Locating the Cisterna Magna in a Pig Model: A Surgical Method to Access the Cisterna Magna in a Pig Model for Direct Cannulation

Overview

In this video, we describe a method to access the cisterna magna in a pig model. The cisterna magna is a preferred site for studies concerning cerebrospinal fluid.

Protocol

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

1. Animal Preparation

1. Sedate a pig by intramuscular (i.m.) injection of tiletamine (3.75 mg/kg) and zolazepam (3.75 mg/kg) and dexmedetomidine (37.5 μg/kg). Wait for it to become unconscious.
2. Prepare an intravenous line by inserting a 20 G cannula into the ear vein.
   NOTE: Make sure the cannula is in the vein by injecting 5-10 mL of saline through the cannula. If the vein has been missed, this will be noticeable by small edema in the ear tissue.
3. Intubate the pig to ensure that the breathing rate can be regulated throughout the surgery.
   NOTE: Ensure successful intubation by applying pressure on the pig's thorax and confirm that forcibly expired air is coming out of the intubation tube.
4. Attach the intubation tube to a ventilator set to a breath rate of 14 breaths/min.
5. Connect a pulse oximeter and cuff to the tail to monitor the heart rate (HR), blood pressure (BP), and oxygen saturation (sats). Insert a rectal thermometer to monitor the core temperature.
6. Prepare an IV bag of ketamine (5 mg/kg/min), midazolam (0.25 mg/kg/min), and fentanyl (2.5 μg/kg/min), in saline and begin to infuse through the ear vein at approximately 2 drops/s.
   NOTE: Throughout the surgery, the infusion rate may need to be increased or decreased based on the animal's vitals.
7. With the pig in the prone position, palpate the back of the head and neck of the animal to locate and mark the occipital crest and spine of the first thoracic vertebrae and the base of each ear.
8. Draw a straight line between the crest and the vertebrae along the longitudinal axis. Draw two lines from the crest to the base of each ear by following the base of the skull (Figure 1A).
9. Check that the animal is in a deep sleep by carefully clamping the tail and watching for the absence of a tail reflex.
   NOTE: If the animal is still reflexive, the anesthetic infusion rate should be incrementally increased until the animal no longer exhibits a reflex.

2. Surgery

NOTE: All through the surgery, it is necessary to have at least one assistant to suction the light bleeding and cauterize any severed vessels.

1. Using a scalpel with a # 21 blade, make a dermal incision along the longitudinal line down to the muscle.
2. Extend two perpendicular dermal incisions further along the shoulders, 10-15 cm in length.
3. From the occipital crests, make dermal incisions along the line down to the base of each ear.
4. Gripping the skin corners formed at the occipital crest with anatomical forceps, carefully separate the skin from the underlying muscle by lightly running the scalpel blade over the fascia, moving from the rostral to caudal. Once the skin has been resected following each of the five incisions, parts of the trapezius muscles should then be visible.
5. Make a longitudinal incision with the scalpel, approximately 1 cm deep, where the trapezius comes together at the midline.
   NOTE: When cutting through the muscles, there is an increased propensity for bleeding, so the cauterizer should be ready. If a larger vessel is severed, one person should quickly compress it with the gauze, while the other person uses the cauterizer.
6. Using a combination of straight and curved surgical forceps, perform blunt dissection working along the longitudinal cut in the muscles. This will separate the bellies of the trapezius, as well as the underlying semispinalis capitus biventer muscle.
7. Sever any persisting muscle fibers with a scalpel and continue blunt dissection until semispinalis capitus complexus becomes visible.
8. Sever the origins of the trapezius and semispinalis capitus biventer muscles along the posterior aspect of the skull. Carefully separate them longitudinally with the scalpel performing blunt dissection until the semispinalis capitus complexus is fully visible.
9. Retract the trapezius and semispinalis capitus biventer muscles using self-retaining retractors.
10. Where the bellies of the semispinalis capitus complexus come together in the midline, make a longitudinal incision with the scalpel approximately 1 cm deep.
    NOTE: Be aware for any additional bleeding here. Bleeding can be managed using a combination of cotton swabs and cauterization.
11. Using surgical forceps, perform a blunt dissection working along the longitudinal cut between the muscle bellies until the dorsal aspect of the atlas (CI) is palpable.
12. Sever the origins of the semispinalis capitus complexus muscles along the posterior aspect of the skull and separate it longitudinally from the underlying vertebrae by scalpel and blunt dissection.
13. Retract the semispinalis capitus complexus muscles using another set of self-retaining retractors.
14. Using a scalpel, carefully remove any remaining tissue overlying the region where the atlas meets the skull base.
15. Placing one arm under the animal's neck and one finger at the juncture of the atlas and skull, simultaneously elevate the head and flex the neck while palpating with the finger to reveal the cisterna magna using the other hand.
    NOTE: The cisterna magna is recognizable when palpating as a strong elastic structure with a small amount of rebound as pressure is released with the finger.

Representative Results

Figure 1. Cisterna magna cannulation in pigs. (A) Pig prepped prior to the start of the surgery and marked where dermal incisions will be performed starting from the occipital crest (OC) then posterior to thoracic vertebrae (TV) and lateral to each ear base (EB). (B) Head in the relaxed position with the trapezius, semispinalis capitus biventer and semispinalis capitus complexus muscles retracted, thus exposing cisterna magna (CM). (C) Head flexed manually to increase access to CM for cannulation and injection. (D) Close-up image of a cannula inserted into CM after injection and fixed in place with the dental cement (DC). (E) Dorsal, ventral, and lateral brain surfaces, respectively, after fluorescent imaging with accompanying structural white light images. Areas of interest that are visible at these surfaces include the interhemispheric fissure (IHS), temporal lobe (TL), and lateral fissure (LF). (F) Structural white light image of the artery and veins on the brain surface. (G) Fluorescent image of (F) showing the tracer distribution along the surface artery. (H) Macroscopic slices from the anterior and posterior cerebral regions show two-dimensional tracer dispersion and distribution in fissures (LF, IHS) and subcortical structures like the striatum (STR) and hippocampus (HPC). (I-J). Confocal images showing the tracer in the PVS, bounded by lectin-stained endothelial cells internally and AQP4 on astrocyte foot processes externally. (K-L). Confocal images showing the tracer in the PVS, bounded by endothelial cells internally with astrocyte foot processes stained for glial fibrillary acidic protein (GFAP) visible forming an outer boundary. (M) Confocal image showing the tracer in the PVS around an arteriole stained for smooth muscle actin (SMA) with tracer also visible in and around, surrounding the brain parenchyma. CM, cisterna magna; DC, dental cement; EB, ear base; GFAP, glial fibrillary acidic protein; HPC, hippocampus; IHS, interhemispheric fissure; LF, lateral fissure; OLB, olfactory bulb; OC, occipital crest; STR, striatum; TL, temporal lobe; TV, thoracic vertebrae.

Materials

Name	Company	Catalog Number	Comments
Anatomical forceps	NA	NA
Marker pen	NA	NA
No. 21 Scalpel blade	Agnthos	BB521
No. 4 Scalpel handle	Agnthos	10004-13
Self-retaining retractors	NA	NA
Surgical forceps	NA	NA