Method and Apparatus for the Automated Delivery of Continuous Neural Stem Cell Trails Into the Spinal Cord of Small and Large Animals.

BACKGROUND: Immature neurons can extend processes after transplantation in adult animals. Neuronal relays can form between injected neural stem cells (NSCs) and surviving neurons, possibly improving recovery after spinal cord injury (SCI). Cell delivery methods of single or multiple bolus injections of concentrated cell suspensions thus far tested in preclinical and clinical experiments are suboptimal for new tract formation. Nonuniform injectate dispersal is often seen due to gravitational cell settling and clumping. Multiple injections have additive risks of hemorrhage, parenchymal damage, and cellular reflux and require additional surgical exposure. The deposition of multiply delivered cells boluses may be uneven and discontinuous. OBJECTIVE: To develop an injection apparatus and methodology to deliver continuous cellular trails bridging spinal cord lesions. METHODS: We improved the uniformity of cellular trails by formulating NSCs in hyaluronic acid. The TrailmakerTM stereotaxic injection device was automatized to extend a shape memory needle from a single-entry point in the spinal cord longitudinal axis to "pioneer" a new trail space and then retract while depositing an hyaluronic acid-NSC suspension. We conducted testing in a collagen spinal models, and animal testing using human NSCs (hNSCs) in rats and minipigs. RESULTS: Continuous surviving trails of hNSCs within rat and minipig naive spinal cords were 12 and 40 mm in length. hNSC trails were delivered across semi-acute contusion injuries in rats. Transplanted hNSCs survived and were able to differentiate into neural lineage cells and astrocytes. CONCLUSION: The TrailmakerTM creates longitudinal cellular trails spanning multiple levels from a single-entry point. This may enhance the ability of therapeutics to promote functional relays after SCI.

Internal decompression of the acutely contused spinal cord: Differential effects of irrigation only versus biodegradable scaffold implantation.

Severe spinal cord injury leads to hemorrhage, edema and elevated tissue pressures that propagate ischemia. Liquefactive necrosis of damaged tissue eventually results in chronic cavities due to a wound healing process lacking adhesive contractile cells. Biomaterials can potently influence wound healing responses. Internal decompression (ID) refers to pial opening, allowing spontaneous extrusion and irrigation of fluid necrotic debris relieving pressure and resulting in a space for biomaterial scaffold insertion. After thoracic contusions, rats were randomized to: contusion only, contusion + ID and contusion + ID + PLGA-PLL scaffold implantation, to test for neuroprotection and endogenous repair over 3 months. ID alone reduced inflammatory activity, cavity volume, and increased tissue sparing. Scaffold biodegradation produced delayed ingrowth of inflammatory and other cells resulting in endogenously derived laminin-rich tissue, marked reduction in cavitation and presence of tissue remodeling macrophages. Extensive recruitment of Schwann cells into adjacent spared white matter occurred, greatest in scaffold-implanted animals. Despite tissue preservation with myelin repair, no groups differed significantly in open field locomotion. However, across all rats, spared epicenter tissue and locomotor outcomes were correlated. Scaffold-implanted animals showed no obvious toxicity. To study the clinical feasibility, timing and indications for scaffold implantation, Göttingen minipigs underwent ID and were implanted with scaffolds 4, 6, and 24 h after T10 contusion. High intra-spinal tissue pressures fell to pre-injury levels after ID and scaffold implantation. Extrusion of necrotic debris left sufficient space for a sized scaffold. These results provided the preclinical rationale for a current clinical study of biomaterial scaffold implantation into the human injured spinal cord.

Biodegradable scaffolds promote tissue remodeling and functional improvement in non-human primates with acute spinal cord injury.

Tissue loss significantly reduces the potential for functional recovery after spinal cord injury. We previously showed that implantation of porous scaffolds composed of a biodegradable and biocompatible block copolymer of Poly-lactic-co-glycolic acid and Poly-l-lysine improves functional recovery and reduces spinal cord tissue injury after spinal cord hemisection injury in rats. Here, we evaluated the safety and efficacy of porous scaffolds in non-human Old-World primates (Chlorocebus sabaeus) after a partial and complete lateral hemisection of the thoracic spinal cord. Detailed analyses of kinematics and muscle activity revealed that by twelve weeks after injury fully hemisected monkeys implanted with scaffolds exhibited significantly improved recovery of locomotion compared to non-implanted control animals. Twelve weeks after injury, histological analysis demonstrated that the spinal cords of monkeys with a hemisection injury implanted with scaffolds underwent appositional healing characterized by a significant increase in remodeled tissue in the region of the hemisection compared to non-implanted controls. The number of glial fibrillary acidic protein immunopositive astrocytes was diminished within the inner regions of the remodeled tissue layer in treated animals. Activated macrophage and microglia were present diffusely throughout the remodeled tissue and concentrated at the interface between the preserved spinal cord tissue and the remodeled tissue layer. Numerous unphosphorylated neurofilament H and neuronal growth associated protein positive fibers and myelin basic protein positive cells may indicate neural sprouting inside the remodeled tissue layer of treated monkeys. These results support the safety and efficacy of polymer scaffolds in a primate model of acute spinal cord injury. A device substantially similar to the device described here is the subject of an ongoing human clinical trial.

An assessment of the antinociceptive efficacy of intrathecal and epidural contulakin-G in rats and dogs.

Contulakin-G is a novel conopeptide with an incompletely defined mechanism of action. To assess nociceptive activity we delivered Contulakin-G as a bolus intrathecally (0.03, 0.1, 0.3, 3 nmol) or epidurally (10, 30, 89 nmol) in rats. Intrathecal Contulakin G significantly decreased Phase II and, to a lesser degree, Phase I paw flinching produced by intradermal formalin. Intrathecal and epidural doses of ED50s were 0.07 nmol and 45 nmol, respectively, giving an epidural/intrathecal ED50 ratio = 647). In dogs, intrathecal Contulakin-G (50-500 nmoL) produced a dose-dependent increase in the thermally evoked skin twitch latency by 30 min after administration, as did morphine (150 and 450 nmol). Epidural morphine (750 and 7500 nmol), but not epidural 1000 nmol Contulakin-G, also significantly decreased skin twitch in dogs. No changes in motor function were seen in any rats or dogs receiving these doses of Contulakin-G. In dogs, no physiologically significant dose-dependent changes in motor function, heart rate, arterial blood pressure, or body temperature were found. Contulakin-G is a potent antinociceptive drug when delivered intrathecally with no observable negative side effects in rats or dogs and may provide an alternative to opioid spinal analgesics.

Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity.

MuO-conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible. We show for the first time that synthetic MrVIB blocks rat NaV1.8 sodium channels and has potent and long-lasting local anesthetic effects when tested in two pain assays in rats. Furthermore, MrVIB can block propagation of action potentials in A- and C-fibers in sciatic nerve as well as skeletal muscle in isolated preparations from rat. Our work provides the first example of analgesia produced by a conotoxin that blocks sodium channels. The emerging diversity of antinociceptive mechanisms targeted by different classes of conotoxins is discussed.

Postischemic administration of CGX-1051, a peptide from cone snail venom, reduces infarct size in both rat and dog models of myocardial ischemia and reperfusion.

CGX-1051 is a synthetic version of a peptide originally isolated from the venom of cone snails. In the present studies, we tested the potential cardioprotective effect of CGX-1051 in a rat and dog model of myocardial ischemia/reperfusion. CGX-1051 was administered 5 minutes before reperfusion as intravenous bolus doses of 30, 100, and 300 microg/kg. Infarct size (IS) is reported as IS/area at risk (AAR). In the rat, the vehicle control group had an IS/AAR of 59.8+/-2.1%. Postischemic administration of CGX-1051 at doses of 30, 100, and 300 microg/kg resulted in an IS/AAR of 52.6+/-4.2%, 34.6+/-5.6% (P<0.05), and 40.8+/-5.2% (P<0.05), respectively. In the dog, the vehicle control group had an IS/AAR of 18.8+/-1.7%. Postischemic administration of CGX-1051 at doses of 30, 100, and 300 microg/kg resulted in an IS/AAR of 16.9+/-2.5%, 8.4+/-2.9% (P<0.05) and 9.9+/-2.4% (P<0.05), respectively. These results demonstrate that administration of CGX-1051 at a clinically relevant time point results in a dose-dependent reduction in IS in both rats and dogs.

Conantokins: peptide antagonists of NMDA receptors.

Conantokins are small peptides (17-27 amino acids) found in the venoms of cone snails (Conus sp.) that inhibit the activity of N-methyl-D-aspartate (NMDA) receptors. Unlike most of the peptides characterized from cone snail venom that contain multiple disulfide bridges, conantokins are linear peptides that possess a high degree of alpha-helicity in the presence of divalent cations, and contain gamma-carboxyglutamic acid residues. Four naturally occurring conantokins have been identified and characterized to date, conantokin-G, conantokin-T, conantokin-R, and conantokin-L. The most extensively characterized, conantokin-G, is selective for subtypes of NMDA receptors containing the NR2B subunit. The conantokins have been synthesized and characterized in a number of animal models of human pathologies including pain, convulsive disorders, stroke, and Parkinson's disease. The potential pharmacological selectivity of the conantokins, coupled with their efficacy in preclinical models of disease and favorable safety profiles indicate that these peptides represent both novel probes for NMDA receptor function as well as an important class of compounds for continued investigation as human therapeutics.