Vernon W. Lin

Peripheral nerve grafts and aFGF restore partial hindlimb function in adult paraplegic rats

The purpose of this study was to evaluate the degree of functional recovery in adult rats with completely transected spinal cord following experimental treatment regimens that include implantation of peripheral nerve segments and local application of acidic fibroblast growth factor (aFGF). Rats were randomly divided to five groups: (1) spinal cord transection, (2) spinal cord transection and aFGF treatment, (3) spinal cord transection and peripheral nerve grafts, (4) spinal cord transection, aFGF treatment, and peripheral nerve grafts, and (5) sham control (laminectomy only). The locomotor behavior of all rats was analyzed by the Basso, Beattie and Bresnahan (BBB) open field locomotor test over the six months survival time. Immunohistochemisty for neurofilament protein, and somatosensory (SSEP) and motor evoked potentials (MEP) were used to evaluate axon growth across the damage site following the different treatments. The results show four principal findings: (1) Only the combination of peripheral nerve grafts and aFGF treatment improved hindlimb locomotor function after spinal cord transection. (2) The SSEP and MEP demonstrated electrophysiological evidence of both sensory and motor information crossing the damaged site, but only in the combined nerve grafts and aFGF treatment rats. (3) Immunostaining demonstrated neurofilament positive axons extending through the graft area and into distal end of spinal cord, but only in the group with combined nerve grafts and aFGF treatment. (4) Retransection of group 4 rats eliminated the behavioral recovery, MEP, and SSEP responses, indicating that the improvement of hindlimb locomotor activity came from supraspinal control. These results demonstrate the ability of the repair strategy combining peripheral nerve grafts and aFGF treatment to facilitate the regeneration of spinal ascending and descending tracts and also recovery of motor behavior following spinal cord injury.

Magnetic coil design considerations for functional magnetic stimulation

Our studies have demonstrated effective stimulation of the bladder, bowel, and expiratory muscles in patients with spinal cord injury using functional magnetic stimulation, However, one limitation of the magnetic coils (MC) is related to their inability to specifically stimulate the target tissue without activation of surrounding tissue. The primary goal of this study was to determine the governing parameters in the MC design, such as coil configuration, diameter, and number of turns in one loop of the coil. By varying these parameters, our approach was to design, construct, and evaluate the induced electric field distributions of two sets of novel MCs. Based on the slinky coil design, the first set of coils was constructed to compare their abilities in generating induced electric fields for focal nerve excitation. The second set of coils was built to determine the effect that changes in two parameters, coil diameter and number of turns in one loop, had on field penetration. The results showed that the slinky coil design produced more focalized stimulation when compared to the planar round coils. The primary-to-secondary peak ratios of the induced electric field from slinky 1 to 5 were 1.00, 2.20, 2.85, 2.62, and 3.53. We also determined that coils with larger diameters had better penetration than those with smaller diameters. Coils with less number of turns in one loop had higher initial field strengths; when compared to coils that had more turns per loop, initial field strengths remained higher as distance from the coil increased. In our attempt to customize MC design according to each functional magnetic stimulation application and patients of different sizes, the parameters of MC explored in this study may facilitate designing an optimal MC for a certain clinical application.

NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O2*-)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO-O2*- interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91phox and P67phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.

High intensity magnetic stimulation over the lumbosacral spine evokes antinociception in rats

OBJECTIVES High intensity magnetic stimulation (MS) applied over the skin can painlessly depolarize superficial and deep nerves and we aimed to evaluate the effectiveness of MS of spinal nerves in evoking a potent analgesic response. METHODS The MS was administered to adult male Sprague-Dawley rats using a Cadwell MES-10 high-speed magnetic stimulator. A Peltier device and von Frey fibers were used to determine heat and mechanical nociceptive responses of the rats. RESULTS A brief (5 min) course of MS over the rats lumbosacral spine produced a long-lasting (30-40 min) and robust (80-90% maximum possible effect) hindpaw antinociceptive effect to both mechanical and heat stimuli. Spinal cord transected rats had intact hindpaw nociceptive withdrawal responses, but transection eliminated MS evoked antinociception, indicating a critical extrasegmental component in the mechanism of MS antinociceptive action. The opiate receptor antagonist naloxone (5 mg/kg, i.p.) completely blocked MS evoked antinociception, demonstrating an opioidergic mechanism for MS antinociception. The alpha(2) adrenoceptor antagonist atipamezole (5 mg/kg, i.p.) slightly reduced the MS antinociceptive response to heat and had no effect on MS antinociception for mechanical stimuli. CONCLUSIONS These data indicate that MS can evoke a robust, long-lasting antinociceptive effect, which requires an intact supraspinal pathway and is opioidergic mediated.

AFGF promotes axonal growth in rat spinal cord organotypic slice co-cultures.

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15-P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.

Re-growth of catecholaminergic fibers and protection of cholinergic spinal cord neurons in spinal repaired rats

The extent of re‐growth of catecholaminergic fibers, the survival of cholinergic neurons and the degree of autonomic dysreflexia were assessed in complete spinal cord‐transected adult rats that received a repair treatment of peripheral nerve grafts and acidic fibroblast growth factor (aFGF). The rats were randomly divided into three groups: (1) sham control group (laminectomy only); (2) spinal cord transection at T8 (transected group); and (3) spinal cord transection at T8, followed by aFGF treatment and peripheral nerve graft (repaired group). The spinal cords and brains of all rats were collected at 6 months post‐surgery. Immunohistochemistry for tyrosine hydroxylase (TH) and dopamine‐β‐hydroxylase (DBH), and fluoro‐gold (FG) retrograde tracing were used to evaluate axon growth across the damage site, and immunocytochemistry for choline acetyl transferase (ChAT) was used to evaluate cholinergic neuronal cell survival following the injury and treatment. When comparing with the transected group, the repaired group showed: (1) lower elevation of mean arterial pressure during colorectal distension; (2) retrogradely labeled neurons in the hypothalamus, zona incerta, subcoeruleus nuclei and rostral ventrolateral medulla following application of FG below the repair site; (3) the presence of TH‐ and DBH‐labeled axons below the lesion site; (4) higher numbers of ChAT‐positive neurons in ventral horn and intermediolateral column near the lesion site. We conclude that peripheral nerve graft and aFGF treatments facilitate the re‐growth of catecholaminergic fibers, also protect sympathetic preganglionic neurons and spinal motor neurons, and reduce autonomic dysfunction in a T‐8 spinal cord‐transected rat model.

Functional magnetic stimulation facilitates gastrointestinal transit of liquids in rats

The purpose of this study was to investigate the effect of a relatively novel technology, functional magnetic stimulation (FMS), on gastrointestinal transit of liquids in rats. Orogastric gavage with technetium‐99 solution was used to assess gastric emptying and gastrointestinal transit time in 92 rats. FMS was performed over the anterior cervical and/or dorsal thoracolumbar regions using a figure‐8 coil. Stimulation protocols were 1, 2, or 4 h in length. FMS accelerated gastric emptying and decreased gastrointestinal transit time. The acceleration was dependent on the stimulation parameters used as well as on the duration of the protocol; high levels of FMS produced a quicker effect, whereas lower levels were effective at later times. This study provides evidence that FMS could be an alternative or adjunct therapy to treat disorders in gastrointestinal motility.

Optimal arrangement of magnetic coils for functional magnetic stimulation of the inspiratory muscles in dogs

In an attempt to maximize inspiratory pressure and volume, the optimal position of a single or of dual magnetic coils during functional magnetic stimulation (FMS) of the inspiratory muscles was evaluated in twenty-three dogs. Unilateral phrenic magnetic stimulation (UPMS) or bilateral phrenic magnetic stimulation (BPMS), posterior cervical magnetic stimulation (PCMS), anterior cervical magnetic stimulation (ACMS) as well as a combination of PCMS and ACMS were performed. Trans-diaphragmatic pressure (Pdi), flow, and lung volume changes with an open airway were measured. Transdiaphragmatic pressure was also measured with an occluded airway. Changes in inspiratory parameters during FMS were compared with 1) electrical stimulation of surgically exposed bilateral phrenic nerves (BPES) and 2) ventral root electrical stimulation at C5-C7 (VRES C5-C7). Relative to the Pdi generated by BPES of 36.3/spl plusmn/4.5 cm H/sub 2/O (Mean /spl plusmn/ SEM), occluded Pdi(s) produced by UPMS, BPMS, PCMS, ACMS, and a combined PCMS + ACMS were 51.7%, 61.5%, 22.4%, 100.3%, and 104.5% of the maximal Pdi, respectively. Pdi(s) produced by UPMS, BPMS, PCMS, ACMS, and combined ACMS + PCMS were 38.0%, 45.2%, 16.5%, 73.8%, and 76.8%, respectively, of the Pdi induced by VRES (C5-C7) (48.0/spl plusmn/3.9 cm H/sub 2/O). The maximal Pdi(s) generated during ACMS and combined PCMS + ACMS were higher than the maximal Pdi(s) generated during UPMS, BPMS, or PCMS (p<0.05). ACMS alone induced 129.8% of the inspiratory flow (73.0/spl plusmn/9.4 L/min) and 77.5% of the volume (626/spl plusmn/556 ml) induced by BPES. ACMS and combined PCMS + ACMS produce a greater inspiratory pressure than UPMS, BPMS or PCMS. ACMS can be used to generate sufficient inspiratory pressure, flow, and volume for activation of the inspiratory muscles.