Een gepolijste en gewapend verdund-schedel Venster voor de lange termijn beeldvorming van de hersenen van muizen
Summary
We presenteren een methode om een beeldvorming raam in de muis schedel die millimeter overspant en is stabiel gedurende maanden zonder ontsteking van de hersenen vormen. Deze methode is zeer geschikt voor longitudinale studies van de bloedstroom, cellulaire dynamiek en cel / vasculaire structuur met behulp van twee-foton microscopie.
Abstract
In vivo beeldvorming van corticale functie vereist optische toegang tot de hersenen, zonder verstoring van de intracraniële omgeving. We presenteren een methode om een gepolijste en versterkte verdund schedel (poorten) venster te vormen in de muis schedel die enkele millimeters in diameter overspant en is stabiel voor maanden. De schedel verdund 10 tot 15 urn dik een draagbare boor optische helderheid bereiken en vervolgens bedekt met cyanoacrylaatlijm en een dekglas: 1) voorzien stijfheid, 2) remmen been hergroei en 3) te verminderen lichtverstrooiing onregelmatigheden op het botoppervlak. Omdat de schedel niet wordt doorbroken, is een ontsteking die het proces wordt bestudeerd van invloed kunnen zijn sterk verminderd. Imaging tot een diepte van 250 pm onder de corticale oppervlak kan worden bereikt met behulp van twee-foton laser scanning microscopie. Dit venster is zeer geschikt voor cerebrale doorbloeding en cellulaire functie te bestuderen in zowel onder narcose en wakker preparaten. Het biedt verder de opprima gelegenheid om cel-activiteit met behulp van optogenetics te manipuleren of de bloedstroom in gericht schepen verstoren door bestraling van circulerende fotosensitizers.
Protocol
Discussion
Twee-foton imaging door een ports raam vereist transmissie via de verdunde bot en de duur, dat het laserlicht verzwakt en voegt optische aberraties op grotere diepte 8. Ondanks dit nadeel kan beeldvorming diepte van 250 pm onder het oppervlak pial worden bereikt met 900 nm excitatie. Grotere diepten beeldvorming kan in principe mogelijk meer excitatiegolflengten 13. Een groot voordeel van deze methode is de afwezigheid van corticale ontsteking die tijdelijk zou kunnen bestaan in methoden met …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk werd ondersteund door de American Heart Association (Post-doctorale beurs te Ays) en de National Institutes of Health (MH085499, EB003832 en OD006831 naar DK). Wij danken Beth Friedman en Pablo Blinder voor commentaar op het manuscript.
Materials
| Agent | Route of delivery | Dose for mouse | Duration | Notes | Source | Ref Ref |
| Pentobarbital (Nembutal) | IP | 90 μg/g | 15-60 min | Narrow safety margin. Work up to proper dose of anesthesia slowly. Supplement 10 % of induction dose as required. | 036093; Butler Schein | 7 |
| Ketamine (Ketaset) mixed with Xylazine (Anased) | IP | 60 μg/g (K)
10 μg/g (X) (mix in same syringe) |
20-30 min | Xylazine is co-injected as a muscle relaxant and analgesic. Supplement only Ketamine at 50% of induction dose as required. | (K) 010177, (X) 033198; Butler Schein | 7 |
| Isoflurane (Isothesia) | Inhalation | 4% mean alveolar concentration (MAC) for induction; 1-2% MAC for maintenance | 4-6 h. | Provided in mixture of 70% oxygen and 30% nitrous oxide. Prolonged anesthesia leads to slow recovery. | 029403; Butler Schein | 26 |
Table 1. Suggested anesthetic agents for survival studies.
| ITEM | COMPANY | CATALOG # / MODEL |
| Betadine | Butler Schein | 6906950 |
| Buprenorphine (Buprenex) | Butler Schein | 031919 |
| Fluorescein isothiocyanate dextran, 2 MDa molecular weight | Sigma | FD2000S |
| Isopropyl alcohol | Fisher | AC42383-0010 |
| Lactated Ringer’s Solution | Butler Schein | 009846; |
| Lidocaine solution, 2 % (v/v) | Butler Schein | 002468 |
| Saline | Butler Schein | 009861 |
| Surgical Milk | Butler Schein | 014325 |
| Texas Red dextran, 70 kDa molecular weight | Invitrogen | D1864 |
| Maxizyme | Butler Schein | 035646 |
| DISPOSABLES | ||
| Carbide burrs, 1/2 mm tip size | Fine Science Tools | 19007-05 |
| Cottoned tip applicators | Fisher Scientific | 23-400-100 |
| Cover Glass, no. 0 thickness | Thomas Scientific | 6661B40 |
| Cyanoacrylate glue | ND Industries | 31428 H04308 |
| Gas duster | Newegg | N82E16848043429 |
| Grip cement powder | Dentsply | 675571 |
| Grip cement solvent | Dentsply | 675572 |
| Insulin syringe, 0.3 mL volume with 29.5 gauge needle | Butler Schein | 018384 |
| Nut and bolt to secure the head | Digikey | Nut, H723-ND; bolt, R2-56X1/4-ND |
| Opthalmic ointment | Butler Schein | 039886 |
| Scalpel blades | Fisher Scientific | 12-460-448 |
| Screws, self-tapping #000 | J.I. Morris Company | FF000CE125 |
| Silicone aquarium sealant | Perfecto Manufacturing | 31001 |
| Tin oxide powder | Mama’s Minerals | EQT-TINOX |
| EQUIPMENT | ||
| Glass scribe | Fisher Scientific | 08-675 |
| Dissecting microscope | Carl Zeiss | OPMI-1 FC |
| Electric powered drill | Foredom or Osada | K.1020 (Foredom) or EXL-M40 (Osada) |
| Electrical razor | Wahl | Series 8900 |
| Forceps, Dumont no. 55 | Fine Science Tools | 11255-20 |
| Feedback regulated heat pad | FHC | 40-90-8 (rectal thermistor, 40-90-5D-02; heat pad, 40-90-2-07) |
| Isoflurane vaporizer | Ohmeda | IsoTec4 |
| Pulse oximeter | Starr Life Sciences | MouseOx |
| Screwdriver, miniature | Garret Wade | 26B09.01 |
| Stereotaxic frame | Kopf Instruments | Model 900 (with mouse anesthesia mask and non-rupture ear bars) |
| Surgical scissors, blunt end | Fine Science Tools | 14078-10 |
| Ultrasonic cleaner | Fisher Scientific | 15-335-30 |
Table 2. List of specific reagents, disposables and equipment.
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