Transverse Processes

All procedures were approved by the Canton of Vaud’s Committee on Animal Experimentation (Switzerland), in accordance with Swiss Federal Law on Animal Welfare and International Association for the Study of Pain guidelines [51 (link)].
The spared nerve injury (SNI) model of neuropathic pain was previously described in rats [32 (link),52 ] and mice [36 (link)]. Briefly, adult C57BL/6 J mice (Charles River, L’Arbresle, France) were anesthetized with 1.5% isoflurane and after exposure of the sciatic nerve, the common peroneal and tibial nerves were ligated together with a 6.0 silk suture (Ethicon, Johnson and Johnson AG, Zug, Switzerland) and transected. In the SNI variant (SNIv_(cp,t)) [36 (link)] the ligation and transection were performed on the sural nerve, leaving the common peroneal and tibial nerves intact. The incision was closed in distinct layers (muscle and skin). Sham surgery was performed similarly except for the nerve ligation and transection.
Spinal nerve ligation (SNL) surgery was adapted from the procedure described by Kim and Chung [31 (link)], and transposed to mice. Briefly, after skin and muscle incision the L5 transverse process of vertebra was exposed and carefully removed. The L4 and L5 spinal nerves were exposed and the L5 spinal nerve was tightly ligated. The incision was closed in distinct layers (muscle and skin).

Surgical complexity influences risk of adverse occurrences. When comparing different surgeons, hospitals, or devices, the extent and nature of the spinal surgery may be a confounding factor. To control for variations in spinal procedures, we developed a quantitative index to rate the invasiveness of surgery.
We based the index on three fundamental elements of spinal procedures: decompression, fusion, and instrumentation of individual vertebrae. Combinations of these three elements on different vertebrae, when combined with surgical approach (anterior or posterior), can be useful in describing many spinal operations. Each operated vertebra can be assigned a score of 0 to 6, based on how many of six procedural elements were performed at that level: anterior decompression, anterior fusion, anterior instrumentation, posterior decompression, posterior fusion, and posterior instrumentation.
We scored the six constituent procedure components using the following definitions:
(1) Anterior decompression: 1 unit for each vertebra requiring partial or complete excision of the vertebral body or the disc caudal to that vertebra.
(2) Anterior fusion: 1 unit for each vertebra that has graft material attached to or replacing that vertebral body.
(3) Anterior instrumentation: 1 unit for each vertebral body that has screws, plate, cage, or structural graft attached to its vertebral body or replacing its vertebral body.
(4) Posterior decompression: 1 unit for each vertebra requiring laminectomy or foraminotomy at the foramen caudal to its pedicle and/or discectomy at the disc caudal to that vertebral body.
(5) Posterior fusion: 1 unit for each vertebra that has graft material on its lamina, facets, or transverse processes.
(6) Posterior instrumentation: 1 unit for each vertebra that has screws, hooks, or wires attached to its pedicles, facets, lamina, or transverse processes.
Each of the six procedure elements can thus be assigned an integer value corresponding to the number of vertebrae on which that procedural component was performed. We also defined a composite "Spine Surgery Invasiveness Index" as the sum of the six procedural element scores for a given surgery. We developed a graphical grid for coding each surgery (Figure 2).
A surgeon-investigator or a trained research assistant completed the surgical procedure grid based on the treating surgeon's operative report. To determine if this grid method could be reliably used in routine clinical documentation, we made available a medical record form to allow surgeons to record the spinal procedure using the grid format in their immediate hand-written brief operative note. Using the treating surgeon's dictated operation report as the reference, we assessed the reliability of invasiveness coding by comparing the surgeons with the two researchers for fifty consecutive cases.

All rats received enrofloxacin (5 mg/kg), carprofen (5 mg/kg) and morphine (2.5 mg/kg) subcutaneously pre-operatively. To broaden generalizability, both a spinal and long bone (femur) application were performed. For spinal applications, rats were placed prone on a prewarmed surgery table under general anesthesia [17 (link)]. The iliac crest and spinous processes were identified and a midline incision was performed 1 cm cranially from the iliac crest. The paraspinal musculature was dissected laterally to expose the transverse processes of L4–5. A 0.8 mm hole was drilled in the transverse process and a stainless-steel screw (1*5 mm), attached via a poly(ε-caprolactone) connecting piece to a polyurethane (PU) catheter (internal diameter 0.6 mm, UNO, Zevenaar, Netherlands) was inserted, so that the catheter opening faced the bone surface (Fig. 1). Sterile saline solution was dripped next to the hole during drilling for cooling. The dead volume of catheters was pre-filled with the appropriate bupivacaine solution to ensure infusion of precise volumes. The catheter was tunneled subcutaneously towards an exit-point between the shoulders and fixed to the skin with a suture. Bupivacaine infusion was initiated after confirmed absence of motor deficits (i.e., following observed movement of tail and paws).Fig. 1

Schematic representation of experimental setup. Screws with a catheter attached were implanted in either the spine or femur. Catheters were then tunneled subcutaneously (dotted line) and exited between the shoulders. A pump was connected to the catheter to allow controlled infusion of bupivacaine solutions. Representative MicroCT images for the two surgical locations are shown

For femoral applications, rats were positioned laterally on the surgery table [18 ]. A longitudinal incision was performed lateral to the femur. The vastus lateralis muscle was separated from the femur by blunt dissection. A 0.8 mm hole was drilled in the femoral shaft and a stainless-steel screw attached to a catheter was inserted. The catheter was tunneled as in the spinal application.

Patients in the PVB and RIB groups were intervened by ultrasound-guided nerve block in the lateral position with local anesthesia. The PVB was performed using the in-plane technique with a linear 4–10 MHz ultrasound probe (LOGIQe, GE Healthcare, Waukesha, WI., U.S.A.). At the parasagittal view, subcutaneous tissues, T5 transverse processes, superior costotransverse ligament (SCTL), and pleura were visualized. An 18 G block needle was inserted vertically or slightly caudally into the paravertebral space (PVS) under the guidance of ultrasound. After the penetration of the SCTL, a slight aspiration was performed to ensure the avoidance of vessels or pleura. Then, 1–2 ml of normal saline was injected into the PVS, the pressure of which pushed down the pleura. The position of the needle tip was confirmed, and 0.4% ropivacaine (Zhejiang Xianju Pharmaceutical Co., Ltd., Zhejiang, China) at 3 mg/kg was injected into the PVS.
A linear 4–10 MHz ultrasound probe (LOGIQe, GE Healthcare, Waukesha, WI, U.S.A.) was placed on the medial border of the scapula between the 4th and 5th rib of the patients in the RIB group. In the ultrasound image, the trapezius muscle, rhomboid muscle, intercostal muscles, pleura, and lung were identified. Under the aseptic condition, an 18 G block needle was inserted laterally in the plane of the T5 level guided by an ultrasound probe with an in-plane technique. The vessel injection should be confirmed negative through aspiration, and 1–3 ml of normal saline was injected to divide the rhomboid and intercostal muscle, and 0.4% ropivacaine at 3 mg/kg was injected into the deep layer of the rhomboid muscle.

Skeletal muscle and adipose tissue parameters were retrieved from routinely obtained chest CT scans at ED presentation or admission. All CT scans were obtained without the application of IV contrast. Total cross-sectional area (CSA) of the pectoralis major and minor muscles was measured bilaterally at the level of the fourth thoracic vertebra. Additionally, CSA of skeletal muscle, VAT, and SAT was demarcated at the level of the first lumbar vertebra (L1). The muscles analyzed at the L1 level included the psoas, erector, spinae, quadratus lumborum, transversus abdominis, external and internal oblique, and rectus abdominis. At both levels, following previously described methods, a single transverse image at the most cranial slide with both vertebral transverse processes clearly visible was used (Fig 1).^{12 ,13} (link) If the selected image was of poor quality, had artefacts, or did not fully depict tissue of interest, the specific slice or missing tissue was considered as a missing value and was not analyzed. CSA of these structures were quantified by one trained assessor, blinded to clinical outcomes, based on pre-established Hounsfield units (HU) thresholds (skeletal muscle, –29 to 150 HU; SAT, –190 to –30 HU; and VAT, –150 to –50 HU).²⁰ (link)^,21 (link) Boundaries were corrected manually when necessary. All analyses were performed with Slice-O-Matic software version 5.0 (Tomovision).Figure 1

A-D, Representative examples of selected CT scan slices at the fourth thoracic vertebra level (A) with demarcated pectoralis major and minor muscle (B) and at first lumbar vertebra (L1) level (C) with demarcated L1 muscle, visceral, and subcutaneous adipose tissue (D).