Interesting SfN 2008 Presentations

[Steven Edwards]

Random presentations that I found interesting and promising from this year's Society for Neuroscience meeting will be posted here. A bonus this year: funding information was provided with most (if not all) presentations. (Name in parentheses is the person who presented at SfN.)

This first post will cover research funded by the PVA.

Novel compounds that overcome inhibition of CNS regeneration (Andrea L Peterson)
The failure of central nervous system (CNS) neurons to regenerate following spinal cord injury (SCI) leads to irreversible paralysis and sensory deficits. Traumatic insults to the spinal cord lead to the deposition of inhibitory myelin debris and the formation of a glial scar, which contains a large number of regeneration inhibitors. Prominent among these inhibitors is a family of astrocyte produced proteoglycans known as chondroitin sulfate proteoglycans (CSPGs). Attempts to achieve regeneration of long axon tracts by overcoming inhibitory signals from myelin and CSPGs have met with limited success, partially owing to a lack of detailed knowledge about the signaling mechanisms underlying inhibitory CSPG signals. One way to advance our knowledge and promote novel therapies for SCI is to identify chemical compounds with the ability to overcome regeneration inhibition.
Our lab has undertaken a “chemical genetics” approach, screening a library of novel triazine compounds to identify those that promote neurite growth on inhibitory substrates. We initially identified four “hit” compounds based on their ability to increase neurite outgrowth on an inhibitory substrate of CNS myelin. Interestingly, these compounds also increase neurite outgrowth from several different classes of CNS neurons on a substrate of inhibitory CSPGs, suggesting that they act through a signaling pathway that is common to both classes of inhibitors. Most excitingly, one of the compounds, F05, promotes acute regeneration of severed dorsal column axons in vivo. Studies of potential mechanisms revealed that the compounds do not act through cAMP, protein kinase C, or the EGF receptor. However, compound F05 does affect microtubule dynamics in cultured hippocampal neurons, suggesting a potential mechanism through which regeneration could be affected. Elucidation of the mechanism of action of these compounds should provide insight into mechanisms of regeneration inhibition, and may lead to novel therapeutic strategies for SCI.

• Funded by:
• Veterans of America, Grant # 2445
• Craig H. Neilsen Foundation
• Wallace H. Coulter Foundation

Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the cns (John Dill)
Axonal regeneration is minimal after the CNS injuries in adult mammals and the medical treatments to recover the neurological loss due to axon disconnection are extremely limited. The nonpermissive environment and reduced intrinsic growth capacity are principally attributed to the failure for axonal elongation. In this report, we studied the role of glycogen synthase kinase-3 (GSK-3) inactivation on neurite and axon growth of adult neurons via combined in vitro and in vivo approaches. We found that the major CNS inhibiting substrates including chondroitin sulfate proteoglycans (CSPG) could inactivate protein kinase B (Akt) and activate GSK-3β signals in neurons. GSK-3 inactivation with pharmacologic inhibitors enhances neurite outgrowth of dorsal root ganglion neurons derived from adult mice or cerebellar granule neurons from postnatal rodents cultured on CNS inhibitors. Application of GSK-3 inhibitors stimulates axon formation and elongation of mature neurons whether in presence or absence of inhibitory substrates. Systemic application of GSK-3 inhibitor lithium to spinal cord-lesioned rats suppresses the activity of this kinase around lesion. Treatments with GSK-3 inhibitors including a clinical dose of lithium to rats with thoracic spinal cord transection or contusion injuries induce significant descending corticospinal and serotonergic axon sprouting in caudal spinal cord and promote locomotor functional recovery. Our studies suggest that GSK-3 signal is an important therapeutic target for promoting functional recovery of adult CNS injuries and that administration of GSK-3 inhibitors may facilitate the development of an effective treatment to white matter injuries including spinal cord trauma given the wide use of lithium in humans.

• Funded by:
• Paralyzed Veterans of America

The temporal expression of S100B and GFAP in cerebrospinal fluid following acute human spinal cord injury (Cody M Mann)
Background: Glial fibrillary acidic protein (GFAP) and S100B have been used as biological markers of injury severity in traumatic brain injury. However, little is known about the temporal release of GFAP and S100B after spinal cord injury in humans or about the correlation of these protein levels with injury severity.
Objective: To evaluate the concentration of S100B and GFAP within the cerebrospinal fluid (CSF) of human patients with acute SCI, and document the temporal pattern of change over 72 hours.
Methods: Human patients within 48 hours of sustaining an SCI were enrolled in a clinical trial in which a lumbar intrathecal catheter was inserted to drain CSF. CSF samples were drawn at 8 hour intervals, centrifuged, and frozen at the bedside. The drains were left in for 72 hours. An ELISA kit was used to evaluate the level of S100B and GFAP in the CSF.
Results: S100B and GFAP were elevated within 24 hours after SCI. The concentrations of these factors decreased over the 72 hours that the drain was inserted.
Discussion: This study describes the temporal release of S100B and GFAP after human spinal cord injury. This information may serve as a tool to help with the classification of injury severity and prediction of neurologic outcome, and may also be useful as a biological method of monitoring the effect of drug therapies for SCI.

• Funded by:
• Vancouver Coastal Health Research Institute
• Rick Hansen Man in Motion Research Foundation
• Michael Smith for Health Research
• Paralyzed Veterans of America

Increased expression of 5'-ectonucleotidase and the adenosine A2B receptor in chronic spinal cord injury (Raymond J Grill)
Trauma to the spinal cord destroys or alters tissue through mechanical and biochemical mechanisms. These injury-induced alterations in the neuronal, glial and vascular environment include glial scarring, failed regenerative growth of motor pathways and the potential for development of devastating neuropathic pain conditions. Nucleotides and nucleosides are important mediators of cellular activity in both normal and pathological conditions. Adenosine is one such modulator that is normally present in low concentrations in the extracellular space where it acts through four G-protein-linked receptors. Injury has been shown to significantly elevate extracellular levels of adenosine through several mechanisms including the metabolism of cell-released ATP via enzymes called ectonucleotidases, the release of intracellular stores through specific transporters, and uncontrolled release through membrane disruption. Following injury, the role of adenosine has generally been considered to be benign; producing neuroprotective effects. However, injury-induced adenosine has also been linked with astroglial activation, influencing astrocyte proliferation and morphological characteristics of gliosis (Liberto et al. 2004). Adenosine levels increase dramatically, but transiently, following SCI (McAdoo et al, 2000), hitting a peak between 30 to 60 minutes post-injury, then returning to baseline levels. Despite the transient increase in adenosine following SCI, we have recently detected elevated immunoreactivity (-IR) of the low-affinity adenosine A2B receptor co-localized with the astrocytic marker GFAP in- and around the site of spinal contusion in adult rats one-to-three months post-SCI. A2B-IR was also detected in cells of the central canal bordering the cavity. Interestingly, A2B+/GFAP+ cells appear to stream from the canal region to the contusion cavity. Elevated 5’-ectonucledotidase (5-NT)-IR co-localized to both reactive astrocytes and blood vessels in- and around the lesion cavity in chronic SCI. While the presence of elevated extracellular adenosine has yet to be demonstrated in the chronically-injured cord, our current data suggest that adenosine may play a role in influencing pathological processes such as the glial scar in chronic SCI.

• Funded by:
• NIH Grant NS049409
• Paralyzed Veterans of America #2511
• Mission Connect, a project of the TIRR Foundation

[Steven Edwards]

Next: Christopher and Dana Reeve Foundation.

Effects of chondroitinase ABC treatment following peripheral nerve grafting into a chronic cervical contusion injury site (Veronica J. Tom)
Our lab has previously shown that aspirating necrotic tissue from a chronic cervical (C) contusion injury site in preparation for apposition of one end of a peripheral nerve graft (PNG) did not further injure the spinal cord nor produce additional functional impairments. Furthermore, following glial cell line-derived neurotrophic factor treatment (GDNF), axons readily regrew into the graft. We wanted to determine if treating the normally inhibitory PNG-spinal cord interface with chondroitinase ABC (ChABC) would promote these chronically injured, regenerating axons to emerge from the PNG into host spinal cord tissue in two grafting models. In the first one, necrotic tissue within a chronic (8 week) C5 hemicontusion cavity was removed by aspiration. GDNF and ChABC or vehicle were microinjected rostral and caudal to the injury site and saturated gelfoam was used to treat the cavity before inserting a segment of predegenerated PN to span the length of the cavity. The second grafting model tested whether there is increased axonal outgrowth from a PNG that bridges a chronic contusion site and a distal, acute, ChABC-treated injury site. Necrotic tissue within a chronic (8 week) C5 hemicontusion cavity was removed by aspiration. The lesion cavity was treated with GDNF-soaked gelfoam and GDNF was microinjected rostral to the injury site before one end of a predegenerated PNG was inserted into the cavity. Three weeks later, a dorsal quadrant (DQ) lesion was made at C7 and treated with microinjections of ChABC or vehicle prior to apposition of the distal end of the PNG. Microinjections were repeated 3 days later. The PN grafts in both experimental paradigms will be infused with BDA to label axons regenerating through the graft to determine if ChABC increases the number and/or length of axons exiting the grafts and if these axons form synaptic contacts with host neurons. Possible anatomical correlates for behavioral improvement in ongoing studies will be reported.

• Funded by:
• NIH NS26380 (JDH)
• Christopher and Dana Reeve Foundation (VJT)

NgR(310)ecto-Fc protein promotes axon growth and recovery in chronic spinal cord injury (Xingxing Wang)
Myelin limits axonal growth and neurological recovery after spinal cord injury (SCI). Three myelin-associated inhibitory proteins,
Nogo-A,Myelin-Associated
Protein (MAG), and Oligodendrocyte Myelin Glycoprotein (OMgp) bind to an axonal Nogo-66 receptor (NgR) protein to collapse axonal growth cones and stop axonal extension. Previous work have demonstrated that a soluble fragment of the NgR containing the ligand binding domain (NgR (310)ecto-Fc) blocks myelin inhibition of axon outgrowth, improves neurological recovery in rats with a mid-thoracic dorsal hemisection or contusion injury when administered within several days of SCI. Here we provide evidence that blockade of myelin inhibition can stimulate axonal growth and recovery in chronic SCI.
A cohort of 46 rats survived thoracic spinal cord contusion injuries, and exihibited stable motor scores without fluctuation between 6-12 weeks after injury. The average BBB score at 12 weeks was 7.9 ± 0.1, meaning that the majority of rats were capable of hindlimb movement, but not weight support. After intracerebroventricular (i.c.v.) catheter placement, the animals received either NgR (310)ecto-Fc or control IgG protein for 12 weeks at a dose of 0.29mg/kg/d. All animal handling, behavioral scoring and histological analysis were performed by personnel without knowledge of the treatment group. The initial BBB scores were identical in the two groups. The improvement of each animal’s BBB score in the NgR (310)ecto-Fc treated group was significantly greater than in the control group. Seven of NgR (310)ecto-Fc treated animals converted to weight bearing, while only one control rat did so over this three month period. In histological examination, increased 5-HT fiber length was observed in the lumbar spinal cord after treatment of chronic SCI. Corticalspinal tract (CST) axon tracing showed a trend towards rostral CST growth in treated animals.
Our results demonstrate that NgR (310)ecto-Fc treatment of chronic spinal contusion improves locomotor recovery long after spontaneous neurological recovery has ceased. The axonal growth stimulated by NgR (310)ecto-Fc therapy is associated with substantial neurological benefits in the chronic rat spinal contusion model.

• Funded by:
• the Christopher and Dana Reeve Foundation
• the Wings for Life Foundtion
• the Dr. Ralph and Marion Falk Medical Research Foundation

Oligodendrocytes illuminated: lighting up the path to differentiation (Sheila Rosenberg)
Effective communication within the nervous system is dependent on the transmission of neuronal action potentials. The efficient conduction of these electrical signals along axons is greatly enhanced by the presence of the myelin sheath. Glial cells known as oligodendrocytes are responsible for the myelination of axons in the central nervous system. These terminally differentiated cells are derived from oligodendrocyte precursor cells (OPCs). During development, OPCs migrate and proliferate along axon tracts before undergoing a temporally synchronized transition into differentiated oligodendrocytes. Using cocultures of purified sensory neurons and cortical OPCs, we previously showed that OPCs must reach a critical density along axons before differentiation will begin. Our findings suggest that differentiation results from spatial and geometric constraints that occur when a critical density of OPCs is reached. Do these constraints induce physical changes in cell size and shape that are responsible for the transition from an OPC to an oligodendrocyte? We employed live-imaging time-lapse microscopy to examine the events that lead to the onset of oligodendrocyte differentiation. By observing the migratory and proliferative patterns of OPCs, we can better understand how packing constraints are established along axons. In addition, the expression of a fluorescent protein driven by a differentiation-specific promoter allows us to visualize the relationship between intercellular interactions and the initiation of differentiation. It is our hope that these studies will help elucidate the manner in which biophysical changes contribute to the onset of oligodendrocyte differentiation in an axonal niche.

• Funded by:
• NIH CBM Training Grant
• National Multiple Sclerosis Society Career Transition Award (TA 3008A2/T)
• Christopher Reeve Foundation (CB2-0606-2)
• Baxter Foundation Award

The impact of tizanidine on reflex mechanical properties in spinal cord injury (Mehdi M. Mirbagheri)
Several medications, such as tizanidine, have been developed to reduce spasticity. This agent has been shown to be as effective as other antispastic medications (such as baclofen) and it is usually better tolerated, with milder side effects. Several studies have investigated the effects of tizanidine on spasticity using clinical measures such as the Ashworth scale and the pendulum test. Since no consistent link between these clinical measures and reflex mechanical properties has been described, the impact of tizanidine on reflex and on muscle mechanical properties has not been identified.
The present study was designed to help us understand the mechanisms of action of tizanidine on spasticity in subjects with incomplete spinal cord injury by quantifying the effects of a single dose of tizanidine (2 mg) on ankle dynamic stiffness and on its intrinsic and reflex components. A series of small and large amplitude perturbations was applied to the spastic ankle muscles, and the resulting torques were recorded. A parallel-cascade system identification method was used to identify intrinsic and reflex contributions to dynamic ankle stiffness at different ankle positions, while subjects remained relaxed.
Our results revealed that stretch evoked joint torque at the ankle decreased significantly following administration of a single dose Using systems identification computational techniques, we found that this reduced torque could be attributed largely to a reduced reflex response, without measurable change in the muscle contribution. As a result, reflex stiffness decreased significantly after using tizanidine. In contrast, there were no significant changes in intrinsic muscle stiffness after the administration of tizanidine. Our findings demonstrate that tizanidine acts to reduce stretch reflex mechanical responses substantially, without inducing comparable changes in intrinsic muscle properties in individuals with spinal cord injury.

• Funded by:
• Christopher Reeve Foundation

GAP-43 UTRs containing the HuD-binding site are needed for axonal mRNA transport and modulate axonal outgrowth (Soonmoon Yoo)
Upregulation of GAP-43 expression has long been associated with neurite growth and regeneration in mammalian nervous system. The Elav protein HuD binds to the 3’UTR of GAP-43 mRNA contributing both to stability and translation of this mRNA. GAP-43 mRNA and HuD have been shown to localize to neurites of PC12 cells (Smith et al., 2004) and we have detected GAP-43 mRNA in axons of adult DRG neurons in culture and to sciatic nerve in vivo. Considering this subcellular localization, we asked whether the 3'UTR of GAP-43 mRNA is needed for its axonal transport. We generated GFP reporters containing the full length, 5' proximal, or 3' distal segments of GAP-43 3’UTR (898-1483, 898-1053, and 1067-1281, resp.). 898-1483 and 1067-1281 constructs showed much stronger axonal GFP signal in transfected DRG neurons compared with the 898-1053 construct. Axonal GFP signals from the full length and distal segment reporters also showed rapid, translation-dependent recovery of fluorescence after photobleaching (898-1053 showed no significant recovery). These data indicate that nucleotides 1067-1281 are necessary and sufficient for transport of GAP-43 mRNA into sensory axons. Over expression of these localizing GAP-43 constructs also altered growth cone morphology and decreased axon length in cultured DRG neurons. Since nucleotides 1067-1281 of GAP-43 mRNA include the HuD binding site, we asked whether increasing HuD availability might account for the effects of overexpresing the GAP-43 3'UTR. Overexpression of either full length HuD or HuD RRM 3 alone increased growth cone size and axonal outgrowth.

• Funded by:
• NIH Grant R01-NS041596
• NIH Grant R01-NS30255
• the Christopher & Dana Reeve Paralysis Foundation
• the Dr. Miriam and Sheldon Adelson Medical Research Foundation

Effect of immunosuppressive therapy on human CNS derived stem cell (hCNS-SC) survival and differentiation in spinal injured C57/BL6 mice (Brian J Cummings)
We previously reported that human CNS stem cells grown as neurospheres (HuCNS-SC) survive, predominantly differentiate into oligodendrocytes, some neurons and few astrocytes, and promote locomotor recovery in immunodeficient NOD-scid mice (Cummings et al., PNAS 2005). However, some level of immunosuppressive therapy will be necessary should these cells be used clinically. The objective of the current study was to evaluate the effect of immunosuppressive agents on engraftment and differentiation potential of these cells in an immunosufficient rodent model of spinal cord injury (SCI). Adult female C57/BL6 mice received a moderate thoracic contusion SCI, followed by transplantation with 75,000 HuCNS-SC either 9-days post-injury (dpi) or 60-dpi. Mice were immunosuppressed with either FK506 alone or in combination with an anti-CD4 antibody for 12 or 16 weeks (9-dpi groups) or 12 weeks (60-dpi groups) post-transplantation. Combined FK506/anti-CD4 antibody treatment was required for survival of human cells in this xenogenic mouse model; no engraftment was observed in either the 9-dpi or 60-dpi groups receiving FK506 alone. In contrast, all 9-dpi animals (7 out of 7) receiving combined FK506/anti-CD4 therapy exhibited successful engraftment, while 54% of 60-dpi animals (6 out of 11) receiving combined FK506/anti-CD4 therapy exhibited successful engraftment. Confocal microscopy for cell fate revealed that the engrafted human cells exhibited differentiation predominantly along an oligodendrocyte lineage (APC-CC1 positive). Doublecortin-positive human cells were also detected. Human GFAP-positive cells were observed immediately adjacent to the lesion epicenter. To study the effect of immunosuppressant withdrawal, immunosuppressant therapy was discontinued in four mice from the 9-dpi transplantation cohort 4 weeks prior to sacrifice. Following immunosuppressant withdrawal, human cells were still detected in two of four mice. Stereological quantification of the total number of engrafted human cells is in progress. In summary, HuCNS-SC successfully engrafted and exhibited the potential to differentiate into oligodendrocytes, neurons, and astrocytes in immunosuppressed C57BL/6 mice, similar to our previous work in immunodeficient NOD-scid mice. In addition, there was no evidence of excessive proliferation, tumor formation, or substantial contribution to glial scar formation by the transplanted HuCNS-SC. Together, these data support the clinical validity of HuCNS-SC transplantation for SCI from the perspective of cell fate and response to the injured CNS niche in the presence of immunosuppressant drug treatment.

• Funded by:
• NIH R01NS049885 (AJA)
• Christopher & Dana Reeve Foundation Animal Core
• Stem Cells Inc

Light induced return of respiratory function following spinal cord injury through the activation of channelrhodopsin 2 (Warren J Alilain)
Paralysis of motor function is a major consequence of spinal cord injury (SCI). Following high cervical SCI, respiratory deficits can result through interruption of descending pre-synaptic inputs to respiratory motor neurons in the spinal cord. Expression of the green algae channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of pre-synaptic inputs. We hypothesized that after infecting adult rat spinal neurons in and around the phrenic motor pool to express ChR2, light stimulation would restore respiratory motor function in cervically hemisected adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. The robust rhythmic activity we observed persisted long after light stimulation had ceased suggesting that photostimulation can potentiate denervated phrenic neurons, possibly via the crossed phrenic pathway, which circumvents the lesion but is normally ineffective or latent. This recovery was accomplished through a unique form of respiratory plasticity, and adaptation which strongly augmented diaphragmatic activity. In particular, photostimulation first led to enhanced, seizure-like diaphragmatic activity in a unique pattern that adapted over time into a pattern much closer to normal breathing. Interesting effects of ipsilateral activity on the contralateral, intact side, suggested a role for interneurons, which can traverse the midline. Furthermore, the induction of this plasticity and recovery was abolished by the NMDA receptor antagonist MK-801, suggesting that this plasticity is NMDA receptor activation dependent. This is the first evidence that ChR2 can be used as a means to restore function after traumatic lesioning in the central nervous system. Specifically, these data suggest a novel, minimally invasive therapeutic avenue to either exercise denervated circuitry or restore motor function following SCI, and the capacity of the respiratory system to employ a form of spinal “learning” when activity is driven maximally via a light switch.

• Funded by:
• NIH Grant NS 25713 (JS)
• Christopher and Dana Reeve Foundation (WJA)

Bladder inhibition or excitation by electrical perianal stimulation in the chronic SCI cat (Jicheng Wang)
Objective: To test the hypothesis that perianal electrical stimulation in chronic spinal cord injured (SCI) cats could induce frequency dependent inhibitory or excitatory reflex bladder responses. Methods: The experiments were conducted after at least 4-5 weeks following spinal cord transection at the T9-T10 level. Electrical stimulation was applied via a pair of hook electrodes to the perianal skin area in 3 awake female chronic SCI cats. A double lumen balloon catheter was inserted through the urethra into the bladder to monitor bladder pressure and infuse saline (2-4 ml/min). Results: Under isovolumetric conditions electrical perianal stimulation at frequencies between 3 Hz and 10 Hz significantly inhibited large amplitude reflex bladder activity induced by bladder distension above the micturition volume threshold. However, stimulation at frequencies between 20 Hz and 50 Hz induced large amplitude bladder contractions when bladder volume was below the micturition volume threshold. Inhibitory stimulation (7 Hz) significantly increased bladder capacity 40±10% when it was applied continuously during cystometrograms (CMG). The optimal excitatory stimulation (30 Hz) induced large amplitude (greater than 25 cm H2O), long duration (greater than 20 sec) bladder contractions at a wide range of bladder volume (10-90% of bladder capacity). Conclusions: This study revealed that activation of pudendal afferent fibers by perianal electrical stimulation could induce frequency dependent reflex bladder responses in awake chronic SCI cats, indicating that a possible non-invasive treatment based on perianal electrical stimulation could be developed to restore both continence and micturition functions for SCI people.

• Funded by:
• NIH Grant DK068566
• NIH Grant DK077783
• Christopher and Dana Reeve Foundation

Monoaminergic modulation of locomotion facilitated by epidural stimulation (ES) in spinal rats (Pavel Musienko)
Lumbosacral circuits that coordinate stepping are under strong descending monoaminergic influence. After a complete spinal cord transection, descending serotonin (5-HT), noradrenaline (NA), and dopamine (DA) containing fibers are interrupted and the control of stepping becomes severely limited. However, post-synaptic monoamine receptors remain present on spinal interneurons and motoneurons. Thus, application of monoamines to lumbosacral segments can alter synaptic and cellular properties and stepping patterns. Our aim was to investigate the relative contribution of selected monoamine receptor subtypes in modulating reflex properties and locomotor activity facilitated by ES in adult spinal rats. Administration of the broad, but predominantly 5-HT2a agonist quipazine improved extension during stance, and the 5-HT1a,7 agonist 8-OHDPAT facilitated locomotor rhythm and coordination. Conversely, the 5-HT2 antagonist ketanserine significantly reduced extensor activity and severely impaired stepping. 5-HT1a,7 antagonists (WAY 100.635+SB-269970) marginally depressed flexor activity, but blocked the facilitating effect of 5-HT1a,7 agonists. The alpha-2 NA agonist clonidine suppressed locomotor rhythm elicited by ES. In contrast, injection of the alpha-1 NA agonist methoxamine or the alpha-2 NA antagonist yohimbine modestly facilitated locomotion. Administration of the DA1,5 antagonist SCH-23390 powerfully increased muscle tone and consequently impaired the coordination between antagonist muscles. In turn the DA1,5 agonist SKF-81297 drastically, although non-specifically, ameliorated locomotion. With the aim to develop an intervention to promote vigorous locomotion during neurorehabilitation, we evaluated the potential of combining agonists. The combination of 5-HT2a, 5-HT1a,7, DA1,5 agonists and ES was strongly synergistic, and promoted well-coordinated, full weight-bearing plantar stepping in adult spinal rats. These results demonstrate the relative contribution of monoamine receptor subtypes to stepping in adult spinal rats when facilitated by ES. Moreover, synergistic combinations of specific monoamine agents and ES offer a unique opportunity to engage efficiently the lumbosacral circuits during chronic neurorehabilitation after a severe spinal cord injury in mammals.

• Funded by:
• University of Zurich, NCCR Neural repair and plasticity
• NIH NS 16333
• Christopher and Dana Reeve Foundation VEC-2007
• RFBR 08-04-00688
• RFBR-CRDF Grant 07-04-91106 (CNF)

Combinatory strategies to facilitate neurorehabilitation and promote recovery of stepping function after a complete spinal cord injury in adult rats (Gregoire Courtine)
After a complete mid-thoracic spinal cord transection (ST), adult rats show minimal ability to generate coordinated stepping. In the present study, we showed that a combination of Quipazine (a predominantly 5-HT2 agonist) and 8-OHDPAT (5-HT1a,7 agonist) with epidural stimulation (ES) at the L2 and S1 spinal segments can promote stable locomotor rhythm on a treadmill with plantar placement, interlimb coordination, and up to 50% of weight bearing as early as one week post-ST. Thus using a combination of serotonin agonists and specific, multi-loci ES, we could engage the lumbosacral circuits in an effective rehabilitative procedure within a few days after ST. After 8 weeks of chronic sensorimotor training using this combinatory intervention, ST rats displayed full weight-bearing coordinated stepping ability, enhanced efficacy of monosynaptic input to flexor and extensor motoneurons, and lar muscle masses. In contrast, the chronic absence of weight bearing and locomotor activity for 8 weeks resulted in a significant decline in the stepping capacity observed one week post-injury in non-treated ST rats. Rats that were step trained under ES or 5-HT agonists alone showed limited improvement compared to rats that received the combinatory intervention. In a terminal experiment, trained and non-trained spinal rats were stepped for 45 min under the combinatory intervention and then returned to their cages for 1 h before intracardiac perfusion. Lumbosacral spinal sections were stained for c-fos immunoreactivity. The number of c-fos+ neurons was markedly higher in non-trained than in both trained and non-injured rats. These results suggest that activity-dependent mechanisms can reinforce the efficacy within specific sensorimotor circuits, resulting in substantial improvements of function after a complete spinal cord injury in adult rats.

• Funded by:
• Craig Nielsen Foundation 20062668
• NIH NS 16333
• Christopher and Dana Reeve Foundation VEC-2007
• RFBR-CRDF Grant 07-04-91106

Improvements in functional outcome measures after human spinal cord injury from a multi-center network providing Locomotor Training (Susan J Harkema)
The NeuroRecovery Network (NRN) is a network of specialized centers that provide standardized activity-based therapy (Locomotor Training, LT) based on current scientific and clinical evidence for people with spinal cord injury (SCI). The objectives are to maximize the availability of the standardized rehabilitative care provided to people with SCI and provide quantitative measures of their recovery. Seven NRN Centers* maintain a database that includes demographics, severity of impairments, balance, level of walking function, health and quality of life, and cost and reimbursement of activity-based therapy following SCI. Methods: The seven NRN Centers evaluated 201 individuals with SCI (AIS C or D) who received LT. Linear mixed effects (LME) models were fit to the Berg Balance Scale (BBS), Six Minute Walk (6MW), and Ten Meter Walk (10MW) data. The LME models are suitable for longitudinal data in which repeated measurements on subjects are correlated. Models were parameterized with intercept and slope fit for each phase grouping. The phases of recovery are: phase I-unable to stand or walk; phase 2 able to stand but not walk; and phase III able to stand and walk. This parameterization allows for the testing of differences among the phases at enrollment, the significance of the rate of improvement shown by each phase group, and differences in the rate of improvement for the phase groups. Random intercepts and slopes were included to account the added variation of repeated measurements on subjects. The models for walking measures (6MW and 10MW) had additional fixed effects terms accounting for the type of assistive device used. Results: Patients receiving LT improved in the three functional outcome measures of interest (BBS, 6MW, 10MW). The phase groupings significantly differed at enrollment in scores on the BBS (p < .0001), 6MW (p < .0001), and 10MW (p < .0001). The BBS had statistically significant (p < .0001) rates of improvement for all phase groupings and also were significantly (p = .01) different among the phases. Phase 2 had the greatest improvement and phase 1 had the least improvement. Phase 2 and 3 groupings had statistically significant rates of improvement in the 6MW (p < .0001) and 10MW (p < .0001) and these rates were significantly different among all three phases; 6MW p = .01), and 10MW p = .006). Phase 3 had the greatest improvement and phase 1 had the least improvement. These results demonstrate functional improvements with LT can occur after SCI when provided with a standardized intervention protocol across multiple rehabilitation sites.

• Funded by:
• Christopher and Dana Reeve Foundation, and Centers for Disease Control and Prevention Grant U10/CCU220379

Acute transplantation of human neural stem cells after spinal cord injury (Mitra J Hooshmand)
We have previously shown that transplantation of multipotent human central nervous system stem cells grown as neurospheres (HuCNS-SC) into NOD-scid mice at 9 days post injury (dpi) promotes locomotor recovery, HuCNS-SC integrate into the host spinal cord as myelinating and synapse-forming cells, and cell survival is necessary to maintain locomotor recovery (Cummings PNAS 2005). Additionally, linear regression analysis of the number of engrafted HuCNS-SC vs. the number of errors on a horizontal ladder beam task revealed a correlation between these variables, suggesting that survival and engraftment are directly related to a quantitative measure of locomotor recovery. We observed no evidence for alteration of the host niche by HuCNS-SC. Currently, the literature suggests that shortening the delay between spinal cord injury (SCI) and cell transplantation could impede the ability of neural stem cells to differentiate into neurons and oligodendrocytes and thus limit recovery. However, numerous studies have shown that neural cells could also promote recovery by alternative mechanisms via modification of the host niche. We therefore hypothesized that acute HuCNS-SC transplantation might promote recovery via alternative mechanisms. HuCNS-SC transplanted into injured mice at 0dpi survived and migrated predominantly towards the injury epicenter, and a significant number of human cells near the lesion differentiated into astrocytes as evidenced by staining for human-specific GFAP. These findings were in contrast to the 9dpi paradigm where HuCNS-SC migrated rostrally and caudally away from the injury site, and only 3% of engrafted cells differentiated into astrocytes. Interestingly, HuCNS-SC that migrated away from the epicenter in the present study exhibited oligodendrocytic and neuronal morphologies similar to that observed at 9dpi. The total number of engrafted HuCNS-SC, determined by unbiased stereology in immunostaining for a human-specific cytoplasmic marker, was comparable between 0- (210,000) and 9-dpi (145,000) paradigms. Immediate transplantation of HuCNS-SC did not alter either locomotor recovery or mechanical allodynia profiles. Furthermore, stereological quantification for the astroglial scar and total lesion volumes revealed no differences in transplanted vs. control. Collectively, our data comparing 0- and 9-dpi paradigms suggest that local cues, defined here by time of transplant, affect cell fate and recovery. In vitro studies may provide insight into signals present in the acutely injured host microenvironment that could alter HuCNS-SC fate and migration.

• Funded by:
• NIH R01NS049885 (AJA)
• Christopher and Dana Reeve Foundation
• StemCells, Inc.

Effect of intermittent fasting following spinal cord injury in mice (Femke Streijger)
Previously, we reported that every-other-day-fasting (EODF) started after an incomplete cervical spinal cord injury, improved functional recovery in rats. EODF treated animals showed a dramatic reduction in lesion size and enhanced sprouting of the corticospinal tract. Furthermore, EODF treatment lowered fasting blood glucose levels, increased beta-hydroxybutyrate levels, dampened the cellular inflammatory response, and enhanced astrocyte alignment at the lesion surface. These mechanisms may play a direct role in the neuroprotective effects of EODF. More recently we found that EODF is also beneficial if started after a thoracic spinal cord contusion in rats.
In this study, we wanted to test whether EODF was also beneficial in mice when started after a moderately severe thoracic spinal cord forceps compression injury. Following injury, EODF mice ate 63% more on days they had access to food, as did mice fed ad libitum. Beta-hydroxybutyrate levels exhibited a ~3-fold increase on fasting days in the EODF treated mice, while glucose concentration was ~20% decreased compared to the ad libitum animals. During the first two-weeks after injury, EODF treated mice appeared acutely ill and showed dull fur, piloerection, and reduced mobility. In contrast ad libitum fed mice only exhibited minimal changes in physical appearance after injury. The body weight after spinal cord injury in the EODF group remained significantly below those of the ad libitum group. Unlike to our observations in rats, no differences in the BMS locomotor scores were observed between mice treated with EODF versus ad libitum fed mice. The horizontal ladder results revealed slightly more hindlimb errors in the EODF fed mice.
Thus, intermittent fasting for 24-hours has no beneficial effects on the behavioural recovery after thoracic spinal cord compression injury in mice. While the effects on ketone and glucose levels of mice and rats were similar, mice appeared more sensitive than rats to dietary restriction when combined with spinal cord injury. We speculate that excessive stressors due to spinal cord injury negated possible beneficial effects of dietary restriction in mice.

• Funded by:
• Craig H. Neilsen Foundation
• Christopher and Dana Reeve Foundation
• CIHR of Canada

Effect of immunosuppression on the engraftment and fate of transplanted human neural stem cells in a constitutively immunodeficient mouse model of spinal cord injury (Chris J Sontag)
We have previously demonstrated locomotor recovery in a NOD-scid mouse model of spinal cord injury (SCI) that received human CNS stem cells grown as neurospheres (HuCNS-SC). The differentiated cells predominantly became oligodendrocytes and neurons, with very few assuming an astrocytic fate (Cummings, PNAS 2005). NOD-scid mice are constitutively immunodeficient, lacking a normal T-cell, B-cell, and complement response. As such, NOD-scid mice provide an excellent experimental model to assess the potential of transplanted human cell populations to engraft and promote histological and locomotor recovery in the absence of confounds due to a xenograft rejection response. However, from a clinical perspective it is clear that allogeneic transplantation of therapeutic human cell populations will require administration of immunosuppressants such as Tacrolimus (FK506), Cyclosporine A (CsA), or Sirolimus (Rapamycin), at least initially. These compounds all affect immunophilin ligand signaling; in addition to altering T-cell activation, they also alter BMP and other signaling pathways that have the potential to affect the proliferation and/or differentiation of both transplanted and endogenous stem cells within the CNS. To evaluate the effect of these immunosuppressants on HuCNS-SC independent of rejection due to a xenogeneic barrier, HuCNS-SC were transplanted into the parenchyma of 50kd contusion-injured NOD-scid mice 9 days post-SCI, in the presence or absence of immunosuppressant drugs. FK506, CsA, Rapamycin, or vehicle was administered 2 days prior to transplantation (i.e., beginning 7 days post-SCI) and daily after transplantation until sacrifice. All animals received BrdU 2 days after transplantation and weekly thereafter. Locomotor recovery was assessed at 1, 7, and 13 weeks post-transplantation via open-field BMS testing, horizontal ladder beam testing, and CatWalk gait analysis. Immunosuppressant administration did not affect recovery of locomotor function determined by BMS testing. Spinal cords were dissected, sectioned and immunostained using human-specific cytoplasmic and nuclear markers in combination with cell lineage markers (GFAP, ß-tubulin III, doublecortin, Olig2, CC-1/APC) to quantify cell engraftment, migration, and fate using unbiased stereological methods.

• Funded by:
• NIH R01NS049885
• NIH T32 NS007444-7
• Stem Cells Inc.
• Christopher & Dana Reeve Foundation Animal Core

Do T-cells mediate NT-3 induced axonal plasticity after spinal cord injury? (Qin Chen)
Over-expression of Neurotrophin-3 (NT-3) promotes axonal sprouting in the acutely injured but not chronically injured spinal cord in immunocompetent rats. In contrast, NT-3 does not induce the same effect in the immunosuppressed rats with acute spinal cord injury. Moreover, NT-3 enhances neuroplasticity in chronically injured spinal cords of immunocompetent rats when the immune responses are re-activated by an i.p. injection of lipopolysaccharide (LPS). Quantitative FACScan and immunohistochemical studies demonstrated that lesioning the CST activated, and LPS re-activated, microglia and CD4 (+) T-cells in the acutely lesioned and chronically lesioned rats, respectively. The number of activated CD4 (+) T-cells stimulated by the wound healing process was less in the immunosuppressed rats compared to that of immunocompetent rats. These findings suggested that immune-mediated wound healing is required for NT-3 to induce neuroplasticity. To further test whether T-cells mediate the neuroplasticity we measured NT-3-induced axonal sprouting in athymic nude rats (rnu/rnu) that lack mature and functional T-cells. Rats received a unilateral lesion of the corticospinal tract (CST) at the level of the medulla. Two weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene (Adv.NT-3) targeted to the motoneurons by retrograde transport. Adv.LacZ was used as a control vector. At 35 days post-lesion the axonal sprouting was measured in rnu/rnu rats and their heterozygous littermates (rnu/+) treated with Adv.NT-3 or Adv.LacZ. The amount of axonal sprouting in the rnu/+ rats treated with Adv.NT-3 was significantly greater than that of rnu/rnu rats treated with Adv.NT-3. There was no difference in the number of sprouting axons among the rnu/+ rats treated with Adv.LacZ and the rnu/rnu rats treated with Adv.NT-3 or Adv.LacZ groups. These data demonstrate that in the absence of mature T cells over-expressed NT-3 will not induce axonal sprouting in rats with acutely injured spinal cords. This finding suggests that T cells-mediated immune activation is required for NT-3 induced axonal growth.

• Funded by:
• Christopher and Dana Reeve Foundation
• Misson Connect

Mechanisms of STAT3-dependent astrocyte reactivity and scar border formation using a new in vitro injury model (Ina B. Wanner)
Reactive astrogliosis is a typical feature of CNS injury, yet little is known on the role of astrocytes in early stages of scar border formation and its molecular underpinnings. After injury, a neuro-protective wound boundary of glia limitans and tightly interwoven astrocyte processes forms. We have established a new in vitro injury model using original injury factors: mature astrocytes are traumatized by mechanical stretch and confronted with meningeal fibroblasts. These stimuli create scar-like lawns (in press). In this study we aimed to identify how STAT3, signal transducer and activator of transcription 3, regulates astrocyte reactivity using STAT3-astrocyte deficient mice that show defective scar borders after spinal cord injury (submitted). Our cultures had two astrocyte phenotypes, polygonal cells and process-bearing, bushy astrocytes with microvilli. We focus on the latter because they resemble the territorial “tiling” of mature protoplasmic astrocytes in vivo. In wild-type cultures, neighboring processes of these cells interlock. In STAT3(-/-) cultures, about 90 % of this subpopulation were astrocytes with malformed processes without the interlocking pattern. We asked if such malformed cells were deficient in their ability to respond to injury stimuli. Wildtype astrocytes reproducibly respond to stretch and fibroblast addition with drastic shape changes. They contracted and formed elongated processes creating borders. Time-lapse imaging showed new process formation and dynamic microvilli. Upon cell contraction the cytoskeleton fasciculated and this led to bright GFAP-signals in reactive cells. STAT3(-/-) astrocytes had lower levels of GFAP protein and displayed a less organized cytoskeleton. Knockout astrocytes failed to form organized “reactive sites” in response to the injury stimuli. Processes often overlapped and cell-cell contacts were rare. Moderate stretching caused a three-fold increase in cell death in STAT3(-/-) astrocytes. We have started to identify proteins that could account for the drastic shape changes underlying astrocyte reactivity and are impeded in the knockout. Our work determines cellular and molecular changes of mature astrocytes in response to trauma and non-neural cell infiltration and shows how STAT3 signaling is critical for those changes. The data suggest that regulating cell-cell contacts and cell adhesion molecules is important for the beneficial early stages of scar border formation and seems not to influence the expression of scar-associated glial axon growth inhibitors that are detrimental for regeneration.

• Funded by:
• Christopher and Dana Reeve Foundation
• The Miami Project to Cure Paralysis

[Steven Edwards]

Next: The Miami Project.

Improvement of locomotor performance following transplantation of autologous Schwann cells in a non-human primate with chronic spinal cord injury (Andres L Maldonado)
Introduction: Radiofrequency lesioning (RF) can be used as a stereotaxic model of spinal cord injury (SCI) to create a lesion cavity surrounded by a demyelinated penumbra. We tested the effects of intracavity autologous Schwann cell (SC) transplantation and treadmill training on locomotor performance (LP) in a non-human primate (NHP) subjected to a RF lesion of the medullary pyramid.
Methods: Two monkeys (Macaca fascicularis) were used. One served as an uninjured control (NHP1). A second, NHP2, underwent RF lesion of the medullary pyramid 2.3 y before transplantation. The right pyramid was targeted. RF was delivered at 80° C for 75 seconds. Magnetic resonance imaging (MRI) was performed. SCs were purified from the sural nerve using the mitogens forskolin (2µM) and heregulin (2.5 nM) and cryopreserved for 2.3 years. Cells were recovered and expanded for 3 passages before transplantation. Cultures were 90% positive for p75 and S-100. SCs were exposed to a GFP lentivirus at an MOI of 100. Three million SCs in 32 µl were injected into the lesion.
LP was tested for 11 weeks prior to, and 24 weeks after transplantation. Behavioral sessions consisted of 15-minute periods at progressively increasing speeds twice weekly. Video clips were analyzed using Peak Motus Software and several kinematic parameters of LP were studied. Inter-animal and pre- to post-transplantation comparisons were performed.
Results: Quadriparesis was observed immediately after the RF injury. A partial recovery was observed over two weeks and a hemiplegia was evident thereafter. MRI showed an extensive lesion of the pyramid and olivary nucleus. LP did not change during the pre-transplantation testing period (11 weeks). Paw/hand dragging, absence of coordination, and lack of weight support were prominent. During the post-transplantation period (24 weeks) a progressive improvement was observed. Parameters including hip height, ankle inclination, extent and variability of hip, knee, and ankle range of motion and cycle phase duration were significantly improved as compared to pre-transplantation. These parameters progressively normalized as compared to similar measures from the non-lesioned primate. Maximum recovery was reached at week 24 post-injury and behavioral testing was discontinued for approximately 4 weeks. Subsequent behavioral testing has shown neither further improvement nor loss of recovery.
Conclusion: Following SC transplantation we observed progressive recovery of LP in a NHP with a chronic pyramidal RF lesion. Stability of the improvement after discontinuing training suggests structural repair as opposed to only a training effect. Replication studies are ongoing.

• Funded by:
• The Miami Project to Cure Paralysis

Transplants of purified embryonic motoneurons into adult rat peripheral nerve improve muscle reinnervation and function (Robert Matheson Grumbles)
Spinal motoneurons die after spinal cord trauma and in several neurological diseases. Motoneurons need to be replaced to prevent irreversible degradation of denervated skeletal muscle. Embryonic day 14-15 (E14-15) ventral spinal cord cells transplanted into adult rat tibial nerve reinnervate muscle, reduce muscle atrophy and restore function (Thomas et al., Neurophysiol. 84:591-595, 2000). Here we evaluated whether purification of the cell preparation for motoneurons prior to transplantation would improve axon regeneration, muscle reinnervation, and recovery of function. The sciatic nerve of adult Fischer rats was transected to induce denervation. The proximal stump was ligated and sutured into hip muscles to prevent regeneration into the distal nerve stump. One week later, E14-15 ventral spinal cord cells were dissociated and separated on a differential density gradient using centrifugation. Ventral spinal cord cells enriched in motoneurons (n=200,000) or cells dissociated using standard techniques (1 million) were injected into the distal tibial nerve stump 10-15 mm proximal to its entry into the medial gastrocnemius muscle. Rats were euthanized ten weeks after transplantation. Compared to the standard dissociated ventral spinal cord cell preparation, the purified transplants had more choline acetyl transferase and neuron-specific nuclear protein-positive neurons, higher numbers of myelinated axons in the nerve to the medial gastrocnemius muscle, greater muscle reinnervation, more muscles that functioned (96 % versus 70 %) and stronger reinnervated motor units. Both types of transplants reduced muscle atrophy significantly. These results suggest that transplantation of enriched populations of motoneurons into peripheral nerve can enhance restoration of muscle innervation and function.

• Funded by:
• NIH Grant NS39098
• The Miami Project to Cure Paralysis

Schwann cells improve survival of embryonic neurons transplanted into the axotomized tibial nerve and muscle reinnervation (Mitsuhiro Enomoto)
Denervation of limb muscles is common in neurodegenerative diseases and trauma that involve death of spinal motoneurons. Without intervention, the muscles will continue to degenerate, compromising their rehabilitation. Transplantation of embryonic ventral spinal cord cells into peripheral nerve near denervated muscles is one strategy to restore innervation when no other peripheral axons are available for reinnervation. This neural transplantation strategy rescues denervated muscle through the re-establishment of neuromuscular junctions. Stimulation of the transplants with low intensity current produces muscle contractions for several hours (Thomas et al. J Neurophysiol 84:591-595, 2000). But in general, the reinnervated muscles are weak and the neuron survival is poor. Since Schwann cells provide factors important for neuron survival and axon growth, we hypothesized that concurrent transplantation of Schwann cells and embryonic neurons would increase neuron survival, axon regeneration, muscle reinnervation and function. Schwann cells were isolated from adult peripheral nerve. Four groups of adult Fischer rats underwent unilateral tibial nerve section to induce widespread muscle denervation. One week later, cells were transplanted into the distal nerve stump. Transplants included either: 1) 1 million dissociated embryonic day 14-15 (E14-15) ventral spinal cord cells in 10 µl L15 medium and 500,000 Schwann cells; 2) E14-15 cells and Schwann cells transduced with a lentivirus that expresses a multifunctional neurotrophin, NT-3/D15A, a modified human NT-3 that can bind and activate both trkB and trkC receptors (Urfer et al., EMBO J 13:5896-5909, 1994); 3) E14-15 cells alone; or 4) only medium without cells. Ten weeks after transplantation, electrical stimulation of the transplants resulted in muscle contractions. E14-15 cell transplants with Schwann cells had more neuron-specific nuclear protein-positive neurons than cell transplants alone, but the neuron count was similar between the two groups that included Schwann cells. Reductions in muscle atrophy occurred in all groups that involved cell transplantation. These data suggest that suboptimal neurotrophic support and axon growth substrates limit neuron survival in peripheral nerve.

• Funded by:
• NIH Grant NS30398
• The Miami Project to Cure Paralysis

Activity dependent labeling of brainstem monoaminergic and non-monoaminergic neurons during mesencephalic locomotor region evoked fictive locomotion in the cat (Brian R Noga)
Mesencephalic locomotor region (MLR) stimulation is thought to evoke locomotor activity by activation of reticulospinal neurons of the brainstem (Noga et al. J Neurophysiol 90:1464-78, 2003). However, work in our laboratory has also shown that during MLR evoked locomotion, spinal monoamine release is widespread extrajunctionally and is dynamically regulated on a timescale of seconds or less (Noga et al. 2007, Soc Neurosci Abstr Vol. 33:924.13). To account for this spinal release, descending monoaminergic neurons should also be activated during locomotion. In the present study we immunocytochemically co-localized serotonin or dopamine-beta-hydroxylase (DBH) with the activity-dependent label c-fos in sections obtained from the mesencephalon and medulla of paralyzed decerebrate cats, capable of walking with electrical stimulation of the MLR. Animals subject to the locomotor task (3-4.5 hours) showed abundant c-fos labeled cells in the cuneiform nucleus (site of stimulation), parabrachial nuclei, periaqueductal grey (PAG), locus ceruleus/subceruleus, magnocellular, gigantocellular and lateral tegmental fields, postpyramidal nucleus of the raphe (midline) and parapyramidal region, medial vestibular nuclei, dorsal motor nucleus of the vagus, n. tractus solitarius and lateral reticular nucleus/rostral ventrolateral medulla. Many of these neurons were innervated by serotonergic fibers. Control animals, not subject to locomotion, showed relatively few labeled neurons in these areas. Many locomotor-activated neurons in the parabrachial nuclei and locus ceruleus/subceruleus were found to be positive for DBH and occasionally for serotonin. In the medulla, the majority of serotonergic neurons within the parapyramidal region were positive for c-fos, in contrast to the relatively few observed in the midline postpyramidal nucleus of the raphe. These results demonstrate the anatomical/functional basis for spinal monoamine release during evoked locomotion and indicate that the primary serotonergic neurons activated by MLR stimulation are those found in the parapyramidal region of the medulla (~P7-8, posterior level). Furthermore, they indicate that in addition to activation of monoaminergic and reticulospinal neurons for the production of locomotion, MLR stimulation also activates vestibular, cardiovascular and respiratory centers in parallel. Serotonergic neurons within the parapyramidal region may be activated by a direct projection from the MLR and/or possibly via the PAG.

• Funded by:
• NIH Grant R01 NS046404
• Miami Project to Cure Paralysis
• State of Florida

Combination therapy after severe contusive spinal cord injury (SCI) using environmental enrichment (EE) and serotonergic hNT2.19 cell transplantation (Alice M Holohean)
5-hydroxytryptamine (5-HT) has been shown to modulate both locomotor and sensory pathways in the spinal cord. The disruption of descending 5-HT innervation after severe contusive spinal cord injury (SCI) leads to the loss of 5-HT immunoreactivity in both the dorsal and ventral horns and has been correlated with the severity of motor dysfunction (Dabney et al., 2004). To replace 5-HT input lost after injury, we derived a cell line from the NT2 neuronal line that secretes 5-HT, called hNT2.19. After differentiation, hNT2.19 cells are phenotypically large, multipolar, immunostain for anti-5HT and have a basal release rate of > 200 ρmol of 5-HT/105 cells/10 min, measured using electrochemical HPLC. Differentiated, viable or non-viable control hNT2.19 cells, 1x106 (2.5x105cells/injection), were transplanted bilaterally, into the dorsal columns, both rostral (T6) and caudal (T10), 1 wk after severe contusive SCI (25 mm weight drop using the NYU impact device) to adult female Sprague Dawley nude rats. After transplantation, animals were returned to either normal or EE cages. Motor testing, performed weekly before and following SCI, included open field testing using the BBB and associated subscore analysis. At 8 wks post- transplantation, a gridwalk error test and footprint analysis of stride length, degree of foot rotation and base of support were measured. Viable cell transplant therapy or EE conditioning groups both showed similar BBB score and subscore improvement over injury alone (p<0.001). Greater improvement (p<0.01) in BBB scales were sustained with the combination of both treatments than from either treatment alone. Reduced foot fall errors and improvements in stride length and degree of foot rotation were also seen in the viable transplant or EE groups, but the combined treatment produced no further improvement. There was no improvement in any group for base of support. The motor improvement seen in the current study may be due to a combination of neuroprotective effects produced by the release of the neurotropin BDNF during EE conditioning (Berrocal et al., 2007) and low doses of the neuromodulator 5-HT from cell transplant therapy.

• Funded by:
• Department of Veterans Affairs grant B4862R
• The Miami Project to Cure Paralysis

Baclofen weakens motor units paralyzed by spinal cord injury (Christine K Thomas)
Baclofen, a GABAb agonist, is used clinically on a long term basis to reduce the hyperreflexia, muscle tone and involuntary muscle activity associated with spasticity but the long-term effects of baclofen on skeletal muscle health are unclear. The aim of our study was to determine whether chronic use of baclofen changes the contractile properties of human thenar motor units paralyzed by cervical spinal cord injury (SCI). Single motor axons to thenar motor units were stimulated intraneurally at 1-100 Hz. The evoked electromyographic activity (EMG) and force were recorded. Results from individuals with chronic cervical SCI (>1 yr) who have or have not taken baclofen since injury were compared to data from uninjured control subjects. Paralyzed motor unit properties were independent of injury duration and level. Compared to control units, chronic cervical SCI resulted in longer twitch contraction times, stronger twitch forces, higher twitch/tetanic force ratios, and more fatigable motor units irrespective of whether baclofen was or was not used. However, subjects taking baclofen had significantly weaker tetanic forces (median: 35 mN; force without baclofen: 45 mN), longer twitch half relaxation times, and reached half maximal force at lower frequencies than control units (median force: 100 mN). EMG duration, an estimate of muscle conduction velocity, was comparable for each group of paralyzed units and longer than for control units. In contrast, axon conduction velocities differed across groups and were slowest for those subjects not taking baclofen and fastest for control units. These data suggest that chronic use of baclofen prolongs muscle relaxation and exacerbates weakness of paralyzed motor units, changes that are likely to have functional effects. As an example, uninjured individuals often use 5-10 % of their muscle capacity (0.5-1 N) to perform daily activities. Approximately 30 paralyzed units would have to be stimulated to produce 1 N of force in subjects taking baclofen, but only 20 units in subjects not taking baclofen, and 10 units in control muscles. Baclofen-induced motor unit weakness may make the whole muscle more prone to fatigue because more paralyzed units would need to be excited during pattern electrical stimulation to produce the same muscle force as paralyzed units from individuals not taking baclofen, or units from uninjured controls.

• Funded by:
• NIH Grant NS30226
• Swedish Medical Research Council
• The Miami Project to Cure Paralysis

Mechanisms of STAT3-dependent astrocyte reactivity and scar border formation using a new in vitro injury model (Ina B. Wanner)
Reactive astrogliosis is a typical feature of CNS injury, yet little is known on the role of astrocytes in early stages of scar border formation and its molecular underpinnings. After injury, a neuro-protective wound boundary of glia limitans and tightly interwoven astrocyte processes forms. We have established a new in vitro injury model using original injury factors: mature astrocytes are traumatized by mechanical stretch and confronted with meningeal fibroblasts. These stimuli create scar-like lawns (in press). In this study we aimed to identify how STAT3, signal transducer and activator of transcription 3, regulates astrocyte reactivity using STAT3-astrocyte deficient mice that show defective scar borders after spinal cord injury (submitted). Our cultures had two astrocyte phenotypes, polygonal cells and process-bearing, bushy astrocytes with microvilli. We focus on the latter because they resemble the territorial “tiling” of mature protoplasmic astrocytes in vivo. In wild-type cultures, neighboring processes of these cells interlock. In STAT3(-/-) cultures, about 90 % of this subpopulation were astrocytes with malformed processes without the interlocking pattern. We asked if such malformed cells were deficient in their ability to respond to injury stimuli. Wildtype astrocytes reproducibly respond to stretch and fibroblast addition with drastic shape changes. They contracted and formed elongated processes creating borders. Time-lapse imaging showed new process formation and dynamic microvilli. Upon cell contraction the cytoskeleton fasciculated and this led to bright GFAP-signals in reactive cells. STAT3(-/-) astrocytes had lower levels of GFAP protein and displayed a less organized cytoskeleton. Knockout astrocytes failed to form organized “reactive sites” in response to the injury stimuli. Processes often overlapped and cell-cell contacts were rare. Moderate stretching caused a three-fold increase in cell death in STAT3(-/-) astrocytes. We have started to identify proteins that could account for the drastic shape changes underlying astrocyte reactivity and are impeded in the knockout. Our work determines cellular and molecular changes of mature astrocytes in response to trauma and non-neural cell infiltration and shows how STAT3 signaling is critical for those changes. The data suggest that regulating cell-cell contacts and cell adhesion molecules is important for the beneficial early stages of scar border formation and seems not to influence the expression of scar-associated glial axon growth inhibitors that are detrimental for regeneration.

• Funded by:
• Christopher and Dana Reeve Foundation
• The Miami Project to Cure Paralysis

The mucolytic ambroxol, a sodium channel blocker, attenuates neuropathic spinal cord injury pain in rats (Shyam Gajavelli)
Spinal cord injury (SCI) patients display symptoms of neuropathic pain including hypersensitivity to cutaneous stimuli. Functional deficits accompanying SCI complicates satisfactory treatment of neuropathic SCI pain, since many analgesic drugs are accompanied by motor deficits. Ideally, a drug with minimal side-effects is desired for treating neuropathic SCI pain. Na+ channels found in the peripheral nervous system have been shown to mediate neuronal hyperexcitability following injury. These channels are ideal analgesic drug targets since they are expressed primarily in sensory afferents and do not have the adverse side-effects associated with blocking CNS Na+ channels. Ambroxol, a mucolytic approved for use in the E.U., has demonstrated to be antinociceptive in rat models of peripheral neuropathic pain and has also demonstrated to potently block the tetrodotoxin-resistant Na+ channel (Nav 1.8) found in the dorsal root ganglion. The effect of this drug on neuropathic SCI pain was tested in a previously described SCI model. A mid-thoracic segment of the spinal cord was compressed for 60 seconds with a micro-vascular clip. Four weeks after injury, rats demonstrated robust decreases in hind paw withdrawal thresholds, indicating tactile hypersensitivity. Ambroxol significantly increased withdrawal thresholds, suggesting an antinociceptive effect. It is possible that both CNS as well as peripheral Na+ channels modulate ambroxol’s efficacy. To determine a role of peripheral Na+ channels in SCI pain, a non-subtype selective Na+ channel blocker with limited accessibility to the CNS (QX 314) was also tested in these rats. QX 314 did not alter withdrawal thresholds, suggesting that Na+ channels in the CNS are important in neuropathic SCI pain. It is possible that ambroxol blocks Na+ channels in the CNS as well as in nociceptors, but the exact site of action is not known. The role of peripheral Na+ channels in neuropathic SCI remains to be elucidated.

• Funded by:
• The Miami Project to Cure Paralysis
• The Craig H. Neilsen Foundation

Comparison of repeated treatment with a cannabinoid receptor agonist and analgesic drugs on neuropathic spinal cord injury pain in the rat (Jacqueline Sagen)
Neuropathic pain is a serious complication that accompanies spinal cord injury (SCI). In different neuropathic pain states, some drugs require repeated dosing before analgesic efficacy is achieved (e.g. carbamazepine, amitriptyline). Other drugs, such as opiates, appear to diminish in efficacy over time. Cannabinoids (CB) have been used with degrees of success to treat various clinical pains. The antinociceptive effects of the CB receptor agonist WIN 55,212-2 (WIN) were evaluated with repeated administration in rats with a SCI and in intact rats without a SCI. In addition, drugs that have been used to treat neuropathic pain were administered in SCI rats. The SCI consisted of exposing the mid-thoracic spinal cord and a micro-vascular clip was applied to the cord for one min. Four weeks after compression, the hind paw sensitivity to mechanical stimulation (withdrawal threshold) was tested. Following baseline withdrawal threshold measurement, rats were injected (s.c.) twice daily with either vehicle or WIN, morphine, gabapentin, carbamazepine or amitriptyline (once daily) for seven days. In a separate group of non-injured rats, the effect of WIN, injected twice daily, on responses to acute noxious heat stimulation (latency) was also measured. Four weeks after SCI, rats displayed robust hind paw hypersensitivity to innocuous mechanical stimulation. WIN dose-dependent increased withdrawal thresholds on both the first and last day of treatment. By contrast, morphine efficacy was significantly attenuated by the last treatment day. Similarly, in uninjured rats, the dose-dependent increase in response latency to noxious heat on the first day of WIN treatment was significantly diminished by the fifth day of treatment. Neither carbamazepine nor amitriptyline was antinociceptive on either the first or last days of treatment. The efficacy of a mid-efficacious dose of gabapentin neither increased nor decreased by the last day of treatment. The WIN data suggest that a change in efficacy of a CB receptor agonist given repeatedly over time depends on the pain state. The treatment strategy used with this SCI model did not uncover “latent” efficacy with initially ineffective drugs. Sustained CB treatment may be practical for clinical SCI pain. Overcoming negative attitudes of regulatory agencies will be needed in allowing patients access to this unique class of analgesic drug.

• Funded by:
• The Miami Project to Cure Paralysis
• NIH Grant NS61172

[Steven Edwards]

Next: Mission Connect.

Molecular mechanisms of CNS node of Ranvier formation (Yasuhiro Ogawa)
In myelinated nerve fibers, action potential conduction depends on polarized axonal membrane domains including axon initial segments and nodes of Ranvier. High densities of voltage-gated Na+ (Nav) channels at the axon initial segment initiate action potentials, whereas nodal Nav channels mediate the currents necessary for rapid and efficient action potential conduction along the axons. However, despite their importance, the molecular mechanisms underlying formation of these domains are not fully understood. Several mechanisms have been proposed to mediate the accumulation of nodal molecules including: 1) restriction of channel mobility by paranodal junctions, 2) clustering and recruitment of nodal proteins by axonal cytoskeletal scaffolds such as ankyrinG, and 3) Nav channel clustering induced by a soluble, glial derived factor. In the peripheral nervous system, the glial derived extracellular matrix (ECM) molecule called gliomedin interacts with axonal cell adhesion molecule neurofascin (NF) 186, and initiates clustering of the nodal molecules. In contrast, in the central nervous system (CNS), gliomedin is not present in the nodal region. Nevertheless, we show here that a specialized ECM is assembled at CNS nodes of Ranvier, and that a component of this ECM, a chondroitin sulfate proteoglycan called brevican, binds directly to NF 186, suggestive of a role similar to gliomedin. To test whether interactions between NF186 and the ECM could account for recruitment of NF186 to CNS nodes, we introduced NF186 truncated constructs into layer II/III pyramidal neurons, and observed nodes in corpus callosum. We found that the NF186 ectodomain alone (i.e. no cytoskeletal scaffold binding) was sufficient to target this construct to CNS nodes. However, while deletion of the NF186 ectodomain (i.e. no ECM binding) resulted in failure of clearance from internodal axon, the truncated NF186 was still clustered at nodes. Furthermore, a careful developmental analysis indicated that paranodal junctions formed before the clustering of NF186 and the accumulation of nodal brevican. These results suggest that a single mechanism cannot account for the assembly of CNS nodes of Ranvier. We propose that paranodal, ECM, and axonal cytoskeletal interactions all contribute to CNS node of Ranvier formation, and can compensate for one another.

• Funded by:
• NIH grant NS044916
• Dr. Miriam and Sheldon G. Adelson Medical Research Foundation
• National Multiple Sclerosis Society
• Mission Connect

Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons (Kenneth H Minor)
Axon growth inhibitory chondroitin sulfate proteoglycans (CSPGs) and myelin associated molecules are widely accepted as major impediments to axon regeneration within the injured adult central nervous system (CNS). Two potential means of overcoming the effects of these inhibitors are to lower their levels within the injured CNS or to block neuronal sensitivity to these molecules. We have demonstrated that spinal infusion of the small leucine rich proteoglycan, decorin can suppress the levels of multiple scar associated inhibitory CSPGs, an effect that likely contributed to the ability of axons to cross decorin treated spinal cord injuries (Davies et al., 2004). However in light of data from Koprivica et al., (2005) that showed that blocking epidermal growth factor receptor (EGFR) activity on neurons promoted neurite extension on both inhibitory CSPGs and myelin, there remained the possibility of a direct effect of decorin on the ability of neurons to grow on these inhibitors via decorin inhibition of EGFR activity (Csordas et al., 2000). We have therefore conducted an in vitro analysis of the effects of decorin on neurite extension by adult dorsal root ganglion (DRG) neurons grown on substrates of inhibitory CSPGs or myelin membranes mixed with laminin. Decorin treated DRG neurons grown on a CSPG / laminin substrate for 48hrs showed a ~14.5 fold increase in average neurite length per neuron compared to untreated controls (av. 218µm +decorin: av. 15µm untreated). Decorin treated DRG neurons grown on a myelin / laminin substrate for 18hrs showed a ~4.8 fold increase in average neurite length per neuron compared to untreated controls (av. 160µm +decorin: av. 33µm untreated). Analysis of EGFR protein levels showed a ~45% reduction in decorin treated adult DRG neurons grown on the CSPG rich substrate. Decorin therefore represents a novel combination therapy for promoting axon regeneration via suppression of inhibitory scar tissue formation and directly boosting the ability of axons to grow within CSPG or myelin rich tissues of the injured CNS.

• Funded by:
• Mission Connect: A project of the TIRR Foundation
• Lone Star Paralysis Foundation

IL-1 receptor antagonist attenuates mechanical allodynia and central sensitiation via decreased glial activation and IL-1β production after spinal cord injury (Claire E Hulsebosch)
Current treatment for SCI includes early intervention with steroid and non-steroidal therapies, both controversial. One promising intervention is to target specific inflammatory pathways, since inflammation is significantly increased after a CNS injury. We tested whether intrathecal treatment (750 ng/ml, 1 µl/hr delivery rate, Alzet pump 1003D) for 3 days after SCI with an FDA approved agent used in rheumatoid arthritis, IL-1 Receptor Antagonist (IL-1ra, Kineret®), would attenuate mechanical allodynia and central sensitization of dorsal horn neurons; and if mechanisms of action included inhibition of glial activation and cytokine production. SCI is modeled in our laboratory by using a contusion injury (IH Impactor: 150 kdynes, 1 sec dwell) which results in consistent development of mechanical allodynia in every animal (non-noxious stimuli becomes noxious) that persists the life of the animal. Male Sprague-Dawley rats (225-240 g) were injured by contusion at spinal segment T10 and received IL-1ra or vehicle treatment and followed for 35 days or more. Behavioral, immunocytochemical and eletrophysiological studies were performed. IL-1ra treated were compared to vehicle treated, SCI untreated groups and sham controls. We report statistically significant reductions in mechanical allodynia, dorsal horn neuronal hyperexcitability to tactile stimuli (central sensitization), decreased GFAP and IL-1beta production. Thus, pro-inflammatory receptor antagonists should be useful therapeutic agents for central neuropathic pain syndromes such as that after SCI.

• Funded by:
• NIH grant NS39161
• NIH grant NS 11255
• Mission Connect of TIRR Foundation
• Dunn and West Foundations
• Mr. Liddell Chair

Free radical scavenger attenuates below level central neuropathic pain following spinal cord injury (Young Seob Gwak)
Spinal cord injury induces overproduction of reactive oxygen species that contribute to neuronal secondary damages as well as potentiates nociceptive transmission. In this study, we compared the intrathecal and systemic treatment of free radical scavenger on modulating below level central neuropathic pain following spinal contusion injury. Rats with spinal cord injury induced by T10 moderate contusion injury (IH device, Body Weight 249 ± 2 g, displacement 864 ±17.5 μm, 154 ± 1.1 kdyne Force, 122 ± 0.3 m/s velocity and 1 sec dwell time) showed below level neuropathic pain behaviors, such as mechanical allodynia, cold and thermal hyperalgesia on in both hindlimbs, over 40 days after injury. To scavenge reactive oxygen species, phenyl-N-tert-butylnitrone (PBN, free radical scavenger), was administrated immediately after spinal cord injury and for 7 consecutive days by either intrathecal (i.t.) or systemic (i.p.) injections. The spinal cord injury with PBN treatment group (100 mg i.p. and 3 mg i.t.) showed attenuation of mechanical allodynia and thermal hyperalgesia, but not cold hyperalgesia, compared to sham and vehicle controls (p<0.05). In addition, spinal cord injury induced neuronal hyperexcitability of wide dynamic range neurons in the lumbar spinal dorsal horn. Topical administration of PBN (3 mg) significantly attenuated neuronal hyperexcitability whereas 1 mg PBN did not show significant effects (p<0.05). The present data suggest that reactive oxygen species contribute to the persistence of central neuropathic pain below the lesion level after spinal cord injury.

• Funded by:
• NIH NS11255
• NIH NS39161
• Dunn and West Foundations
• Mission Connect of TIRR Foundation

Aquaporin 1 in spinal cord injury (Julieann C Lee)
The role of water channel aquaporin 1 (AQP-1) in uninjured or injured spinal cords is unknown. AQP-1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP-1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant 4 to 8 fold increases in AQP-1 levels persisting up to 11 months post-contusion, not only at the site of injury (T10), but also in cervical and lumbar segments, a novel finding. Given that the anti-oxidant melatonin significantly decreased SCI-induced AQP-1 increases, and that hypoxia inducible factor Hif-1 alpha was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP-1 increases after SCI. Interestingly; AQP-1 levels were not affected by long-lasting hypertonicity that significantly increased astrocytic AQP-4, suggesting that the primary role of AQP-1 is not regulating isotonicity in spinal cords. Based on our results we propose novel roles for AQP-1 in injured spinal cords: (a) in neuronal swelling, since AQP-1 was increased in all surviving neurons after SCI, (b) in formation of fluid-filled cysts and (c) in the development of the neuropathic pain after SCI. We have shown that decreased AQP-1 in melatonin-treated SCI rats correlated with decreased AQP-1 immunolabeling in dorsal horn sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP-1 and chronic neuropathic pain after SCI.
Supported by Mission Connect, a project of TIRR Foundation; NIH NINDS and John Sealy Memorial Endowment Fund for Biomedical Research.

• Funded by:
• Mission Connect Grant
• NIH NINDS Grant
• John Sealy Memorial Endowment Fund for Biomedical Research

[Steven Edwards]

Next: Craig H. Neilsen Foundation

Novel compounds that overcome inhibition of CNS regeneration (Andrea L Peterson)
The failure of central nervous system (CNS) neurons to regenerate following spinal cord injury (SCI) leads to irreversible paralysis and sensory deficits. Traumatic insults to the spinal cord lead to the deposition of inhibitory myelin debris and the formation of a glial scar, which contains a large number of regeneration inhibitors. Prominent among these inhibitors is a family of astrocyte produced proteoglycans known as chondroitin sulfate proteoglycans (CSPGs). Attempts to achieve regeneration of long axon tracts by overcoming inhibitory signals from myelin and CSPGs have met with limited success, partially owing to a lack of detailed knowledge about the signaling mechanisms underlying inhibitory CSPG signals. One way to advance our knowledge and promote novel therapies for SCI is to identify chemical compounds with the ability to overcome regeneration inhibition.
Our lab has undertaken a “chemical genetics” approach, screening a library of novel triazine compounds to identify those that promote neurite growth on inhibitory substrates. We initially identified four “hit” compounds based on their ability to increase neurite outgrowth on an inhibitory substrate of CNS myelin. Interestingly, these compounds also increase neurite outgrowth from several different classes of CNS neurons on a substrate of inhibitory CSPGs, suggesting that they act through a signaling pathway that is common to both classes of inhibitors. Most excitingly, one of the compounds, F05, promotes acute regeneration of severed dorsal column axons in vivo. Studies of potential mechanisms revealed that the compounds do not act through cAMP, protein kinase C, or the EGF receptor. However, compound F05 does affect microtubule dynamics in cultured hippocampal neurons, suggesting a potential mechanism through which regeneration could be affected. Elucidation of the mechanism of action of these compounds should provide insight into mechanisms of regeneration inhibition, and may lead to novel therapeutic strategies for SCI.

• Funded by:
• Veterans of America, Grant # 2445
• Craig H. Neilsen Foundation
• Wallace H. Coulter Foundation

Labeling regenerating axons in the injured newt spinal cord (Katherine A Zukor)
Unlike adult mammals, adult newts are able to functionally recover after spinal cord injury. This recovery requires severed long distance projection axons to regenerate through the injury site and make functional connections with downstream targets. In order to study how this occurs, it is necessary to be able to distinguish such regenerating axons from local circuit or degenerating axons. Using methods adapted from similar studies done in zebrafish, two axon tracers, a small molecular weight biotinylated dextran amine (BDA, 3000 MW, lysine fixable, Invitrogen, D71235) and biocytin (Pierce, 28022), were tested for their ability to specifically label axons for relatively long distances in the spinal cord of the adult newt, Notophthalmus viridescens. A piece of gel foam was soaked in a 10% solution of BDA or 5% solution of biocytin and applied to a fresh spinal cord transection site. Twenty-four hours later about 4 mm of spinal column was harvested for analysis. The tracers were labeled on transverse and longitudinal paraffin sections with streptavidin-Cy5 conjugates (SA-Cy5, Invitrogen, SA1011) and visualized via confocal microscopy. BDA was found to work well as an axon tracer in the newt spinal cord, while biocytin was not. BDA appears to specifically label only axons when viewed in cross sections and travels at least 3 mm from the application site in 24 hours when viewed in longitudinal sections. When applied about 2 mm rostral or caudal to a fresh spinal cord transection or crush injury, the tracer is unable to travel across the injury before axons have had a chance to regenerate. These data demonstrate that BDA can be used to label regenerating axons. Future experiments will use this method to determine when axons regenerate, where they are at each stage of regeneration, and what pathways they choose in order to reach functional targets. The nature of the extracellular environment they migrate through will be characterized in order to determine how much influence permissive and inhibitory molecules appear to have in enabling and guiding axon regeneration. Since the inhibitory nature of the mammalian central nervous system is thought to be a primary reason why spinal cord regeneration fails, it is expected that the extracellular environment of the injured newt spinal cord will not contain many growth cone inhibitors. These experiments will shed light on how nature has solved the problem of spinal cord regeneration in an adult vertebrate and may provide insights into how to improve spinal cord regeneration in mammals.

• Funded by:
• The Craig H. Neilsen Foundation

The effects of BDNF, NT-3 and D15A lentiviral gradients on neural precursor grafts in the injured spinal cord (Joseph F Bonner)
Spinal cord injury (SCI) is a devastating condition characterized by the disruption of long axonal tracts and cell death. We have been studying the possibility of using neural stem cells to replace lost neurons and repair damaged neural pathways. Our previous studies have shown that neuronal and glial restricted precursors (NRP and GRP respectively) can be harvested from E13.5 spinal cords of transgenic alkaline phosphatase (AP) rats, and that the AP marker can be used for reliable graft detection and fate analysis. We found that after transplantation in the injured spinal cord, the NRP/GRP differentiate into neurons and glial, respectively. Our focus in the current studies is to create a functional neuronal relay in a dorsal columns injury model. In this model, successful neural repair would allow sensory information to be relayed from injured host axons, through the grafted neurons to target neurons in the dorsal column nuclei. This approach shifts the challenge of long distance regeneration from adult injured neurons to embryonic neurons, testing the premise of neuronal replacement by neural stem cells. We hypothesize that induction of axonal growth from grafted NRP can be accomplished by generating directional neurotrophin gradients using lentiviral vectors.
We transplanted NRP/GRP acutely into a dorsal column hemi-section injury and a week later injected the lentiviral vectors 5mm rostral to the injury site. The vectors diffuse through the intact tissue, infecting host cells and creating a neurotrophin gradient that is high at the viral injection site and low near the injury site. We then examined whether such gradients induce axonal outgrowth from the grafted NRP. We compared the effects of 3 different lentiviral gradients, BDNF, NT-3 and D15A, which is a mutated form of NT-3 with bioactivity at both the trkB and trkC receptors. We analyzed the ability of these vectors to create neurotrophin gradients by ELISA, and by measuring process outgrowth from NRP/GRP grafts. Our results show that neurotrophin gradients in the spinal cord can be produced using lentivirus and that these gradients can promote directional axonal outgrowth from NRP/GRP grafted at the injury site. The analysis of different neurotrophin gradients will allow examination of their specificity and ultimately allow the induction of directional axonal growth towards putative targets. This is an important building block in the formation of a novel relay after spinal cord injury.

• Funded by:
• NIH Grant NS055976
• Craig H Neilsen Foundation
• Drexel University Spinal Cord Center

Comparison of Polymer Scaffolds in Rat Spinal Cord; A step towards quantitative assessment of combinatorial approaches to spinal cord repair (Bingkun Chen)
Spinal cord injury (SCI) results in permanent injury of axons, neurons and glial cells. Regrowth of axons is essential for repair and functional recovery after spinal cord injury. Biodegradable polymers can simultaneously provide a scaffold for cell delivery and a reservoir for sustained release of therapeutic factors. In this study, we are using tissue engineering with biodegradable polymer scaffolds to bridge the gap, for future use as a platform that will allow quantitative comparison of the effect of delivery of multiple cell types and therapeutic factors. We have demonstrated that biodegradable polymer scaffolds loaded with growth-promoting cells support axon regeneration. Optimal polymer type to support regeneration remains unknown. Thoracic (T9/10) spinal cords of adult female rats were transected and immediately implanted with schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly (caprolactone fumarate) (PCLF) or oligo (poly (ethylene glycol) fumarate) (OPF) hydrogel. Scaffolds loaded with solubilized basement membrane preparation (without schwann cells) were used for negative controls. Behavioral assessment was conducted to examine functional recovery after using the BBB locomotor rating scale. One month after injury and scaffold implantation, tissue was harvested, frozen and the scaffold/cord cut into transverse 8 micrometer sections. Morphometric analysis of neurofilament-stained sections at rostral, midlevel, and caudal scaffold were performed. All of the polymers supported axonal growth. Quantitative, retrograde axonal tracing with fast blue demonstrated that axons from rostral cord grew up to 15 mm caudal to the scaffold. The model provided a quantitative basis for assessing individual or combined therapeutic strategies that will form the basis for future combinatorial approaches to spinal cord repair.

• Funded by:
• EB02390
• Neilsen Foundation

Inhibition of myosin II activity reduces axonal retraction after spinal cord injury (Kengo Ogata)
The retraction of injured spinal cord axons is one of the earliest responses to spinal cord injury (SCI). Axons displaced from the site of injury likely is a disadvantage for future regeneration. We have previously shown that axonal retraction following severing in vitro depends on myosin II activity. In this study we tested the hypothesis that direct or indirect inhibition of myosin II activity will result in reduction in the extent of axon retraction after SCI in vivo. SCI (hemisection) was made in adult Sprague-Dawley rats at cervical level 3(C3) by gentle aspiration. The Myosin II inhibitory reagent Blebbistatin or the RhoA-kinase inhibitors Y27632 were delivered to the injury site with gel foam immediately following injury. DMSO (instead of blebbistatin) and PBS (instead of Y27632) were administered to the vehicle control animals. 7 days before injury all rats were microinjected with biotinylated dextran amine (BDA) into the red nucleus and medullary reticular formation to label the rubrospinal and reticulospinal tracts anterogradely. Animals were perfused at 7 and 28 days after injury. By image analysis we measured the distance of BDA-labeled axon terminals from the lesion site and the mean distance of all measured terminals was calculated. The extent of retraction (mean distance from lesion site) for Blebbistatin treatment groups was significantly less than that of control groups (mean 331 vs. 425 um and 351 vs. 477 um, respectively). These results indicate that axonal retraction can be influenced by direct inhibition of myosin II activity or indirectly thru the inhibition of the RhoA kinase pathway. This suggests the possibility that a neuroprotective therapeutic strategy can lead to an increase in the potential for axonal regeneration.

• Funded by:
• Craig H. Neilsen Foundation

Effects of riluzole and isradipine on reflex excitability and strength in human chronic spinal cord injury (Renee D. Theiss)
The abnormal excitability of spinal neurons and circuitry provides a novel target for intervention in human chronic spinal cord injury (SCI). Studies in animal models have shown that persistent currents, such as sodium and calcium, profoundly influence neuronal excitability, and the recovery of these currents in spinal neurons of animals with spinal cord injury coincides with the appearance of spastic reflexes. Inspired by these animal models, we hypothesize that reflex hyperexcitability in human chronic SCI could be controlled by modulating the intrinsic excitability of spinal neurons. To test this hypothesis, we examined the effects of two pharmacological agents, riluzole and isradipine, on reflex excitability in subjects with chronic (> 1 yr) incomplete SCI. In animal models, both drugs decrease neuronal excitability through persistent currents; riluzole inhibits persistent sodium and isradipine blocks persistent calcium. Prior to and following single-dose, oral administration of 50mg riluzole, 5mg isradipine, or placebo, flexion withdrawal reflexes and hip-movement-induced extensor spasms were elicited, and subjects performed maximum volitional contractions (MVCs). Flexion reflexes were elicited via electrical stimulation (10 pulses, 200 Hz, 1 ms duration, 20s inter stimulus interval) of variable intensity at the medial arch. Extensor spasms were elicited by ramp-and-hold (15s hold) hip extension movements from ~50deg of flexion to ~10deg of extension at 60deg/s. Isometric MVCs were generated about the ankle (plantar- and dorsi-flexion) and hip (flexion and extension). Lower extremity joint torques and electromyographic activity were recorded to quantify all responses. Preliminary data in seven subjects with chronic, incomplete SCI indicated a significant decrease (2-way ANOVA, p = 0.0003, F = 14.9) in peak dorsiflexion torque during the flexion withdrawal responses in the post-test - pretest comparisons between the riluzole and placebo conditions for all stimulus intensities. On average, riluzole administration decreased peak torque amplitude by approximately 20%. Riluzole administration produced minor changes in peak ankle MVCs compared to placebo. Preliminary data of isradipine administration in three of the above seven subjects have not shown appreciable changes in flexion withdrawal reflex amplitude. These results suggest a contribution of persistent sodium currents but not persistent calcium currents in regulating flexion reflex amplitude. Whether the changes in reflexive behaviors induced by these drugs will facilitate or hamper recovery during rehabilitation is still unknown.

• Funded by:
• C.H. Neilsen Foundation Grant 2780 (RDT)

Amphetamine enhanced motor training improves recovery of forelimb function following cervical contusion injury (Laura Krisa)
Ischemic injury to the sensorimotor cortex in rats results in motor impairments, including forelimb movements used in reaching and grasping. Motor training combined with d-amphetamine (AMPH) administration has been shown to increase dendritic arborization of cortical motor neurons, enhance monoaminergic function and ameliorate these motor deficits. A unilateral cervical contusion located between spinal cord cervical levels three and four (C3/4) also produces deficits in skilled forelimb reaching and grasping. We tested the hypothesis that a combination therapy of motor training and AMPH administration would enhance recovery of forelimb function through its action on descending monoaminergic pathways. Male rats were trained on single pellet and staircase reaching tasks six weeks prior to undergoing a unilateral cervical contusion injury (175 kilo dynes) at C3/4. Three days following surgery, rats were performance matched and assigned to either the AMPH (1mg/kg) + motor training group or saline group. Motor training began 10-14 days following injury and consisted of two daily 15-minute sessions of tasks that required repetitive forelimb reaching and grasping. Forelimb function was assessed in single pellet and staircase reaching tasks. Locomotor function was tested by grid walking and in the open field for 13 weeks post contusion. Following sacrifice, spinal cord sections were examined for extent of tissue sparing and the presence of dopamine-β-hydroxylase (DBH) labeled axons. Animals receiving combination therapy performed significantly better than control animals in the single pellet reaching task, evident at nine weeks following injury. The motor training emphasized skilled forelimb reaching and this was the function that recovered. No significant differences were observed between groups in the staircase reaching, in which grasp function is emphasized. Locomotor tests showed a deficit with a partial recovery but no differences between groups. Thus, this combination therapy did not transfer to behavioral tests that engage different forelimb muscles and movements. We conclude that combining AMPH with motor training increased skilled forelimb reaching function following a unilateral cervical contusion.

• Funded by:
• CENT grant (J.S.S.)
• The Craig H. Neilsen Foundation (J.S.S.)
• NIH NS055976

Every-other-day fasting started after thoracic spinal cord contusion injury improves functional recovery (Ward T. Plunet)
Previously we found that therapeutic every-other-day fasting (fasting started after injury) improved functional recovery, reduced lesion size, and increased beta-hydroxybutyrate and full-length/truncated trk B ratio in a rat cervical spinal cord injury model. In this experiment we tested if therapeutic EODF would improve recovery after a low thoracic contusion injury. Four groups of adult male Sprague Dawley rats were studied: Control (ad libitum fed), Post-EODF (EODF started after injury), Pre-EODF (EODF started 3 weeks prior to injury), and a Pair-Fed control group (receiving the same total grams of food as the EODF groups but provided with food portions every day). The open field locomotion behavioural test revealed that both the Pre-EODF (at 48, 64 and 70 days post injury) and Post-EODF groups (64 and 70 days post injury) performed significantly better than either the control group or the Pair-Fed control group. On the horizontal ladder task (irregular rungs changed each testing period) there was a trend (p = 0.11) for the Post-EODF group making fewer errors than either the control group or the Pair-Fed control group at 72 days post injury. The CATWALK device revealed several differences in footprint analysis among groups. The Pre-EODF group reached a higher regularity index (measurement of limb coordination) than either the control group or the Pair-Fed control group, and the Post-EODF group had a higher index than the control group. A higher percentage of animals in both the Pre and Post EODF groups displayed the normal range of forelimb and hindlimb swing duration. Additionally, both EODF groups showed significantly smaller abnormalities in hindlimb to forelimb placements. Spared white and gray matter host tissue, along with 5-HT sprouting distal to the injury are being accessed and will be reported. In summary, either therapeutic EODF started after injury or Pre-EODF can improve functional recovery after a low thoracic spinal cord contusion injury as revealed by multiple behavioral measurements. This behavioral recovery is not due to simple calorie restriction or weight difference because the Pair-Fed group did not perform better than the control group in any parameter and on several measurements the EODF groups were better than both the control and Pair-Fed groups. Therapeutic EODF is a simple treatment that displays robust moderate effects in two different animal spinal cord injury paradigms making it an interesting candidate for clinical translation.

• Funded by:
• Craig H. Neilsen
• Canadian Institutes of Health Research (CIHR)

Strategies to overcome the inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocyte progenitor cells (Donna J Osterhout)
Formation of a glial scar after spinal cord injury (SCI) creates a significant barrier to regeneration and functional recovery. Expression of chondroitin sulfate proteoglycans (CSPGs) occurs quickly after injury, although the deposition of individual CSPG molecules occurs at various times. Neurocan, for example, appears early, while phosphacan is expressed weeks after the initial trauma. The inhibitory effects of CSPGs on neuronal sprouting are well documented, and we have demonstrated that specific CSPGs can also inhibit process outgrowth and differentiation of oligodendrocyte progenitor cells (OPCs).
In this study, we examined the effects of CSPGs on OPC migration in vitro. Normally, OPCs plated on laminin display a bipolar morphology extending long processes; however if CSPGs are added to the culture, the processes retract. Time-lapse microscopy shows that addition of CSPGs to OPCs immediately arrests their migration on the substrate and the cells begin to retract their processes within 30 minutes.Addition of the enzyme chondroitinase (cABC), which cleaves the glycosaminoglycan chains from the CSPG protein core, completely reverses this effect. Furthermore, the addition of a Rho kinase inhibitor also neutralizes the inhibition, suggesting that the CSPGs interact with a surface receptor that activates the Rho kinase pathway. Treatment with the neurotrophin NT-3 or dibutyryl cAMP also overcomes the inhibitory effects of CSPGs, stimulating OPC differentiation. Taken together, these data suggest the glial scar inhibits OPC migration and differentiation. Moreover, treatment strategies that promote axonal regeneration may also enhance remyelination, which is important for recovery of motor function.

• Funded by:
• NYS DOH SCIRB C022046, C020931
• Craig H. Neilsen Foundation

Characterization of spinal and supraspinal input to phrenic motor circuit in the normal and injured spinal cord of adult rat (Michael A Lane)
Previous studies have demonstrated spontaneous recovery of phrenic motoneuron (PhMN) and diaphragm activity following C2 hemisection (C2HMx). Despite progressive improvements in breathing, chronic PhMN and ventilation deficits remain. Using retrograde transneuronal pseudorabies virus (PRV) tracing, we recently demonstrated a population of cervical pre-phrenic interneurons (pPhINs) in uninjured and injured (2 weeks post-C2HMx) rats.
Characterization of the segmental circuitry and bulbospinal projections that may influence post-injury changes in PhMN function was pursued in the present study. The number and distribution of labeled cells was examined 1-12 weeks post-C2HMx. All experiments were performed in accordance with NIH guidelines and institutional IACUC approval. Plethysmography was used to assess respiratory function over this period and terminal neurophysiological assessment of phrenic nerve activity was made in a subset of animals. For tracing studies, the diaphragm was surgically exposed in anesthetized animals, and the phrenic circuit was transynaptically labeled by administering PRV to the left hemidiaphragm. Animals were perfuse-fixed 48-72 hours post-PRV. Sections (40μm) of the cervical spinal cord and medulla were labeled with antibodies to PRV. Temporal labeling was observed in PhMNs (48hrs), pPhINs (56-64 hours) and cells within the medulla (64-72hrs). To characterize the neurotransmitter phenotype of pPhINs, immunocytochemical studies using antibodies against GABA, Glycine, 5-HT and choline acetyltransferase were performed.
Lastly, we examined whether pPhINs could serve as propriospinal relay between the medulla and PhMNs in post-C2HMx. In a sub-group of animals, injections of biotin dextran amine (10,000 kDa) were made into the ventral respiratory group (VRC) of the medulla, to label descending projections. Ten days later, PRV was administered to the left hemidiaphragm and animals perfused 64 hours post-PRV. Studies are underway to examine the relative input from the VRC ipsi- and contralateral to the PRV labeled PhMNs in normal and injured rat spinal cord.
The present study demonstrated that labeling in the reticular nuclei of the medulla was observed prior to labeling in the VRC. Dual-tracing also suggests that pPhINs may represent a propriospinal relay between then VRC and PhMNs. Initial neurotransmitter profiling suggests some PRV-positive pPhINs around the central canal are cholinergic. While contribution of interneurons to pathways associated with plasticity are being established, it is possible that some of these cells modulate recovery associated with the CPP.

• Funded by:
• NIH/NINDS RO1 NS054025-01
• Anne and Oscar Lackner Endowed Chair (PJR)
• Craig H. Neilsen Foundation (MAL)

The mucolytic ambroxol, a sodium channel blocker, attenuates neuropathic spinal cord injury pain in rats (Shyam Gajavelli)
Spinal cord injury (SCI) patients display symptoms of neuropathic pain including hypersensitivity to cutaneous stimuli. Functional deficits accompanying SCI complicates satisfactory treatment of neuropathic SCI pain, since many analgesic drugs are accompanied by motor deficits. Ideally, a drug with minimal side-effects is desired for treating neuropathic SCI pain. Na+ channels found in the peripheral nervous system have been shown to mediate neuronal hyperexcitability following injury. These channels are ideal analgesic drug targets since they are expressed primarily in sensory afferents and do not have the adverse side-effects associated with blocking CNS Na+ channels. Ambroxol, a mucolytic approved for use in the E.U., has demonstrated to be antinociceptive in rat models of peripheral neuropathic pain and has also demonstrated to potently block the tetrodotoxin-resistant Na+ channel (Nav 1.8) found in the dorsal root ganglion. The effect of this drug on neuropathic SCI pain was tested in a previously described SCI model. A mid-thoracic segment of the spinal cord was compressed for 60 seconds with a micro-vascular clip. Four weeks after injury, rats demonstrated robust decreases in hind paw withdrawal thresholds, indicating tactile hypersensitivity. Ambroxol significantly increased withdrawal thresholds, suggesting an antinociceptive effect. It is possible that both CNS as well as peripheral Na+ channels modulate ambroxol’s efficacy. To determine a role of peripheral Na+ channels in SCI pain, a non-subtype selective Na+ channel blocker with limited accessibility to the CNS (QX 314) was also tested in these rats. QX 314 did not alter withdrawal thresholds, suggesting that Na+ channels in the CNS are important in neuropathic SCI pain. It is possible that ambroxol blocks Na+ channels in the CNS as well as in nociceptors, but the exact site of action is not known. The role of peripheral Na+ channels in neuropathic SCI remains to be elucidated.

• Funded by:
• The Miami Project to Cure Paralysis
• The Craig H. Neilsen Foundation

[Steven Edwards]

Next: New York

New mechanisms in neurogenic differentiation of embryonic stem cells (ESC) (Daniel K Kehoe)
ESCs have the potential to become a limitless source of tissue for cell therapies and drug testing. This study focuses on better understanding the specific signals directing the differentiation of ESCs to neural progeny. We further examine the differentiation of ESCs in a bioreactor as a way of producing neuronal cells in quantities suitable for clinical therapies. The integrative nuclear fibroblast growth factor (FGF) signaling (INFS) has been show to mediate the activation of genes thereby coaxing the differentiation of umbilical cord blood cells and human neuronal precursors to neuronal cells. Here, we investigated if a similar mechanism can be utilized for the neurogenic commitment of mouse ESCs (mESCs). We found that treatment of mESCs with agents such as retinoic acid (RA) and cyclic adenosine monophosphate (cAMP) activates INFS, in part by nuclear translocation of an FGF receptor. Immunopositivity for glial fibrillary acidic protein (GFAP) revealed an increased propensity of mESCs for astrocyte differentiation upon stimulation with cAMP. In contrast, RA prompted the differentiation towards neurons as shown by enhanced expression of tyrosine hydroxylase and β3-tubulin. To that end, the localization of the RA receptor in differentiating stem cells in various stages of neuronal differentiation was also studied. These findings may lead to the development of methods for directed differentiation of ESCs to neuronal cells with high efficiency. For such methods to be utilized in clinical therapies, ESC-derived neurons must be generated in high quantities. Hence, we also examine if neuronal differentiation of hESCs can be performed in stirred suspension bioreactors. Cells formed neurosphere-like aggregates in the bioreactor and in a serum-free medium. A transition was noted to neuroepithelial cells and then radial glial cells. Finally, neuron-like cells emerged upon plating of the neurospheres. Differentiated cells displayed increased gene/protein levels of Pax6, Nestin, β3-tubulin, MAP2 and GABA compared to undifferentiated hESCs, and similar levels of ectoderm genes when compared to cells differentiated exclusively in static culture. This scalable system has the ability to produce mass quantities of neurospheres. In conjunction with an INFS-based method for directing the differentiation of these cells further to neuronal cells, this stem cell culture modality may facilitate the production of cellular material suitable for therapies against neurodegenerative diseases.

• Funded by:
• IRDF, SUNY, Buffalo
• NYSTEM
• NYSTAR

The role of axonal translation in developing axons during synapse formation (Ramiro D Almeida)
An important feature of axons during neuronal development is the presence of mRNAs and translational machinery. This allows for the phenomenon of local translation. Local translation of a variety of axonal mRNAs have been described in recent years, with evidence for local translation having roles in LTP, LTD, axonal guidance and growth cone collapse. During development mRNAs are transported to both dendrites and axons where they regulate neuronal growth. Local translation has been better described in dendrites of cultured mammalian neurons, where the requirement for local translation in different models of learning and synaptic plasticity has been addressed in recent years.
Unlike dendritic local protein translation axonal protein synthesis has not been object of intense studies in the past years. During development axons navigate through a complex environment to form specific connections with the appropriate target cell. Although to date several mRNAs have been identified in axons and growth cones of developing neurons no role has been attributed to local mRNA translation in synapse formation in mammalian cells.
One prominent axonal transcript is the beta-actin mRNA. Beta-actin mRNA has roles in mediating responses to axonal guidance cues and stimuli that induce axonal turning. Here we show that local translation of beta-actin plays a role in synapse formation. Beta-actin is translated in axons, and accumulates at presynaptic sites. Selective targeting of axonal beta-actin mRNA interferes with presynaptic differentiation. Together, these results suggest that local translation has a role in synapse formation.

• Funded by:
• NYS SPINAL CORD RESEARCH PROGRAM, Postdoctoral/Mentored Scientist Development SCIRB06-01

Regulation of neurogenesis by nuclear FGF receptor-1 (FGFR1) using nanoparticle-mediated gene transfer into adult brain (Ewa K Stachowiak)
The present study was undertaken to elucidate the role of FGF receptor-1 (FGFR1) in development of the neural stem/progenitor-like cells (NS/PC), using nanoparticle-based non-viral gene delivery. Cell surface FGFR1 stimulated proliferation of cultured nondifferentiated NS/PC. In contrast, FGFR1 that translocates to the cell nucleus stimulated NS/PC differentiation and associated gene activities. To determine the role of nuclear FGFR1 in NS/PC development in the brain we used amine-functionalized ORMOSIL nanoparticles as a novel non-viral vector for in vivo gene transfection. Monodispersed nanoparticles, surface functionalized with amino groups were synthesized for electrostatic complexation with plasmid DNA. Stereotaxic injections of nanoparticles complexed with plasmid pEGFP (Nanoplex) into mouse brain lateral ventricle allowed us to visualize the extensive transfection and expression of enhanced green fluorescent protein in NS/PC-like cells of the subventricular zone. An in vivo fiber optic-based confocal bioimaging showed, in live animals, the transfection and retention of viability of the cells. Transfection of a plasmid expressing the nucleus targeting form of FGFR1 inhibited proliferation and induced neuronal differentiation by cells in subventricular zone and adjacent rostral migratory stream, followed by migration into olfactory bulbs, cortex and striatum. Thus stimulation of nuclear FGFR1 signaling allows us to control the proliferation and differentiation of the NS/PCs in the adult brain. The ORMOSIL nanoparticles provide an effective gene delivery platform that may be used to elucidate and control molecular gene mechanisms that regulate development of brain NS/PCs

• Funded by:
• IRDF, SUNY, Buffalo
• NYSTEM

The role of tissue plasminogen activator in axonal regeneration of a mouse spinal cord injury model (Noreen Bukhari)
The National Spinal Cord Injury Database estimates that 11,000 new cases of spinal cord injury (SCI) occur in the United States each year with a prevalence of 253,000 persons. Acute administration of corticosteroids to suppress the body’s inflammatory response is the most widely used medical treatment for SCI. This treatment highlights two important points for researchers: the body’s secondary response is the more debilitating effect of the injury and new therapeutic approaches must focus on treating the chronic form of the injury. Chondroitinase ABC (ChABC) is a bacterial enzyme that has consistently been shown to reduce the secondary damage called glial scar and enhance axonal plasticity. However, the mechanism underlying this repair remains unclear.
Our group has previously reported that ChABC enhances the interaction of the extracellular serine protease, tissue plasminogen activator (tPA) and its downstream product, plasmin, with the extracellular matrix molecules of the glial scar in in vitro and ex vivo models of SCI. We now test the contribution of this serine protease to ChABC promoted axonal repair using mice deficient in tPA. We hypothesized that tPA acts downstream of ChABC to promote axonal plasticity after SCI. We test the role of tPA in the survival and neurite outgrowth of primary cortical neurons in an in vitro ChABC treated glial scar. We also evaluate tPA's role as a downstream effector of ChABC action in an in vivo model of mouse SCI. Immunohistochemistry is used to visualize the glial scar and axon tracing to measure neurite outgrowth. These studies are intended to elucidate the mechanism of action of a leading therapy against spinal cord injury, and thereby help in the drug development of the bacterial compound.

• Funded by:
• NYS SCIRP C020929

Unilateral cervical Spinal Cord Injury (SCI): Comparison of injuries induced with the IH vs. NYU/MASCIS devices, and a novel method for assessing distal forelimb functional recovery (Karen A. Irvine)
Previous work characterized a model of unilateral cervical contusion injury in the rat using the NYU/MASCIS device to create both mild (6.25mm) and moderate (12.5mm) injuries (Gensel et al., J.Neurotrauma 2006; 23: 36-54). This model has some advantages over thoracic SCI, e.g. cervical injuries are more common than thoracic injuries in humans, and unilateral injuries require minimal animal care post-injury. In the present study, we used the Infinite Horizon (IH) device to produce mild and moderate injuries to match behavioural recovery and histological outcome to those created by the NYU/MASCIS device. A force of 75 kdyn and 100 kdyn using the IH device was comparable to impacts of 6.25mm and 12.5mm on the NYU/MASCIS device respectively.
In addition to the behavioural outcomes previously used (paw placement, grooming, Catwalk), we developed a new measure for assessing recovery of distal forelimb function following cervical contusion SCI, the Irvine, Beattie, Beattie and Bresnahan score (IBBB). Video footage of the rats eating differently shaped cereals prior to and after SCI were analysed and the features of recovery of forelimb use, such as object control, object support, presence of abnormal flailing movements, digital extension, digital movement, grasping technique, and postural compensation, over time were assessed. Preliminary results indicate that the IBBB score continues to detect improvements in forelimb recovery whilst the other behavioural scores reach plateau. Furthermore, recovery assessed using the IBBB score, like that seen in the grooming and paws placement behavioural tests, was sensitive to injury severity. This task has a number of advantages: (1) rats do not need to be food deprived prior to the task in order for them to perform (2) it takes advantage of an innate behaviour therefore the rats do not need to be extensively trained (3) the score could potentially be adjusted to analyze recovery of forelimb function in other rodents or species.

• Funded by:
• NIH Grant NS31193
• NIH Grant NS49881
• NYS Co19772

cAMP and polyamines overcome inhibition by MAG by activating Cdk5 via increased expression of p35 regulated by activation of eIF5A (Huifang He)
The inhibitory molecules associated with myelin are one of the major obstacles to successful axon regeneration after CNS injury. Previously, we have shown that elevating intracellular levels of cyclic AMP (cAMP) can block the inhibition by MAG and myelin. Elevation of cAMP results in up-regulation of arginase I (ArgI) and subsequent increased synthesis of polyamines. Up-regulation of ArgI or priming neurons with the polyamine putrescine blocks the inhibition of axonal growth by MAG/myelin. Here we show that Cyclin-dependent kinase 5 (Cdk5) is required for db-cAMP and putrescine to overcome inhibition. The effect of db-cAMP and putrescine in overcoming inhibition by MAG is abolished in the presence of an inhibitor of Cdk5, Roscovitine. Neurons infected with dominant negative Cdk5 HSV viruses are not able to overcome inhibition by MAG in the presence of db-cAMP. Moreover, db-cAMP and putrescine up-regulate expression of p35, the neuronal specific activator for Cdk5. This in turn induces the kinase activity of Cdk5. Furthermore, we show that putrescine up-regulates p35 protein by hypusine modification of eIF5A, and this hypusination is necessary for putrescine to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines encourage regeneration after CNS injury.

• Funded by:
• NIH-SNRP
• NIH-RCMI
• NIH-NS37060
• NIH-NS41073
• NYSDOH,C018614

Strategies to overcome the inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocyte progenitor cells (Donna J Osterhout)
Formation of a glial scar after spinal cord injury (SCI) creates a significant barrier to regeneration and functional recovery. Expression of chondroitin sulfate proteoglycans (CSPGs) occurs quickly after injury, although the deposition of individual CSPG molecules occurs at various times. Neurocan, for example, appears early, while phosphacan is expressed weeks after the initial trauma. The inhibitory effects of CSPGs on neuronal sprouting are well documented, and we have demonstrated that specific CSPGs can also inhibit process outgrowth and differentiation of oligodendrocyte progenitor cells (OPCs).
In this study, we examined the effects of CSPGs on OPC migration in vitro. Normally, OPCs plated on laminin display a bipolar morphology extending long processes; however if CSPGs are added to the culture, the processes retract. Time-lapse microscopy shows that addition of CSPGs to OPCs immediately arrests their migration on the substrate and the cells begin to retract their processes within 30 minutes.Addition of the enzyme chondroitinase (cABC), which cleaves the glycosaminoglycan chains from the CSPG protein core, completely reverses this effect. Furthermore, the addition of a Rho kinase inhibitor also neutralizes the inhibition, suggesting that the CSPGs interact with a surface receptor that activates the Rho kinase pathway. Treatment with the neurotrophin NT-3 or dibutyryl cAMP also overcomes the inhibitory effects of CSPGs, stimulating OPC differentiation. Taken together, these data suggest the glial scar inhibits OPC migration and differentiation. Moreover, treatment strategies that promote axonal regeneration may also enhance remyelination, which is important for recovery of motor function.

• Funded by:
• NYS DOH SCIRB C022046, C020931
• Craig H. Neilsen Foundation

[lunasicc42]

thank you for this

[Steven Edwards]

Next: Maryland

Role of Smad2/3 in TGF-β mediated regulation of chondroitin sulfate proteoglycans in astrocytes (Bala TS Susarla)
Spinal cord injury (SCI) often leads to permanent paralysis due to the inability of axons to regenerate. This regenerative failure is due in part to the inhibitory environment of the glial scar at the lesion site. Traumatic injury induces expression of the cytokine transforming growth factor beta (TGF-β). Expression of the chondroitin sulfate proteoglycans (CSPGs), which are highly inhibitory to axonal re-growth are also upregulated after SCI. As TGF-β induces expression of several core CSPG proteins in glial cells, it is thought that TGF-β may be responsible for upregulating CSPG expression after SCI. However, the mechanisms through which TGF-β regulates CSPG expression are not known. We have started to examine the role of Smad proteins (Smad2 and Smad3) in TGF-β-mediated regulation of CSPG expression in murine astrocytes, oligodendrocyte progenitor cells as well as after spinal cord injury in vivo. We have utilized Smad3 null mice in addition to lentivirally expressed shRNA specific for Smad2 or Smad3. Smad3 null mice showed less deposition of CSPGs than did wild type mice after dorsal hemisection. Smad specific-lentiviral (LV) shRNA reduced either Smad2 or Smad3 expression by 70-80% in primary murine astrocytes. Preliminary studies suggest that LV-Smad2 shRNA inhibited TGF-β-induced upregulation of the CSPG core proteins neurocan and phosphacan in astrocytes. Our data suggest that inhibition of Smad signaling after SCI may be one mechanism through which deposition of the inhibitory CSPGs may be prevented.

• Funded by:
• Maryland Spinal Cord Injury Research Board Grant FHA07-001

[Steven Edwards]

Next: Canada

Transplantation of PDGF responsive precursors following spinal cord injury (Jason R Plemel)
Spinal cord injury results in massive oligodendrocyte death and subsequent demyelination resulting in long-term white matter defects. The development of cell replacement strategies might be a means to replace lost oligodendrocytes and remyelinate denuded axons. However, the optimal precursor population remains to be determined. We used a neurosphere culture system to characterized PDGF-responsive precursor (PRP) from the ventral forebrain of embryonic day 14 mice and demonstrated that they could generate oligodendrocytes, neurons and astrocytes. PRPs differ from the EGF/FGF-responsive neural stem/progenitor cells, commonly used in transplantation paradigms, particularly in their capacity for astroglial differentiation in vitro. Previous reports have demonstrated that human fetal EGF/FGF-responsive neural stem/progenitor cells rarely generate oligodendrocytes, whereas human PRPs isolated from embryonic and adult sources retain their capacity to generate oligodendrocytes, indicating that PRPs might be a better source of human oligodendrocyte precursor cells.
Therefore, we tested the potential of PRPs isolated from GFP-expressing transgenic mice to remyelinate axons in the injured rat spinal cord. One week after a moderate thoracic (T9) spinal cord contusion in adult male Sprague Dawley rats, PRPs were transplanted rostrally and caudally to the lesion site. PRPs integrated into host tissue and approximately one third of them developed into the typical phenotype of mature branched oligodendrocytes expressing the marker APC-CC1. Evidence that the transplanted cells myelinate is demonstrated by the close association of GFP and myelin basic protein by confocal microcopy. Another hallmark of myelination is the expression and appropriate localization of potassium channels (e.g. Kv1.2) in the (juxta)paranodal axon. Expression of Kv1.2 immunoreactivity surrounded by GFP-positive (PRP) processes was indicative of typical formation of nodes of Ranvier by the myelin forming PRPs. Transplantation studies in Shiverer mice are underway to confirm the myelinating capacity of PRPs. Survival of transplanted PRPs was comparable to that seen in other studies using similar precursor cells following spinal cord compression injury (Karimi-Abdolrezaee et al., 2006). However, unlike those previous studies, transplantation of PRPs post-contusion did not increase the amount of global myelination or improve functional recovery. These findings indicate that PRPs are a novel source of myelinating precursor cells, but in our injury paradigm these cells were insufficient to promote functional recovery.

• Funded by:
• CIHR
• Michael Smith Foundation of Health Research
• NeuroScience Canada

Schwann cells differentiated from skin-derived precursors survive and contribute to repair after transplant into chronically injured spinal cord (Joseph Samuel Sparling)
Cellular transplantation is increasingly considered as a treatment option for spinal cord injury (SCI) although the optimal type and source of cell as well as mode of transplantation is still unknown. We have previously demonstrated that Schwann cells generated from skin-derived precursors (SKP-SCs) harvested from mouse back skin promote repair and functional recovery when transplanted into the contused rat spinal cord 1 week after injury. Since SKPs are readily accessible in the skin of adults, one of the main advantages of SKP-SCs is their potential for autologous transplantation. However, autologous transplantation of SKP-SCs would require several weeks of cell amplification in culture, so delaying transplantation for 1 week post-injury is not a clinically feasible time frame for autologous transplantation of these cells. Hence, the goal of the present work was to assess the therapeutic potential of SKP-SCs in a more clinically relevant chronic injury setting. We transplanted (>500,000) cells directly into the lesion sites of contused rats one month after SCI. In order to promote cell survival, migration out of the lesion, and repair processes in general, some of these animals received combinatorial treatments including: pre-treatment of cells with Lithium chloride or neuregulin, injections of Chondroitinase ABC in the lesion prior to cell transplantation, and systemic treatment of rats with Simvastatin (1 mg/kg/day for 2 weeks) to mitigate inflammation. Open field locomotor scores revealed no significant gain of function at 4 weeks post-transplant - consistent with our previous work that showed functional gains only after 8 weeks. Importantly, none of the animals lost further locomotor function due to the delayed transplantation surgery. All treatment groups demonstrated cell survival, which in many rats included rostro-caudal bridges spanning the extent of the lesion site to leave only small cavities. These bridges were often well integrated into the host spinal cord with minimal astrocyte hypertrophy. They were also frequently filled with axons, many of which were myelinated by SKP-SCs. SKP-SCs were also found within the host spinal cord where they appeared to remyelinate denuded axons. The transplanted SKP-SC stimulated a dramatic increase in the presence of endogenous Schwann cells which participated in the formation of the lesion site bridges as well as the remyelination of demyelinated axons in the host rim. Quantitative analyses of these effects are currently under way. In conclusion, these initial data indicate that transplantation of SKP-SC into the chronically injured spinal cord appears to be a viable approach for spinal cord repair.

• Funded by:
• Stem Cell Network of Canada

Fibroblasts are a major source of netrin-1 in the injured mouse spinal cord (K Adam Baker)
Netrin-1 is a bifunctional chemotropic guidance cue, attracting some axons and repelling others. UNC5 homologue netrin receptors are required for the repellant response to netrin-1. They are the predominant netrin-1 receptors expressed by neurons in the mature spinal cord, consistent with the conclusion that netrin-1 is a myelin-associated inhibitor of axon regeneration. Recent reports have presented apparently conflicting findings, either concluding that netrin-1 expression is upregulated in the adult mouse CNS following injury, or that netrin-1 expression is immediately decreased and remains below control levels at a site of injury in the adult rat spinal cord. We sought to clarify these results by examining the consequences of spinal cord injury in adult mice heterozygous for a LacZ reporter gene under the transcriptional control of the endogenous netrin-1 promoter. Cervical dorsal hemisectionswere performed intransgenic mice and expression of β-gal at the injury site was assessed 8 days following injury. Netrin-1 was found to be expressed by a subset of cells in the developing glial scar. Immunohistochemical double labeling detected netrin-1 gene expression by fibroblasts and a subset of astrocytes but not by microglia/macrophages. Examination of β-gal expression in cultures of mixed glial cells derived from transgenic mice detected netrin-1 expression by cell types consistent the findings obtained in vivo. These results provide evidence for limited expression of netrin-1 by cells associated with the glial scar in the adult mouse CNS. The differences in netrin-1 expression detected in rat and mouse spinal cord may be consistent with previously described species-specific differences in the response of rats and mice to spinal cord injury.

• Funded by:
• Canadian Institutes of Health Research
• Fonds de la Recherche en Sante du Quebec
• Multiple Sclerosis Society of Canada

[Steven Edwards]

Next: Roman Reed SCI Research Fund

A combination of neurotrophin-3 and conditioning lesions allows sensory axon regeneration across a spinal cord lesion site, target reinnervation and formation of new synapses (Laura A Taylor-Alto)
Neurotrophin gradients beyond sites of spinal cord injury (SCI) and peripheral nerve conditioning lesions promote growth of ascending sensory axons into and for short distances beyond lesion sites. In the present study, we tested whether combinatorial treatment, including both conditioning lesions and neurotrophin gradients, could promote growth of lumbar dorsal root ganglia (DRG) sensory axons from a cervical dorsal column injury site into the nucleus gracilis, where these axons normally terminate. Adult rats underwent C1 dorsal column wire knife lesions to transect ascending sensory axons, followed by grafts of autologous bone marrow stromal cells into the lesion site. Concurrently, lentiviral vectors expressing NT-3 were injected into the nucleus gracilis to promote axon growth toward denervated target neurons. Some subjects also received peripheral nerve conditioning lesions one week prior to spinal cord lesions. Four weeks after treatment, a combination of anterograde and retrograde axonal tracing revealed regenerating sensory axons in the nucleus gracilis in animals that received lentiviral neurotrophin-3 (NT-3) delivery with or without conditioning lesions; in contrast, regenerating axons were not observed in the target nucleus after injections of control, GFP-expressing, lentiviral vectors. In animals treated with NT-3 delivery and conditioning lesions, axon density in the nucleus gracilis was significantly greater than in any other group. Confocal and ultrastructural examination of tissue from animals that received combinatorial treatment indicated axon-dendrite apposition and synaptic contacts formed by regenerated axons within the nucleus gracilis. Synapses were found on dendritic arborizations of gracilis neurons, similar to synapses commonly observed in the intact nucleus gracilis. These findings reveal that neurotrophic factor expression within denervated target regions combined with augmentation of neuronal growth programs at the cell soma can induce regeneration and reinnervation of the original target nucleus after SCI.

• Funded by:
• California Roman Reed SCI Research Fund
• International Spinal Research Trust
• Adelson Medical Research Foundation
• NIH Grant NS 54883
• VA

Inhibitory and excitatory spinal locomotor networks are differentially activated after spinal cord transection and locomotor training in adult rats (Ronaldo M Ichiyama)
Locomotor training has been demonstrated to change the electrophysiological, morphological, and pharmacological properties of motoneurons after a complete spinal cord transection. Information about changes in interneuronal populations, however, is scarce. We investigated the activation pattern of spinal interneurons and motoneurons in step-trained adult spinal rats. Forty rats received a complete mid-thoracic spinal cord transection and were implanted with epidural stimulation (ES) electrodes at spinal segments L2 and S1. Starting one week after surgery, ten rats were trained daily (5 days/week) 30 min/day for 8 weeks to step bipedally under ES and quipazine (a 5-HT2 agonist) administration (i.p., 0.3 mg/kg) using an upper body weight support apparatus. The remaining rats were not trained and received either quipazine only (n=10), ES only (n=10), no treatment (n=5), or no ES implant (n=5). Nine weeks post- lesion all rats were tested for bipedal locomotor capacity. On the last day of the study, each rat stepped for 60 min under ES and quipazine and then returned to their cage for another 60 min before transcardiac perfusion (4% paraformaldehyde). Spinal cords were prepared for FOS-immunohistochemistry (IHC), and successive sections of spinal segments L2, 3, 4, 5, 6 and S1 were labelled for GAD67, Glycine, ChAT or VGluT1. Our preliminary results demonstrate that step-trained rats showed significant improvements in locomotor behavior, i.e., greater step height and length, better coordination and consistency of stepping than either of the non-trained groups. FOS IHC revealed that locomotor training selectively activates a subset of spinal interneurons to perform a locomotor task. The specific patterns of activation of inhibitory and/or excitatory neurons suggest that locomotor training facilitates and reinforces specific neuronal networks that are sufficient to engage the locomotor circuitry.

• Funded by:
• California Roman Reed Fund
• Christopher and Dana Reeve Foundation VEC-2007

Bladder impairment varies with comparable contusion injuries at different spinal levels (Brian T David)
Bladder function is regarded as one of the most important consequences of a spinal cord injury (SCI) (Anderson (2004), J. Neurotrauma, 21, 1371-83) but is rarely assessed in animal studies of SCI. Here, we use a simple outcome measure (volume of retained urine) to assess bladder dysfunction over time following moderate contusion injuries at 3 different thoracic levels.
Thirty female Sprague-Dawley rats were used. Ten rats received a 200 kD contusion injury from an Infinite Horizon device (IH device) at either the first (T1), fourth (T4), or ninth thoracic vertebral level (T9). The volume of urine retained in the bladder was measured by anesthetizing the animals with isoflurane, expressing the bladder, and weighing the urine daily for fourteen days. To compare bladder deficits with the degree of impairment of hindlimb motor function, locomotion was assessed using the BBB open field rating scale.
Rats with contusions at T4 and T9 exhibited bladder impairments reflected by increased urine retention from 1-12 days post injury. In contrast, rats with contusions at T1 exhibited minimal deficits. Lesion size and overall functional impairment was comparable between groups based on quantitative assessments of lesion area at the epicenter and BBB scores. Moreover, a radial analysis of sparing of different fiber pathways revealed no differences in sparing of different funiculi between the groups.
Possible explanations for the relative sparing of bladder function following contusion injuries at T1 include sparing of a critical descending pathway or sparing of a spinal circuit between T1 and T4 that is able to mediate reflex voiding in rats.

• Funded by:
• NIH-NO1-NS-3-2353
• Roman Reed Spinal Cord Injury Research Fund of California

Ampakines enhance axonal outgrowth by cortical neurons (Ching-Yi Lin)
There is an abundance of evidence that neurotrophic factors can support upper motor neuron survival after damage to axons in the central nervous system (CNS) and can facilitate axonal growth in vitro and in vivo. However, the question of how these effects can be harnessed to facilitate neuronal survival and axonal growth following CNS injury remains. The present studies tested if positive modulators of AMPA-type glutamate receptors (ampakines), which have been shown to increase neuronal BDNF expression in vitro and in vivo, also stimulate axonal growth. Rat cortical neurons were cultured onto a poly-L-lysine coated glass substrate in the ‘somal well of a Microfluidic Culture Platform (MCP) (Park et al., Nat. Protoc. 1:2128-36, 2006), which includes fluidic-isolation compartments (i.e., somal and axonal wells) separated by a grooved bridge region into which neurites can grow. Ampakines were applied to the MCP somal well, the axonal well, or both, and the length of neurite elongation into the bridge region was evaluated after 1-4 days of ampakine exposure. Treatments with the ampakines CX614 and CX929 significantly increased neurite outgrowth. These effects were not site- (soma vs axon) specific and were comparable across doses tested (0.1 - 10 µM for CX614 and 0.5 to 50 µM for CX929). While increases in mature BDNF protein were associated with ampakine treatment of dissociated cortical neurons, the growth promoting effects of ampakine treatment were not blocked by addition of the BDNF scavenger, TrkB-Fc. However, the Trk signaling antagonist K252a attenuated ampakine effects on neurite length most reliably after application to the somal well. These results demonstrate that ampakines enhance axonal growth and suggest that the growth response is mediated, at least in part, by increases in the signaling of multiple neurotrophins (e.g., BDNF and NGF). These results further support the possibility that ampakines might be used to facilitate reactive axonal growth following CNS damage.

• Funded by:
• This project is supported by the Roman Reed Spinal Cord Injury Research Fund of California

Assessment of axon regeneration and behavioral improvement after spinal cord injury in OMgp/MAG/Nogo triple knockout mice (Jae K Lee)
Oligodendrocyte myelin glycoprotein (OMgp), myelin associated glycoprotein (MAG), and Nogo are thought to be three major inhibitors of axon regeneration present in adult mammalian CNS myelin. However, the lack of compelling in vivo evidence for axon regeneration in the respective single knockout mice studied thus far raises the possibility of functional redundancy between the three inhibitors. In this study, we addressed this issue by using 7-9 week old female triple knockout mice that lack all three myelin inhibitors, OMgp, MAG, and Nogo-A,B,C (triple KO). Baseline behavioral assays are consistent with a possible redundancy between the three inhibitors during development. After a dorsal hemisection at vertebral level T8, however, both wild-type and triple KO mice showed similar degrees of locomotor recovery during 6 weeks post-injury as assessed by the BBB and BMS scales. We are currently analyzing BDA-labeled corticospinal axons to determine the axon regenerative phenotype in the triple KO mice.

• Funded by:
• NINDS Grant R01NS054734
• NINDS F32 NS056697
• Roman Reed Fund RR04-111