Minocycline protects motor but not autonomic neurons after cauda equina injury.

Conus medullaris/cauda equina injuries typically result in loss of bladder, bowel, and sexual functions, partly as a consequence of autonomic and motor neuron death. To mimic these injuries, we previously developed a rodent lumbosacral ventral root avulsion (VRA) injury model, where both autonomic and motor neurons progressively die over several weeks. Here, we investigate whether minocycline, an antibiotic with putative neuroprotective effects, may rescue degenerating autonomic and motor neurons after VRA injury. Adult female rats underwent lumbosacral VRA injuries followed by a 2-week treatment with either minocycline or vehicle injected intraperitoneally. The sacral segment of the spinal cord was studied immunohistochemically using choline acetyltransferase (ChAT) and activated caspase-3 at 4 weeks post-operatively. Minocycline increased the survival of motoneurons but not preganglionic parasympathetic neurons (PPNs). Further investigations demonstrated that a larger proportion of motoneurons expressed activated caspase-3 compared to PPNs after VRA injury and indicated an association with minocycline's differential neuroprotective effect. Our findings suggest that minocycline may protect degenerating motoneurons and expand the therapeutic window of opportunity for surgical repair of proximal root lesions affecting spinal motoneurons.

Functional reinnervation of the rat lower urinary tract after cauda equina injury and repair.

Conus medullaris and/or cauda equina forms of spinal cord injury commonly result in a permanent loss of bladder function. Here, we developed a cauda equina injury and repair rodent model to investigate whether surgical implantation of avulsed lumbosacral ventral roots into the spinal cord can promote functional recovery of the lower urinary tract. Adult female rats underwent sham surgery (n = 6), bilateral L5-S2 ventral root avulsion (VRA) injury (n = 5), or bilateral L5-S2 VRA followed by an acute implantation of the avulsed L6 and S1 ventral roots into the conus medullaris (n = 6). At 12 weeks after operation, the avulsed group demonstrated urinary retention, absence of bladder contractions and external urethral sphincter (EUS) electromyographic (EMG) activation during urodynamic recordings, increased bladder size, and retrograde death of autonomic and motoneurons in the spinal cord. In contrast, the implanted group showed reduced urinary retention, return of reflexive bladder voiding contractions coincident with EUS EMG activation, anatomical reinnervation of the EUS demonstrated by retrograde neuronal labeling, normalization of bladder size, and a significant neuroprotection of both autonomic and motoneurons. In addition, a positive correlation between motoneuronal survival and voiding efficiency was observed in the implanted group. Our results show that implantation of avulsed lumbosacral ventral roots into the spinal cord promotes reinnervation of the urinary tract and return of functional micturition reflexes, suggesting that this surgical repair strategy may also be of clinical interest after conus medullaris and cauda equina injuries.

Novel repair strategies to restore bladder function following cauda equina/conus medullaris injuries.

Trauma to the thoracolumbar junction or lumbosacral spine may result in a conus medullaris or cauda equina syndrome. In both conditions, symptoms typically include paraparesis or paraplegia, sensory impairment, pain, as well as bladder, bowel, and sexual dysfunctions. We present in this review a series of neural repair strategies that have been developed to address the unique features and challenges of subjects with a conus medullaris or cauda equina syndrome. We address, in particular, neural repair strategies that may have a translational research potential to restore bladder function. Recent animal injury models have suggested that a progressive retrograde death of both autonomic and motor neurons may contribute to the neurological deficits in subjects with conus medullaris and cauda equina injuries. For subjects with acute injuries, we present novel strategies to promote neuroprotection, axonal regeneration, and functional reinnervation of the lower urinary tract. For subjects with chronic injuries, we discuss new approaches to replace lost autonomic and motor neurons. A brief discussion on a variety of outcome measures that may be suitable to evaluate the function of the lower urinary tract in rodent neural repair models is also provided.

Improved detection of fluorogold-labeled neurons in long-term studies.

Fluorogold (FG) is a widely used neuroanatomical tracer. However, because FG-labeled neurons become undetectable over time, its use has been limited in long-term studies. We investigated whether the detection of FG in retrogradely labeled neurons in long-term studies can be improved by immunohistochemistry (IHC) using an antibody to FG. We performed intraperitoneal injections of a FG solution to retrogradely label all parasympathetic preganglionic neurons (PPNs) and motoneurons (MNs) in the S1 spinal cord segment in adult rats. At 1, 6, and 12 weeks after the tracer injection, sections were immunohistochemically processed for FG and choline acetyltransferase (ChAT), an endogenous marker for all PPNs and MNs. Stereological counts demonstrated no cell loss of FG-labeled PPNs and MNs at 6 and 12 weeks. Cell size measurements showed that FG-immunolabeled neurons were smaller at 12 weeks, but not at 6 weeks. However, it is likely that there was no neuronal atrophy, but loss/degradation of the dye at a timepoint between 6 and 12 weeks, as ChAT-immunolabeled neurons showed no cell size reduction at 12 weeks. Our results suggest that the use of an antibody against FG improves the detection of FG for reliable neuronal counts and that the dye is not toxic to the retrogradely labeled neurons. We conclude that FG-labeling is a useful tool to determine neuronal counts in long-term studies, but should be used cautiously for neuronal size measurements.

A single re-implanted ventral root exerts neurotropic effects over multiple spinal cord segments in the adult rat.

Spinal cord injuries, particularly traumatic injuries to the conus medullaris and cauda equina, are typically complex and involve multiple segmental levels. Implantation of avulsed ventral roots into the spinal cord as a repair strategy has been shown to be neuroprotective and promote axonal regeneration by spinal cord neurons into an implanted root. However, it is not well known over what distance in the spinal cord an implanted ventral root can exert its neurotropic effect. Here, we investigated whether an avulsed L6 ventral root acutely implanted into the rat spinal cord after a four level (L5-S2) unilateral ventral root avulsion injury may exert neurotropic effects on autonomic and motor neurons over multiple spinal cord segments at 6 weeks postoperatively. Using retrograde labeling techniques and stereological quantification methods, we demonstrate that autonomic and motor neurons from all four lesioned spinal cord segments, spanning more than an 8 mm rostro-caudal distance, reinnervated the one implanted root. The rostro-caudal distribution suggested a gradient of neurotropism, where the axotomized neurons closest to the implanted site had the highest probability of root reinnervation. These results suggest that implantation of a single ventral root may provide neurotropic effects to injured neurons at the site of lesion as well as in the adjacent spinal cord segments. Our findings may be of translational research interest for the development of surgical repair strategies after multi-level conus medullaris and cauda equina injuries, in which fewer ventral roots than spinal cord segments may be available for implantation.

Use-dependent modulation of inhibitory capacity in the feline lumbar spinal cord.

The ability to perform stepping and standing can be reacquired after complete thoracic spinal cord transection in adult cats with appropriate, repetitive training. We now compare GAD(67)A levels in the spinal cord of cats that were trained to step or stand. We confirmed that a complete spinal cord transection at approximately T12 increases glutamic acid decarboxylase (GAD)(67) in both the dorsal and ventral horns of L5-L7. We now show that step training decreases these levels toward control. Kinematic analyses show that this downward modulation is correlated inversely with stepping ability. Compared with intact cats, spinal cord-transected cats had increased punctate GAD(67) immunoreactivity around neurons in lamina IX at cord segments L5-L7. Compared with spinal nontrained cats, those trained to stand on both hindlimbs had more GAD(67) puncta bilaterally in a subset of lamina IX neurons. In cats trained to stand unilaterally, this elevated staining pattern was limited to the trained side and extended for at least 4 mm in the L6 and L7 segments. The location of this asymmetric GAD(67) staining corresponded to the motor columns of primary knee flexors, which are minimally active during standing, perhaps because of extensor-activated inhibitory interneuron projections. The responsiveness to only a few days of motor training, as well as the GABA-synthesizing potential in the spinal cord, persists for at least 25 months after the spinal cord injury. This modulation is specific to the motor task that is performed repetitively and is closely linked to the ability of the animal to perform a specific motor task.

Titanium mesh implantation--a method to stabilize the spine and protect the spinal cord following a multilevel laminectomy in the adult rat.

The development of clinically relevant larger spinal cord injury models is in part limited by the possibility of a widened or multilevel laminectomy causing a spinal cord injury from an unstable spine or from compression of the spinal cord by adjacent soft tissues. In the adult rat, we have developed a method to protect the spinal cord and stabilize the spinal column using a titanium mesh implant following a bilateral, multilevel lumbar laminectomy. For this purpose, bilateral and expanded L1-4 laminectomies were performed with or without the use of a titanium mesh to protect the spinal cord and stabilize the spine. Without titanium mesh protection, the rats developed a severe paraparesis or paraplegia, urinary retention, gross anatomical signs of cord compression, and motoneuron loss. In the titanium mesh treatment group, the rats typically maintained a normal gait and lower urinary tract function, normal gross anatomical features of the spinal cord, and normal motoneuron counts. We propose that the use of a titanium mesh implant may assist in the development of clinically relevant larger spinal cord injury and repair models.

Autonomic and motor neuron death is progressive and parallel in a lumbosacral ventral root avulsion model of cauda equina injury.

Injuries to the cauda equina of the spinal cord result in autonomic and motor neuron dysfunction. We developed a rodent lumbosacral ventral root avulsion injury model of cauda equina injury to investigate the lesion effect in the spinal cord. We studied the retrograde effects of a unilateral L5-S2 ventral root avulsion on efferent preganglionic parasympathetic neurons (PPNs) and pelvic motoneurons in the L6 and S1 segments at 1, 2, 4, and 6 weeks postoperatively in the adult male rat. We used Fluoro-Gold-prelabeling techniques, immunohistochemistry, and quantitative stereologic analysis to show an injury-induced progressive and parallel death of PPNs and motoneurons. At 6 weeks after injury, only 22% of PPNs and 16% of motoneurons remained. Furthermore, of the neurons that survived at 6 weeks, the soma volume was reduced by 25% in PPNs and 50% in motoneurons. Choline acetyltransferase (ChAT) protein was expressed in only 30% of PPNs, but 80% of motoneurons remaining at 1 week postoperatively, suggesting early differential effects between these two neuronal types. However, all remaining PPNs and motoneurons were ChAT positive at 4 weeks postoperatively. Nuclear condensation and cleaved caspase-3 were detected in axotomized PPNs and motoneurons, suggesting apoptosis as a contributing mechanism of the neural death. We conclude that lumbosacral ventral root avulsions progressively deplete autonomic and motor neurons. The findings suggest that early neuroprotection will be an important consideration in future attempts of treating acute cauda equina injuries.

Glial reactions in a rodent cauda equina injury and repair model.

In the adult rat, an avulsion injury of lumbosacral ventral roots results in a progressive and pronounced loss of the axotomized motoneurons. A subsequent acute implantation of an avulsed ventral root into the spinal cord has neuroprotective effects. However, it has not been known whether a surgical implantation of an avulsed ventral root into the spinal cord for neural repair purposes affects intramedullary glial and microglial reactions. Here, adult female Sprague-Dawley rats underwent a unilateral L5-S2 ventral root avulsion injury with or without acute implantation of the L6 ventral root into the spinal cord. At 4 weeks postoperatively, immunohistochemistry using primary antibodies to GFAP (astrocytes), Ox-42 (microglia), and ED-1 (macrophages) was performed at the L6 spinal cord segment, and quantified using densitometry. Our results show that a lumbosacral ventral root avulsion injury induces an activation of astrocytes, microglia, and macrophages in the ventral horn. Interestingly, an acute implantation of an avulsed root into the white matter does not significantly affect the activation of glial cells or macrophages in the ventral horn. We speculate that neuroprotective and axonal growth promoting benefits of the combined glial and microglial/ macrophage responses may outweigh their potential negative effects, as previous studies have shown that implantation of avulsed roots is a successful strategy in promoting reinnervation of peripheral targets.

Differential distribution of growth associated protein (GAP-43) in the motor nuclei of the adult rat conus medullaris.

GAP-43 is normally produced by neurons during developmental growth and axonal regeneration, but it is also expressed in specific regions of the normal adult nervous system. We studied the protein expression of GAP-43 within the conus medullaris portion of the spinal cord in adult male rats. Immunohistochemistry for choline acetyltransferase (ChAT) was first performed to identify specific efferent autonomic and motor nuclei in lumbosacral segments of the spinal cord. Adjacent sections were then processed for GAP-43 immunoreactivity (IR). We show GAP-43 IR in the superficial portion of the dorsal horn, the intermediolateral nucleus, and the dorsal commissural tract. We also demonstrate a differential distribution of GAP-43 IR between different motor nuclei of the conus medullaris. Using densitometry, the most prominent GAP-43 IR was detected in the dorsolateral and dorsomedial motor nuclei, which represent the human Onuf's nucleus homologue. Confocal microscopy of double immunofluorescent labeling for ChAT and GAP-43 demonstrate GAP-43 IR in the neuropil of the autonomic and motor nuclei, and many of the GAP-43 IR arbors are in close apposition with the efferent cholinergic neurons. We note that the efferent neurons of both the autonomic and somatic nuclei, which are ultimately responsible for the integrated normal control of the lower urinary tract, bowel and sexual functions, are heavily innervated by GAP-43 enriched projections. We speculate that these functionally related neurons retain a physiological GAP-43-associated synaptic plasticity throughout adult life.