Guidelines for the conduct of clinical trials for spinal cord injury (SCI) as developed by the ICCP panel: clinical trial outcome measures.

An international panel reviewed the methodology for clinical trials of spinal cord injury (SCI), and provided recommendations for the valid conduct of future trials. This is the second of four papers. It examines clinical trial end points that have been used previously, reviews alternative outcome tools and identifies unmet needs for demonstrating the efficacy of an experimental intervention after SCI. The panel focused on outcome measures that are relevant to clinical trials of experimental cell-based and pharmaceutical drug treatments. Outcome measures are of three main classes: (1) those that provide an anatomical or neurological assessment for the connectivity of the spinal cord, (2) those that categorize a subject's functional ability to engage in activities of daily living, and (3) those that measure an individual's quality of life (QoL). The American Spinal Injury Association impairment scale forms the standard basis for measuring neurologic outcomes. Various electrophysiological measures and imaging tools are in development, which may provide more precise information on functional changes following treatment and/or the therapeutic action of experimental agents. When compared to appropriate controls, an improved functional outcome, in response to an experimental treatment, is the necessary goal of a clinical trial program. Several new functional outcome tools are being developed for measuring an individual's ability to engage in activities of daily living. Such clinical end points will need to be incorporated into Phase 2 and Phase 3 trials. QoL measures often do not correlate tightly with the above outcome tools, but may need to form part of Phase 3 trial measures.

Leukemia inhibitory factor enhances the regeneration of transected rat sciatic nerve and the function of reinnervated muscle.

The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L+F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L+F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L+F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L+F-treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function.

Midkine (MK), a retinoic acid (RA)-inducible gene product, produced in E. coli acts on neuronal and HL60 leukemia cells.

We have shown previously that (i) retinoic acid (RA), an anti-neoplastic agent, activates the midkine (MK) gene in mammalian embryonic carcinoma cells, and that (ii) the MK of 118 amino acids, purified from L cells, induces neurite outgrowth of mammalian embryonic brain cells. In this paper, we describe an unconventional strategy for the purification of a fully active MK from E. coli with a high yield. The MK was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein. The MK fusion protein extracted from the bacterial inclusion bodies with guanidine-HCl was renatured, refolded slowly and cleaved by thrombin at the site where the GST links to the MK. The purified free MK, like RA, induced neurite outgrowth from central neurons of the mouse spinal cord, and suppressed the growth of human HL60 leukemia cells in vitro. Unlike RA, however, the MK did not induce granulocytic differentiation of HL60 cells. Furthermore, the MK supported the survival of an NGF-insensitive sensory neuron subpopulation(s) from chicken embryo dorsal root ganglion. Thus, the actions of the MK and leukemia inhibitory factor (LIF) are surprisingly similar. There is no sequence similarity between MK and LIF, however, and unlike MK, LIF production does not appear to be RA-inducible.