Stereotaktisk Infusion af Oligomert amyloid-beta i musen Hippocampus
Summary
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.
Abstract
Alzheimers sygdom er en neurodegenerativ sygdom, der påvirker den aldrende befolkning. Et centralt neuropatologisk træk ved sygdommen er den overproduktion af amyloid-beta og aflejring af amyloid-beta plaques i hjernen regioner af de ramte individer. Gennem årene har forskerne skabt mange Alzheimers sygdom musemodeller, der forsøger at kopiere amyloid-beta patologi. Desværre kun de musemodeller selektivt efterligne sygdomstilstande funktioner. Neuronal død, en fremtrædende virkning i hjernen hos patienter med Alzheimers sygdom, er mærkbart mangler i disse mus. Derfor har vi og andre ansat en metode til direkte infusion opløselige oligomere arter af amyloid-beta – former for amyloid-beta, der har vist sig at være mest giftige for neuroner – stereotaksisk ind i hjernen. I denne rapport anvender vi mandlige C57BL / 6J mus til at dokumentere denne kirurgisk teknik til at øge amyloid-beta-niveauer i en udvalgt område af hjernen. Deninfusion mål er gyrus dentatus i hippocampus, fordi denne hjerne struktur, sammen med den basale forhjerne, der er forbundet med det cholinerge kredsløb, udgør en af de områder af degeneration i sygdommen. Resultaterne af opløftende amyloid-beta i den tandede gyrus via stereotaktisk infusion afsløre stigninger i neurontab i gyrus dentatus inden for 1 uge, mens der er en ledsagende stigning i celledød og cholinerg neuron tab i den lodrette led af det diagonale bånd af Broca af den basale forhjerne. Disse virkninger observeres op til 2 uger. Vores data tyder på, at den nuværende amyloid-beta infusion model giver en alternativ musemodel til at løse region specifik neuron død på kort sigt. Fordelen ved denne model er, at amyloid-beta kan være forhøjet i en rumlig og tidsmæssig måde.
Introduction
Amyloid plaque indskud, som er sammensat af amyloid-beta (Ap 1-42), er et centralt element i patologien af Alzheimers sygdom (AD). Talrige undersøgelser har vist, at høje eller toksiske niveauer af rekombinant oligomert AB 1-42 fremkalde neuronal død, synaptisk dystrofi, tab og dysfunktion; samt indlæring og hukommelse underskud 1-4. Hjerneregioner berørte inkluderer hippocampus, cortex, og subkortikale strukturer såsom basale forhjerne og amygdala 5,6. Til dato er der flere transgene musemodeller, der forsøger at simulere Ap 1-42 patologi af AD. Afhængigt af stammen disse dyr vise sig at være nyttige i behandlingen af udvalgte patologiske træk ved AD. Desværre, med undtagelse af 2 transgene linjer, APP23 og 5XFAD, disse mus aldrig replikere fuldt neuronal tab, et centralt aspekt af AD. Selv med den neuronale tab observeret i APP23 og 5XFAD, den neuronale død obseraba var subtile, alder afhængig, og isoleret til nogle få udvalgte områder 7,8.
Den direkte infusion af oligomert Ap 1-42 i vildtype mus hjerne giver en fremragende in vivo-model, som replikerer den neuronale død aspekt af amyloidopathy 1,9,10. I modsætning til de almindeligt anvendte transgene musemodeller den oligomere Ap 1-42 infusion model er ideel til akut opløftende Ap 1-42 niveauer i en rumlig og tidsmæssig måde. Fordelen ved at anvende vildtype-mus for denne model undgår potentielle erstatninger og bivirkninger fra mutationerne introduceret i transgene mus linjer. Tidligere undersøgelser har vist, at infusion af toksiske niveauer af Ap 1-42 i hippocampus fremkalder neurondød i nærheden af injektionsstedet inden for 1 uge 1. Øvrigt i overensstemmelse med den observation, at Ap 1-42 er toksisk for cholinerge neuroner 11 den basale forhjernecholinerge neuron (BFCN) population, der rager til hippocampus er faldet 20-50% inden for 7-14 dage efter beta-amyloid infusion 1,10 i mus, effektivt giver mulighed for undersøgelser af isolerede neuronal kredsløb i hjernen. Siden BFCN projekt ipsilaterally til tandede gyrus af hippocampus 12, for det meste kontrol / køretøj og oligomere Ap 1-42 løsninger kan injiceres på begge sider af hjernen giver sammenligninger mellem venstre og højre hjernehalvdel 1.
I denne rapport vil vi give en detaljeret kirurgisk og injektion metode til voksne vildtype C57BL / 6J mus. Denne musestamme er valgt på grund af sin omfattende brug i forskning. Teknisk set kan enhver hjerne region målrettes til infusion, men her vil vi bruge gyrus dentatus i hippocampus som målet at illustrere teknikken.
Protocol
Representative Results
Discussion
For at opnå en succesfuld Ap 1-42 injektion forsøgslederen eller kirurg skal: 1) Brug aseptisk teknik; 2) identificere hjernen område af interesse med nøjagtige koordinater korrekt; 3) være i stand til korrekt fastgøre musen i stereotaktisk ramme med hjernen nivelleret i AP og ML akse; 4) har evnen til at betjene mikromanipulator med præcision; 5) sikre korrekt postoperativ pleje. Hvis følges disse vigtige trin musen skulle overleve operationen med ingen observerbar infektion.
<p class="jove_conte…Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Institute of Neurologiske og Stroke give NS081333 (til CMT), Alzheimers Association tilskud NIRG-10-171721 og National Institute of Mental Health tilskud MH096702 (til UH), og National Institute on Aging-finansierede Alzheimers sygdom Research Center på Columbia University pilot tilskud AG008702 (til YYJ og JB).
Materials
| 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 |
| Buprenex (0.3mg/ml) | Henry Schein Medical | 1217793 | 0.1 mg buprenex 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 |
| NU4 Antibody (Oligomeric Amyloid Antibody) | Gift from William Klein/Northwestern U. | not available | 1:2000 dilution |
| Anti-Amyloid Oligomeric Antibody (Polyclonal Rabbit) | EMD Millipore | AB9234 | May be used in place of Nu4; needs to be tested by the end user |
| 6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody) | Biolegend | sig-39320 | 1:1000 dilution |
| ChAT Antibody (Polyclonal Goat) | Millipore | AB144P | 1:100 dilution |
| DeadEnd Fluorometric TUNEL system | Promega | G3250 | Follow manufacturer's directions for use |
| Prolong Gold Antifade Reagent with DAPI | Invitrogen | P36935 | Use when coverslipping slides |
| Fluorogold | Fluorochrome, LLC | not available | 2% solution |
| Absolute Ethanol (200 proof) | Fisher Scientific | BP2818-4 | For making 70% ethanol for sanitizing and disinfecting |
| Novex 10-20% Tricine gel | Life Technologies | EC6625BOX | For separating Aβ1-42 |
| Novex Tricine SDS Running Buffer (10X) | Life Technologies | LC1675 | For running 10-20% Tricine gels |
| Novex Tris-Glycine Transfer Buffer (25X) | Life Technologies | LC3675 | For transferring 10-20% Tricine gels |
| SuperSignal Western Blot Enhancer | Thermo Scientific | 46640 | For enhancing Aβ1-42 signal; follow manufacturer's protocol |
| Protran BA79 Nitrocellulose Blotting Membrane, 0.1 μm | GE Healthcare Life Sciences | 10402088 | For transferring 10-20% Tricine gels |
| Xcell SureLock Mini-Cell | Life Technologies | EI0001 | Electrophoresis aparatus for running 10-20% Tricine gels |
| 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. 2.5×1.5mm; 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. 7.25×7.25in) | 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 |
References
- Baleriola, J., et al. Axonally Synthesized ATF4 Transmits a Neurodegenerative Signal across Brain Regions. Cell. 158 (5), 1159-1172 (2014).
- Haass, C., Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nature reviews. Molecular cell biology. 8 (2), 101-112 (2007).
- Knowles, J. K., et al. The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo. The Journal of neuroscience : the official journal of the Society for Neuroscience. 29 (34), 10627-10637 (2009).
- Troy, C. M., et al. beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation. Journal of neurochemistry. 77 (1), 157-164 (2001).
- Crews, L., Rockenstein, E., Masliah, E. APP transgenic modeling of Alzheimer’s disease: mechanisms of neurodegeneration and aberrant neurogenesis. Brain structure & function. 214 (2-3), 111-126 (2010).
- Gotz, J., Ittner, L. M. Animal models of Alzheimer’s disease and frontotemporal dementia. Nature reviews. Neuroscience. 9 (7), 532-544 (2008).
- Calhoun, M. E., et al. Neuron loss in APP transgenic mice. Nature. 395 (6704), 755-756 (1998).
- Oakley, H., et al. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 26 (40), 10129-10140 (2006).
- Jean, Y. Y., et al. Caspase-2 is essential for c-Jun transcriptional activation and Bim induction in neuron death. The Biochemical journal. 455 (1), 15-25 (2013).
- Sotthibundhu, A., et al. Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor. The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (15), 3941-3946 (2008).
- Kar, S., Quirion, R. Amyloid beta peptides and central cholinergic neurons: functional interrelationship and relevance to Alzheimer’s disease pathology. Progress in brain research. 145, 261-274 (2004).
- Leranth, C., Frotscher, M. Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers. The Journal of comparative neurology. 289 (2), 304-314 (1989).
- Fa, M., et al. Preparation of oligomeric beta-amyloid 1-42 and induction of synaptic plasticity impairment on hippocampal slices. Journal of visualized experiments : JoVE. (41), (2010).
- Paxinos, G., Franklin, K. B. J. . The Mouse Brain in Stereotaxic Coordinates. , (2001).
- Gage, G. J., Kipke, D. R., Shain, W. Whole animal perfusion fixation for rodents. Journal of visualized experiments : JoVE. (65), (2012).
- Currle, D. S., Monuki, E. S. Flash freezing and cryosectioning E12.5 mouse brain. Journal of visualized experiments : JoVE. (4), (2007).
- Jin, M., et al. Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proceedings of the National Academy of Sciences of the United States of America. 108 (14), 5819-5824 (2011).
- Lambert, M. P., et al. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proceedings of the National Academy of Sciences of the United States of America. 95 (11), 6448-6453 (1998).
- Masters, C. L., Selkoe, D. J. Biochemistry of amyloid beta-protein and amyloid deposits in Alzheimer disease. Cold Spring Harbor perspectives in medicine. 2 (6), a006262 (2012).
- Chakrabarti, M., et al. Estrogen receptor agonists for attenuation of neuroinflammation and neurodegeneration. Brain research bulletin. 109C, 22-31 (2014).
- Cirrito, J. R., et al. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. The Journal of neuroscience : the official journal of the Society for Neuroscience. 23 (26), 8844-8853 (2003).
- Puzzo, D., et al. Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (53), 14537-14545 (2008).
- Puzzo, D., et al. Endogenous amyloid-beta is necessary for hippocampal synaptic plasticity and memory. Annals of. 69 (5), 819-830 (2011).
- Akpan, N., et al. Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke. The Journal of neuroscience : the official journal of the Society for Neuroscience. 31 (24), 8894-8904 (2011).
- Fulmer, C. G., et al. Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination. The Journal of neuroscience : the official journal of the Society for Neuroscience. 34 (24), 8186-8196 (2014).
- Thakker-Varia, S., et al. The neuropeptide VGF is reduced in human bipolar postmortem brain and contributes to some of the behavioral and molecular effects of lithium. The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (28), 9368-9380 (2010).
- Greferath, U., et al. Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice. The European journal of neuroscience. 12 (3), 885-893 (2000).
- Brashear, H. R., Zaborszky, L., Heimer, L. Distribution of GABAergic and cholinergic neurons in the rat diagonal band. Neurosciences. 17 (2), 439-451 (1986).
- Clarke, D. J. Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study. Brain research. 360 (1-2), 349-354 (1985).
- Garcia-Osta, A., Alberini, C. M. Amyloid beta mediates memory formation. Learning & memory. 16 (4), 267-272 (2009).
