Regular ArticleAssessing behavioural function following a pyramidotomy lesion of the corticospinal tract in adult mice
Introduction
Following injury to the adult mammalian central nervous system (CNS), axonal regeneration and plasticity is extremely limited. Most of the functional deficits resulting from injury to the spinal cord are caused by the interruption of descending and ascending axonal tracts, and the persistence of these deficits is due to their failure to regenerate (Schwab and Bartholdi, 1996, Houle and Tessler, 2003, Selzer, 2003). This failure is thought to be due to the combination of a limited inherent regenerative capacity of the axons (Schwab and Bartholdi, 1996, Neumann and Woolf, 1999); an insufficiency of trophic factors (Widenfalk et al., 2001, Jones et al., 2001) and the presence of growth inhibitory molecules, such as chondroitin sulphate proteoglycans (Fawcett and Asher, 1999, Silver and Miller, 2004) and myelin-associated neurite growth inhibitors (Schwab, 2002, McGee and Strittmatter, 2003, Filbin, 2003). Although a number of recent studies have demonstrated a capacity for some spinal systems to spontaneously sprout in the adult (Raineteau and Schwab, 2001, Weidner et al., 2001, Bareyre et al., 2004), the fact is that there is limited functional recovery following CNS injury and there is a great potential for enhancing CNS plasticity with the aim of promoting functional improvements.
One recent focus has been to study intact systems following CNS injury, with the aim of enhancing plasticity of spared fibres. A number of studies have investigated whether intact corticospinal tract (CST) projections can be induced to sprout into the denervated side of the spinal cord following a unilateral pyramidotomy lesion. This lesion transects one half of the CST at the level of the pyramids in the brainstem, rostral to the spinal cord. The reactions of the uninjured tract projecting in the spinal cord can then be studied following various intervention strategies aimed at promoting plasticity. Sprouting of the intact CST has been observed in this lesion model following treatments which neutralise inhibitory factors associated with CNS myelin (Vanek et al., 1998, Thallmair et al., 1998, Z'Graggen et al., 1998, Raineteau et al., 1999, Raineteau et al., 2002, Blochlinger et al., 2001, Bareyre et al., 2002) and treatments which promote growth, such as delivery of neurotrophic factors (Zhou and Shine, 2003, Zhou et al., 2003). Pyramidotomy results in deficits in voluntary motor function of the forepaws (Steward et al., 2004, Lacroix et al., 2004) and impairments in forelimb function such as limb use asymmetry (Thallmair et al., 1998, Z'Graggen et al., 1998), precise stepping (Z'Graggen et al., 1998, Metz and Whishaw, 2002), somatosensory function (Thallmair et al., 1998), locomotion and gait (Metz et al., 1998, Fanardjian et al., 2001) and skilled paw reaching (Whishaw et al., 1993, Thallmair et al., 1998, Z'Graggen et al., 1998, Weidner et al., 2001). However, all these studies have used a rat model and there has been a distinct lack of studies describing this lesion in adult mice, other than to look at the glial reaction caused by this injury (Leong et al., 1995) and no one, to our knowledge, has investigated behavioural deficits after pyramidotomy in mice. Given the increasing use of genetically modified mice, it would be beneficial to develop a murine pyramidotomy model and to characterise a number of reliable behavioural outcome measures for assessing functional deficits. This model would be valuable for future studies examining the effects of gene deletion or other strategies aimed at promoting plasticity following CNS injury.
Here, we develop a mouse pyramidotomy lesion model and assess functional impairments in the following behavioural paradigms: rearing, grid walking, tape removal, CatWalk gait analysis and staircase pellet reaching. The rearing test is specifically designed to encourage use of the forelimbs for vertical exploration of a cylinder in rats (Napieralski et al., 1998, Liu et al., 1999, Schallert et al., 2000, Soblosky et al., 2001, MacLellan et al., 2002, Woodlee et al., 2005), and mice (Baskin et al., 2003, Wells et al., 2005). This test assesses the animals' voluntary use of the forelimbs for upright postural weight bearing support (Baskin et al., 2003) and thus highlights limb use asymmetries. The grid walking test assesses deficits in voluntary motor control and limb movements involved in precise stepping, co-ordination and accurate paw placement (Z'Graggen et al., 1998, Napieralski et al., 1998, Metz et al., 2000, Soblosky et al., 2001, Ma et al., 2001, Schucht et al., 2002, MacLellan et al., 2002, Selak and Fritzler, 2004). Grid walking also requires sensory feedback for limb placement (Baskin et al., 2003) and thus assesses deficits in sensorimotor function (Z'Graggen et al., 1998, Merkler et al., 2001, Zhang et al., 2002, Menet et al., 2003). The tape removal test is particularly sensitive to somatosensory deficits (Thallmair et al., 1998) and has been used in both rats (Thallmair et al., 1998, Schallert et al., 2000, Bradbury et al., 2002, Onifer et al., 2005) and mice (Wells et al., 2005). The CatWalk is a novel test of gait which has the benefit of measuring a number of locomotor-related assessments simultaneously (Hamers et al., 2001) and has been used in various experimental paradigms with rats (Hamers et al., 2001, Lankhorst et al., 2001, van Meeteren et al., 2003, Vrinten and Hamers, 2003, Vogelaar et al., 2004) and mice (Vogelaar et al., 2004). The staircase pellet reaching test assesses accurate forepaw reaching and forelimb motor control and has a history of use in rats (Montoya et al., 1991, Whishaw et al., 1997, Nikkhah et al., 1998, Colbourne et al., 2000, MacLellan et al., 2002, Samsam et al., 2004) but less in mice (Baird et al., 2001). This task was chosen because unilateral lesions of the CST are known to abolish directed forepaw retrieval by the ipsilateral paw (Keyvan-Fouladi et al., 2003).
Here, we report the development of a unilateral pyramidotomy model in mice and the methodology for a number of reliable behavioural tests to assess functional deficits following this lesion.
Section snippets
Animals and surgery
Adult male C57BL/6 mice (Harlan UK Ltd., 20–25 g, 6–8 weeks old) were used in these studies and all surgical procedures were performed in accordance with U.K. Home Office regulations (Animals Scientific Procedures Act, 1986). Mice were anaesthetised with a mixture of medetomidine (0.5 mg/kg) and ketamine (75 mg/kg) and sterile precautions were used throughout. Surgical procedures were adapted from previous studies using rats to perform a pyramidotomy lesion in mice, whereby a unilateral lesion
Histological assessment of lesion
Gross anatomy revealed a transection in the right pyramidal tract of the brainstem in lesioned mice (Fig. 1A). Apart from a small ventral uncrossed component, the CST decussates below the level of the lesion; therefore, a lesion of the right pyramidal tract will denervate the CST on the left side of the spinal cord (illustrated in schematic, Fig. 1B). The extent of the pyramidotomy lesion was determined using immunostaining for protein kinase Cγ (PKCγ) in the brainstem and spinal cord (Fig. 1).
Discussion
In the present study, we have established a consistent and reliable pyramidotomy lesion model in adult mice. We selected a number of behavioural tests to evaluate effects on both motor and sensory behaviours and assessed them on their ability to consistently measure deficits in forelimb behaviours following this lesion. We conclude that the rearing, tape removal, grid walking and CatWalk tests translate well to mice.
Conclusion
We have developed a robust, reproducible and consistent model for assessing a unilateral pyramidotomy lesion of the CST in mice. We have also developed sensitive and reliable behavioural outcome measures for assessing deficits following this lesion. The majority of tests revealed lesion effects on forepaw function, to varying degrees, with the rearing, grid walking, tape removal and CatWalk tests in particular revealing these effects throughout the testing period. The development of a
Acknowledgments
This work was supported by the Medical Research Council, The International Spinal Research Trust, and the Wellcome Trust. The authors would like to thank Prof. Patrick Doherty for invaluable input and John Grist and Jonathan Ramsey for technical support.
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