Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Summary

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Abstract

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Introduction

Standard stereotaxic surgery to target brain sites in mice¹ commonly involves fixation of the skull using a set of ear bars and a mouth bar. Coordinates are then estimated based on reference atlases^2,3, and skull landmarks, namely, bregma (the point where the sutures of the frontal and parietal bones come together) or lambda (the point where the sutures of the parietal and occipital bones come together; Figure 1A,B). Through a burr hole into the skull above the estimated target, the target region can then be reached, either for delivery of ....

Protocol

The author declares that the protocol follows the guidelines of the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center.

1. Preparation of surgical instruments and stereotaxic frame

NOTE: The surgery is performed under aseptic conditions. Sterility is maintained using the sterile tip technique.

1. Install the stereotaxic arm with a micropipette or syringe filled with an injectable of choice (adeno-associated .......

Discussion

Standard stereotaxic surgery commonly relies on skull landmarks to calculate the coordinates of target sites in the CNS¹. Target sites are then accessed via burr holes that are drilled through the skull¹. This method is not ideal for the caudal brainstem as target sites are located distant from the skull landmarks in the anteroposterior and dorsoventral planes² and as the anatomy of the skull and overlying muscles make access challenging

Materials

Name	Company	Catalog Number	Comments
Alcohol pad	Med-Vet International	SKU: MDS090735Z	skin preparation for the prevention of surgical site infection
Angled forceps, Dumont #5/45	FST	11251-35	only to grab dura
Betadine pad	Med-Vet International	SKU:PVP-PAD	skin preparation for the prevention of surgical site infection
Cholera toxin subunit-b, Alexa Fluor 488/594 conjugate	Thermo Fisher Scientific	488: C34775, 594: C22842	Fluorescent tracer
Clippers	Wahl	Model MC3, 28915-10	for shaving fur at surgical site
Electrode holder with corner clamp	Kopf	1770	to hold glass pipette
Flowmeter	Gilmont instruments	model # 65 MM	to regulate flow of isoflurane and oxygen to mouse on the surgical plane
Fluorescent microspheres, polystyrene	Thermo Fisher Scientific	F13080	Fluorescent tracer
Heating pad	Stoelting	53800M	thermoregulation
Induction chamber with port hook up kit	Midmark Inc	93805107 92800131	chamber providing initial anasthesia
Insulin Syringe	Exelint International	26028	to administer saline and analgesic
Isoflurane	Med-Vet International	SKU:RXISO-250	inhalant anesthetic
Isoflurane Matrix VIP 3000 vaporizer	Midmark Inc	91305430	apparatus for inhalant anesthetic delivery
Laminectomy forceps, Dumont #2	FST	11223-20	only to clean dura
Medical air, compressed	Linde	UN 1002	used with stimulator & PicoPump for providing air for precision solution injection
Meloxicam SR	Zoo Pharm LLC	Lot # MSR2-211201	analgesic
Microhematocrit borosilicate glass pre calibrated capillary tube	Globe Scientific Inc	51628	for transfection of material to designated co-ordinates
Mouse adaptor	Stoelting	0051625	adapting rat stereotaxic frame for mouse surgery
Needle holder, Student Halsted- Mosquito Hemostats	FST	91308-12	for suturing
Oxygen regulator	Life Support Products	S/N 909328, lot 092109	regulate oxygen levels from oxygen tank
Oxygen tank, compressed	Linde	USP UN 1072	provided along with isoflurane anasthesia
Plastic card	not applicable	not applicable	any firm plastic card, cut to fit the stereotactic frame (e.g. ID card)
Pneumatic PicoPump ( or similar)	World Precision Instruments (WPI)	SYS-PV820	For precision solution injection
Saline, sterile	Mountainside Medical Equipment	H04888-10	to replace body fluids lost during surgery
Scalpel handle, #3	FST	10003-12	to hold scalpel
Scissors, Wagner	FST	14070-12	to cut polypropylene suture
Spring scissors, Vannas 2.5mm with accompanying box	FST	15002-08	scissors only to open dura, box to elevate body
Stereotactic micromanipulator	Kopf	1760-61	attached to electrode holder to adjust position based on co-ordinates
Stereotactic 'U' frame assembly and intracellular base plate	Kopf	1730-B, 1711	frame for surgery
Sterile cotton tipped applicators	Puritan	25-806 10WC	absorbing blood from surgical field
Sterile non-fenestrated drapes	Henry Schein	9004686	for sterile surgical field
Sterile opthalmic ointment	Puralube	P1490	ocular lubricant
Stimulator & Tubing	Grass Medical Instruments	S44	to provide controlled presurred air for precision solution injection
Surgical Blade #10	Med-Vet International	SKU: 10SS	for skin incision
Surgical forceps, Extra fine Graefe	FST	11153-10	to hold skin
Surgical gloves	Med-Vet International	MSG2280Z	for asceptic surgery
Surgical microscope	Leica	Model M320/ F12	for 5X-40X magnification of surgical site
Suture 5-0 polypropylene	Oasis	MV-8661	to close the skin
Tegaderm	3M	3M ID 70200749250	provides sterile barrier
Universal Clamp and stand post	Kopf	1725	attached to stereotactic U frame and intracellular base plate
Wound hook with hartman hemostats	FST	18200-09, 13003-10	to separate muscles and provide surgical window