A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

Summary

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Abstract

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 μm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 μm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

Protocol

1. Preparing for Surgery ⁱ

1. Clean the surgical tools by sonicating in a mixture of Maxizyme and Surgical Milk in an ultrasonic cleaner. Autoclave the surgical tools before each experiment.
2. Ensure that all necessary reagents and disposables are available. A list of reagents and disposables is provided in Table 2. Reagents and disposables that come in contact with exposed tissue should be sterile, when possible.
3. Induce anesthesia. Typical anesthetics suitable for survival studies a.......

Discussion

Two-photon imaging through a PoRTS window requires transmission through the thinned bone and the dura, which attenuates the laser light and adds optical aberrations at greater depths ⁸. However, despite this drawback, imaging depths up to 250 μm below the pial surface can be achieved with 900 nm excitation. Greater imaging depths may in principle be possible with longer excitation wavelengths ¹³. A major advantage of this method is the absence of cortical inflammation that might exist transiently.......

Materials

Name	Company	Catalog Number	Comments
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

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