Reliable evaluation of functional recovery is necessary in order to study spontaneous functional improvements over time after SCI. Assessment of function is also useful in evaluating the beneficial effects of a therapy used in SCI models. Therefore, proper selection of appropriate functional assessment methods in animal studies is crucial to properly interpret the results. The
recovery of function is mainly assessed with behavioural testing and more rarely by electrophysiology.
1.8.1 Behavioural testing
Final functional outcome following SCI depends on the extent of neuronal
damage, loss of white matter and reorganization of the remaining neural tissue.
Behavioural assessments help to determine the lesion severity, location and extent of spontaneous recovery and also document improvements in response to therapeutic intervention. However, it is important to choose a suitable
behavioural technique to match the relevant hypothesis of the experiment (Basso, 2004). Ideally the behavioural responses in animal models of SCI should be relevant to those seen in human patients with SCI (Sedy et al., 2008).
Some of these tests are simple and require little specialist training, while others need specialist training and sophisticated equipment. Muir and Webb divided behavioural tests according to the type of data collected. These are end point measures, kinematic measurements and kinetic measurements (for review see (Muir and Webb, 2000).
1) End point: these are measures in which animals are required to accomplish a particular goal and are scored on their ability to achieve it.
2) Kinematic: these describe qualitative measures where movements of the whole body and body segments relative to each other and/or an external frame of reference are measured and compared.
3) Kinetic: these tests help to quantify or describe the force produce by a limb or limbs, for example during weight support or force generated during pulling a
force transducer such as a grip strength meter (for review see (Muir and Webb, 2000).
Behavioural tests can also be divided into motor, sensory, sensory-motor, autonomic and reflex based test, according to the predominant sensorimotor systems involved in a particular behaviour. However, it is very difficult to differentiate one system involvement in a particular behavioural outcome since multiple systems normally contribute to all functions.
1.8.1.1 Motor tests
These tests primarily assess skeletal muscle functions that are not involved in locomotion. There are many tests that look at specific aspects like the inclined plane test which evaluates the animal’s ability to maintain body position on a board that is continually raised by increasing the angle (Pearse et al., 2005).
Similarly, the limb hanging test utilizes the grasping function of the paws
(Pearse et al., 2005) and forelimb asymmetry tests assess asymmetries produced by a variety of spinal cord injuries (Gensel et al., 2006). Food pellet reaching tests evaluate the ability to use the forelimbs to reach, grasp and retrieve a food pellet after cervical injuries (Whishaw, 2000). Grip strength tests assess neuromuscular function by sensing the peak amount of force generated by animals grasping a bar (Pearse et al., 2005, Anderson et al., 2009a) and will be discussed in more detail in the third chapter of this thesis.
1.8.1.2 Sensory tests
Purely sensory function in animal models of SCI is difficult to evaluate. However, changes in sensory function indicative of pain after SCI can be evaluated by observing exaggerated responses in animal behaviour to normally innocuous stimuli. For example, von Frey’s filament test is used to evaluate the degree of mechanical allodynia (Gris et al., 2004). In the von Frey test calibrated filaments of increasing force are applied to the plantar surface of the forelimb or hindpaw and a positive response is seen if the paw is briskly withdrawn (Gris et al., 2004).
Increased sensitivity is indicated by withdrawal from less stiff filament than normal. In addition, use of a pre-heated plate for hot plate-based tests and noting the time of response in the form of a withdrawal can detect enhanced
sensitivity to thermal stimuli (Gale et al., 1985). Altered responses can be accompanied by licking, vocalization, overgrooming and aggression (Sedy et al., 2008).
1.8.1.3 Sensory-motor tests
These tests assess coordination and functional integration in sensory and motor systems. In the rope walking test animals are trained to run over the horizontal oriented rope (Kim et al., 2001) and in the beam walking test rats are trained to run over different diameter beams (Hicks and D'Amato, 1975, Metz et al., 2000).
Animals are videotaped and the number of errors (slips and falls) made by the animal while crossing are counted. Similarly the horizontal ladder walking (grid walking) test is very sensitive in evaluating sensory-motor coordination
(Behrmann et al., 1992, Metz et al., 2000). In the horizontal ladder walking test animals walk along a horizontal ladder with variable rung spacing (Metz and Whishaw, 2002, Chan et al., 2005). Runs are video recorded as for rope and beam walking and then the animals limb placement on the rungs evaluated. This will also be discussed in further detail later in the thesis.
1.8.1.4 Locomotor tests
Locomotor test are those in which the locomotor apparatus, the forelimbs and hindlimbs are tested after SCI. These tests are mostly in the form of an ordinal rating scale. The Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) is the most commonly used test for assessing locomotion in spinal cord injured rats. It scores animals’ hindlimb kinematic ability on a scale of 0 to 21, where 0 indicates no hindlimb motor function while 21 represents function in an uninjured rat (Basso et al., 1995). This scoring system does not require animal training, scoring can be learned quickly and reliably, and results can be
compared with other labs (Basso et al., 1995). Similarly for forelimb locomotor assessment a forelimb assessment scale (FLAS) has been devised. The FLAS is a modification of the BBB score and it has a scoring from 0 showing no forelimb motor function to 17 for uninjured animal (Anderson et al., 2009a).
1.8.2 Electrophysiological testing
Electrophysiology is the study of the electrical properties of cells and tissues. In vivo electrophysiology represents a way of directly quantifying function and changes in motor and sensory pathways after SCI reliably. Also the
electrophysiology can be focussed on a particular tract and fibres, and therefore changes in a single pathway can be examined. In addition, training of animals is not required as in behavioural assessment methods. In addition,
electrophysiological methods are also used in evaluation of therapies used in SCI (Bradbury et al., 2002, Toft et al., 2007). However, these techniques require expensive instruments and extensive personnel training and an ability to interpret recordings at the time they are recorded to deal with any technical issues (Blight, 1992).
1.8.2.1 Cord dorsum potential
Cord dorsum potential (CDP) recording is a technique used to measure the postsynaptic electrical activity evoked in the spinal cord. CPDs reflect the depolarization of interneurons or primary afferents fibres in the dorsal horn (Willis and Coggeshall, 2004). The characteristics and amplitude of CDPs reflect the level of activation and strength in the local circuits due to stimulation of ascending pathways (if stimulation from a peripheral nerve) or due to
descending pathways (for example stimulation of corticospinal system).These potentials can be recorded directly from the surface of the cord, from the skin overlying the vertebral column (Sedgwick et al., 1980) and also from the
epidural space (Shimoji et al., 1977).
CDPs can be used to assess the functional abilities of spinal pathways after SCI (Sedgwick et al., 1980) and monitor the function perioperatively (Ahn and Fehlings, 2008). It provides a sensitive and reliable method for observing the outcomes of experimental SCIs and also for assessing the effects of potential therapies and is therefore useful in SCI research (Bradbury et al., 2002, Riddell et al., 2004, Toft et al., 2007). Using a crush injury model of the cervical cord of rats, application of chondroitinase ABC was shown to improve CDP potentials evoked by CST axons below the injury. This finding correlated with anatomical and behavioural evidence (Bradbury et al., 2002). Similarly after a dorsal column
lesion with application of OECs, improvements in CDPs evoked by stimulation of fibres in dorsal roots were recorded. This was attributed to a neuroprotective action (Toft et al., 2007). Hence, assessing CDPs in different SCI models can be very useful to evaluate changes in the circuitry in the vicinity of the lesion and can also help in evaluating the therapeutic effects of different interventions on the spinal circuits.
1.8.2.2 Somatosensory evoked potentials
Somatosensory evoked potentials (SEPs) are a series of waves that can be recorded from the surface of the somatosensory cortex. SEPs reflect the sequential activation of neural structures along the somatosensory pathway in response to electrical stimulation of peripheral nerve (Sedgwick et al., 1980).
SEPs provide direct quantitative measurements of the functional ability of the ascending somatosensory pathways. They are used for the diagnosis and characterisation of neurological abnormalities in humans (Small et al., 1978, Dietz and Curt, 2006). SEPs are also used for monitoring the condition of the spinal cord intraoperatively (Grundy, 1983). This sort of direct feedback on the state of neural networks and pathways allows surgeons to safely perform
complicated surgeries, such as resection of tumours and reconstructive procedures (Malhotra and Shaffrey, 2010). SEPs have been also used to assess the integrity of the spinal cord in animal models of SCI. Changes in the
amplitudes and latencies of SEPs are correlated with injury severity and
behavioural outcomes (Fehlings et al., 1989, Nashmi et al., 1997). Thus it can be another way of quantitatively analysing the effects of therapeutic interventions in SCI models.