Behavioural analysis

Open field test of locomotion:

The Basso, Beattie, Bresnahan (BBB) score is a way of measuring locomotor function in hindlimbs following SCI (Basso et al., 1996). As long as the method is carried out consistently, the BBB score can be used to compare results between laboratories and to compare the effectiveness of different treatments. The scale ranges from the highest score of 21(uninjured animals with normal locomotion) to 0 (complete paralysis). Typically following SCI, a curve is obtained which reaches a plateau at various scores, characteristic of injury severity. A subscore can further be calculated from the data collected from the BBB score (Lankhorst et al., 1999). This can be informative when a BBB recovery curve reaches an artificial plateau at the time where stepping and coordination is regained, although recovery of finer aspects of paw placement may be overlooked.

Animals were allowed to acclimatise preoperatively to the testing environment in an open field, which was a circular plastic enclosure (Fig. 2.3; width 88 cm, depth 30 cm; Water Features On-Line, Tyne & Wear, UK). This was considered to be an optimum size for at least four continuous steps at the time when the animals began walking, to give an accurate assessment of coordination. Animals were introduced to the open field on at least three separate occasions, either in cage groups or individually. This was essential for the

avoidance of fear behaviour (urination, freezing, crouching during locomotion and dragging

61 hind limbs) especially at later recovery time points after SCI, which could give an

inaccurate assessment of locomotor recovery. During testing, each rat was allowed to walk continuously in the open field for 4 min, while characteristics of hind limb movement (ankle, knee and hip) were marked on a sheet (Appendix 3). The rat was then removed from the open field, which was then wiped with 50 % ethanol solution and allowed to dry before the next rat was introduced. This was to help prevent the spread of any urinary infections and to remove any „fear scent‟ that might have been present in the urine and could affect behaviour of the animal (Basso, 2004). A BBB score was assigned for each hind limb using the locomotor rating scale (Appendix 4) and the score for both hind limbs was averaged.

Following surgery, the animals‟ locomotor function was monitored every day using the open field test for the first 7 days and then at 10, 14, 21 and 28 days post-operation.

Figure 2.3 Open field test. Animals were allowed to acclimatise to the testing environment before SCI. Each animal was observed for 4 min and movements related to recovery of hind limb and locomotor functions were recorded.

2.3.1 Measurement of bladder function

Data on bladder function were collected on the care sheets (Appendix 1). Each day for 7-10 days following SCI, it was noted whether manual expression was required and whether the abdomen was wet, indicating incontinence. Bladder width and length measurements were

62 taken post-mortem after perfusion. The bladder was dabbed with paper towel and

measurements taken with a ruler. The area was calculated from these.

Ultrasound

Bladder volume was measured using a high resolution portable digital ultrasound system (Sonosite® MicroMaxx®; BCF Innovative Imaging, Livingston, Scotland, UK) with a SLA/13-6 MHz 26 mm linear array transducer as described by Al-Izki et al. (2009). The ultrasound system acquires a high-resolution two-dimensional image from which

volumetric calculations were made. For bladder measurements, the abdomens were shaved and an ultrasound gel (Alpha tube ultrasound scanning gel; BCF Innovative Imaging) was applied. The baseline reading was taken while the animal was anaesthetized preceding the SCI surgery. Following SCI, on days 3, 5 and 8, the awake animal was gently restrained, and the transducer was first placed longitudinally against the animal to capture the maximum bladder length and depth (Fig. 2.4). Next; the transducer was rotated 90° to capture the maximum width of the bladder. The volume of bladder urine was automatically calculated by the ultrasound imaging software.

Figure 2.4 Images used to calculate bladder volume. The dotted line indicates the width (D¹) depth (D²) and width (D³) measurements used to calculate the volume.

63 2.3.2 Measurement of mechanical hypersensitivity

For behavioural assessment of tactile hypersensitivity readings were taken using an automated plantar aesthesiometer („von Frey‟: Ugo Basile, Italy). This consists of a movable actuator that lifts a blunt probe at a controlled force rate onto the plantar paw surface (Fig. 2.5). This rate was set at 1.7 grams/second with a cut off weight of 50 grams and latency of 30 seconds. This experimental set-up benefits from the removal of

observational bias, human error, and variable force applied by the investigator using manual von Frey hairs. The mechanical aesthesiometer has greater accuracy (to nearest 0.1 gram) and the weight incurred on the plantar paw increases at a linear rate from 0 to 50 g, whereas manual hair numbers have an exponential force increase, with large force

differences at higher hair values between adjacent hairs. Rats were acclimatised and a baseline reading taken, and then tested post operatively. The probe made contact with the same plantar area, and increased force until the foot was voluntarily withdrawn. A positive response was determined as a single voluntary flexion withdrawal of the foot, with or without foot licking or biting, from the meshed flooring during the increasing weight of the probe. Each hind limb was tested 3 times, with an interval of 5 minutes to prevent

habituation to the stimulus. Post-operative tests were performed on days 7, 14, 21 and 28.

Figure 2.5 Behavioural testing of the rat plantar hind paw. Diagram modified from Takahashi et al. 2003. The area for mechanical testing is at the border of the L4 and L5 dermatomes, avoiding paw pads, as indicated by the red circle.

64 2.4 Tissue harvesting for immunohistochemical analysis

Perfusion fixation and embedding of rat tissue

At specific time points following injury, animals were euthanised using CO2 asphyxiation and placed on a plastic board inside a fume hood. Once the absence of corneal or forepaw reflexes was confirmed, a midline incision was made along the chest. Scissors were used to cut the abdominal wall, from mid abdomen to the xiphisternum. The xiphisternum was then grasped with forceps and the diaphragm trimmed away from the ribcage. The ribs were cut on either side of the chest cavity, exposing the heart. The sternum and ribs were then held back with forceps to allow easy access to the heart. The fixative delivery system was gravity-fed and the tap opened slightly to allow a slow flow of saline (0.9% NaCl). A cannula was then inserted into the left ventricle and clamped in place. The tap was opened to allow full flow of saline and the right atrium cut. Once all the blood had been washed from the animal, judged by the lightening of the liver to a pale red-yellow colour, the tap was switched over to allow ~300 mL fixative (4% paraformaldehyde, pH 7.4) to flow through.

Tissue was dissected and post fixed for 2 hr, then cryoprotected in 20% sucrose (in phosphate buffer, 0.1M pH 7.4) for at least 3 days. A segment of liver tissue of

approximately 1cm³ and 5 mm segments containing the spinal cord lesion site, rostral and caudal segments, and the equivalent levels in naїve and laminectomy groups were

embedded in OCT Embedding Medium (VWR, Lutterworth), frozen on dry ice, and stored at −80 ºC for subsequent processing for immunohistochemistry.

Cryosectioning of Embedded Rat Tissue

At least 10 minutes before use, the Leica CM 1900 cryostat (Leica, Milton Keynes, U.K.) was set to hold both the chamber and chuck at –12-20 °C and the temperature left to

stabilise. Tissues were moved from the -80 ºC freezer and left in the cryostat chamber for 5-10 minutes to warm up to -12-20 ºC before being mounted on the chuck. Rat spinal cord or liver sections were mounted onto the chuck using OCT Embedding Medium (VWR, Lutterworth, U.K.), which was left to freeze for 5-10 minutes before sectioning. 15 μm

65 thick transverse sections were cut for spinal cord, 10 μm for liver and transferred onto Superfrost slides (VWR, Lutterworth, U.K), and left to dry for at least 20 min. Sections were cut serially so that each slide contained a selection of sections throughout the lesion and were stored in cryoprotectant (120 ml ethylene glycol, 200 ml PBS, and 200 g sucrose) at -20 ºC until required for general histological staining or immunohistochemistry (IHC).