Using templated agarose scaffolds to promote axon regeneration through sites of spinal cord injury.

The past 30 years of research in spinal cord injury (SCI) have revealed that, under certain conditions, some types of axons are able to regenerate. To aid these axons in bridging the lesion site, many experimenters place cellular grafts at the lesion. However, to increase the potential for functional recovery, it is likely advantageous to maximize the number of axons that reach the intact spinal cord on the other side of the lesion. Implanting linear-channeled scaffolds at the lesion site provides growing axons with linear growth paths, which minimizes the distance they must travel to reach healthy tissue. Moreover, the linear channels help the regenerating axons maintain the correct mediolateral and dorsoventral position in the spinal cord, which may also improve functional recovery by keeping the axons nearer to their correct targets. Here, we provide a protocol to perform a full spinal cord transection in rats that accommodates an implanted scaffold.

Motor axonal regeneration after partial and complete spinal cord transection.

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.

PTEN deletion enhances the regenerative ability of adult corticospinal neurons.

Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. We found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons was lost and this was accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminished neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhanced compensatory sprouting of uninjured CST axons and enabled successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possessed the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury.

Matrix metalloproteinase-7 facilitates immune access to the CNS in experimental autoimmune encephalomyelitis.

BACKGROUND: Metalloproteinase inhibitors can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Matrix metalloproteinase-9 (MMP-9) has been implicated, but it is not clear if other MMPs are also involved, including matrilysin/MMP-7 - an enzyme capable of cleaving proteins that are essential for blood brain barrier integrity and immune suppression. RESULTS: Here we report that MMP-7-deficient (mmp7-/-) mice on the C57Bl/6 background are resistant to EAE induced by myelin oligodendrocyte glycoprotein (MOG). Brain sections from MOG-primed mmp7-/-mice did not show signs of immune cell infiltration of the CNS, but MOG-primed wild-type mice showed extensive vascular cuffing and mononuclear cell infiltration 15 days after vaccination. At the peak of EAE wild-type mice had MMP-7 immuno-reactive cells in vascular cuffs that also expressed the macrophage markers Iba-1 and Gr-1, as well as tomato lectin. MOG-specific proliferation of splenocytes, lymphocytes, CD4+ and CD8+ cells were reduced in cells isolated from MOG-primed mmp7-/- mice, compared with MOG-primed wild-type mice. However, the adoptive transfer of splenocytes and lymphocytes from MOG-primed mmp7-/- mice induced EAE in naïve wild-type recipients, but not naïve mmp7-/- recipients. Finally, we found that recombinant MMP-7 increased permeability between endothelial cells in an in vitro blood-brain barrier model. CONCLUSION: Our findings suggest that MMP-7 may facilitate immune cell access or re-stimulation in perivascular areas, which are critical events in EAE and multiple sclerosis, and provide a new therapeutic target to treat this disorder.

Electrically evoked locomotor activity in the turtle spinal cord hemi-enlargement preparation.

We assessed the locomotor capacity of the left half of the spinal cord hindlimb enlargement in low-spinal turtles. Forward swimming was evoked in the left hindlimb by electrical stimulation of the right dorsolateral funiculus (DLF) at the anterior end of the third postcervical spinal segment (D3). Animals were held by a band-clamp in a water-filled tank so that hindlimb movements could be recorded from below with a digital video camera. Left hindlimb hip and knee movements were tracked while electromyograms (EMGs) were recorded from left hip and knee muscles. In turtles with intact spinal cords, electrical stimulation of the right D3 DLF evoked robust forward swimming movements of the left hindlimb, characterized by rhythmic alternation between hip flexor (HF) and hip extensor (HE) EMG discharge, with knee extensor (KE) bursts occurring during the latter part of each HE-off phase. After removing the right spinal hemi-enlargement (D8-S2), DLF stimulation still evoked rhythmic locomotor movements and EMG bursts in the left hindlimb that included HF-HE alternation and KE discharge. However, post-surgical movements and EMG bursts had longer cycle periods, and movements showed lower amplitudes compared to controls. These results show that (1) sufficient locomotor CPG circuitry resides within the turtle spinal hemi-enlargement to drive major components of the forward swim motor pattern, (2) contralateral circuitry contributes to the excitation of the locomotor CPG for a given limb, and (3) a sufficient portion of the descending DLF pathway crosses over to the contralateral cord anterior to the hindlimb enlargement to activate swimming.

Location of spinal cord pathways that control hindlimb movement amplitude and interlimb coordination during voluntary swimming in turtles.

We performed mechanical lesions of the midbody (D2-D3; second to third postcervical spinal segments) spinal cord in otherwise intact turtles to locate spinal cord pathways that 1) activate and control the amplitude of voluntary hindlimb swimming movements and 2) coordinate hindlimb swimming with the movement of other limbs. Pre- and postlesion turtles were held by a band clamp around the carapace just beneath the water surface in a clear Plexiglas tank and videotaped from below so that kinematic measurements could be made of voluntary forward swimming with motion analysis software. Movements of the forelimbs (wrists) and hindlimbs (knees and ankles) were tracked relative to stationary reference points on the plastron to obtain bilateral measurements of hip and forelimb angles as functions of time along with foot trajectories. We measured changes in limb movement amplitude, cycle period, and interlimb phase before and after spinal lesions. Our results indicate that locomotor command signals that activate and regulate the amplitude of voluntary hindlimb swimming travel primarily in the dorsolateral funiculus (DLF) at the D2-D3 level and cross over to drive contralateral hindlimb movements. This suggests that electrical stimulation of the D3 DLF, which was previously shown to evoke swimming movements in the contralateral hindlimb of low-spinal turtles, activated the same locomotor command pathways that the animal uses during voluntary behavior. We also show that forelimb-hindlimb coordination is maintained by longitudinal spinal pathways that are largely confined to the ventrolateral funiculus (VLF) and mediate phase coupling of ipsilateral limbs, presumably by interenlargement propriospinal fibers.

Crossed commissural pathways in the spinal hindlimb enlargement are not necessary for right left hindlimb alternation during turtle swimming.

We examined the coordination between right and left hindlimbs during voluntary forward swimming in adult red-eared turtles, before and after midsagittal section of the spinal cord hindlimb enlargement (segments D8-S2) or the enlargement plus the first preenlargement segment (D7-S2). Our purpose was to assess the role of crossed commissural axons in these segments for right-left hindlimb alternation during voluntary locomotion. Midsagittal splitting severed commissural fibers and separated the right and left halves of the posterior spinal cord. Adult turtles (n = 9) were held by a band clamp around the shell in a water-filled tank while digital video of forward swimming was recorded from below and computer analyzed with motion analysis software. In a subset of these animals (n = 5), we also recorded electromyograms from hip extensor and/or hip flexor muscles on both sides. Surprisingly, splitting spinal segments D8-S2 or D7-S2 did not affect the strength of out-of-phase coordination between right and left hindlimbs, although hindlimb movement amplitudes were reduced compared with presurgical controls. These results show that commissural axons in the hindlimb enlargement and preenlargement cord are not necessary for right-left hindlimb alternation during voluntary swimming. We suggest that alternating propriospinal drive from the right and left sides of the forelimb enlargement maintains the out-of-phase coordination of right and left hindlimbs in the bisected-cord preparation. Our data support the hypothesis that descending propriospinal (forelimb-hindlimb) and crossed commissural (hindlimb-hindlimb) spinal cord pathways function together as redundant mechanisms to sustain right-left hindlimb alternation during turtle locomotion.