Kelli Sharp

Calcium channel alpha-2-delta-1 protein upregulation in dorsal spinal cord mediates spinal cord injury-induced neuropathic pain states.

&NA; Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI‐induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non‐noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage‐gated calcium channel alpha‐2‐delta‐1 subunit (Cavα2δ‐1) proteins is effective in the management of SCI‐induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Cavα2δ‐1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI‐induced dysregulation of spinal Cavα2δ‐1 plays a contributory role in below‐level allodynia development in a rat spinal T9 contusion injury model. We found that Cavα2δ‐1 expression levels were significantly increased in L4–6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI‐induced Cavα2δ‐1 protein upregulation by intrathecal Cavα2δ‐1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI‐induced Cavα2δ‐1 upregulation in spinal dorsal horn is a key component in mediating below‐level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients. Spinal cord contusion injury caused increased calcium channel Cavα2δ‐1 subunit expression in dorsal spinal cord that contributes to neuropathic pain states.

Thrombospondin-4 Contributes to Spinal Sensitization and Neuropathic Pain States

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Regenerative Growth of Corticospinal Tract Axons via the Ventral Column after Spinal Cord Injury in Mice

Studies that have assessed regeneration of corticospinal tract (CST) axons in mice after genetic modifications or other treatments have tacitly assumed that there is little if any regeneration of CST axons in normal mice in the absence of some intervention. Here, we document a previously unrecognized capability for regenerative growth of CST axons in normal mice that involves growth past the lesion via the ventral column. Mice received dorsal hemisection injuries at thoracic level 6–7, which completely transect descending CST axons in the dorsal and dorsolateral column. Corticospinal projections were traced by injecting biotinylated dextran amine (BDA) into the sensorimotor cortex of one hemisphere either at the time of the injury or 4 weeks after injury, and mice were killed at 20–23 or 46 d after injury. At 20–23 d after injury, BDA-labeled CST axons did not extend past the lesion except in one animal. By 46 d after injury, however, a novel population of BDA-labeled CST axons could be seen extending from the gray matter rostral to the injury into the ventral column, past the lesion, and then back into the gray matter caudal to the injury in which they formed elaborate terminal arbors. The number of axons with this highly unusual trajectory was small (∼1% of the total number of labeled CST axons rostral to the injury). The BDA-labeled axons in the ventral column were on the same side as the main tract and thus are not spared ventral CST axons (which would be contralateral to the main tract). These results indicate that normal mice have a capacity for CST regeneration that has not been appreciated previously, which has important implications in studying the effect of genetic or pharmacological manipulations on CST regeneration in mice.

Bilateral cervical contusion spinal cord injury in rats

There is increasing motivation to develop clinically relevant experimental models for cervical SCI in rodents and techniques to assess deficits in forelimb function. Here we describe a bilateral cervical contusion model in rats. Female Sprague-Dawley rats received mild or moderate cervical contusion injuries (using the Infinite Horizons device) at C5, C6, or C7/8. Forelimb motor function was assessed using a grip strength meter (GSM); sensory function was assessed by the von Frey hair test; the integrity of the corticospinal tract (CST) was assessed by biotinylated dextran amine (BDA) tract tracing. Mild contusions caused primarily dorsal column (DC) and gray matter (GM) damage while moderate contusions produced additional damage to lateral and ventral tissue. Forelimb and hindlimb function was severely impaired immediately post-injury, but all rats regained the ability to use their hindlimbs for locomotion. Gripping ability was abolished immediately after injury but recovered partially, depending upon the spinal level and severity of the injury. Rats exhibited a loss of sensation in both fore- and hindlimbs that partially recovered, and did not exhibit allodynia. Tract tracing revealed that the main contingent of CST axons in the DC was completely interrupted in all but one animal whereas the dorsolateral CST (dlCST) was partially spared, and dlCST axons gave rise to axons that arborized in the GM caudal to the injury. Our data demonstrate that rats can survive significant bilateral cervical contusion injuries at or below C5 and that forepaw gripping function recovers after mild injuries even when the main component of CST axons in the dorsal column is completely interrupted.

Long-Distance Migration and Colonization of Transplanted Neural Stem Cells

A recent study reported remarkable survival and exuberant axon outgrowth from transplants of neural stem cells (NSCs) in a fibrin matrix with growth factors that were grafted into a complete spinal cord transection site in rats (Lu et al., 2012). As part of the NIH-supported replication project (Facilities of Research Excellence-Spinal Cord Injury), we repeated key parts of that study. Because this is a surgical intervention that may depend on skills that require extensive experience, we felt that the goals of the replication would be best served if the same surgeon performed the lesion surgeries and transplants.

Salmon fibrin treatment of spinal cord injury promotes functional recovery and density of serotonergic innervation.

The neural degeneration caused by spinal cord injury leaves a cavity at the injury site that greatly inhibits repair. One approach to promoting repair is to fill the cavity with a scaffold to limit further damage and encourage regrowth. Injectable materials are advantageous scaffolds because they can be placed as a liquid in the lesion site then form a solid in vivo that precisely matches the contours of the lesion. Fibrin is one type of injectable scaffold, but risk of infection from blood borne pathogens has limited its use. We investigated the potential utility of salmon fibrin as an injectable scaffold to treat spinal cord injury since it lacks mammalian infectious agents and encourages greater neuronal extension in vitro than mammalian fibrin or Matrigel®, another injectable material. Female rats received a T9 dorsal hemisection injury and were treated with either salmon or human fibrin at the time of injury while a third group served as untreated controls. Locomotor function was assessed using the BBB scale, bladder function was analyzed by measuring residual urine, and sensory responses were tested by mechanical stimulation (von Frey hairs). Histological analyses quantified the glial scar, lesion volume, and serotonergic fiber density. Rats that received salmon fibrin exhibited significantly improved recovery of both locomotor and bladder function and a greater density of serotonergic innervation caudal to the lesion site without exacerbation of pain. Rats treated with salmon fibrin also exhibited less autophagia than those treated with human fibrin, potentially pointing to amelioration of sensory dysfunction. Glial scar formation and lesion size did not differ significantly among groups. The pattern and timing of salmon fibrins effects suggest that it acts on neuronal populations but not by stimulating long tract regeneration. Salmon fibrin clearly has properties distinct from those of mammalian fibrin and is a beneficial injectable scaffold for treatment of spinal cord injury.

Calcium channel α2δ1 proteins mediate trigeminal neuropathic pain states associated with aberrant excitatory synaptogenesis.

Background: Factors mediating orofacial neuropathic pain are not well defined. Results: Trigeminal nerve injury-induced calcium channel α2δ1 protein up-regulation in trigeminal ganglia and spinal complex correlated with enhanced spinal presynaptic neurotransmission, excitatory synaptogenesis, and orofacial pain states. Conclusion: This neuroplasticity may mediate orofacial neuropathic pain states by enhancing dorsal horn excitatory synaptic neurotransmission. Significance: This reveals a mechanism underlying orofacial neuropathic pain states. To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Characterization of Ectopic Colonies That Form in Widespread Areas of the Nervous System with Neural Stem Cell Transplants into the Site of a Severe Spinal Cord Injury

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1. Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

A Straight Alley Version of the BBB Locomotor Scale

We describe here an alternative procedure for assessing hindlimb locomotor function after spinal cord injury that uses the BBB scale, but tests animals in a reward-baited straight alley rather than an open field. Rats were trained to ambulate in a straight alley and habituated to the open field typically used for BBB open field testing. Three groups of rats were tested. Sprague-Dawley rats received either 200 kD (n=19) or 300 kD contusions (n=9) at T9 with the Infinite Horizon device. Fisher rats (n=8) received moderate contusions (12.5 mm) at T8 with the NYU impactor. BBB scores were assessed at different post-injury intervals in the open field and the straight alley, and scores were compared by correlation analyses. BBB scores in the open field vs. the straight alley were highly correlated (r=0.90), validating the use of the straight alley for locomotor assessment. Rats exhibited a larger number of bouts of continuous steps in the straight alley vs. the open field (termed passes), providing more opportunities to score hindlimb use and coordination over the 4 min testing interval. Comparisons of scores across days revealed higher day-to-day correlations in the straight alley vs. the open field (r(2) values of 0.90 and 0.74 for the straight alley and open field respectively), revealing that the straight alley yielded more reliable scores.