Complete Transection and Dural Closure Surgeries
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. Adult female Sprague-Dawley rats (250–300 g) were anesthetized with 80 mg/kg ketamine (Fort Dodge Animal Health) and 5 mg/kg xylazine (Ben Venue Laboratories) via intraperitoneal injection. Akwa Tears lubricant opthalamic ointment (Akron, Inc.) was used to prevent drying of the eyes during surgery.
Hair was clipped from each animal’s back, and the site of incision was disinfected using Povidone-Iodine Scrub Swabsticks (Professional Disposables, Inc.). The heating pad temperature was constantly maintained at 37°C during surgery. The operation was performed using a sterile technique with the aid of a Zeiss OPMI-6 surgical microscope. A 2-cm midline incision was made along the T-7 to T-10 spinous processes. The thoracolumbar fascia and paraspinal musculature were incised along the spinous processes and retracted. A laminectomy was performed at the level of T9–10. The dura mater was incised 5 mm longitudinally using a microforceps and micro scissors. Hemostasis was obtained with the application of light pressure with a cotton swab until the bleeding stopped. The spinal cord was then cut transversely using a microsurgical knife. The completeness of the transection was confirmed by passing a microhook between the 2 cut ends. The muscles, subcutaneous tissue, and skin were sutured closed in 1 group of 11 animals, while in the other 2 groups, the dura was also closed by either a running 10–0 prolene suture in 12 rats, or using a topical 0.1-ml high molecular weight hyaluronic acid gel (Daltons, Healon; Pharmacia) in 4 rats. The completeness of dural closure was confirmed by observing CSF filling the subdural space, none of which leaked through the closed dura.
Subpial Transection Surgeries
In the second part of this study, we sought to compare the effects of complete and subpial transection on glial scar and cyst formation. In 1 group of animals (9 rats) complete transection followed by dural closure using sutures was performed as described above. In the second group of animals (8 rats), subpial transections were performed. A laminectomy was performed and the spinal cord was approached from a lateral orientation. Under direct observation of the dorsal vein, a microsurgical knife was placed horizontally just beneath the vein and directed all the way to the other side. The knife was then rotated, cutting the spinal cord in a single turn, keeping the dorsal spinal vasculature (both arterial supply and venous drainage) intact (). The dura was also closed with sutures.
Fig. 1 Artist’s schematic representation of subpial surgical transection technique. Under direct observation of the dorsal vein, a microsurgical knife is placed horizontally just beneath the vein and directed all the way to the other side. The knife (more ...)
Postoperatively, animals were given buprenorphine 0.05 mg/kg subcutaneously for pain for the first 48 hours, Lactated Ringer’s solution was administered as needed, and gentamycin was given (Schering-Plough) intramuscularly for 3–7 days to prevent infection. For the duration of the experiment, bladder voiding and observation of the animals was performed twice daily. Topical antibiotic ointments were used to treat decubital ulcers.
Spinal Cord Tissue Harvesting
Animals from the dural closure study were killed 1-month postsurgery, while those from the subpial transection study were killed 2 hours and 3 weeks postsurgery. All animals were killed with intramuscular injections of pentobarbital (Fort Dodge Animal Health), and fixation was performed by transcardial perfusion with 4% paraformaldehyde. The spinal columns were removed en bloc and postfixed in 4% paraformaldehyde for 48 hours. The spinal cords (including the transected area plus 1 cm above and below the lesion) were then removed and postfixed in 4% paraformaldehyde for an additional 24–48 hours before being embedded in paraffin, and 8-μm longitudinal serial sections were taken. The longitudinal sections were oriented in the sagittal plane. Throughout the entire sagittal spinal cord, 12-to-15 × 8–μm sections were collected for analysis at 150-μm intervals. Complete transection of the spinal cord was observed in every case. Sectioning was performed using a Reichert Jung tabletop microtome.
Masson trichrome staining was performed on tissue sections obtained from all animal groups. Briefly, sections were washed in tap water and then stained with hematoxylin for 10 minutes. After rinsing, sections were stained with Biebrich scarlet for 5 minutes, rinsed, and placed in phosphotungstic/phosphomolybdic acid for 10 minutes. Sections were then transferred directly into Aniline blue for 5 minutes, rinsed, and placed in 1% acetic acid for 1 minute. After rinsing, the sections were dehydrated, cleared, and coverslipped. All reagents were from Sigma-Aldrich.
Image acquisition was performed using a Zeiss AxioCam loaded on a Zeiss Axio Imager Z1 microscope, and Zeiss KS400 software was used to determine the percentage normal cord, scar formation, and cyst formation. These areas are outlined in . The thick black line delineates the area within which the measurements were taken. This area excluded scarring extrinsic to the spinal cord. Cysts were identified as fluid-filled cavities (surrounded by thin black lines), while scar was identified as spinal cord tissue infiltrated with collagen (blue tissue), because collagen is not normally found in the spinal cord. The normal spinal cord is delineated by a thin red line. All analyses were carried out by blinded observers.
Fig. 2 Photomicrograph showing a representation of technique used for determining the percentage normal cord, scar formation, and cyst formation on trichrome-stained spinal cord sections. Zeiss KS400 software was used to determine the volume of the spinal cord (more ...)
The 1-way ANOVA was used, with post-hoc analysis performed using the Bonferroni test for the first dural closure study. The Student unpaired t-test was used to analyze the subpial transection data. Values of p ≤ 0.05 were considered statistically significant. Commercially available software (Graph Pad Prism 4) was used for all analyses.