
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15	For collecting brain sections
VWR Micro Cover Glass 24 X 50 mm	VWR	48393241	For mounting microscope slides
Thermo Scientific Nalgene Syringe Filter 0.2 μm	Fisher Scientific	194-2520	For sterilizing saline solution
Sterile dual tip skin markers by Viscot Medical	Medline	VIS1422SRL91	For marking coordinates on the skull