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Preliminary work was conducted in mice. Stimulators were implanted into 6 BL6 mice, however, 3 of the stimulators failed within 2 days of implantation. The

experiments were therefore moved to rats, in which a larger implantable stimulator, which has been used more extensively, could be used. This allowed options in the stimulation patterns. As continuous stimulation results in high muscular atrophy (Ferguson et al. 1989), a burst pattern was selected. Bone deposition is not a fast process with a maximal response to an established loading protocol using high forces being achieved 4-8 days after the initial load (Forwood et al. 1996).

Therefore, as this protocol used a smaller load, a period of 28 days was selected, as this would allow both muscle and bone ample time to show a response.

Nine male, eight week-old Wistar rats (weights 228-282g) underwent surgical implantation of miniature neuromuscular stimulators (Russold and Jarvis, 2007). It was not possible to conduct a power analysis as this was the first experiment to look at bone morphology in response to electrical stimulation, and therefore it was not possible to estimate the effect. Given the ethical implications of animal work it was decided that 9 was the largest sample size justifiable. Under general isoflurane

120 anaesthesia (Isoflurane 2%, O2 49%, NO2 49%) rats were injected with analgesia

(intramuscular Buprenorphine, 0.3mg/ml) and antibiotic (subcutaneous Baytril, 25mg/ml). Incisions were made in the left flank for stimulators to be implanted in to the peritoneal cavity, and sutured in place. The electrodes were passed

subcutaneously into the hindlimb and sutured either side of the left common

peroneal nerve. The incision was sutured closed; a further intramuscular injection of Buprenorphine was delivered along with subcutaneous saline to replace fluid. Rats were kept warm at all times with a heat pad. Stimulators were switched on by

passing a magnet over the implant. Stimulators delivered 0.2ms pulses at 100 Hz for a total of 200ms every 30s, resulting in a total of 9.6 minutes of stimulation per day. Each 200ms burst of stimulation at 100Hz caused a very brief but fused (tetanic, near maximum force) contraction of the muscles in the antero-lateral compartment of the leg (Fig.4.1). The right hand side was left as a contra-lateral control. Animals were free to ambulate throughout the stimulation period and were checked daily for any behavioural disturbance, weight loss and to ensure stimulators were working. Over this time period all animals were considered to be showing normal behaviour. After 28 days of stimulation the animals were euthanized by CO2 asphyxiation. All

experiments received ethical approval and were carried out in strict accordance with the Animals (Scientific Procedures) Act of 1986. To evaluate any possible systemic or surgical effects on the contra-lateral control limb, three additional age, sex and strain matched rats were analysed as controls (no surgery or specific loading regimes).

4.2.2 Imaging

Hindlimbs from 6 experimental and the 3 control animals were removed, and stored in 10% phosphate-buffered formal saline (PBFS). Hindlimbs were imaged along with a hydroxyapatite phantom using the Metris X-tek custom 320kV bay system

(University of Manchester). Data were reconstructed at 40µm resolution (isometric voxels). For standard microCT, x-ray tube parameters of 75kV and 200µA were used. As conventional microCT imaging does not provide muscular detail (see section 2.2.2 and Fig.4.2) limbs were stained with I2KI. To control for concentration-

dependent specimen shrinkage (see Chapter 3) all samples were immersed in the same concentration solution (9% I2KI, dissolved in PBFS) for the same time period

(9 days). Specimens were then imaged with microCT for a second time (90Kv; 130uA; 40µm). Hindlimbs from the remaining three experimental animals were subjected to muscle histology (see below).

121 Figure 4.1 A three-dimensional reconstruction demonstrating the muscles which were stimulated by the miniature implantable stimulator. Stimulation of the common peroneal nerve resulted in contraction of tibialis anterior, extensor digitorm longus, peroneus longus and peroneus brevis, all marked with a *.

Figure 4.2 Iodine-enhanced microCT compared with standard microCT. Transverse cross-sections of the same mouse hindlimb imaged using a) iodine- enhanced micro-CT, and b) standard micro-CT.

122 4.2.3 Macroscopic analysis

Relative volumes of stimulated muscles tibialis anterior (TA) and extensor digitorum longus (EDL) were calculated from contrast enhanced microCT images using the stereological method of Volume Est plugin for ImageJ (Merzin, 2008; Schneider et al., 2012).

The maximum force production capability of TA was estimated using I2KI microCT

data. Average fascicle lengths were calculated from 20 fascicles throughout TA. Physiological cross-sectional area was calculated by dividing average muscle fascicle lengths by muscle volume (Alexander and Vernon 1975). Muscle force estimates were then calculated by multiplying PCSA with muscle stress value of 0.3 N mm-2 (Strait et al., 2005). Force estimates correspond well with measured values for male Wistar rat TA (Maas et al. 2001).

MicroCT data provides a wealth of different factors which could be measured (see section 2.2.2). Initially, regional measurements were made along the length of the bone to allow targeted further analysis. BMD and cortical thickness measurements in particular were selected as they address the two main ways in which a bone can adapt; through its structure or composition. Regional cortical thickness and bone mineral density (BMD) data were obtained from microCT data using the BoneJ plugin for ImageJ (Doube et al., 2010; Schneider et al., 2012). To capture regional differences, 10 evenly distributed sites were sampled along the posterior and

anterior surfaces of the tibia. For BMD measurements, greyscale values from the rat data were compared with greyscale values of a hydroxyapatite phantom by

regression analysis. The greyscale values in the rat tibia were then calibrated for hydroxyapatite concentration.

4.2.4 Geometric morphometrics

To establish the control and experimental tibiae with geometries closest to the mean form, geometric morphometrics (GMM) was used. Twelve homologous landmarks and four curves were identified on each tibia (n=6 control, n=6 stimulated, Table 4.1) using Landmark (Wiley et al. 2005). Landmark data were then analysed using Procrustes superimposition and principal components analysis in MorphoJ (Klingenberg, 2011).

123 Table 4.1 Tibial landmarks used for geometric morphometrics

Number Landmark description

Point 1 Lowest most anterior point on proximal epiphysis