Scott Sayers

Carbon filament implants promote axonal growth across the transected rat spinal cord

Significant regeneration of neural pathways following complete transection of the spinal cord has yet to be demonstrated. In the present study, we analyzed the ability of carbon filaments to function as a scaffold for the regrowth of injured axons in the rat spinal cord. Through the use of three different axonal tracing methods, severed spinal axons were observed growing on and between carbon filaments implanted into the completely transected rat spinal cord. Carbon filaments, by providing a favorable adhesive surface and also a possible guiding function, may prove useful in the treatment of spinal cord injury.

Preparation of brain-derived neurotrophic factor-and neurotrophin-3-secreting schwann cells by infection with a retroviral vector

One reason that the central nervous system of adult mammals does not regenerate after injury is that neurotrophic factors are present only in low concentrations in these tissues. Recent studies have shown that the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) acts to encourage the regrowth of motor and sensory fibers after spinal cord injury. Other studies have reported that the regrowth of axons after injury was enhanced by the implantation of Schwann cells, which normally secrete BDNF and NT-3. The purpose of the present study was to genetically modify Schwann cells to secrete increased amounts of BDNF or NT-3 by infection with a retroviral vector. Retroviral vectors were constructed by the ligation of BDNF or NT-3 cDNA to the LXSN vector. Viruses were generated from the plasmid forms of the vectors by transient transfection of PA317 amphotrophic retroviral packaging cells. Viruses were harvested and used to infect the human Schwann cell line designated NF-1T. Northern blot analysis of poly (A+) RNA prepared from Schwann cells that were infected with BDNF- or NT-3-containing virus showed the presence of BDNF or NT-3 mRNA. An enzyme-linked immunosorbent assay (ELISA) for BDNF and NT-3 was performed on media the cells were grown in, and on cellular extracts prepared from the BDNF- and NT-3-infected Schwann cells. The ELISA results demonstrated that the Schwann cells were secreting increased levels of immunologically active BDNF or NT-3. Immunocytochemical staining of these cells revealed the presence of these two neurotrophic factors located in perinuclear granules. These neurotrophic factor-secreting Schwann cells are currently being evaluated for their efficacy in the treatment of spinal cord injury.

Hypertrophy of Neurons Within Cardiac Ganglia in Human, Canine, and Rat Heart Failure: The Potential Role of Nerve Growth Factor

Background Autonomic imbalances including parasympathetic withdrawal and sympathetic overactivity are cardinal features of heart failure regardless of etiology; however, mechanisms underlying these imbalances remain unknown. Animal model studies of heart and visceral organ hypertrophy predict that nerve growth factor levels should be elevated in heart failure; whether this is so in human heart failure, though, remains unclear. We tested the hypotheses that neurons in cardiac ganglia are hypertrophied in human, canine, and rat heart failure and that nerve growth factor, which we hypothesize is elevated in the failing heart, contributes to this neuronal hypertrophy. Methods and Results Somal morphology of neurons from human (579.54±14.34 versus 327.45±9.17 μm2; P<0.01) and canine hearts (767.80±18.37 versus 650.23±9.84 μm2; P<0.01) failing secondary to ischemia and neurons from spontaneously hypertensive rat hearts (327.98±3.15 versus 271.29±2.79 μm2; P<0.01) failing secondary to hypertension reveal significant hypertrophy of neurons in cardiac ganglia compared with controls. Western blot analysis shows that nerve growth factor levels in the explanted, failing human heart are 250% greater than levels in healthy donor hearts. Neurons from cardiac ganglia cultured with nerve growth factor are significantly larger and have greater dendritic arborization than neurons in control cultures. Conclusions Hypertrophied neurons are significantly less excitable than smaller ones; thus, hypertrophy of vagal postganglionic neurons in cardiac ganglia would help to explain the parasympathetic withdrawal that accompanies heart failure. Furthermore, our observations suggest that nerve growth factor, which is elevated in the failing human heart, causes hypertrophy of neurons in cardiac ganglia.

Carbon filaments provide support and directionality to growing rat fetal spinal cord explants.

Spinal cord explants obtained from 15 to 17-day-old fetal rats were cultured on bundles of 5-7 microns diameter carbon filaments attached to the bottom of Petri dishes. After a 3 week incubation period, the cultures were fixed and observed by light, scanning, and transmission electron microscopy. Neurites and glial processes were found to be growing both on and between the carbon filaments. The carbon filaments appeared to provide a biocompatible scaffold which promoted adhesion and gave directionality to the growing cell processes. These properties may make carbon filaments a suitable substrate for in vivo implantation into the damaged spinal cord.

Distribution of alpha 1 subunit isoform of (Na,K)-ATPase in the rat spinal cord

Three isoforms of the alpha subunit of (Na,K)-ATPase have been identified in the rat central nervous system. Using a probe specific for the alpha 1 isoform, mRNA levels were measured from five sections of the rat spinal cord using slot blot techniques. Assigning a value of 1 to the slope obtained from the cervical section, the upper thoracic section was 2.6 times higher; the midthoracic section was 4.5 times higher; the lower thoracic section was 2.6 times higher; and the lumbar section was 1.7 times higher. The results suggest that alpha 1 isoform mRNA levels are not uniform throughout the spinal cord. In situ hybridization techniques showed that alpha 1 isoform mRNA was diffusely abundant in glial and central canal ependymal cells, while labeled neurons were localized exclusively in lateraily located anterior horn neurons in cervical, thoracic, and lumbar segments and in ventromedial neurons in mid-thoracic spinal cord. Also, dorsal root ganglia neurons were extensively labeled at all segments.

Acetylcholine receptor gene expression in skeletal muscle of chronic ethanol-fed rats

The expression of the neuromuscular acetylcholine receptor (AChR) alpha-subunit gene was evaluated in soleus muscles from an animal model of chronic alcoholism. At 8 weeks of age, test rats were placed on a nutritionally complete liquid diet containing 6.7% ethanol (v/v). Age- and weight-matched control rats were pair-fed an isocaloric liquid diet. After a 16-week diet period, soleus muscles were obtained and total RNA and poly(A)+ RNA were isolated. Muscle RNA levels from ethanol-fed and control rats were comparable. AChR alpha-subunit mRNA was detected by hybridization of muscle poly(A)+ RNA with a 32P-labeled, complementary riboprobe. The steady-state level of AChR alpha-subunit mRNA was reduced by 39% (p less than 0.001) in soleus muscles from the ethanol-fed rats as compared to pair-fed controls. These results suggest that the expression of the AChR alpha-subunit gene is down-regulated after chronic ethanol exposure at a transcriptional or posttranscriptional level.

Evaluation of Bipolar Permaloc TM Electrodes for Direct Bladder Stimulation

Purpose: A bladder control system for spinal cord injured (SCI) patients is needed that can be implanted with minimally invasive methods. New Permaloc bipolar electrodes consisting of 6 mm helical, wire, stimulating surfaces separated by 3 mm and a polypropylene securing barb (Synapse Biomedical Inc) were developed for this application. They are implanted on the bladder wall with a 16 gauge needle, a minimally invasive method. Methods: Seven swines were anesthetized, the lower urinary tract exposed and instrumented with pressure transducers. Four Permaloc TM electrodes were implanted following identification of effective bladder wall stimulation sites next to the ureters and dorsal neurovascular bundle. Bladder stimulation to induce high pressures was conducted at 40 Hz, 400 � s pulses, 5 s stimulation periods and a high stimulating current of 40 mA. Results: At the high stimulating current peak bladder pressures were low, ranging from 12±2 to15±3 cm H20, insufficient to induce urination. Urethral sphincter contractions occurred during high bladder pressure. A spinal reflex role for high sphincter pressures during stimulation was shown by similar high pressures recorded during a bladder squeeze test without stimulation. Conclusions: Stimulation with Permaloc TM bipolar electrodes at high currents produced insufficient bladder pressures for urination. Further modifications of the electrode such as greater separation of the bipolar stimulating surfaces or changes in the testing methods such as alternative animal models are needed to induce high bladder pressures without side effects.

Respiratory responses to stimulation of abdominal and upper-thorax intercostal muscles using multiple Permaloc ® electrodes

Stimulation of abdominal and upper-thoracic muscles was studied with the long-term goal of improved respiratory care for spinal cord injury (SCI) patients. A 12-channel stimulator and multiple surface and implanted Permaloc electrodes were evaluated in five anesthetized canines. Abdominal stimulation with 100 mA using four bilateral sets of surface electrodes placed on the midaxillary line at the 7th through 13th intercostal spaces and with a closed airway at a large lung volume produced an expiratory tracheal pressure of 109 +/- 29 cm H2O (n = 2, mean +/- standard error of the mean). Similar high pressures were induced with implanted electrodes at the same locations. Upper-thoracic stimulation with 40 mA and four sets of implanted electrodes ventral to the axilla induced inspiratory pressures of -12 +/- 2 cm H2O (n = 5). Combined extradiaphragmatic pacing with an open airway produced a tidal volume of 440 +/- 45 mL (n = 4). The robust respiratory volumes and pressures suggest applications in SCI respiratory care.

Neuroprosthetics for SCI Bladder Management: The Argument for Direct Bladder Stimulation

Implantable neuroprosthetic systems are an important area of practice and research in urinary care for individuals with spinal cord injury (SCI). These devices need to manage three lower urinary tract conditions: urethral sphincter contractions during bladder contractions, an underactive bladder producing poor voiding responses, and neurogenic detrusor overactivity causing urinary incontinence. Two neuroprosthetic approaches have addressed these conditions: sacral anterior root stimulation (SARS) and direct bladder wall stimulation (DBWS). The SARS approach is commercialized for SCI bladder management as the Brindley-Finetech Bladder Control System and is available in Europe. Limitations of this device include invasive surgery and the need for rhizotomy of sacral dorsal (sensory) nerve roots. The DBWS implants produced daily voiding in many SCI individuals, however, clinical use was discontinued primarily because of technical concerns with stimulators and electrodes as well as some cases of poor voiding responses and side effects. These limitations are reviewed as well as efforts to return DBWS to clinical investigations using Permaloc® Systems (Synapse Biomedical Inc., Oberlin OH). This new neuroprosthetic platform includes mapping and intramuscular electrodes as well as multilead cables and new stimulator devices.

Stimulation of abdominal and upper thoracic muscles with surface electrodes for respiration and cough: acute studies in adult canines

Objective: To optimize maximal respiratory responses with surface stimulation over abdominal and upper thorax muscles and using a 12-Channel Neuroprosthetic Platform. Methods: Following instrumentation, six anesthetized adult canines were hyperventilated sufficiently to produce respiratory apnea. Six abdominal tests optimized electrode arrangements and stimulation parameters using bipolar sets of 4.5 cm square electrodes. Tests in the upper thorax optimized electrode locations, and forelimb moment was limited to slight-to-moderate. During combined muscle stimulation tests, the upper thoracic was followed immediately by abdominal stimulation. Finally, a model of glottal closure for cough was conducted with the goal of increased peak expiratory flow. Results: Optimized stimulation of abdominal muscles included three sets of bilateral surface electrodes located 4.5 cm dorsal to the lateral line and from the 8th intercostal space to caudal to the 13th rib, 80 or 100 mA current, and 50 Hz stimulation frequency. The maximal expired volume was 343 ± 23 ml (n=3). Optimized upper thorax stimulation included a single bilateral set of electrodes located over the 2nd interspace, 60 to 80 mA, and 50 Hz. The maximal inspired volume was 304 ± 54 ml (n=4). Sequential stimulation of the two muscles increased the volume to 600 ± 152 ml (n=2), and the glottal closure maneuver increased the flow. Conclusions: Studies in an adult canine model identified optimal surface stimulation methods for upper thorax and abdominal muscles to induce sufficient volumes for ventilation and cough. Further study with this neuroprosthetic platform is warranted.