This research was supported by a grant of the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: H14C04660000).

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and with the Animal Care and Use Committee of the Institutional Animal Care and Use Committee of KIST (Seoul, Korea).
1. Animal Preparation
<ol>
	<li>Prepare a group of 7-week-old male ICR mice (or C57BL/6).</li>
	<li>Let the mice go through an acclimation process for 3-7 days after transport to restore homeostasis. In general, place a group of 4-5 mice in each cage and maintain them at 22-23 °C and 40% humidity, with a 12/12 hr light/dark cycle. Provide water and food ad libitum. 
	Note: The acclimation process is critical to allow recovery from the stress of shipping and adaptation to new housing, food, water, and smell as well as a new cage and light cycle. In this experiment, mice were housed in Individually Ventilated Cages (IVCs) located in the Specific Pathogen Free (SPF)-grade facility</li>
	<li>Weigh each mouse and exclude subjects whose weight deviates ± 10% from the average.</li>
	<li>Divide the subjects into two groups: a vehicle-injected group (Aβ(-)) and an Aβ-injected group (Aβ(+)). If the experiment is to test the efficacy of a particular drug candidate, divide the mice into four groups: (1) Aβ(-)/drug(-), (2) Aβ(-)/drug(+), (3) Aβ(+)/drug(-), and (4) Aβ(+)/drug(+).</li>
</ol>
2. Aβ Peptide Preparation
<ol>
	<li>Treat the Aβ peptides, derived through chemical synthesis5 or from commercial sources, with pre-chilled 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to dissolve any aggregates and to make the peptides homogenous6.
	<ol>
		<li>Sonicate the Aβ/HFIP solution in a water-bath sonicator for 5 min (at RT), gently vortex, and incubate the solution for 30 min at RT. Remove the HFIP completely via evaporation with nitrogen and store the homogenous Aβ at -80 °C until use.</li>
	</ol>
	</li>
	<li>Prepare the Aβ monomers: make 1 mM Aβ dimethylsulfoxide (DMSO) stock and dilute it 10-fold in phosphate buffered saline (PBS) (100 µM Aβ, 10% DMSO, 90% PBS.)</li>
	<li>Prepare Aβ oligomers.
	<ol>
		<li>Incubate the Aβ monomer solution (containing Aβ(1-42) or Aβ(1-40)) (step 2.2.) at 37 °C for 3 or 7 days, respectively.</li>
		<li>Place the Aβ solution in a sealed tube in a zippered bag containing a wet paper towel to provide a humidified environment to prevent water evaporation during the incubation period.</li>
	</ol>
	</li>
	<li>Confirm prepared Aβ oligomers via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with photo-induced cross-linking of unmodified proteins6.</li>
	<li>Prepare the control PBS solution containing 10% DMSO for the vehicle ICV injection.</li>
</ol>
3. Syringe Preparation
<ol>
	<li>Prepare a microsyringe with a 26 G stainless steel needle for ICV injection.</li>
	<li>Sterilize the syringe in autoclave with all the other apparatus that will be used for surgical procedure. After the autoclave, place the apparatus under the UV light for 20 min.</li>
	<li>Wrap the needle of the microsyringe with Parafilm to adjust its length to 3.8 mm to enable maximum accuracy in the depth of the injection into the brain (Figure 1A). (For B6 mice, adjust syringe needle to 3.6 mm). 
	Note: Compression of the Parafilm will lead to the depth of the injection exceeding 3.6 mm ventral. To prevent the Parafilm from being compressed during needle insertion, densely wrap the needle multiple times.</li>
	<li>Wash the inner space of the syringe with 70% ethanol, twice.</li>
	<li>Sonicate the syringe and the needle in a water-bath sonicator for 30 min at RT.</li>
	<li>Wash the inner space of the syringe with 70% ethanol, twice.</li>
	<li>Rinse out the syringe with distilled water and dry it completely in a fume hood for longer than 30 min. Leave the clean syringe under a UV light for 20 min before starting the Aβ injection.</li>
</ol>
4. Aβ-injected Mouse Model Preparation
Note: Clean the operating field with a disinfectant to maintain sterile conditions and sterilize all surgical instruments for ICV injection using 70% ethanol and UV exposure.
<ol>
	<li>Anesthetize the mouse by intraperitoneal injection of a mixture of xylazine (20 mg/kg) and tiletamine·HCl/zolazepam·HCl (80 mg/kg) before the ICV injection. Place the mouse on a warm mat to maintain its body temperature while anesthetized and confirm adequate anesthesia by assessing the foot-pinch response.</li>
	<li>Apply sterile PBS drops to both eyes to prevent corneal dryness during the procedure (Figure 1B).</li>
	<li>Prepare two mirrors by drawing multiple straight vertical lines on their surfaces in case of difficulty injecting the syringe perpendicularly.</li>
	<li>Place one mirror (M1) right next to the body of the mouse and the other (M2) in front of the head. Keep M1 parallel to the imaginary midline between the two eyes of the mouse and arrange M2 perpendicular to M1, so that the two planes form a 90° angle (Figure 1C). Place M1 on the left side of the mouse if the researcher is right-handed. Place M1 on the right side of the mouse if the researcher is left-handed.</li>
	<li>Spray 70% ethanol onto the middle of the forehead of the mouse and rub it with dry cotton swabs. Do not wet too large of an area of the forehead to avoid decreasing the body temperature due to evaporation of the alcohol.</li>
	<li>Wipe the same area on the forehead with 2% chlorhexidine solution using a clean cotton swabs. Repeat scrubbings with alcohol and chlorohexidine for total of three times each. 
	Note: If necessary, shave hair on the forehead of the mouse in order to minimize the possibility of contamination</li>
	<li>Locate bregma.
	<ol>
		<li>Using the thumb and index finger, tightly hold down the skin of the forehead, directly above the two eyes to the degree that both eyes protrude. Drag the skin back to make the forehead taut and minimize skull movements under skin.</li>
		<li>Form an invisible triangle by assigning three vertices: the two eyes of the mouse and the point where the thumb and index finger meet, the bregma. (Figures 1B, Red arrow: bregma)</li>
	</ol>
	</li>
	<li>Locate the injection point with measuring tape: -1.0 ± 0.06 mm posterior to bregma, 1.8 ± 0.1 mm lateral to the sagittal suture, and 2.4 mm in depth (Figure 1D, blue stars: injection points in each hemisphere). (For B6 mice: -0.9 mm posterior, 1.7 mm lateral, and 2.2 mm in depth)</li>
	<li>Fill the syringe with 10 µl Aβ solution or vehicle with excess solution in order to avoid accidental air injection.</li>
	<li>Place the syringe at the injection point (described in protocol 4.7). Make the syringe perpendicular to the plane of injection point. Align reflected images of the syringe in both mirrors with the drawn lines on the mirrors from a fixed perspective (Figure 1C).</li>
	<li>Begin inserting the needle until the parafilm wrapping reaches the skin.</li>
	<li>Keep the hand holding syringe steady and use other hand to inject 5 µl of the Aβ solution or vehicle, slowly over 5 sec without pausing. Make sure the syringe remains perpendicular throughout. After completing the injection, wait 3 to 5 sec before removing the syringe for diffusion.</li>
	<li>Assuming the original triangle position, exert moderate pressure to protect the skull from any unnecessary movements. Remove the syringe without tilting.</li>
	<li>Place the Aβ-injected mouse on warm pad for recovery and maintain its sternal recumbency. Do not leave the mouse unattended until it regains consciousness and do not return the mouse to a cage containing non-surgical mice until it has fully recovered.</li>
	<li>Wash the syringe according to steps 3.3-3.6.</li>
	<li>Postoperatively monitor the mobility of the mice and check for any sign of infection or illness in the mice for 5-7 days.</li>
</ol>
<img alt="Microsyringe injection setup, measurement technique, rodent anatomy schematic, research experiment." src="/files/ftp_upload/53308/53308fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Aβ Injection into the ICV region. (A) Parafilm-wrapped syringe needle compared to an unmodified syringe needle; (B) PBS drops onto eyes to prevent dryness; the triangle formed on the forehead of mouse with thumb, index finger, and the two eyes of mouse as well as the imaginary midline equidistant from each eye; (C) two mirrors (M1 and M2) with multiple vertical lines drawn on the surfaces to assist perpendicular injection of the syringe needle; (D) the triangle with the injection points, indicated as blue stars (-1.0 ± 0.06 mm from bregma and 1.8 ± 0.1 mm sagittally) on each hemisphere of mouse; Green arrow: parafilm, Red arrow: bregma <a href="https://www.jove.com/files/ftp_upload/53308/53308fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Confirmation of Aβ Injection by Y-maze and Brain Analyses
<ol>
	<li>Assess the working memory of Aβ-injected mice by Y-maze tests3,7,8
	<ol>
		<li>Wait 3 days after the injection for the onset of memory deficits.</li>
		<li>Perform the Y-maze tests using a black plastic maze with three identical arms labeled A, B, and C, each of which branches out at a 120° angle from the center of the maze (width x length x height, 10 cm x 40 cm x 12 cm).</li>
		<li>Place a mouse at the start arm (arm A) and allow the subject to freely explore the maze for 8 min.</li>
		<li>Measure the number of entries into each arm and calculate the alternation rate of each subject3,9.</li>
	</ol>
	</li>
	<li>Directly examine postmortem brains to check if the injection targeted the ICV region appropriately.
	<ol>
		<li>Anesthetize the mouse by the same procedure as outlined in section 4.1. Then sacrifice the mouse by cervical dislocation.</li>
		<li>Decapitate the mouse and remove the skin to expose the skull using surgical scissors. Remove tissues and muscles from the cranium.</li>
		<li>Make cuts on the lambdoid suture (between the parietal and interparietal bones) without damaging the brain. Remove the occipital and interparietal bones, then the parietal and frontal bones. Remove the cranial bone using forceps and isolate the brain from the skull by lifting up the meninges.</li>
		<li>Wash the brain in chilled sterile saline and cut the ICV region of the brain in the coronal direction with a surgical knife (Figure 2).</li>
		<li>Confirm the injection region based on the trace of the needle insertion on the brain tissue (Figure 2). Check if the needle trace has reached one of ventricles. If the needle trace does not meet or pass through the ventricles, exclude that subject's data from analysis of the experimental results.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The most important step in this protocol is the ICV injection of A&#946;. This protocol is designed to inject A&#946; into the ICV region of mice without stereotactic instruments11,12. Before starting an experiment, a preliminary period of practice injections with a blue dye instead of A&#946; should take place to achieve sufficient dexterity. Immediately after the injection, it is necessary to check whether the pigment injection was performed properly. Carefully take out the brain and check whether the approp...

Given that amyloid-&#946; (A&#946;) is a pathological hallmark of Alzheimer&#39;s disease (AD), the development of AD animal models has focused on neural overexpression of A&#946;. Because mutations in amyloid precursor protein (APP) or presenilin (PS) lead to disturbance of A&#946; equilibrium and ultimately to the pathogenesis of familial AD1, mouse models involving APP or PS gene mutations have been generally accepted. Among the wide range of transgenic mice, prototypical mouse models include the following: TG2576, PDAPP, APP/PS1 and APP23. In the brain, these mice generally exhibit A&#946; aggregation and eventually senile plaques; plaque formation is followed by significant cognitive impairment such that they show poor performance in behavioral tests of learning and memory. The generation of using transgenic mice that naturally mimic human AD pathology has thus contributed to AD research society by allowing us to monitor the progression of the disease. However, using transgenic mice is uneconomical and time-consuming because it takes months for the mice to develop A&#946; plaques and even longer to show A&#946;-induced synaptic or behavioral abnormalities2,3. Originally developed as an alternative to overcome the shortcomings of transgenic mouse models, non-transgenic models are also commonly used due to their distinct advantages. Pathogen-induced AD models can be produced by the direct injection of A&#946; into the brain, whereas AD-like cognitive deficits can also be triggered by other chemical and physical means-such as the injection of neurotoxic compounds such as scopolamine, the induction of lesions in cognition-related areas such as the hippocampus, or by cortical damage4. However, the non-pathogenic induction of cognitive impairment does not accurately reflect the fundamental pathophysiology of AD; instead, it only mimics its symptomatic outcomes. In contrast, a pathogen-induced AD model, the A&#946;-injected mouse model, can not only show AD-like behavioral abnormalities but can also exhibit A&#946; pathology, the common feature shared by familial and sporadic AD.
Despite the difficulty to visualize A&#946; plaques in the brain tissue, the largest benefit of the A&#946;-injected model that makes it attractive for AD investigation is its controllability. Researchers can weed out the individual differences in mouse models that can lead to erroneous data in drug-related studies. Timely drug treatment is enabled depending on the mechanism of the candidate drug; to elaborate, an inhibitor of A&#946; aggregation can be applied before the injection of A&#946;. Additionally, investigators can assume that the pathogenic transformation that arises after A&#946; injection is derived from the A&#946; exposure because the other factors are tightly controlled, including individual differences.
In this protocol, a vivid description of how to induce an AD-like phenotype in normal mice via A&#946; intracerebroventricular (ICV) injection without stereotactic instruments is presented. Minimizing insertion-provoked damage to brain tissue is essential to prevent the possibility of structural damage and lesion-induced inflammation. A lack of skill in mouse handling leads to unexpected neuronal injury. Furthermore, techniques that enable the appropriate angle and depth to be achieved during the injection are especially important to circumvent frequent mistakes. In addition to a detailed, vivid explanation of the ICV injection, the reliability of the model produced by the following protocol is also illustrated in the following sections. The following protocol could be a reliable and easily understood tool that contributes to AD research, thereby providing a steppingstone that could ultimately lead to a meaningful discovery for AD society.

<table><tbody><tr><td>ICR mouse</td><td>Orientbio</td><td></td><td>male, 6-8 weeks, 27-29 g of body weight</td></tr><tr><td>C57BL/6 mouse</td><td>Orientbio</td><td></td><td>male, 6-8 weeks, 21-23 g of body weight</td></tr><tr><td>Amyloid-beta1-42</td><td>in house synthesis</td><td></td><td>stock concentration: 1 mM/DMSO, injected concentration: 100 &amp;mu;M/10% DMSO and 90% PBS</td></tr><tr><td>ICV injection syringe (26s gauge)</td><td>Hamilton</td><td>80308</td><td></td></tr><tr><td>Evans blue dye (EBD)</td><td>abcamBIochemicals</td><td>ab120869</td><td>1% EBD in PBS</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>PBS</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Gradi-GelTM II Gradient PAGE Analysis Kit</td><td>ELPiS Biotech</td><td>EBS-1056</td><td>15% Gel</td></tr><tr><td>Precision Plus ProteinTM Dual Xtra Standards</td><td>Bio-Rad</td><td>161-0377</td><td></td></tr><tr><td>Silver-Staining Kit</td><td>GE-Healthcare</td><td>17-1150-01</td><td></td></tr><tr><td>LabDiet</td><td>Orientbio</td><td>5053</td><td></td></tr></tbody></table>

intracerebroventricular injection of amyloid-&#946; peptides in normal mice to acutely induce alzheimer-like cognitive deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

This section illustrates examples of the results that can be obtained by confirmation of Aβ aggregation and Y-maze assessment of memory deficits. Using the full-length Aβ(1-42) peptide of 42 amino acids, mixture of Aβ monomers, oligomers, and fibrils (Figure 3) was produced. Through the HFIP-induced monomerization step, relatively homogeneous monomers (Lane B) were obtained. After the 7 day incubation, diverse sizes of Aβ aggregates (Lane C) developed. Trimers and tet...

Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-ÃŽÂ² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

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

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

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

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42.0K Views.  Korea Institute of Science and Technology.  De amyloid-β (Aß) -injected diermodel kan de toediening van een bepaalde hoeveelheid en de soort van Ap-fragmenten en vermindert de individuele verschillen binnen elke studiegroep. Dit protocol beschrijft de intracerebroventriculaire (ICV) injectie van Ap zonder stereotactische instrumenten, waardoor de productie van Alzheimer-achtige gedragsafwijkingen bij normale muizen. 

Video: Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

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

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

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.

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 ...

Behavior

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biology

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

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

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

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

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

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

Medicine

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

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

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

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

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

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

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

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

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

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

Source: Sajadi, A., et al. <a href="https://app.jove.com/v/54215">Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for the continuous infusion of amyloid-beta (A&#946;) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of A&#946; peptide-specific antibodies coupled with the infusion of soluble A&#946; oligomers (A&#946;o) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

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

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

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

Source: Xiaoguang, W., et al. <a href="https://app.jove.com/v/56157">Establishment of a Valuable Mimic of Alzheimer&#39;s Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for injecting amyloid-beta peptides, aluminum chloride, and a neuroinflammatory growth factor, which collectively aids in toxic amyloid plaque formation in the hippocampus region. This causes memory impairment in the rat, characteristic of Alzheimer&#39;s disease.

Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

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