2 METHODS

2.1 Animals

Adult male Sprague-Dawley rats (350-400 g body weight, purchased from Taconic, Denmark) housed in standard cage with food and water ad libitum and maintained under the 12 hours’ light- dark cycle at a constant environmental temperature of 21°C (humidity: 65%) were randomly assigned to experimental groups. Animals losing more than 10% body weight following the injury were excluded from the study. All procedures were approved by the research animal ethics board of Uppsala County (permits C101/13 and C165/14) and performed according to the rules and regulations of the Swedish Board of Agriculture.

2.2 Surgery and histology

Controlled cortical impact (CCI) was performed as previously described (Clausen et al., 2011). Briefly, anesthesia was induced by isoflurane (4% in air) and maintained with 1.2% isoflurane in a 70% nitrous oxide/30% oxygen, delivered via a nose cone. Core body temperature was maintained at 37±0.3 ºC by a heating pad (CMA150, CMA, Stockholm, Sweden). Subcutaneous local anesthesia was injected (bupivacaine; Marcain®, AstraZeneca, Sweden). A craniotomy, 5×6mm2, was centered at bregma 0.5 mm and 3.5 mm lateral to the midline over the right sensorimotor cortex. With the rats in the stereotaxic frame, CCI brain injury was induced by a CCI-device (VCU Biomedical Engineering Facility, Richmond, Virginia, USA) using a 4.0 mm diameter piston, and producing a 1.0 mm compression of the brain at a speed of 2.4 m/s and 100 ms duration (Figure 1a-d). The impactor was perpendicular to the exposed cortex. After the injury, the bone flap was replaced and the wound was closed with interrupted sutures. Sham-injured animals underwent identical surgery and anesthesia without receiving the CCI. Animal weight and wound healing were monitored daily post-surgery.
Three days following the brain injury, rats were sacrificed with overdose of pentobarbital and the brains were dissected. Frozen brains were cut into 14 μm thick coronal sections using a cryostat (HM500, Microm GmbH, Walldorf, Germany) and mounted on Superfrost+ object glasses (Histolab, Gothenburg, Sweden). Following H&E staining (Histolab, Gothenburg, Sweden), digital images of the sections were acquired using a stereo microscope (Zeiss Stemi 2000-C; Zeiss Gmbh, Göttingen, Germany) equipped with a digital camera (Mcm5c; Zeiss Gmbh, Göttingen, Germany).
The injury-induced loss of brain tissue measured in each hemisphere with the SectionToVolume software (Hanell, Hedin, Clausen & Marklund, 2012). The lesion area (mm2) was calculated by outlining missing cortical tissue for each section taken at 0.5 mm intervals, and lesion volume (mm3) determined by multiplying the sum of the contused areas obtained from each section by the distance between sections (0.5 mm). The tissue loss in the injured (right) and contralateral (left) hemisphere after the CCI was 13.03 ± 4.14 and 0.01 ± 0.00. mm3 (mean ± S.E.M.; n = 5), respectively. Almost no tissue was lost in the sham operated group (n = 3) in both hemispheres (0.01 ± 0.00 mm3).
To visualize the architecture of the brain structures around the lesion site, 4 brains from CCI group and 3 brains from sham-operated group were cut into 50 µm thick sections with a sliding microtome (Microm HM450, ThermoScientific, Germany) connecting to a freezing unit (Microm KS34, ThermoScientific, Germany) and every fourth section was stained with Toluidine blue (Sigma-Aldrich). Microphotographs were taken with a conventional light microscope (Leica DM6000B, Leica Microsystems, Germany) and processed with Adobe Photoshop CC (version 19). No damage for the cortices that underwent sham injury was revealed. For the CCI rats, the center of the injury was in the desired point in the cortex. The extent of injury ranged 2 – 4 mm rostrocaudally, 2 – 4 mm mediolaterally, and 1.5 – 2 mm in depth. In most CCI rats the damaged tissue was lost during the staining process, leaving a hole in the damaged region; or sometimes retained after staining (Figure 1d).

2.3 Spinal cord transection

The animals were anesthetized with sodium pentobarbital (I.P.; 60 mg/kg body weight, as an initial dose and then 6 mg/kg every hour). After measurement of postural asymmetry, the rats were placed on a stereotaxic frame to maintain the body temperature at 37 ± 0.3 ºC by a heating pad connected by a rectal probe (CMA150, CMA, Stockholm, Sweden). A laminectomy at the thoracic T2-T3 level was carried out, and the spinal cord was completely transected using a pair of fine scissors. Local infiltration of 3.5 mg/ml lidocaine (Xylocaine) with 2.2 μg/ml adrenaline was used to reduce nociceptive input during surgery. A piece of Spongostan (Medispon® (MDD sp. zo.o., Toruń, Poland) was placed between the rostral and caudal stumps of the spinal cord. The completeness of the transection was confirmed by (i) inspecting the cord during the operation to ensure that no spared fibers bridged the transection site and that the rostral and caudal stumps of the spinal cord are completely retracted; and (ii) examining the spinal cord in all animals after termination of the experiment. After completion of all surgical procedures, the wounds were closed by the 3-0 suture (AgnTho’s, Sweden) and rats were kept under infrared radiation lamp to maintain body temperature during monitoring of postural asymmetry.

2.4 Visual measurement of postural asymmetry

The magnitude of postural asymmetry (MPA) and the side of the flexed limb were assessed as described previously (Bakalkin & Kobylyansky, 1989). Briefly, the measurements were performed under pentobarbital (60 mg/kg, i.p.) anesthesia. The level of anesthesia was titrated to produce the most consistent measurements and was characterized by a barely perceptible corneal reflex and a lack of overall muscle tone. The rat was placed in ventral decubitus (prone) position on a millimeter-paper sheet. To minimize effects of tactile stimulation during the analysis three threads were glued to the nails of the middle three toes of each limb. The hindlimbs were gently pulled at the threads for 5-10 mm to reach the same level, then set free and the MPA was measured in millimeters as the length of the projection of the line connecting symmetric limb points (digits 2-4) on the longitudinal axis of the rat (Figure 1e,f). The procedure was repeated six times in immediate succession, and the mean asymmetry value for a given rat was calculated and used in statistical analyses. A limb displaying shorter projection was regarded as flexed.