SCI model
Uni/bilateral
Region
Species: Strain
References
Contusion
Weight-drop
Bilateral
Cervical/thoracic
Dog
Rat: Sprague-Dawley
Mouse: C57BLC
(47)
(50)
(62)
Weight-drop (NYU)
Thoracic
Rat: Long-Evans
(48)
Weight-drop, electromagnetic spinal cord injury device (ESCID)
Bilateral
Thoracic
Rat: Wistar
Mouse: Balbc-B10/PL
(49)
(51)
Infinite horizons (IH) impactor
Cervical or thoracic
Rat: Sprague-Dawley
(52)
(53)
Louisville Injury System Apparatus (LISA)
Bilateral
Thoracic
Rat: Sprague-Dawley
(54)
Compression
Aneurysm clip
Bilateral (unilateral)
Thoracic
Rat: Sprague-Dawley
(55)
Extra/subdural balloon
Bilateral
Thoracic
Rat: Sprague-Dawley Rat: Wistar
(56)
(57)
(58)
Transection
Scissors/microscissors
Bilateral
Cervical
Thoracic
Rat: Wistar
Rat: Sprague-Dawley
(59)
(60)
Hemisection/over-hemisection/partial transection
Lateral hemisection lesion cavity by aspiration
Unilateral
Cervical
Rat: Sprague-Dawley
(61)
Lateral hemisection using microknife
Unilateral
Cervical
Mouse: C57B1/6
(63)
Dorsal hemisection by microscissors
Bilateral dorsal hemisection
Cervical
Mouse: Glast-CreER
(65)
Lateral over-hemisection by iridectomy scissors
Bilateral dorsal columns/unilateral
Cervical/thoracic
Rat: Sprague-Dawley
(64)
Ischemia
Photochemical lesion
Thoracic
Rat: Wistar
(66)
Irradiation lesion
Thoracic/lumbar
Cat
(67)
Vessel ligation
Thoracic/lumbar
Rat: Sprague-Dawley
(68)
Syringomyelia
Quisqualic acid and kaolin injections
Bilateral
Cervical
Rat: Sprague-Dawley
(69)
Clip compression and kaolin injections
Bilateral
Thoracic
Rat: Wistar
(70)
The various injury models all have advantages and disadvantages. It is not possible to conclude that any of them is more relevant to clinical situations in general, since clinical SCI is often the result of a sudden impact of the bone structures transmitted to the spinal cord (contusion), followed by a sustained compression resulting from the dislocation of vertebrae, and sometimes also transection of axons by penetrating bone fragments. In a clinical cohort of patients, the contribution of these factors will vary considerably, and their relative impact is rarely possible to determine in the individual patient. This heterogeneity is a problem in clinical trials, particularly if a novel intervention is to be studied acutely. Rather than arguing for the choice of a particular SCI animal model, referring to supreme clinical relevance, a candidate treatment should be studied in several injury models. In this context, it is also questionable if any added value is achieved by applying a multi-site-one-model design to experimental SCI studies, as has been suggested (71). In our view, positive effects in two or more animal models would give much better support for possible clinical utility of the treatment evaluated.
Choosing a relevant animal model for proof-of-concept depends on the therapeutic mechanism studied; neuroprotection, regeneration, plasticity, or replacement. When potentially neuroprotective drugs are studied, contusion or compression injury models are preferred over transection injuries since they are characterized by extensive secondary injury that can be modulated (i.e., reduced or increased). Since degenerative mechanisms at different spinal levels are likely to be similar, the level of injury is probably of minor importance when assessing the efficacy of a neuroprotective treatment. In contrast, for an elaborate design of conduits guiding regenerative growth, the anatomy of the axonal tracts is crucial, and consequently also the choice of spinal level studied. The neuronal connections that need to be reestablished for improving respiration after a cervical injury are different from those required to restore urogenital functions after a thoracic injury. An anatomically complete transection is the best choice for achieving unequivocal proof of a functional regenerative effect. In this situation, the choice of spinal level is however limited by the fact that animals with complete transection at cervical or high thoracic levels are very difficult to manage and care for, and therefore most often avoided. The choice of model for replacement therapy will depend on the cell type to be replaced. If, for example, cells with the potential to remyelinate axons are evaluated, demyelinated axons must of course be present, surrounding the central lesion of an incomplete injury. If neurons are to be replaced, any type of model can be used.
4 Tools for Functional Analyses
Morphological and stereological methods can be used to quantify a neuroprotective effect of a drug or cell therapy, anatomical tract tracing, and other techniques can help us assess the extent of regenerative growth or anatomical plasticity. Still, every treatment intended for SCI patients has to be proven effective with respect to functional outcome. Functional analyses in SCI animal models are therefore absolutely necessary. The requirements of the tests used will however be different depending on the type of injury as well as the mechanism of the treatment studied.
It is important to recognize that all test situations except those that concern spontaneous locomotion in the animal’s home cage are to some extent stressful. To minimize variation between animals it is crucial to give the animal sufficient time to get accustomed to the test equipment and test situation. If reward is necessary in the test situation, sweets (cereal, syrup, etc.) can be used. Starving the animals to appreciate food pellets is not recommended, since it is important that injured animals have optimal conditions to recover.
4.1 Tests of Open Field Locomotion
A number of functional assessment methods to evaluate hindlimb movement during open field locomotion have been developed during the last decades. The Combined Behavioral Score (CBS) (72) and the Motor Performance Score (MPS) (22) are examples of compound variables based on several tests, either with the results just summed (CBS) or applied in a hierarchical design (MPS). The most widely used test of locomotion in rodents with SCI during the last 15 years is the Basso–Beattie–Bresnahan locomotor score (BBB-score) (73, 74) and variants thereof such as the BBB-subscore (75) and the Basso Mouse Scale (76).
In contrast to CBS and MPS, the BBB-score does not include any other tests; it solely includes a refined qualitative analysis of hindlimb movements, coordination, and trunk stability. Tests of open field locomotion have the important advantage that motor functions ranging from complete paralysis to normal locomotion can be assessed. Although used very extensively, the BBB-score do suffer from some significant limitations. The scoring is by nature subjective and the data is ordinal, nonlinear, but may nevertheless be statistically analyzed with parametric statistical methods according to Scheff et al. (77). In addition, the sensitivity is not sufficient at all parts of the assessment scale. Importantly, the BBB-score is not suitable for cervical injuries, with lack of sensitivity as a particular problem (78). Finally, all parameters of normal locomotion may not be reproduced in different strains of rats. As an example, we have found that paw placement in normal Harlan Sprague-Dawley rnu/rnu immune-deficient rats is different from standard Harlan Sprague-Dawley rats (unpublished observation).
To assess open field locomotion the animal is required to be spontaneously active. To increase the motivation to move, injured and normal rats can be evaluated simultaneously. This helps the observer to identify mild pathological changes in locomotion. Importantly, to keep the observer blinded, the normal rat must not be a rat from a control group, but rather taken from a reference group which is not a part of the study. A “house” or similar hiding place from the animal’s home cage placed on the other side of the open field is a strong incentive for the animal to move. Still, the overall health status of the animal can have effect on the performance, and certain animals also seem to be more affected by the test situation, showing significantly worse performance in the open field test situation compared to the home cage.
4.2 Beam and Grid Walk Tests
There are a number of functional tests that require that the animals can perform stepping. These tests are commonly used for mild-to-moderate thoracic contusion or compression injuries, but can be applied in animals with unilateral cervical injuries, in which the fore- and hindlimbs on one side of the animal are affected, provided some degree of stepping still is present. In the beam walk (or narrow beam walk) test for rats (79), the runway is 1–2 m long beams, width ranging from a few centimeters to less than 1 cm, with a flat surface and a soft mattress or elastic net below. In our lab, the test is performed by having the animal traverse a number of beams with successively decreasing width, each beam three times, until it falls off the beam, makes more foot slips than predetermined as exclusion criteria or refuses to walk the runway (22). The smallest width passed is recorded as the test result. Alternatively, a single beam, which gets successively narrower, can be used, again recording the smallest width passed.
In the grid walk test, the animal has to cross a meshwork (80) or a horizontal ladder (81), the test track typically about 10–30 cm wide and 1–2 m long with holes a few centimeters wide. Slightly raising the end of the track (meshwork or ladder) may increase the motivation for the animals to cross it. The number of paw misplacements and slips (i.e., when a paw slips through a hole in the mesh or between two rungs) is usually recorded. Since a mildly injured (or normal) rat can retract a misplaced paw very fast to correct the mistake, video recording is necessary for the evaluation. A major problem is that severely injured rats drag their hindlimbs without them slipping through the holes. To eliminate this problem, the number of steps when the animal takes a grip of the grid or rung can instead be used as outcome parameter. However, if animals with mild injuries are used, it will be necessary to quantify a large number of correct grips, making it a labor-intensive test.
A variant of the test, which has been used for mice, is to use a shorter ladder in a close to vertical position (82). The position of the ladder ascertains that paralyzed hindlimbs slip between the rungs of the ladder, thus counting of misplaced hindlimbs (or grip of rungs) correlates with motor function. Our experience is that the grid walk test is very sensitive to small functional differences in mildly injured rats, which was also noted by Lankhorst et al. (75) when they compared the grid walk test to BBB. However, since the beam walk and grid walk tests require stepping to have any discriminative power, they can only be used for mild-to-moderate SCI.
4.3 Gait Analysis
As an alternative to subjective scoring of locomotion, gait analysis of SCI animals can be used. The CatWalk (Noldus Information Technology, The Netherlands) or the TreadScan (CleverSys, USA) systems are commercially available alternatives with digital acquisition of foot-prints and automated analysis of a number of parameters such as swing and stance time, stride length, toe spread, track width, etc. Although as a general rule, it is advantageous to use objective quantitative analyses, the rather expensive equipment adds surprisingly little to the analytical power of common locomotion scoring (see, e.g., (83–85)). Gait analysis also requires weight-bearing stepping and thus shares this limitation with the beam- and grid walk tests. Consequently only mild-to-moderate injuries can be studied. Moderately injured animals that for some reason temporarily lose hindlimb weight support have to be assigned a non-quantitative (or “pseudo-quantitative”) result or be excluded from the analysis. Either way, it will lead to biased or skewed data. Thus, these tests have limited use, but certainly add very important objective information in selected SCI models.
4.4 Swimming Tests
Locomotion in paraparetic rodents can also be analyzed using tests of swimming performance, and similar to open field locomotion, scoring systems have been suggested (86, 87), which assess limb movements, coordination, trunk position and stability, tail movement, etc. One of the potential benefits of analyzing SCI animals during swimming is that trunk and hindlimb movement parameters in severely injured animals are more easily detected. In the absence of weight-bearing capacity, weak movements of the hindlimbs may be more easily evaluated in a freely floating animal, while such movements can be difficult to detect during open field locomotion. However, none of the scoring systems have been validated, and according to our experience they are less reliable than BBB and other assessments of locomotion. Importantly, while normal rats consistently get the maximum score in locomotion tests, swimming scores of normal rats vary (see, e.g., (87)).
In addition to scoring movements, kinematic analysis of high speed video recordings of swimming can be used to objectively quantify components of movements such as angular velocity, rather than being limited to a subjective scoring (88, 89). So far, the validity and reliability of these methods for SCI animals have not been studied.
Water at room temperature is a mild stressor particularly to mice. With a platform at the end of the swim tank, most rats will immediately swim to the platform, and climb it to get out of the water. It is our experience that during sessions of repeated swimming, it is important to leave the rat on the platform for some time (10–15 s) and then take it out of the tank to reinforce the reward of reaching the platform. If a rat is immediately brought back from the platform to the other end of the tank for a new trial, some rats will stop swimming after 3–4 trials, something we interpret as learned helplessness.
4.5 Tests of Forelimb Function
As mentioned, from a translational aspect one may argue that cervical injuries, representing about 50% of all SCI, should be specifically modeled. Functional analysis of unilateral cervical injuries requires test methods largely different from those used for lower thoracic and lumbar injuries. The asymmetrical nature of the injury places certain demands on the test, but most importantly, skilled forepaw movements, manual dexterity involving the functions of digital muscles can be studied. Methods to analyze these functions include testing skilled reaching for food pellets (62), removing pieces of adhesive tape applied to the forepaw or forehead (90), and forepaw use during vertical exploration-rearing (91) sometimes called forelimb preference test. Gait analysis or analysis of forelimb use during swimming as well as grid walk test for analysis of ipsilateral forelimb–hindlimb coordination have also been used to evaluate cervical spinal cord injuries. Analysis of these latter tests is complicated by the fact that the cervical injury will affect hindlimb motor function, even if gross motor functions are preserved.
If the involvement of sensorimotor integration is to be excluded from the test, a defined forepaw motor function can be studied using a test of grip strength (92). An important advantage using this type of test is its quantitative nature. As we have seen, most quantitative objective tests are much more complicated than grip tests, while simpler tests with few exceptions rely on subjective nonlinear scoring. In this respect, forepaw grip strength test is exceptionally useful. For a more extensive review on functional effects of cervical SCI in rats, see (93).
4.6 Choice of Tests
The importance of using multiple analysis methods has been nicely illustrated in another disease model. When analyzing locomotor deficits in a mouse model of Huntington’s disease, Pallier and colleagues (94) found that gait abnormalities present in the home cage, did not appear in an automated treadmill task. This illustrates that there can be major differences how non-motor factors affect performance in locomotion tests.
The anatomical substrates for different motor tests are of course different, and this can have surprisingly large consequences. In an interesting study on unilateral cervical injuries in rats, Anderson and colleagues (92) showed that recovery of forepaw motor function after medial or lateral hemisections depended on the test used. While rats with dorsal-medial transection often permanently lost gripping ability but improved in the food retrieval test, rats with lateral transections recovered in grip strength but suffered permanent deficits in food retrieval. Although it is not known if recovery in these tests is due to plasticity or regeneration, one must assume that a truly regenerative treatment tested in a cervical injury model would show very different effectiveness depending on the functional tests applied. Again, this emphasizes the need to use a wide range of functional tests when neuroprotective or regenerative treatments are evaluated after SCI.
While the relevance of SCI models can be inferred from the comparison of the biophysics of the injuries as well the resulting progressive pathological changes in rodents and human patients, the situation is different when it comes to functional evaluation of these injury models. There are neuroanatomical and neurophysiological differences between rodents and human beings that make functional comparison complicated. But it is an even more complex task to determine how a given improvement in rodent hindlimb motor performance can be translated to a functional improvement in human patients.
For validation of tools for functional analyses, the result of the functional assessment is usually correlated with a morphological parameter describing the extent of the lesion. Most commonly this parameter reflects the amount of spared white matter at the epicenter of the injury or the volume of the remaining spinal cord tissue. The rationale for measuring spared white matter is that hindlimb motor function mainly depends on descending (and ascending) axonal tracts in the white matter. If instead spared total spinal cord tissue is used, gray matter including neuronal tissue participating in local reflex circuitry is included. For most experimental injuries there is a close correlation between loss of gray and white matter (see, e.g., (57)). Thus, correlating spared white matter or spared total tissue will often give similar results for thoracic injuries, while gray matter will be relatively more important for loss of function after cervical injuries.
However, correlation of functional outcome with a morphological parameter is not evidence that the test is suitable for all purposes. It tells us how functions are lost with increasing injury severity. For studies on recovery, neuroprotection, or regeneration, some aspects of locomotion may be more affected than others. An example of this led to the introduction of the so-called BBB-subscore (75). These researchers observed that toe clearance and paw position are parameters lost after mild injuries while forelimb–hindlimb coordination is only affected after moderate injuries. However, limb coordination most often does not recover in spite of recovery of other functions (see (95) for discussion). Likewise, certain pathways may be more amenable to drug-enhanced regeneration, which could lead to a recovery of functions that do not match the expected sequence. To avoid the risk of missing important effects, a variety of functional tests should be included in the outcome assessment.
Functional and behavioral tests are not just used as outcome measures for neuroprotective and regenerative treatments, but also to detect side effects of treatments. Substances such as chondroitinase ABC (degrading inhibitory chondroitin sulfate proteoglycans) and Nogo antisera promote regeneration and plasticity, and enhance motor recovery after experimental SCI (96, 97). However, such treatments as well as neurotrophic factor or cell delivery may increase the risk of adverse effects. Among side effects, the greatest concern is probably increased pain, hyperalgesia, or allodynia, i.e., pain perception in response to non-painful stimuli (98, 99). Hence, analysis of pain perception should be added to the functional tests performed. The modalities typically studied are mechanical and thermal, assessed by applying (1) pressure, using calibrated filaments of different dimensions (“von Frey hairs”) which are pressed onto the skin until the filament becomes bent (100, 101); (2) cold, by applying ethyl chloride or acetone on the paw or the shaved skin (102); (3) heat, by placing the rat on a hot plate, or focusing light on the paw or shaved skin (103). Rats react to painful stimuli in several ways, but vocalization has been shown to be the most reliable response. The functional assessment methods here described are summarized in Table 2.
Table 2
Summary of functional assessment methods
Functional test | Function assessed | Uni-/bilateral | SCI type | SCI severity | Species/strain | References | |
|---|---|---|---|---|---|---|---|
Open field locomotion | |||||||
Combined Behavioral Score (CBS) | Sensory–motor function (hindlimb) | Bilateral | Contusion—weight-drop | Mild/moderate/severe | Rat: Sprague-Dawley | (72) | |
Motor performance score (MPS) | Sensory–motor function (hindlimb) | Bilateral | Photochemical lesion/transection | Mild/moderate/severe | Rat: Sprague-Dawley | (22) | |
Basso–Beattie–Bresnahan (BBB) | Open field locomotion (hindlimb) | Bilateral | Contusion—weight-drop(NYU, OSU)/transection | Mild/moderate (contusion) | Rat: Sprague-Dawley/Long-Evans | ||
Severe (transection) | |||||||
Basso Mouse Score (BMS) | Open field locomotion (hindlimb) | Bilateral | Contusion—weight-drop (ESCID, OSU, IH)/transection | Mild/moderate/severe | Mouse: C57BL/6, C57BL/10, B10.PL, BALB/c, C57BL/6 × 129S6 | (76) | |
Grid walk/ladder walk | Sensory–motor function (hind- and fore limb) | Bilateral/unilateral | Dorsal hemisection | Mild/moderate | Rat: Sprague-Dawley | (80) | |
Compression injury | Mouse: C57BL/6J | (81) | |||||
(82) | |||||||
Beam walk | Sensory–motor function | Bilateral/unilateral | Cortical lesion | Mild/moderate | Rat: Charles River | (79) | |
Photochemical lesion/transection | Rat: Sprague-Dawley | (22) | |||||
Gait analysis | |||||||
CatWalk/TreadScan | Sensory–motor function (hind- and forelimb) | Bilateral/unilateral | Contusion—weight-drop (NYU)/dorsal hemisection | Mild/moderate | Rat: Wistar | (83) | |
Contusion-LISA | Rat: Wistar | (84) | |||||
Mouse: C57Bl/6 | (85) | ||||||
Swimming tests | |||||||
LSS | Sensory–motor function (hind- and forelimb) | Bilateral/unilateral | Contusion—weight-drop (NYU) | Mild/moderate/severe | Rat: Sprague-Dawley | (86) | |
Kinematics | Dorsal or lateral hemisection | Moderate/severe | Rat: Sprague-Dawley | (88) | |||
Rat: Lewis | (89) | ||||||
Mouse: C57BL/6 | Other tests | ||||||
Forelimb reaching test | Sensory–motor function (forelimb) | Bilateral/unilateral | Contusion—weight-drop | Moderate | Rat: Sprague-Dawley | (62) | |
Forelimb preference test | Sensory–motor function (forelimb) < div class='tao-gold-member'>
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