Name: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus (Video) | JoVE Journal
Uploaded: 2015-06-17T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Learn stereotaxic infusion of oligomeric amyloid-beta 1-42 into the mouse hippocampus dentate gyrus. This method enables precise study of amyloid-beta neurotoxicity and neuronal pathology in vivo.

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5 minutes

This work was supported by National Institute of Neurological Disorders and Stroke grant NS081333 (to CMT), Alzheimer&#8217;s Association grant NIRG-10-171721 and National Institute of Mental Health grant MH096702 (to UH), and National Institute on Aging-funded Alzheimer&#8217;s Disease Research Center at Columbia University pilot grant AG008702 (to YYJ and JB).

Note: For all animal experimentation, Institutional and National guidelines for the care and use of laboratory animals were followed.
1. Prepare Surgical Instruments and Solutions for Surgery
<ol>
	<li>Autoclave all stainless steel surgical instruments.</li>
	<li>Prepare 70% ethanol by diluting 200 proof absolute ethanol with sterile molecular grade deionized distilled water.</li>
	<li>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.
	<ol>
		<li>Remove the plunger and needle from the Hamilton syringe. Air dry the parts in a laminar flow hood O/N.</li>
		<li>Irradiate the needle and syringe with ultraviolet light for 30 min before use.</li>
	</ol>
	</li>
	<li>Prepare saline solution by dissolving NaCl in molecular grade water to a final weight to volume concentration of 0.9%. Sterilize the solution by filtering it through 0.2 &#956;m pore filter.</li>
</ol>
2. Prepare Oligomeric A&#946;1-42
<ol>
	<li>Monomerize and resuspend recombinant human A&#946;1-42 in DMSO to 5 mM exactly as described in the publication of Fa&#39; and others13 .</li>
	<li>Dilute 5 mM A&#946;1-42 with sterile (1x) PBS to 100 &#181;M on the day before surgery.</li>
	<li>Mix the solution well by trituration.</li>
	<li>Incubate A&#946;1-42 solution for 12 hr at 4&#730;C. 
	Note: Control solutions such as diluting DMSO in PBS or scrambled/reverse A&#946;1-42 peptide should be prepared the same way as A&#946;1-42.</li>
</ol>
3. Determine Injection Coordinates
<ol>
	<li>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 interest14. 
	&#8203;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, &#177;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 &#177; 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.</li>
</ol>
4. Stereotaxic Frame Setup
<ol>
	<li>Wear a surgical face mask, clean lab coat, and sterile surgical gloves.</li>
	<li>Wipe down stereotaxic instrument and mouse adaptor with 70% ethanol.</li>
	<li>Lay down sterile surgical drape on the counter.</li>
	<li>Place the stereotaxic instrument with mouse adaptor on top of the sterile surgical drape.</li>
	<li>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.
	<ol>
		<li>Attach the heating pad to the warm water recirculator pump according to manufacturer&#8217;s directions.</li>
		<li>Turn on the warm water recirculator pump and set the temperature to 37 &#176;C, and then wait until it reaches temperature.</li>
	</ol>
	</li>
	<li>Affix the 50 &#181;l Hamilton syringe with a 29 G needle onto the stereotaxic frame by screwing it onto the motorized vertical injecting shaft according to manufacturer&#8217;s directions.</li>
	<li>Turn on the bead sterilizer and wait until it reaches the manufacturer&#8217;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.</li>
	<li>Turn on hot plate and set it to 42 &#176;C and place a clean empty cage atop.</li>
	<li>While the Hamilton syringe is fixed on the stereotaxic frame use the motorized stereotaxic injector to draw up the A&#946;1-42 solution. 
	&#8203;Note: Draw up more than 4 &#956;l of the solution into the syringe and then use the stereotaxic injector to set the injecting volume. Operate the injector according to manufacturer&#8217;s instructions.</li>
</ol>
5. Animal Preparation
<ol>
	<li>Determine the weight of the mouse using the weigh scale.</li>
	<li>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.</li>
	<li>After the mouse is sedated take the hair clipper and shave its head to expose the skin over the skull.</li>
	<li>Place the mouse on top of the heating pad on top of the stereotaxic bed.</li>
	<li>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.</li>
	<li>Position the bilateral ear crossbars into auditory meatus to secure the head. Move each crossbar in until it hits the skull, and then turn the screw to lock it.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Apply a drop of eye cream or eye drop to the eyes to keep them moist. Make sure the eyes are moist throughout the surgical procedure.</li>
	<li>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.</li>
</ol>
6. Surgery and Infusion
<ol>
	<li>Use a scalpel to make a 2-3 mm incision in the midline of the scalp, and then use a 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 &#8220;The Mouse Brain in Stereotaxic Coordinates&#8221;14.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Record the DV coordinate.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Use the micromanipulator to return the Hamilton syringe needle tip back to the bregma dot.</li>
	<li>Record the starting AP position.</li>
	<li>Calculate the ending AP position by subtracting 2.00 mm from the starting AP coordinate.</li>
	<li>Use the micromanipulator to reposition the Hamilton syringe needle to the final AP coordinate.</li>
	<li>Record the current ML coordinate.</li>
	<li>Calculate the left and right ending ML coordinates by adding or subtracting 1.3 mm from the starting ML coordinate.</li>
	<li>Use the micromanipulator to move the Hamilton syringe needle to either the ending ML coordinate.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Swing the Hamilton syringe clear out of the way.</li>
	<li>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.</li>
	<li>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&#8217;t exit the hole.</li>
	<li>Record the starting DV coordinate.</li>
	<li>Calculate the ending DV coordinate by subtracting 2.2 mm from the starting DV coordinate.</li>
	<li>Lower the needle down to the ending DV coordinate.</li>
	<li>Activate the stereotaxic injector pump to pump 4 &#181;l of A&#946;1-42 into the dentate gyrus at a rate of 0.5 &#181;l/min.</li>
	<li>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.</li>
	<li>Move the needle to the other side of the brain and repeat steps 6.13-6.18.</li>
</ol>
7. Animal Removal and Postoperative Care
<ol>
	<li>Unscrew the ear bars and face/nose guard. Remove the mouse from the apparatus.</li>
	<li>Use a student standard pattern forceps to pull close the scalp and then seal the wound with suture.</li>
	<li>Inject 1 ml of sterile saline into the mouse via IP for hydration.</li>
	<li>Inject buprenorphine (0.1 mg/kg) subcutaneously to relieve pain.</li>
	<li>Maintain the mouse in the clean empty cage set on top of 42 &#176;C hot plate until it wakes, then return it to its housing cage with ample food and water.</li>
	<li>Administer buprenorphine via subcutaneous injection every 12 hr for 3 days for pain relief.</li>
</ol>
8. Suture Removal
<ol>
	<li>Anesthetize mouse with ketamine/xylazine cocktail at 100 mg/kg ketamine and 10 mg/kg xlyazine by IP. Note: This is done 7-10 days after surgery.</li>
	<li>Use a student standard pattern forceps and straight fine scissors to remove the suture.</li>
	<li>Inject 1 ml of sterile saline into the mouse via IP for hydration.</li>
	<li>Maintain the mouse in a clean empty cage set on top of 42 &#176;C hot plate until it wakes, and then return it to its housing cage with ample food and water.</li>
</ol>
9. Animal Sacrifice and Tissue Processing
<ol>
	<li>Anesthetize the mouse and perfuse it with 4% paraformaldehyde15, remove the brain, and process it for cryosectioning16. 
	Note: The timing of animal sacrifice depends on the experimenter. However, for our studies we chose 7 and 14 days post surgery.</li>
</ol>

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</ol>

The authors declare that they have no competing financial interests.

To achieve a successful A&#946;1-42 injection the experimenter or surgeon must: 1) use aseptic technique; 2) correctly identify the brain region of interest with accurate coordinates; 3) be able to properly secure the mouse in the stereotaxic frame with the brain leveled in the AP and ML axis; 4) have the ability to operate the micromanipulator with precision; 5) ensure proper post-operative care. If these key steps are followed the mouse should survive the surgery with no observable infection.
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Amyloid plaque deposits, which are composed of amyloid-beta (A&#946;1-42), are a key feature of the pathology of Alzheimer&#8217;s disease (AD). Numerous studies have shown that high or toxic levels of recombinant oligomeric A&#946;1-42 elicit neuronal death, synaptic dystrophy, loss and dysfunction; as well as learning and memory deficits1-4. Brain regions affected include the hippocampus, the cortex, and subcortical structures such as the basal forebrain and the amygdala5,6. To date, there are multiple transgenic mouse models that attempt to simulate the A&#946;1-42 pathology of AD. Depending on the strain these animals prove to be useful in examining select pathological features of AD. Unfortunately, with the exception of 2 transgenic lines, APP23 and 5XFAD, these mice never fully replicate neuronal loss, a key aspect of AD. Even with the neuronal loss observed in APP23 and 5XFAD, the neuronal death observed was subtle, age dependent, and isolated to a few select regions7,8.
The direct infusion of oligomeric A&#946;1-42 into the wild-type mouse brain provides an excellent in vivo model which replicates the neuronal death aspect of amyloidopathy1,9,10. Unlike the commonly utilized transgenic mouse models the oligomeric A&#946;1-42 infusion model is ideal for acutely elevating A&#946;1-42 levels in a spatial and temporal manner. The advantage of using wild-type mice for this model obviates potential compensation or side effects from the mutations introduced in transgenic mouse lines. Past studies have shown that infusing toxic levels of A&#946;1-42 into the hippocampus elicits neuron death in the vicinity of the injection site within 1 week1. Moreover, consistent with the observation that A&#946;1-42 is toxic for cholinergic neurons11 the basal forebrain cholinergic neuron (BFCN) population which projects to the hippocampus is decreased 20-50% within 7-14 days following beta-amyloid infusion1,10 in mice, effectively allowing for the examinations of isolated neuronal circuitry in the brain. Since BFCN project ipsilaterally to the dentate gyrus of the hippocampus12, for the most part control/vehicle and oligomeric A&#946;1-42 solutions can be injected on either side of the brain allowing comparisons to be made between the left and right hemispheres1.
In this report we will provide a detailed surgical and injection methodology for adult wild-type C57BL/6J mice. This mouse strain is chosen because of its wide use in research. Technically, any brain region can be targeted for infusion, however here we will use the dentate gyrus of the hippocampus as the target to illustrate the technique.

<table><tbody><tr><td>Ketamine HCl (100 mg/ml)</td><td>Henry Schein Medical</td><td>1049007</td><td>100 mg ketamine per 1 kg animal</td></tr><tr><td>Xylazine (20 mg/ml)</td><td>Henry Schein Medical</td><td>not available</td><td>10 mg xylazine per 1 kg animal</td></tr><tr><td>Buprenex (0.3 mg/ml)</td><td>Henry Schein Medical</td><td>1217793</td><td>0.1 mg buprenex per 1 kg animal</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>David Teplow/UCLA</td><td>not available</td><td>100 &amp;mu;M; This amyloid was used in the paper</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>Bachem</td><td>H-1368</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-80B</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>Scrambled A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-46B</td><td>Can be used as control peptide for comparing A&amp;beta;1-42</td></tr><tr><td>NU4 Antibody (Oligomeric Amyloid Antibody)</td><td>Gift from William Klein/Northwestern U.</td><td>not available</td><td>1:2,000 dilution</td></tr><tr><td>Anti-Amyloid Oligomeric Antibody&amp;nbsp; (Polyclonal Rabbit)</td><td>EMD Millipore</td><td>AB9234</td><td>May be used in place of Nu4; needs to&amp;nbsp; be tested by the end user</td></tr><tr><td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td><td>Biolegend</td><td>sig-39320</td><td>1:1,000 dilution</td></tr><tr><td>ChAT Antibody (Polyclonal Goat)</td><td>Millipore</td><td>AB144P</td><td>1:100 dilution</td></tr><tr><td>DeadEnd Fluorometric TUNEL system</td><td>Promega</td><td>G3250</td><td>Follow manufacturer&#039;s directions for use</td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>Use when coverslipping slides</td></tr><tr><td>Fluorogold</td><td>Fluorochrome, LLC</td><td>not available</td><td>2% solution</td></tr><tr><td>Absolute Ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818-4</td><td>For making 70% ethanol for sanitizing and disinfecting</td></tr><tr><td>Novex 10-20% Tricine gel</td><td>Life Technologies</td><td>EC6625BOX</td><td>For separating A&amp;beta;1-42</td></tr><tr><td>Novex Tricine SDS Running Buffer (10X)</td><td>Life Technologies</td><td>LC1675</td><td>For running 10-20% Tricine gels</td></tr><tr><td>Novex Tris-Glycine Transfer Buffer (25X)</td><td>Life Technologies</td><td>LC3675</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>SuperSignal Western Blot Enhancer</td><td>Thermo Scientific</td><td>46640</td><td>For enhancing A&amp;beta;1-42 signal; follow manufacturer&#039;s protocol</td></tr><tr><td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &amp;mu;m</td><td>GE Healthcare Life Sciences</td><td>10402088</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>Xcell SureLock Mini-Cell</td><td>Life Technologies</td><td>EI0001</td><td>Electrophoresis aparatus for running 10-20% Tricine gels</td></tr><tr><td>GenTeal Lubricant Eye Gel</td><td>Novartis</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores&lt;br /&gt;&amp;#160;&lt;br/&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Refresh Optive Lubricant Eye Drops</td><td>Allergan</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td></tr><tr><td>Betadine</td><td>Stoelting</td><td>50998</td><td>For sanitizing and disinfecting</td></tr><tr><td>Round/Tapered Spatula&amp;nbsp;</td><td>VWR</td><td>82027-490</td><td>For opening animal mouth</td></tr><tr><td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6 mm; Length 28 mm)</td><td>Fine Science Tools</td><td>18050-28</td><td>Optional; For keeping scalp skin apart during injection</td></tr><tr><td>Straight Fine Scissors (Cutting edge 25 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>14060-11</td><td>For cutting scalp</td></tr><tr><td>#3 Scalpel Handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>#11 Surgical Blade</td><td>Fine Science Tools</td><td>10011-00</td><td>For making scalp incision</td></tr><tr><td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>91100-12</td><td>For holding scalp closed during suturing</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201</td><td>For shaving hair</td></tr><tr><td>T/Pump Warm Water Recirculator&amp;nbsp;</td><td>Kent Scientific&amp;nbsp;</td><td>TP-700</td><td>For warming animal during surgery</td></tr><tr><td>Resusable Warmining Pad (5&quot; x 10&quot;)</td><td>Kent Scientific&amp;nbsp;</td><td>TPZ-0510FEA</td><td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td></tr><tr><td>Cordless Micro Drill</td><td>Stoelting</td><td>58610</td><td>Use 0.8 mm steel burrs to drill holes in the skull</td></tr><tr><td>Lab Standard Stereotaxic Instrument with Mouse &amp;amp; Neonatal Rat Adaptor</td><td>Stoelting</td><td>51615</td><td></td></tr><tr><td>Just for Mouse Stereotaxic Instrument</td><td>Stoelting</td><td>51730</td><td>Can use this in place of Stoelting Cat. #51615</td></tr><tr><td>Quintessential Stereotaxic Injector</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Dry Glass Bead Sterilizer</td><td>Stoelting</td><td>50287</td><td>For sterilizing stainless steel instruments</td></tr><tr><td>Sterile Surgical Drape (18&quot; x 26&quot;)</td><td>Stoelting</td><td>50981</td><td></td></tr><tr><td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td><td>Hamilton Company</td><td>7637-01</td><td>For brain injection; use different syringes for different solutions</td></tr><tr><td>29 G Needle, Small Hub RN NDL</td><td>Hamilton Company</td><td>7803-06</td><td>For attaching to the Hamilton syringe for brain injection</td></tr><tr><td>1 ml BD Tuberculin Syringes</td><td>VWR</td><td>BD309659</td><td>For administering anesthesia and saline</td></tr><tr><td>30 G Needle (0.5&quot;)</td><td>VWR</td><td>BD305106</td><td>For administering anesthesia and saline</td></tr><tr><td>Portable Electronic CS Series Scale (Ohaus)</td><td>VWR</td><td>65500-202</td><td>For weighing animals to determine anesthesia dose</td></tr><tr><td>Hot plate (Top Plate Dims. 7.25x7.25 in)</td><td>VWR</td><td>47751-148</td><td>For warming animals post-surgery</td></tr><tr><td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td><td>Covidien</td><td>S1173</td><td>For closing wound</td></tr><tr><td>Vetbond Tissue Adhesive (3M)</td><td>Santa Cruz Biotechnology</td><td>sc-361931</td><td>Optional: for aiding in wound closure; Use with suture.</td></tr><tr><td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td><td>Fisher scientific</td><td>22-029-488</td><td>For cleaning and drying surgical wound</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific&amp;nbsp;</td><td>12-550-15</td><td>For collecting brain sections</td></tr><tr><td>VWR Micro Cover Glass 24 X 50 mm</td><td>VWR</td><td>48393241</td><td>For mounting microscope slides</td></tr><tr><td>Thermo Scientific Nalgene Syringe Filter 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>194-2520</td><td>For sterilizing saline solution</td></tr><tr><td>Sterile dual tip skin markers by Viscot Medical</td><td>Medline</td><td>VIS1422SRL91</td><td>For marking coordinates on the skull</td></tr></tbody></table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

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.

The present method of preparing human recombinant oligomeric Aβ1-42 yields soluble oligomeric species consisting of monomers, dimers, trimers, and tetramers (Figure 1A). These low molecular weight Aβ1-42 species, but not the fibrils and plaques, have been shown in numerous settings to be most toxic to neurons1,4,9,17-19. To determine whether or not oligomeric Aβ1-42 induces neuron death in the mouse brain Aβ1-42 (4 µl of 100 µM stock solution) was in...

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 ...

The overall goal of this procedure is to stereotaxic inject a Liga meric a beta 1 42, into the dentate gyrus of the hippocampus. This is accomplished by first preparing a liga meric, a beta 1 42 solution. The second step is to determine the injection coordinates.Next surgically, expose the skull of the animal and introduce two holes in it for needle insertion. The final step is to move the syringe needle into its final position and to infuse a beta 1 42. Ultimately, the Stereotaxic Abeta 1 42 injection method is used to examine the toxic effects of Abeta 1 42 in a live animal.The main advantage of this technique over existing transgenic, Abeta 42 mouse models is that oligomeric ALA 42 can be elevated in the special and temporal manner and reliably reproduce the cell death pathology that is lacking in the transgenic mouse models. This method can help answer key questions in the Alzheimer's field, such as how pathological processes spread in Alzheimer's disease. To begin this procedure, shave the head of an anesthetized mouse to expose the skin.Next, place the mouse on a heating pad in a stereotaxic frame. Then secure its incisors in the teeth guard and a nose piece over the face of the mouse. After that, position the ear bars into the auditory mauss in order to secure the head to check.If the head is secured, gently press it and ensure that it doesn't move. Then apply ointment to both eyes to keep the moist. After that, disinfect the surgical site by applying Betadine to the skin over the skull, followed by 70%ethanol with sterile cotton swabs.Perform the alternating Betadine and ethanol cleaning two more times. Now make a two to three millimeter incision in the midline of the scalp with a scalpel. Use straight fine scissors to extend the incision line to about 1.0 centimeters to expose the sagittal suture bgma and lambda of the skull.After that, use forceps to pull the skin apart. Clean the skull with a sterile cotton swab, soaked with sterile saline. Then use a clean, dry cotton swab to dry the skull.Next, mark a dot. On the Bre ma position the Hamilton syringe using the micro manipulator so that the tip of the needle just touches the dot on the bgma. Then record the DV coordinate mark a dot on the Lambda point, make sure that the AP axis of the skull is level by moving the syringe posteriorly to the Lambda point.The tip of the needle should touch the Lambda point as it did the bgma point. After that, record the DV position. The DV coordinates for Bre, MA and Lambda should be within 0.5 millimeters.Now return the needle tip to the BMA dot. Record the starting AP position. Then calculate the ending AP position by subtracting two millimeters from the starting ap.Coordinate and reposition the syringe needle to the final AP coordinate. Next, record the current ML coordinate. Calculate the left and right ending ml coordinates by adding or subtracting 1.3 millimeters from the starting ml.Coordinate and move the syringe needle to the ending ML coordinates. Touch the needle tip to the surface of the skull on both ending ML coordinates, and make sure the DV coordinates are within 0.5 millimeters. While at the ending ml coordinate, pull the needle up, 0.5 to one centimeters over the skull.Mark a dot on the skull with a dark colored extra fine point marker. Repeat this step for the contralateral side of the skull. Then move the syringe away.Drill a hole on either the left or right ml. Coordinate on the skull using a 0.8 millimeter drill bit. Repeat this step for the contralateral side.Afterward, move the syringe to one of the newly introduced holes. Lower the needle to just past the skull, and be careful not to puncture the brain to ensure that the needle has passed the skull. Gently push the needle against the skull to make sure that the needle doesn't come out of the hole.Next, record the starting DV coordinate. Calculate the ending dv. Coordinate by subtracting 2.2 millimeters from the starting DV.Coordinate and lower the needle to the ending db.Coordinate then set the stereotaxic injector pump to infuse four microliters of a beta 1 42 into the dentate gyrus at a rate of 0.5 microliters per minute. After the infusion, let the needle remain in place for an additional minute to minimize backflow of solution out of the injection site. After that, move the needle to the other side of the brain and repeat the procedure.Now, unscrew the ear bars. Use student standard pattern forceps to pull the scalp closed and seal the wound with the suture. Take off the nose guard and remove the mouse.Inject one milliliter of sterile saline into the mouse intitally for hydration and inject buprenorphine at 0.1 milligrams per kilogram subcutaneously for pain relief. Afterward, transfer the mouse to a clean empty cage set on top of a 42 degree Celsius hot plate until it wakes up. Then return the mouse to its housing cage with ample food and water.This figure shows the infusion of a beta 1 42 and vehicle in the respective left and right dentate gyrus of a nine month old mouse. Broken lines depict the injection tracted and the arrows show the collapsed dentate gyrus. Within one week of Abeta 1 42 injection, the dentate gyrus exhibited elevated Abeta 1 42 levels in the molecular layer and the polymorphic cell layer close to the injection site at seven and 14 days.Abeta 1 42 elicited increases in tunnel positive staining and decreases in dentate gyrus thickness, confirming the neurodegenerative effects of Abeta 1 42. Here, a retrograde tracer fluoro gold was co injected with Abeta 1 42 into the dentate gyrus. Seven days following injection, fluorgold was detected in the basal forebrain suggesting the retrograde transport of the label via cholinergic neurons.Fluorgold positive neurons in the basal forebrain were chosen for quantification. Seven days following injection, Abeta 1 42 elicited increases in tunnel and decreases in chat. Positive neurons in the basal four brain Once mastered this technique can be done in approximately 30 minutes if performed properly.Following this procedure, other methods can be used to answer additional questions such as studies of molecular mechanism to address the mechanistic effect of amyloid infusion on the brain. I'd like to thank you for watching our video and I hope that the method will be useful in your experiments.

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

Research

JoVE Journal

Neuroscience

One feature of Alzheimer's Disease is the elevation of Aβ1-42 peptide. Here we provide a protocol for preparing synthetic Aβ1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of Aβ-induced pathology and drug screening.

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

This video demonstrates a technique for the continuous infusion of amyloid-beta (Aβ) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of Aβ peptide-specific antibodies coupled with the infusion of soluble Aβ oligomers (Aβo) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

Controlled Infusion of A&beta; Peptide-Specific Antibodies and A&beta; Oligomers into the Rat Hippocampus

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.

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

Source: Arango-Lievano, M. et. al., Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases. J. Vis. Exp. (2016)This video demonstrates in vivo two-photon imaging to visualize fluorescently labeled amyloid plaques and cerebral vasculature. Blue fluorescence marks plaques, while green fluorescence highlights the cerebral blood vessels. A 3D map of the region of interest and high-resolution mosaic imaging reveal detailed interactions of amyloid plaques with cerebral...

In Vivo Imaging of Cerebral Vessels and Amyloid Plaques in an Alzheimer’s Disease Mouse Model

Source: Slezia, A. et al. Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice. J. Vis. Exp. (2019)This video demonstrates the electrophoretic delivery of gamma-aminobutyric acid (GABA) to inhibit seizure activity in an anesthetized mouse. It outlines the steps involved in probe implantation, neural activity recording, seizure induction, and controlled GABA delivery via a microfluidic ion pump to assess its therapeutic efficacy.

Electrophoretic Delivery of GABA into the Epileptic Focus of an Anesthetized Mouse

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

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

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

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

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

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

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

Learn stereotaxic infusion of oligomeric amyloid-beta 1-42 into the mouse hippocampus dentate gyrus. This method enables precise study of amyloid-beta neurotoxicity and neuronal pathology in vivo.

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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