Materials and Reagents

Pyrex Glass Petri dishes (150 x 20 mm) (Corning, catalog number: 3160-152BO )
Pyrex crystallizing dish (Corning, catalog number: 3140-150 )
Wheaton Coplin staining jars (Sigma-Aldrich, catalog number: S6016 )
Moist tissue paper
Needle
Single edged surgical blade (Sigma-Aldrich, catalog number: S2771 )
Cotton swabs, sterile (6 inch) (Medline Industries, catalog number: MDS202000Z )
Plastic Pasteur pipettes (BRAND, catalog number: 747755 )
Phosphate buffered saline (PBS), pH 7.4 (Sigma-Aldrich, catalog number: P4417 )
Tricaine (MS222) (Sigma-Aldrich, catalog number: E10521 )
Zebrafish Aquarium System water
0.1% cresyl violet solution (Sigma-Aldrich, catalog number: C5042 )
0.1% luxol fast blue solution (Sigma-Aldrich, catalog number: L0294 )
4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
0.5 M EDTA (Sigma-Aldrich, catalog number: E9884 )
Paraffin (Sigma-Aldrich, catalog number: 327212 )
Xylene (Sigma-Aldrich, catalog number: 534056 )
0.05% lithium carbonate solution (Sigma-Aldrich, catalog number: 255823 )
Graded ethanol (Sigma-Aldrich, catalog number: 24102 )
Note: This product has been discontinued.
Glacial acetic acid (Sigma-Aldrich, catalog number: A6283 )

Equipment

Leica rotary microtome (Leica Biosystems Nussloch, model: RM 2125RTS )
Student Dumont #5 forceps (Sigma-Aldrich, catalog number: F6521 )
Micro dissectingspring scissors (Harvard Apparatus, catalog number: 728500 )
Stereozoom dissecting microscope (Olympus, models: SZX7 and SZ51 )

Procedure

Note: The present study was carried out according to the guidelines provided by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environments and Forests, Government of India). Surgery protocol was approved by the Institutional Animal Ethics Committee of our department under registration with CPCSEA (CPCSEA/ORG/CH/REG NO.925/295).

Maintenance of fish
Zebrafish stock population is either bred in our animal house facility or obtained from local pet shop. Fish are kept in separate groups of 15 in the aquatic system maintained at 28 °C on a 14 h light and 10 h dark cycle.
Preparation of surgical plates
106 mm glass Petri dish is used and moist tissue paper is placed in it, where the anesthetized fish is laid laterally (Figure 1a; Video 1). The gill is covered with tissue paper soaked in water or PBS.
Fishes of same size and age (approximately 3-4 cm in length, 4-6 months old) are anaesthetized by dipping them in 0.02% tricaine (MS222) for 3-5 min (long exposure like 10 min or more could affect post-surgical recovery of spinal cord injured fish) at room temperature. It is important to check that fish are completely anaesthetized prior to surgery. After placing the anaesthetized fish on the Petri dish, the body is touched with a needle and absence of twitching movement confirms complete anaesthesia. Petri dish with anaesthetized fish is placed under stereozoom dissecting microscope and surgery is performed under the microscope.
Surgery protocol

Crush injury:

A longitudinal incision is given with a single-edged blade on the second stripe of the fish body at the level of the dorsal fin, which corresponds to the 15/16^th vertebrae (Figure 1b; Video 1).
After making the cutaneous wound (Figure 1c; Video 1), one has to scrape muscle sidewise to reach the vertebral column, which resides at a much deeper level. Blood coming to the wound site can be cleared by using a sterile cotton swab (Figure1d; Video 1).

Figure 1. Making a cutaneous wound before spinal cord injury. a. An anaesthetized adult zebrafish is placed laterally over a moist tissue paper before inflicting spinal cord injury; b. The pigmented stripped region (PR) below dorsal fin (DF) is the area where the cutaneous wound is made; c. Making a cutaneous wound by a sharp blade; d. Clearing the blood from wound area by using a sterile cotton swab; e. Tearing the muscles deep inside the wound using forceps to reach and clearly visualize the spinal cord.

Video 1. Inflicting spinal cord injury in adult zebrafish
Vertebral column is a hard but translucent tissue and spinal cord, encased within the vertebrae, is visible as the meninges are pigmented. After clearing the surrounding tissues, vertebral column is crushed dorso-ventrally with Dumont forceps #5 for 1 sec (Video 1; Figures 2 and 4, where the injury epicentre is marked with an asterisk).

Figure 2. Crush injury to spinal cord using Dumont #5 forceps. a. Holding the forceps in stretch to clearly visualize the spinal cord before performing injury; b. Inflicting crush injury by holding the full spinal cord dorso-ventrally using Dumont #5 forceps for 1 sec; c. Covering the vertebral wound with muscle and skin; d. Higher magnification view of spinal cord inside the wound before injury; e. Higher magnification view of the spinal cord at the wound site during crush injury; f. Higher magnification view of the spinal cord inside the wound after crush injury. Star indicates the injury epicentre.
One can use the bony projections of vertebral column and mark the corresponding fin rays of dorsal fin as a land mark. We cannot perform laminectomy in zebrafish spine, as we do while inflicting SCI in mouse, because the vertebral column is too thin here. After completing injury, surrounding muscle tissues are placed back to cover the injury site and only a single suture can be given through skin to reduce the wound size.
Fishes are returned into shallow water in a Petri dish and water is gently blown over the gills using a plastic pipette so that fish can recover from anaesthesia quickly and can swim on their own. Fishes are finally transferred to larger tanks and allowed to regenerate for a specified period of time at 28 °C.

Transection injury

Similar to crush injury a longitudinal incision is given on the skin laterally with a single-edged blade on the second stripe of the fish body at the level of dorsal fin, which corresponds to the 15/16^th vertebrae.
After carefully scrapping the muscle beneath the wound, spinal cord encased in the vertebral column can be visualised clearly. On reaching the vertebral column deep to the wound site, the spinal cord is completely transected using a micro-dissecting spring scissors (Figures 3 and 4).
As spinal cord is a soft spongy tissue, a sharp cut through the gap between the two vertebrae by the two blades of micro-scissors is enough to ensure complete transection separating the rostral and caudal stumps completely from each other. The injury epicentre is marked with an asterisk in Figure 4c. The injury would lead to paralysis of posterior part of the body. After completing injury procedures, surrounding muscle tissues are placed back to cover the vertebral wound. The suture may be given through the skin (Figure S1) or tissue glue can be used (optional) to reduce the wound size. Fishes are returned to a shallow water tank and water is blown gently over the gills using a plastic pipette so that fish can recover from anaesthesia quickly.
Fishes are finally transferred to larger tanks and allowed to regenerate for a specified period of time at 28 °C.

Figure 3. Transection injury to spinal cord using a micro-scissor. a. Holding the forceps in open position to clearly visualize the spinal cord before inflicting injury; b. Performing transection injury by completely severing the spinal cord into rostral and caudal stumps by using fine spring scissors; c. Covering the vertebral wound with muscle and skin; d. Higher magnification view of the spinal cord before injury; e. Higher magnification view of the spinal cord during transection injury; f. Higher magnification view of the spinal cord after transection injury. Star indicates the injury epicentre.

Figure 4. Uninjured and injured spinal cord after crush and transection injury. a. Higher magnification picture of the uninjured spinal cord. b. Higher magnification picture of the spinal cord after crush injury. c. Higher magnification picture of the spinal cord after transection injury. Star indicates the injured region. SC = Spinal cord, VC = Vertebral column.

Histological analysis to study time course after injury
Luxol fast blue and cresyl violet staining (0.05% lithium carbonate solution and glacial acetic acid are used for differentiation step) is performed on the spinal cord tissue sections according to the standard Klüver-Barrera protocol (Sheehan and Hrapchak, 1980). Both uninjured and injured fishes are dissected, spinal cord taken out, fixed in 4% paraformaldehyde, decalcified in 0.5 M EDTA, passed through graded alcohol and xylene and embedded in paraffin (Hui et al., 2010). Time course analysis of stained section in uninjured cord shows relative distribution of white matter and grey matter as depicted in Figure 5a. Anatomical distribution of neurons in the subependymal and axons and cell bodies in white matter are obvious. Details of the cellular components are discussed in our previous publication (Hui et al., 2010). Here substantial tissue loss is shown at the early stages after crush injury (Figure 5b) with some tissue sparing, a characteristic feature of the crush injury model. Time course cellular response depends on the degree and extent of injury. In this case, as the transection injury is very clean, not much tissue has been lost and the stump tissues are very closely apposed (Figure 5c). Regeneration is very impressive and the time frame does not vary much when compared with crush injury.

Figure 5. Luxol fast blue staining in spinal cord tissue. Histological sections of uninjured spinal cord (a), spinal cord 3 days after crush injury (b) and spinal cord 3 days after transection injury (c) stained with luxol fast blue and cresyl violet. Please note that there are some spared tissues at the injury epicentre in b, whereas two separate stump tissues are clearly visible in c, with no axonal sparing. GM = Grey matter region of spinal cord, WM = white matter region of spinal cord, N = neuron, SEP = sub-ependyma, EP = ependyma. Star indicates the approximate injury epicentre. Scale bar=100 µm (a-c).

Data analysis

Injury protocols were standardized in our laboratory where hundreds of animals have been used for our array analysis (Hui et al., 2014). We could injure 30-40 animals in a day. All the histological analysis with the spinal cord tissues were repeated 3-5 times. The pictures shown here are not repeated in other publications. However, time course analysis of regeneration in zebrafish cord was previously published (Hui et al., 2010, http://www.suklaghosh.com).

Notes

Two very simple yet standardized protocols for inflicting crush and transection injury in adult zebrafish cord have been described. The reproducibility of these injury modalities can be followed by analysing the tissue sections stained with different staining protocols like luxol fast blue and cresyl violet or Mallory’s trichrome staining or even routine hematoxylin/eosin staining.
Staining, particularly using longitudinal section of the spinal cord tissue, enable us to characterize the nature of injury and visualize cellular response(s) at the injury epicentre and the adjoining area after injury and compare with the normal part of the same cord.

Acknowledgments

Both these injury protocols described here are generated at Sukla Ghosh’s laboratory, Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India. Grant support received for the work was from DBT (Govt. of India, BT/PR5489/AAQ/03/245/2004). Dr. Subhra Prakash Hui was recipient of Senior Research Fellowship from CSIR (Govt. of India). The spinal cord crush injury protocol in zebrafish has been adapted from our previous publications (Hui et al., 2010; 2014).

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