Corticospinal tract fibers cross the ephrin-B3-negative part of the midline of the spinal cord after brain injury

doi:10.1016/j.neures.2010.12.004

Neuroscience Research

Volume 69, Issue 3, March 2011, Pages 187-195

https://doi.org/10.1016/j.neures.2010.12.004 Get rights and content

Abstract

The fibers of corticospinal tract (CST), which control fine motor function, predominantly project to the contralateral spinal cord, not recross to the ipsilateral side. Ephrin-B3, which is expressed in the midline of the spinal cord, and its receptor, EphA4, are crucial for preventing CST fibers from recrossing the midline in the developing spinal cord. However, these fibers can cross the midline to the denervated side after a unilateral CST or cortical injury. We determined the reason CST fibers can cross the midline after a cortical injury and the changes in ephrin-B3–EphA4 signaling associated with such a crossing. We first examined axonal sprouting from CST fibers after unilateral ablation of the motor cortex in postnatal and adult mice. CST fibers crossed the midline of the spinal cord after cortical ablation, especially when conducted during the early postnatal period. These fibers were well associated with functional recovery after the injury. We next assessed the mRNA expression of ephrin-B3 and EphA4 before and after the ablation. Surprisingly, no changes were detected in the expression patterns. We found, however, that ephrin-B3 expression in the ventral part of the midline disappeared after postnatal day 9 (P9), but was pronounced along the entire midline before P6. Most of the CST fibers crossed the midline through the ventral region, where ephrin-B3 expression was absent. Our results suggest that ephrin-B3 is not expressed along the entire midline of the spinal cord, and sprouting axons can cross the midline at ephrin-B3-negative areas.

Research highlights

▶ Sprouting axons of the CST can cross the midline at ephrin-B3-negative areas. ▶ Ephrin-B3 is not expressed along the entire midline of the spinal cord. ▶ Ephrin-B3 expression in the midline disappears after postnatal day 9.

Introduction

The corticospinal tract (CST) is a bundle of fibers that originate from neurons in the sensorimotor cortex and predominantly descend in the contralateral side of the spinal cord. This tract controls fine motor function, and damage to this tract causes permanent deficits in it. In mice, most CST fibers cross to the opposite side at the pyramidal decussation, descend in the ventral portion of the dorsal funiculus, and project to the interneurons of the contralateral spinal cord, and do not recross the midline in the spinal cord to innervate the ipsilateral side. It was revealed that the expression of ephrin-B3 in the midline of the spinal cord and that of its receptor, EphA4, in the cortical neurons are crucial for preventing the CST fibers from recrossing the midline in the spinal cord during postnatal development (Dottori et al., 1998, Coonan et al., 2001, Kullander et al., 2001, Yokoyama et al., 2001). Genetic ablation of ephrin-B3 in mice results in bilateral innervation by the CST fibers (Kullander et al., 2001, Yokoyama et al., 2001) and EphA4-knockout mice show an identical phenotype (Dottori et al., 1998, Coonan et al., 2001).

There is considerable evidence that under certain conditions, CST fibers can recross the midline in the spinal cord. For example, middle cerebral artery occlusion (MCAO) induces axonal sprouting across the midline of the spinal cord in rodents (Liu et al., 2007), and treatment with a Nogo receptor antagonist after MCAO increases axonal sprouting and enhances functional recovery (Lee et al., 2004). Further, the sprouting is more pronounced if the lesion is induced during the early postnatal period (Reinoso and Castro, 1989, Kuang and Kalil, 1990, Ono et al., 1990, Takahashi et al., 2009). Axonal sprouting among intact CST fibers on the denervated side of the spinal cord might lead to the formation of an alternative network of motor fibers and contribute to functional recovery after the development of lesions involving the cerebral cortex or CST (Murphy and Corbett, 2009, Benowitz and Carmichael, 2010). However, it remains unknown why CST fibers can cross the midline despite the presence of the ephrin-B3 midline barrier.

In this study, we sought to determine (1) the reason CST fibers can cross the midline of the spinal cord despite ephrin-B3 expression and (2) the effects of a unilateral cortical lesion on ephrin-B3–EphA4 signaling. Although postnatal expression of ephrin-B3 and EphA4 has been investigated (Kullander et al., 2001, Liebl et al., 2003, Benson et al., 2005), the changes in this expression and the relation between this expression and the ability of CST fibers in the spinal cord to cross the midline after a cortical lesion have not yet been evaluated. In the current study, we first confirmed that CST fibers cross the midline of the spinal cord after unilateral cortical ablation in postnatal and adult mice. We next assessed the expression of ephrin-B3 and EphA4 in the mice. We found that ephrin-B3 was not expressed along the entire midline of the spinal cord, and sprouting axons crossed the midline at ephrin-B3-negative areas.

Section snippets

Mice

In this study, we used C57BL/6J mice at postnatal day 9 (Charles River; P9, n = 20) and adulthood (8 weeks, n = 20). The mice were divided into the following categories in each age group: (1) unilateral cortical ablation followed by anterograde tracing of CST (n = 5 for each age group), (2) unilateral cortical ablation followed by behavioral tests (n = 5 for each age group). Sham-operated mice (n = 5 for all the groups) underwent the same surgical procedures, except for the cortical ablation. All the

CST axons crossed the midline of the spinal cord after cortical ablation

We compared the potential for axonal sprouting of the CST following unilateral ablation of the sensorimotor cortex between P9 and adult mice (Fig. 1A). We firstly determined whether the cortical damage led to a complete destruction of the CST. The cervical spinal cord was stained with PKCγ, a marker of the CST, at 1 week after the injury (Bradbury et al., 2002). PKCγ immunoreactivity was present bilaterally in the dorsal CST of the cervical spinal cord in sham-operated mice (data not shown).

Discussion

In this study, we report that ephrin-B3 is expressed only in the dorsal part of the midline in the adult spinal cord, and after the development of a unilateral cortical lesion, the axons originating from intact CST neurons cross to the denervated side of the cervical cord through the ephrin-B3-negative ventral part of the midline. The ephrin-B3 receptor, EphA4, is expressed in the CST axons in neonatal and adult mice. This expression is maintained after the development of a unilateral cortical

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These discrepancies in locomotion have questioned the role that CST plays in rodent locomotor function, although differing locomotor recovery with different mutants might be related to inadequate CST fiber sprouting and connection with caudal targets. Additionally, more regulatory proteins including the axon guidance molecule, ephrinB3, and its receptor EphA4 [99,100], Krüppel-like Factor 7 (KLF7) [101], signal transducer and activator of transcription 3 (STAT3) [102], conventional protein kinases C (cPKC) [103], transcriptional factor Sox11 [104], and the mTOR substrate, S6 Kinase 1 (S6K1) [105] were shown to influence CST axonal sprouting. Again, the amount of CSTs axons detected in the lesion site is very low (Fig. 3D), which indicate that robust regeneration of CSTs to across the lesion site were hard to realize by the existing methods or treatments.
Complete transected spinal cord injury (SCI) severely influences the quality of life and mortality rates of animals and patients. In the past decade, many simple and combinatorial therapeutic treatments have been tested in improving locomotor function in animals with this extraordinarily challenging SCI. The potential mechanism for promotion of locomotor function relies either on direct motor axon regeneration through the lesion gap or indirect neuronal relay bridging to functionally reconnect transected spinal stumps. In this review, we first compare the advantages and problems of complete transection SCI animal models with other prevailing SCI models used in motor axon regeneration research. Next, we enumerate some of the popular bio-scaffolds utilized in complete SCI repair in the last decade. Then, the current state of motor axon regeneration as well as its role on locomotor improvement of animals after complete SCI is discussed. Last, the current approach of directing endogenous neuronal relays formation to achieve motor function recovery by well-designed functional bio-scaffolds implantation in complete transected SCI animals is reviewed. Although facilitating neuronal relays formation by bio-scaffolds implantation appears to be more practical and feasible than directing motor axon regeneration in promoting locomotor outcome in animals after complete SCI, there are still challenges in neuronal relays formation, maintaining and debugging for spinal cord regenerative repair.
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…once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably.
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Citation Excerpt :
Therefore, unlike in development, the midline of the adult CNS seems molecularly virtual and not enriched in specific axon guidance molecules. Nevertheless, all ‘midline’ cues are still highly expressed in the adult brain either by neurons or by glia cells and their expression can be upregulated in sites of injury, in the cortex or spinal cord [106–110]. Likewise, most commissural axon guidance receptors are still expressed by adult neurons.
Commissural neurons project their axons across the midline of the nervous system to contact neurons on the opposite side. Although their existence has been known for more than a century, the function of brain commissures, as well as their diversity and evolutionary advantage, are far from understood. Recent genetic studies in mammals have led to the identification of subsets of commissural neurons, which, in the hindbrain and spinal cord, control the tuning and bilateral coordination of locomotion. The molecular mechanisms and transcriptional programs which specify axonal laterality during development are also now being elucidated. Finally, new studies have confirmed that axonal laterality is plastic and that facilitating the commissural sprouting of axon collaterals might influence functional recovery after brain injury.
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Notably, peak GAP-43 expression in CSC 7 and 14 days after FL-SMC lesion occurs during periods of spontaneous recovery of forelimb function (as indicated by improvements in Montoya Staircase performance between 3 and 14 days post-stroke) and returns to baseline as recovery plateaus. Based on data showing sprouting of axon collaterals from uninjured CST into stroke-denervated gray matter in the spinal cord (DeVetten et al., 2010; Jayaram et al., 2012; LaPash Daniels et al., 2009; Liu et al., 2007, 2008, 2009, 2012; Omoto et al., 2011; Puig et al., 2010; Reitmeir et al., 2011; Starkey et al., 2012; Thomalla et al., 2004; Wang et al., 2012; Yu et al., 2009; Zhang et al., 2010), we postulate that the increases in spinal cord levels of GAP-43 after FL-SMC would primarily reflect sprouting of descending axons from the CST into denervated spinal gray matter. However, given that axonal rewiring likely includes ipsilateral (CST fibers originating in uninjured motor cortex sprouting into stroke affected gray matter) and contralateral (sprouting of spared axons originating in peri-infarct cortex) components, our measures of spinal plasticity provide a summation of structural plasticity at the level of the cervical or lumbar cord incorporating sprouting from CST originating in both cortical hemispheres, as well as neurite outgrowth from interneurons in the spinal cord (as has been reported after spinal cord injury (Bareyre et al., 2004; Vavrek et al., 2006)).
Stroke induces pathophysiological and adaptive processes in regions proximal and distal to the infarct. Recent studies suggest that plasticity at the level of the spinal cord may contribute to sensorimotor recovery after cortical stroke. Here, we compare the time course of heightened structural plasticity in the spinal cord against the temporal profile of cortical plasticity and spontaneous behavioral recovery. To examine the relation between trophic and inflammatory effectors and spinal structural plasticity, spinal expression of brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was measured. Growth-associated protein 43 (GAP-43), measured at 3, 7, 14, or 28 days after photothrombotic stroke of the forelimb sensorimotor cortex (FL-SMC) to provide an index of periods of heightened structural plasticity, varied as a function of lesion size and time after stroke in the cortical hemispheres and the spinal cord. Notably, GAP-43 levels in the cervical spinal cord were significantly increased after FL-SMC lesion, but the temporal window of elevated structural plasticity was more finite in spinal cord relative to ipsilesional cortical expression (returning to baseline levels by 28 post-stroke). Peak GAP-43 expression in spinal cord occurred during periods of accelerated spontaneous recovery, as measured on the Montoya Staircase reaching task, and returned to baseline as recovery plateaued. Interestingly, spinal GAP-43 levels were significantly correlated with spinal levels of the inflammatory cytokines TNF-α and IL-6 as well as the neurotrophin NT-3, while a transient increase in BDNF levels preceded elevated GAP-43 expression. These data identify a significant but time-limited window of heightened structural plasticity in the spinal cord following stroke that correlates with spontaneous recovery and the spinal expression of inflammatory cytokines and neurotrophic factors.
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^☆: This work was supported by a research grant from the Grant-in-Aid for Young Scientists (S) from JSPS.

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Corticospinal tract fibers cross the ephrin-B3-negative part of the midline of the spinal cord after brain injury☆

Abstract

Research highlights

Introduction

Section snippets

Mice

CST axons crossed the midline of the spinal cord after cortical ablation

Discussion

Brain Res. Bull.

Neurobiol. Dis.

Prog. Neurobiol.

Brain Res. Dev. Brain Res.

Brain Res.

Brain Res.

J. Neurosci. Methods

Brain Dev.

Exp. Neurol.

Exp. Neurol.

Neuron

J. Neurosci. Methods

Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth

Proc. Natl. Acad. Sci. U.S.A.

Chondroitinase ABC promotes functional recovery after spinal cord injury

Nature

Motor cortex bilateral motor representation depends on subcortical and interhemispheric interactions

J. Neurosci.

Eph tyrosine kinase receptor EphA4 is required for the topographic mapping of the corticospinal tract

Proc. Natl. Acad. Sci. U.S.A.

Chronic electrical stimulation of the intact corticospinal system after unilateral injury restores skilled locomotor control and promotes spinal axon outgrowth

J. Neurosci.