Surgical Technique for Lumbar Spinal Catheter Insertion in Pigs Enabling Continuous Access to the Thecal Sac in a Terminal Setup

Summary

We present a technique for inserting a lumbar spinal catheter at the L4-L5 level in a 3-month-old Danish Landrace pig as part of a terminal research protocol, enabling continuous infusion or CSF sampling from the thecal sac.

Abstract

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine and human central nervous systems (CNS). However, accessing the cerebrospinal fluid (CSF) in larger pig breeds by conventional lumbar puncture techniques can be challenging due to an oblique orientation of the spinal spinous processes and a limited interlaminar space. Accordingly, an open surgical procedure for inserting a lumbar spinal catheter for continuous CSF sampling at the L4/L5 level in pigs is thoroughly described in this work. After positioning the pig and identifying the anatomical landmarks, a dorsal midline surgical incision is made to expose the spinous processes. By advancing the introducer needle, the spinal catheter is inserted inside the thecal sac of the spinal canal while leaving the bone structures of the spine intact. This method allows continuous infusion into or sampling from the porcine thecal sac with minimal bleeding or CSF leakage. The procedure is simple, time-efficient, and reproducible across different experimental setups, offering significant potential for various pre-clinical studies, including pharmacokinetic research, surgical training, and spinal cord injury models.

Introduction

Animal models are essential when ethical or practical limitations prevent the use of human subjects to investigate diseases or test surgical methods. While rodents are commonly used due to their low cost, their translational relevance is limited by significant differences from humans¹. Pigs, however, offer several advantages compared to rodents, including anatomical and physiological similarities to humans - especially in the context of CNS research^1,2. Canine models have historically served as experimental models for CNS research, but ethical considerations have constrained the use of dogs in recent years³. Furthermore, the comparable size of porcine organs to human enhances their use in surgical research and procedural training⁴. The porcine CNS and spine closely mirror that of humans, with similarities in brain and spinal cord architecture and functionality^1,5,6. Importantly, the dimensions of the vertebral column and spinal canal in pigs make them suitable for various pre-clinical studies^7,8, including surgical procedural training^9,10, drug penetration^11,12,13, and spinal cord injuries¹⁴.

Access to the CSF in porcine models is crucial in many experimental setups. While lumbar puncture provides a method for singular CSF sampling or intrathecal drug administration, repeated lumbar punctures are impractical. They pose a potential risk of intraspinal hematomas, nerve damage, and CSF contamination with blood. In human patients, spinal microcatheters are commonly used for continuous lumbar CSF drainage in aneurismal subarachnoid hemorrhages and should, due to size similarities, be equally suited for continuous CSF sampling in pigs. However, species-specific anatomical differences in pigs present unique challenges for CSF access. For example, the presence of overlapping laminae, ossified ligaments, and abundant epidural adipose tissue makes conventional percutaneous lumbar puncture techniques less reliable¹⁵. In Göttingen minipigs, a minimally invasive percutaneous method has been employed, which enables serial CSF sampling¹⁶. This method relies upon manual identification of the lumbar intervertebral spaces, and the catheterization itself is performed without visualization of the introducer. However, this technique is less suitable for larger pigs, as anatomical variations in vertebral size, spinous processes, and the amount of epidural adipose tissue make percutaneous catheterization more difficult¹⁵. Therefore, more invasive methods involving exposure of the spine may be required in larger porcine models to ensure reliable catheter placement.

The purpose of this manuscript is to describe the surgical procedure for inserting a spinal catheter into the porcine thecal sac at the L4/L5 level. The procedure involves positioning the subject, planning the surgical incision based on anatomical landmarks, and accessing the posterior bone structures of the spine prior to catheterization.

Protocol

Subjects were housed in compliance with local regulations under the approval of the Danish Animal Experiments Inspectorate (license no. 2020-15-0201-00401). Subject information: Domestic swine, female, approximately 40 kg, 3 months of age.

1. Subject housing and preoperative fasting

1. House subjects in groups at 12 h light/dark cycles in approved housing pens for at least 14 days prior to the procedure to ensure proper acclimatization and reduce stress¹⁷.
2. Ensure that subjects have been on a food withdrawal regime for 12 h prior to the planned anesthesia to reduce the risk of regurgitation. If the subjects' diet includes alfalfa or other types of hay, this must be excluded from the diet 2-3 days prior to the procedure, as this can delay gastric emptying time further¹⁸.

2. Anesthesia and monitoring

1. Anesthetize the subject with an intramuscular injection of 2 mL/10 kg bodyweight of a mixture of Ketamine 6.25 mg/mL, Zolazepam 6.25 mg/mL, Tiletamine 6.25 mg/mL, butorphanol 1.25 mg/mL, and xylazine 6.25 mg/mL (Zoletil).
2. Place the subject in a supine position on top of a heating blanket to support thermoregulation.
3. Intubate the subject with a size 6.5 tube¹⁹, and ventilate it mechanically with non-humidified air, a tidal volume of 8-10 mL/kg, and a respiratory rate of 16-22 breaths/min according to the expiratory end-tidal CO₂ concentrations < 6.0 kPa.
   NOTE: CO₂ readings confirm the correct intratracheal location of the tube.
4. Maintain anesthesia by inhalation of 3%-4% vaporized sevoflurane¹⁸.
5. Apply ophthalmic ointments carefully bilaterally to avoid dryness during anesthesia.
6. Ensure a sufficient degree of anesthesia by checking for muscle relaxation and absence of palpebral movement every 10^th min¹⁸.
7. Insert a bladder catheter with a thermometer into the subject's bladder through the urethra¹⁹ to monitor temperature and collect urine in a suitable catheter bag.
8. Insert a peripheral venous catheter in a suitable superficial ear vein by percutaneous puncture and use it for continuous saline (NaCl, 0.9%) infusion, drug infusion, and euthanasia at the end of the study.
9. Insert a femoral artery catheter (6 Fr sheet) in the right femoral artery through a percutaneous puncture. Use this access for continuous invasive blood pressure monitoring.
10. Monitor the subject's vital signs every 5 min throughout the procedure.
    NOTE: Vital signs include pulse, continuous invasive arterial blood pressure, intravesical temperature, and end-tidal CO₂ concentration.

3. Animal positioning

1. Place the subject in a prone position centrally on the operating table. Ensure the spine of the subject is straight to avoid any scoliosis.
2. Place a sandbag beneath the lumbar aspect of the spine to increase the angulation between the laminae.
3. Shave the hair from the surgical site with a trimmer.
4. Apply iodine solution to the surgical site in centrifugal patterns. Repeat this process until the entire surgical site is covered.
5. Tilt the subject slightly to an upright position.

4. Preparation of surgical equipment

1. Prepare the surgical equipment listed in the Table of Materials.

5. Identifying key anatomical landmarks

1. Identify the iliac crest at each side of the subject's lumbar spine and follow the contours medially until the sacrum is identified (Figure 1).
2. Identify the intervertebral space in the midline between the cranial aspect of the sacrum and the spinous process of L6.
3. Identify the spinous processes of L6, L5, and L4 (Figure 1, Figure 2).

6. Exposing the spinous processes

1. Make a midline incision along the spinous processes L4-L6 using scalpel no. 24, cutting through skin and subcutis.
2. Use a monopolar to cauterize small bleeding from superficial veins and arterioles.
3. Wipe off the blood with a surgical swamp and check for active bleeding; use the monopolar accordingly.
   NOTE: It is important to stop even minor bleeding to avoid hematomas.
4. Insert the surgical retractor and expand the opening.
5. Identify the supraspinous ligament dorsal to the spinous processes.
6. Expand the incision gradually with the monopolar along the lateral aspect of the spinous processes until approximately 1 cm of the spinous processes is visible (Figure 3).
   NOTE: If the person performing the procedure is right-handed, one should consider following the subject's right lateral aspect of the spinous process to ease the insertion of the introducer later.
7. Identify the interspinous ligament between L4/L5 (Figure 3).
8. Check for active bleeding and apply the monopolar for cauterizing accordingly.

7. Access to the thecal sac

1. Identify the L4/L5 intervertebral space between the lamina of the spinous processes by manual palpation.
2. Place the introducer with its bevel and lumen oriented in a cranial direction angled towards the L4/L5 intralaminar space (Figure 2, Figure 4, Figure 5).
3. Ensure the introducer is kept at a 30° horizontal and 45° cranial inclination (Figure 5).
   NOTE: Aim towards the intralaminar space between L4/L5.
4. Gradually advance the introducer until gentle resistance is felt; this represents the ligamentum flavum.
   NOTE: The sensation of blunt resistance indicates that the introducer is stopped by the spinous process. If this occurs, retract the introducer 1 cm and advance again at a slightly different inclination.
5. Apply a firm yet very careful pressure and advance the introducer millimeter by milliliter through the flavum ligament until a sudden loss of resistance is felt.
   NOTE: If the subject exhibits motor reflexes in the lumbar musculature or hind legs, it is due to direct contact with the nerve roots and not insufficient anesthesia.
6. Follow each advancement of the introducer by removing the trocar to check for visible CSF flow.
7. Confirm the correct placement of the introducer within the spinal canal by visual confirmation of CSF flow from the introducer after it has penetrated the ligamentum flavum and, subsequently, the dura mater.
   NOTE: Spontaneous flow of CSF may be slow. Confirmation can be expedited by filling the introducer with sterile saline and observing for pulsation.
8. Reinsert the trocar into the introducer to avoid excessive loss of CSF while preparing the catheter.

8. Insertion of catheter into the thecal sac

1. Insert the guidewire into the catheter.
2. Remove the trocar from the introducer.
3. Insert the catheter, containing the guidewire, into the introducer until gentle resistance is felt.
4. Measure 5 cm distally from the introducer and set a mark with the surgical marker.
5. Apply gentle yet firm pressure as the catheter is advanced into the thecal sac until the previously measured mark reaches the introducer.
   NOTE: Due to medullary reflexes, the subject might twitch/move despite being sufficiently anesthetized.
6. Retract the introducer carefully while keeping the catheter in its position.
   NOTE: Apply a firm grip on the catheter as soon as it is visible above the skin to avoid misplacement as the introducer is removed.
7. Remove the guidewire while keeping a firm grip on the catheter at skin level (Figure 6).
8. Attach a 2 mL single-use plastic syringe to the catheter.
9. Confirm location within the spinal canal by aspiration CSF from the catheter.
10. In the case of lacking CSF in the syringe, gently retract the catheter a few millimeters to restore patency.
11. Secure the spinal catheter to the surgical retractor and the skin by tape to avoid misplacement.

9. Administration of lipopolysaccharide

1. Administer a 400 μg of E. coli lipopolysaccharide (LPS) (OH:143) into the central venous sheet.
2. Start a timer.

10. CSF sampling

1. Obtain CSF samples hourly for the following 24 h to measure total leucocyte count, CSF-albumin, and CSF IgG. Draw a maximum of 0.5 mL of CSF in each sample.

11. Euthanasia

1. Administer a bolus of pentobarbital (50 mg/kg) through the peripheral venous catheter.
2. Observe the pulse, blood pressure, and end-tidal CO₂ concentration curves at the respirator for flatline as a confirmation of the cardiac arrest.

Results

The prone positioning of the pig optimizes surgical access to the lumbar vertebrae. The use of supportive sandbags increases the angulation between adjacent lumbar spinous processes, thereby improving access to the spinal canal.

The present study aimed to investigate the inflammatory response within the CSF compartment following intraventricular inoculation with E. coli lipopolysaccharide. A total of 10 pigs underwent the procedure, and subsequent CSF analyses revealed an increase in ...

Discussion

The demonstrated procedure for lumbar catheter insertion for continuous CSF sampling in pigs involves several crucial steps. Firstly, the correct vertebral level must be exposed to ensure optimal conditions for successful catheterization. The porcine spinal cord extends further caudally compared to humans, reaching the S2-S3 level¹⁵, in contrast to the human conus medullaris, which terminates at the L1-L2 level²⁰. Consequently, accessing the lumbar cistern for CSF sampling ...

Materials

Name	Company	Catalog Number	Comments
Adjustable operating table	N/A	N/A
Bair Hugger heater	3M	B5005241003
Bair Hugger heating blanket	3M	B5005241003
Endotracheal tube size 6.5	DVMed	DVM-107860	Cuffed endotracheal tube
Euthasol Vet	Dechra Veterinary Products A/S	380019	phentobarbital for euthanazia, 400mg/mL
Foley Catheter 12F	Becton, Dickinson and Company	D175812E	Catheter with in-built thermosensor
Intravenous peripheral catheter	Avantor	BDAM381344	Size G18
Intravenous sheath	Coris Avanti		Avanti Cordis Femoral Sheath 6F
Monopolar, ForceTriad System	Medtronic
Plastic Syringe, 2 mL	Becton, Dickinson and Company	300928
Primus respirator	Dräger		Respirator with in-built vaporiser for supplementary Sevofluran anesthisa
Self-retaining retractor	World Precission Instruments	501722	Weitlander retractor, self-retaining, 14 cm blunt
Silicone Lumbar Catheter incl. Introducer	Integra	NL8508330
Sterile Saline	Fresnius Kabi	805541	1000 mL
Sterile surgical swaps
Surgical scalpel no 24	Swann Morton	5.03396E+12	Swann Morton Sterile Disposable Scalpel No. 24
Zoletil Vet	Virbac		Medical mixture for induction of anesthesia