XV. Spinal Cord Injury and Related Neurological Disorders

A. General

[296] EMPLOYMENT OF IBM SPEECH RECOGNITION IN USER-BASED REMOTE CONTROL: A PILOT STUDY

David A. Ross, MSEE, MEd
Atlanta VA Medical Center, Rehabilitation R&D (151-R) Decatur, GA 30033; email: davidross@ilinks.net

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Pilot Project #C1712-PA)

PURPOSE--The overall purpose of this 1-year study was to improve the quality of life of veterans with spinal cord injury (SCI) through the development of a device that would give them the ability to verbally control their environment wherever they chose to go: around the home, in their van, their office, friends' homes, public buildings, and on public streets. The specific objective was to evaluate the feasibility of making such a device available to veterans.

METHODOLOGY--Three components comprised the U-VOICE hardware: 1) a Voice Connexion® speech recognition/synthesis PC/104 board (in place of discontinued IBM hardware), 2) a miniature, wearable PC, and 3) a small custom board with Radio Frequency (RF) and Infra-Red (IR) transmitters. RF switch-receivers were employed for control of fans, lights, doorbells, door locks, devices on the wheel chair, cross-walk buttons, parking gates, and so forth. Transmitted IR codes were employed to control home electronics, van doors and lifts, and public elevators.

  No visual display was used, as all actions taken by U-VOICE were initiated by voice command and verbally confirmed via speech synthesis. U-VOICE provided voice assistance during all aspects of training and use. Context-based word recognition software was written to improve word recognition rates, especially for emergency commands.

  Investigators evaluated the prototype, determining speech recognition reliability for normal commands and for emergency commands and the reliability of the IR and RF transmitters under varying indoor and outdoor conditions. Then, with the assistance of a focus group of five professionals and three consumers, they evaluated the usability of the interactive voice-control interface and the usability of the device as a whole.

PROGRESS--This project has been successfully completed.

RESULTS--Word recognition rates for the Voice Connexion hardware alone was a disappointing 84 percent in general, and only 63 percent for emergency words spoken under stress conditions. However, with context-sensitive software the recognition rates improved to 96 and 92 percent respectively. Finally, by changing the emergency command to "alarm, alarm, alarm," a 98.5 percent first-time and a 100 percent second-time recognition rate was achieved.

  RF control was nearly 100 percent reliable at 50 feet (even through walls), and became less so at 75 feet. IR control was reliable at 7 feet or less, but at distances past 10 feet dropped significantly. Also, it was important that U-VOICE be pointed toward the IR receiver. This was inconvenient at times: for this reason, the focus group recommended use of RF control whenever possible.

  The focus group agreed that remote control of devices in all environments was a valuable goal and suggested an extended list of devices for control, including telephones, appliances, home thermostat, computer, teller machines, and point-of-sale payment machines. However, they also thought that voice control alone was too restrictive and awkward at times. Two users argued for puff and sip control as an alternative to, or an addition to, voice control.

FUTURE PLANS--We propose the research and development of a modular Remote Control Interface (RCI) to meet the needs of the larger population of veterans with upper body hand and arm impairments, including veterans with SCI. We hypothesize that a modular combination of 5 or 6 input modes could meet the needs of 95 percent of this population of nearly 10 million veterans.

RECENT PUBLICATIONS FROM THIS RESEARCH

[297] CORTICAL SENSORIMOTOR REORGANIZATION IN SPINAL CORD INJURY: A PILOT STUDY

Joseph B. Green, MD; Yolanda Bialy; Elena Sora; Anthony Ricamato
Rehabilitation Research and Development Center, Edward Hines, Jr., VA Hospital, PO Box 20, Hines, IL 60141

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Pilot Project #B2065-PA)

PURPOSE--This is a pilot project to determine the feasibility of detecting reorganization of motor control in the cerebral cortex following spinal cord injury (SCI).

METHODOLOGY--We are applying the new technology of 128-electrode, high resolution electroencephalography to determine how the motor representation in the cerebral cortex changes by mapping cortical potentials associated with actual, imagined, and attempted movements of the fingers and toes in nondisabled controls and SCI patients. Movements are cued by visual stimuli which trigger the averager. The dipole sources (generators) of the movement related cortical potentials are determined and coregistered with the subjects magnetic resonance images (MRI) of the brain.

PROGRESS--We have recorded and mapped movement related potentials in actual and imagined finger and toe movements in 30 nondisabled subjects and 5 SCI patients.

PRELIMINARY RESULTS--Actual and imagined movement potentials differ in localization in normal controls. Actual movements are associated with potentials, which are more contralateral to the side of finger movement than imagined movement potentials, which tend to be midline in location. This is confirmed in dipole source localization. Data in SCI patients shows that midline potentials generated by attempts to move the paralyzed toes tend to be more contralateral than in normal toe movements, raising the possibility that toe representation may have been absorbed into the hand area.

FUTURE PLANS--More subjects will be recruited with paraplegia and quadriplegia. Ideally we would like to repeat the testing in acute cases to identify when cortical representation changes following SCI. The next step would be to investigate ways to prevent loss of representation of paralyzed limbs.

RECENT PUBLICATIONS FROM THIS RESEARCH

[298] EFFECT OF SUPPORTED STANDING AND UPPER BODY EXERCISE ON LOWER EXTREMITY SPASTICITY IN PERSONS WITH SPINAL CORD INJURY

Linda S. Fehr, MS; Morris A. Fisher, MD; W. Edwin Langbein, PhD
Hines VA Medical Center, Rehabilitation Research and Development Center, Hines, IL 60141; Loyola Medical Center, Department of Medicine, Maywood, IL 60153

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B811-2RA)

PURPOSE--The purpose of this research is to: a) demonstrate that supported standing and/or aerobic upper body exercise (AUBX) significantly alter signs of the upper motor neuron syndrome (UMNS), particularly lower extremity tone and reflexes; and b) analyze neurophysiological measures indicative of altered motor neuron pool excitability and/or presynaptic inhibition and define the relationship between these measures and changes in signs of UMNS following AUBX or standing.

METHODOLOGY--Subjects with spinal cord injury and history of lower limb hypertonicity complete three procedures: a) to test the effect of moderate-intensity AUBX on signs of UMNS, subjects perform 20 min of submaximal wheelchair ergometry exercise; b) to examine the effect of low-intensity activity, subjects complete 40 min of supported standing; c) a timeout (control condition) is included to isolate effects of physical activity from changes occurring during quiet rest and testing procedures alone. Baseline measures of tone and reflexes are followed immediately by an experimental condition (AUBX, standing, or timeout). To examine the temporal pattern of changes in tone and reflexes following the experimental condition, all measurements are repeated immediately following activity or timeout and at 90-min intervals for 3 hrs.

  Tone at the knee is assessed by pendulum drop test (normalized relaxation index or R2n). Electrophysiological measurements include H/M ratios and F wave amplitudes and persistence.

PRELIMINARY RESULTS--Of six subjects completing the experimental protocol, three exhibited substantial improvement in tone following AUBX and standing as evidenced by increased mean R2n with respect to baseline. Mean R2n in this group of "responders" increased 13 and 28 percent immediately following AUBX and standing, respectively. Alternatively, mean R2n declined 11 percent immediately following the timeout period. Even greater relative improvement was observed at 3 hours postactivity: mean R2n for the group was increased 6 percent and 59 percent above baseline following AUBX and standing, respectively, while mean R2n under control conditions had decreased 21 percent. In contrast, the three remaining subjects also experienced improvement in tone following AUBX and standing but in many cases, these improvements were less than those observed under control conditions. In this group of "partial responders," mean R2n was increased 10 and 7 percent immediately following AUBX and standing, respectively, but was increased 9 percent immediately following timeout. Similarly, increases of 11 and 2 percent were seen in mean R2n 3 hours post AUBX and standing, respectively, while an increase of 12 percent was seen 3 hours after the timeout period. With respect to electrophysiological measures, decreased H/M ratios and the facilitation of F waves are consistent with decreased motor neuron pool excitability and decreased recurrent inhibition, respectively. All responders exhibited either decreased H/M ratios or a facilitation of F waves or both following AUBX or standing. In contrast, none of the partial responders exhibited decreasing H/M ratios; and F waves were facilitated in only one of these subjects. In none of the six subjects were decreases in H/M ratios or a facilitation of F waves observed under control conditions.

FUTURE PLANS--Although it is not possible at this juncture to make definitive statements about the potential role of moderate intensity AUBX and supported standing in the treatment of spasticity, the fact that there is consistency of some measures in this small sample is most encouraging. These preliminary data reflect the anticipated complexity of UMNS in SCI and support further study of our hypothesis. Application of our experimental protocol to a larger sample of subjects with SCI should clarify physiological similarities as well as diversity in this population. Such information is expected to be of great value in the effective classification and treatment of these patients.

RECENT PUBLICATIONS FROM THIS RESEARCH

[299] CAUSE FOR MALE INFERTILITY AFTER SPINAL CORD INJURY AND ITS PREVENTION

Hosea F. S. Huang, PhD; Leonard M. Pogach, MD
East Orange VA Medical Center, East Orange, NJ 07019

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B885-RA)

PURPOSE--The fertility rate of men becomes impaired after spinal cord injury (SCI). Analysis of their semen obtained by electro-stimulation reveals a general decrease in sperm count and progressive sperm motility and an increase in number of sperm with abnormal morphology. These findings suggest that defects in spermatogenesis may be responsible for the abnormal semen quality seen in ejaculates of SCI men. The aim of current year was to investigate the causes for the decrease in sperm production and the abnormal sperm parameters after SCI in the rat model.

METHODOLOGY--SCI was induced in male adult rats by surgical transection of spinal cord at the level of T9 vertebra. Animals were sacrificed at various times after the surgery. Testicular tissues were fixed in Bouin's solution and processed for histology or whole mounts of seminiferous tubules.

  Qualitative normalcy of spermatogenesis was determined by the presence and location of each cell type in specific cellular association in each stage of the seminiferous epithelial cycle.

  Quantitative analysis of spermatogonial proliferation was performed by enumerating the number of type A1 spermatogonia and preleptotene spermatocytes in whole mounts of stages VII-IX seminiferous tubules. Results were expressed as cell number per 100 Sertoli cell nucleoli. Quantitative analysis of differentiating spermatogenic cells was achieved by counting the number of preleptotene and pachytene spermatocytes, and step 7 and 19 spermatids in cross sections of stage VII-VIII epithelial tubules. Results were normalized as cell number per 100 Sertoli cell nuclei.

PROGRESS--Spermatogenesis became impaired as early as 3 days after the induction of SCI. Spermatocytes and spermatids were the first cell types to show abnormalities. Spermatogenesis became totally regressed 2 to 3 months after SCI, characterized by the absence of all spermatogenic cells, including the proliferating spermatogonia. This occurred in the presence of normal pituitary-testis hormone axis, suggesting that nonendocrine factors may be involved in the SCI-induced regression of spermatogenesis.

  Preliminary quantitative analysis of spermatogonial proliferation revealed a 25-30 percent decrease in the number of type A1 spermatogonia and preleptotene spermatocytes 4 weeks after SCI. These results indicate an impaired spermatogonial proliferation. Since there was an acute suppression of pituitary-testis hormone axis shortly after the injury, it is postulated that stem cell renewal may be impaired, resulting in the decrease in the number of proliferating spermatogonia. The decrease in spermatogonial proliferation is apparently responsible for the subsequent regression of spermatogenesis.

  Restoration of qualitatively complete, but quantitatively reduced, spermatogenesis was noted in 9 of 18 rats killed 6 mo after injury. These results demonstrate that the SCI related azospermia is reversible. The presence of persisting abnormality in the restored spermatogenesis is consistent with the observations in SCI men. This finding demonstrates that the SCI rat is an appropriate model to study the effect of SCI on human spermatogenesis.

FUTURE PLANS--Because the early spermatogenic lesions after SCI resemble those occuring after testosterone deprivation and/or hypophysectomy, we are currently investigating the possibility of preserving spermatogenesis in the SCI rats by combinations of testosterone and FSH. This experiment is currently underway.

  Because normal Sertoli cell functions are essential for normal spermatogenesis, the regression of spermatogenesis in SCI rats may be attributable to abnormal Sertoli cell function. To examine this possibility, we will examine the effects of SCI on Sertoli cell function, using rats with Sertoli cell enriched testis as a model. This model is produced by X-irradiation of pregnant female rats at the 20th day of gestation. We have irradiated 17 pregnant females and all of them have delivered babies normally. The male pups will be weaned at 20 days of age and will be subjected to SCI operation when they reach 70-80 days of age.

[300] MANUAL WHEELCHAIR USER UPPER EXTREMITY PAIN

Rory A. Cooper, PhD; Michael L. Boninger, MD; Sean D. Shimada, MS; Thomas J. O'Connor, MS; Carmen DiGiovine, BS
Human Engineering Research Laboratories (151R-1), Pittsburgh Veterans Affairs Medical Center, Pittsburgh, PA 15206; Department of Rehabilitation Science and Technology, School of Health and Rehabilitation Science and the Departments of Bioengineering and Mechanical Engineering, School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261; Department of Orthopaedic Surgery, Division of Physical Medicine and Rehabilitation, School of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA 15261

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B869-2RA)

PURPOSE--The incidence of upper limb pain among manual wheelchair users has been estimated to be between 30 and 70 percent, and the probability that they will experience debilitating arm pain increases over time. Although several activities of daily living may contribute to this pain, several studies have implicated the use of manual wheelchairs as a primary contributor. Moreover, recent studies tend to indicate that wheelchair athletes do not experience a higher incidence of pain than their sedentary counterparts. This may be due to the greater care and consideration given to the selection and fitting of wheelchairs by athletes. This study focuses on two issues: the relationship between biomechanical factors, carpal tunnel syndrome, and rotator cuff injury; and cross-sectional changes in arm pain among wheelchair users with increasing years of experience. It is hypothesized that biomechanical factors related to wheelchair design and the wheelchair user's stroke will be identified and related to the incidence of arm pain.

METHODOLOGY--Veterans with thoracic level spinal cord injury are being recruited to participate in this study. Subjects are asked to complete a medical history and pain survey, and each has a unilateral magnetic resonance image and plane radiographs made of the arm. Clinical electromyograms are being used to determine the presence of neuropathies. Biomechanical analyses are performed using a SMARTWheel to collect 3-D force and moment data, and the OptoTrac (Northern Digital) active marker system is being used to collect 3-D motion data. Kinematic and kinetic data are collected in real-time. Anthropometric data are collected, and used with Hanavan's model. The kinematic, kinetic, and anthropometric data are combined to calculate joint moments and forces. These data are also used to calculate several variables which are hypothesized to be able to discriminate between the biomechanics of people with and without clinical symptoms of upper limb pain. Statistical procedures are being used to compare the biomechanics of groups of subjects with and without arm pain. The MRI and EMG data are used in the determination of the two groups, and are being used to investigate the effects of chronic manual wheelchair use on arm joint structures.

PROGRESS--We have constructed a database of eligible subjects and are using it to sort eligible subjects based upon their length of time using a manual wheelchair. All of the necessary instrumentation has been installed and the appropriate interfaces have been developed. Software and hardware have been developed to perform anthropometric data collection using the new equipment. Algorithms for analyzing the data have been developed and additional development is ongoing. Complete data have been collected on a single subject, and data on several others: data collection is ongoing. We were also given recent institutional review board approval to begin a cadaver study to improve our biomechanical models of the upper limb.

PRELIMINARY RESULTS--Preliminary results show that there are likely to be potentially injurious forces present at the wrist during normal manual wheelchair propulsion. We have also performed an analysis of the frequencies distribution of pushrim forces. This has allowed us to select optimal filter frequencies. We have also developed a 3-D equivalent for the center of pressure.

FUTURE PLANS--We plan to continue to collect data on a greater number of subjects and to complete model development. When we have sufficient data, we will examine changes in upper limb joint structure with year of wheelchair use, and determine whether our biomechanical models are effective in discriminating between manual wheelchair users with and without arm pain.

RECENT PUBLICATIONS FROM THIS RESEARCH

[301] RECURRENCE OF BACTERIURIA AND PROGRESS TO SYMPTOMATIC URINARY TRACT INFECTION IN SPINAL CORD-INJURED PATIENTS

Rabih O. Darouiche, MD; Richard Cadle, PharmD; Ki-Soo Kil, PhD; Richard A. Hull, PhD; Colleen Stewart, RN; Daniel M. Musher, MD
Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX 77030

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B807-RA)

PURPOSE--The four objectives of this project were to: 1) analyze the effect of treating asymptomatic episodes of urinary tract infection (UTI) on the progression to symptomatic UTI in spinal cord-injured (SCI) patients who undergo sterile intermittent bladder catheterization; 2) explore the potential relationship between the in-vitro adherence of Klebsiella pneumoniae organisms and recurrence of bacteriuria in these patients; 3) localize the site(s) adjacent to bladder where bacteria may continue to reside despite eradication of bacteriuria by antibiotic therapy; and 4) differentiate between relapse of UTI by same bacterial strain vs reinfection by a different bacterial strain.

METHODOLOGY--Eligible hospitalized patients were randomized to receive either a 1-week course of antibiotic therapy for asymptomatic episodes of UTI (Greater than or equal to 105 cfu of bacteria/ml of urine and >/=104 WBC/ml of urine) or no antibiotic treatment and were monitored for the development of the primary outcome of symptomatic UTI. The in-vitro adherence of recovered K. pneumoniae organisms to human uroepithelial, HEp-2 and buccal cells was correlated to the likelihood of particular bacterial strains to cause recurrence of UTI despite antibiotic therapy. Cultures of potential reservoir sites, including prostate, urethra, perineum, and rectum, were simultaneously obtained with urine cultures from patients randomized to the treatment group. Among those with recurrent UTI, DNA typing of bacterial isolates was done using the polymerase chain reaction (PCR) technique.

PROGRESS--The practicality of this study was proven in 31 evaluable patients so far.

RESULTS--The progression to symptomatic UTI was significantly lower among patients randomized to the treatment (3/16=19 percent) versus the nontreatment group (10/15=67 percent; p=0.007). The adherence of K. pneumoniae to human cells was mediated by type 1 fimbria and correlated with the likelihood of recurrent UTI. Preliminary results suggest that sites adjacent to the bladder, such as prostate, urethra, perineum and rectum, may contribute to recurrence of UTI by acting as potential reservoir sites from which bacteria may migrate again into the bladder following eradication of bacteuiria with appropriate antibiotics. Molecular analysis by PCR showed that the majority of instances of recurrent UTI were due to relapse by the same bacterial strain.

FUTURE PLANS--Patient enrollment will continue to a maximum of 60 evaluable patients. A cost-benefit analysis of the treatment of asymptomatic episodes of UTI in these SCI patients will be done. The findings of this study should also help guide health care providers as to which SCI patients are likely to experience progression from asymptomatic to symptomatic UTI and, therefore, perhaps administer antibiotic therapy for asymptomatic UTI in only those at high risk of progression. If the role of potential reservoir sites is confirmed, it should have an impact on the type and duration of antibiotic therapy for UTI in this population.

RECENT PUBLICATIONS FROM THIS RESEARCH

[302] NATURAL HISTORY AND CLINICAL COURSE OF URINARY TRACT COMPLICATIONS IN PATIENTS WITH SPINAL CORD DYSFUNCTION

Samuel L. Stover, MD; L.K. Lloyd, MD
University of Alabama at Birmingham, Spain Rehabilitation Center, Birmingham, AL 35233

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202-2646

PURPOSE--Analyzing a spectrum of urologic data acquired from a large number of persons with spinal cord injury (SCI) will help clinicians understand the natural history of the urinary tract and its complications following SCI, thus helping clinicians select those prevention and management methods capable of assuring the most positive prognosis.

  This study seeks to: 1) document the natural history and clinical course of urinary tract complications among persons with SCI who utilize various methods of neurogenic bladder management; 2) answer a series of important clinical research questions that can impact future urologic management and improve the medical care and well-being of persons with SCI; and 3) encourage the utilization of the UAB-SCI Urologic Database by other institutions which could provide a much larger cohort of persons with SCI for future collaborative studies.

METHODOLOGY--Data are collected prospectively for each patient admitted to the UAB-Spinal Cord Injury Care System (UAB-SCOTCHES) at admission, discharge, and annually thereafter. In addition, data have been collected retrospectively from chart reviews on 596 patients between 1970 and April 1979. Since 1979, persons who were enrolled retrospectively have been followed prospectively along with the more recently injured persons. Persons constituting the prospective study group (n=1594) were injured and admitted between May 1979 and July 1995. The latter group will continue to grow in size as the project continues. Overall, 2,190 persons have been entered into the project database, although records are only retained if there is adequate follow-up information, which includes 1315 persons.

RESULTS--The database is now available on computer software with quality control computer programs that cross-check the data for out-of-range entries and internal consistency. This increases opportunities to compare data among users since variable definitions and collection method will be uniform.

  During the project year the most extensive analysis accomplished to date was completed on the urology database. A consecutive sample of 1,114 persons with SCI who were injured between 1969 and 1994 were studied. Total and individual effective renal plasma flow (ERPF), which is a measure of renal function, were compared to determine the effect of different bladder management methods on long-term renal function. With very sophisticated methods of data analysis, supervised by the Department of Biostatistics, it was concluded that renal function was adequately preserved in the great majority of persons with SCI and did not appear to be influenced to any great extent by the method of bladder management. These findings have very important clinical implications, and although somewhat unexpected, are very encouraging to the person with SCI and to the clinician trying to decide on the method of bladder management. Extensive studies were also conducted on urologic complications and have been published.

FUTURE PLANS--New patients with SCI are continually added to the study population and data on the large population followed in our clinics are continually added to the database. Some patients have follow-up data for as long as 27 years. Further investigation and analysis of data will continue during the next 2 years. Employers of the research will focus on long-term renal function outcome resulting from various methods of bladder management and other secondary complications of neurogenic bladder management.

RECENT PUBLICATIONS FROM THIS RESEARCH

[303] CAUSES AND COSTS OF UNPLANNED REHOSPITALIZATIONS AMONG PERSONS WITH SPINAL CORD INJURY

Michael J. DeVivo, DrPH
University of Alabama at Birmingham, Spain Rehabilitation Center, Birmingham, AL 35233; email: devivom@sun.rehabm.uab.edu

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202-2646

PURPOSE--There have been several published studies of rehospitalization rates, risk factors for rehospitalization, and associated costs among persons with spinal cord injuries (SCI). However, only limited baseline data on the long-term incidence of a few secondary medical complications such as renal and bladder stones have been published, and the relationship between the occurrence of these secondary complications and subsequent rehospitalizations has not been determined. Moreover, the National Spinal Cord Injury Statistical Center (NSCISC) data set cannot be used for this purpose, because there is no established linkage in that data set between reported occurrences of secondary complications and rehospitalizations. The purpose of this study is to provide baseline data documenting the leading causes of unplanned rehospitalizations among person with SCI and the average costs associated with each cause so that frequent and costly complications can be given higher priority for further study and the effectiveness of techniques to reduce the incidence of complications and hospitalizations can be assessed using rigorous cost-benefit analyses.

  The objectives of this study are: 1) to identify the most frequent causes of unplanned rehospitalizations among persons with SCI, 2) to determine the average length of stay and cost for each cause of unplanned rehospitalization among these persons, and 3) to describe, epidemiologically, the causes and costs of unplanned rehospitalization among these persons.

METHODOLOGY--The basic study design is cross-sectional with a 2-year prospective data collection period. All persons with traumatic SCI currently being followed at the University of Alabama at Birmingham Spinal Cord Injury Care System (UAB-SCICS) are eligible for this study, regardless of how long ago their injury occurred.

  Admission sheets for University Hospital are scanned daily to identify rehospitalizations among person with SCI. Persons returning for clinic visits and outpatient annual evaluations are asked whether they have been rehospitalized at another facility since their last contact with us. When appropriate rehospitalization is identified, medical record and billing information are obtained. ICD9CM codes are used to document the primary cause of rehospitalization. Other complications that may have contributed to the need for rehospitalization are documented as secondary causes.

  The percentage of rehospitalizations, average length of stay, and charges due to each type of secondary complication will be determined. Mean length of stay and charges of each cause of rehospitalization will be compared by using Student's t test. The distribution of causes of rehospitalization will be characterized epidemiologically. The chi-square test will be used to compare the percentages of rehospitalizations due to each cause by time postinjury, age group, gender, race, education level, neurologic level of injury, degree of injury completeness, urban/rural hospital location, marital status, and presence of insurance coverage. When sample sizes for individual causes of rehospitalization permit, multiple linear regression analysis will be conducted to determine the effect of these predictor variables on length of stay and charges for rehospitalizations resulting from that cause.

PRELIMINARY RESULTS--The project began in 1994. As of July 1996, 250 rehospitalizations had occurred. We have begun the process of obtaining medical records for those hospitalizations and now have completed data collection forms for 76 cases.

FUTURE PLANS--Continue data collection and begin analysis when the sample size is sufficient.

[304] SECONDARY CONDITIONS AFTER SPINAL CORD INJURY: RELATIONSHIP TO LIFE ADJUSTMENT

J. Stuart Krause, PhD
Shepherd Center, Atlanta, GA 30309

Sponsor: Shepherd Center, Atlanta, GA 30309; National Center for Medical Rehabilitation Research, National Institute of Health, Bethesda, MD 20892

PURPOSE--The purpose of this study is to identify both prevalence and risk factors for secondary conditions after spinal cord injury (SCI). Key elements of this research include: (1) the utilization of two large SCI samples, one of which oversamples females and minorities, (2) the development of a measure of secondary conditions, and (3) building upon a prominent longitudinal study of life adjustment and SCI.

METHODOLOGY--Participants. Two distinct participant samples were used in this study. The first sample consisted of a large stratified (by gender, race, and age) sample of 723 cases from outpatient files of a large Southeastern rehabilitation hospital. A total of 437 participants who were currently active in the Minnesota Longitudinal Study (MLS) as of 1994 comprised the second participant sample. The same three screening criteria were used for both samples: a traumatic SCI, the injury was at least 2 years duration, and at least 18 years of age at the time of the study.

  Instruments. The Life Situation Questionnaire (LSQ) was developed in 1974 to measure information on multiple aspects of life adjustment. The Secondary Conditions Questionnaire (SCQ) was developed specifically for this study; its two parts request different types of information regarding the same 50 secondary conditions for a total of 100 items. The first part requests epidemiologic information and the second information on the extent to which the condition impacts individuals' lives. Psychometric data, except for test-retest data, is yet to be collected.

  Procedures. The LSQ and SCQ were sent to each potential participant. Follow-up calls and second sets of materials are used to recruit all initial nonrespondents. Participants are offered $20 and a copy of study results as inducements to participate.

PROGRESS--Of the southeastern sample, 579 completed the SCQ, as did 352 of the MLS participants. Data from the two samples has just been processed and entered into a system file.

RESULTS--Preliminary data analyses have just been completed. They were consistent with findings from previous epidemiologic studies, many of which used different methodologies. Analyses to identify risk factors for secondary conditions are currently underway.

IMPLICATIONS--The results of this study will help rehabilitation professionals to develop prevention programs based on knowledge of risk factors related to a wide range of adjustment variables.

FUTURE PLANS--Data analysis and dissemination will be completed over the next 12 months.

RECENT PUBLICATIONS FROM THIS RESEARCH

[305] RACE, GENDER, AGE, AND ADJUSTMENT AFTER SPINAL CORD INJURY: THE SOUTHEASTERN LONGITUDINAL STUDY

J. Stuart Krause, PhD; Carol A. Anson, PhD
Shepherd Center, Atlanta, GA 30309; Klemm Analysis Group Atlanta, GA 30309

Sponsor: Shepherd Center, Atlanta, GA 30309

PURPOSE--The purpose of this study is to identify the relationships between gender, race, age and life adjustment after spinal cord injury (SCI).

METHODOLOGY--Participants. Participants were selected from outpatient files of a Southeastern rehabilitation hospital. All participants had traumatic onset SCI of at least 2 years duration and were a minimum of 18 years of age at the time of the study. A total of 362 individuals participated in the study (63 percent response rate). Overall, 57 percent of the participants were male and 65 percent were Caucasian.

  Instruments. The Multidimensional Adjustment Profile (MAP) was developed specifically for this study and is an updated version of the Life Situation Questionnaire (LSQ). The MAP contains six sections including: (1) biographic and injury-related status, (2) vocational and avocational activities, (3) educational history, (4) psychological adjustment, (5) problems, and (6) health and medical status. Ten scales have been developed from the MAP, nine of which were based on factor analysis of life satisfaction and problems scales.

  Procedures. All participants completed the MAP. Subsamples completed one of three other instruments. Participants were offered $5 as well as descriptive study results as inducements to participate in the study. All materials were obtained by mail.

PROGRESS--The first stage of this study has been completed. Current efforts are focusing on analysis and dissemination of the study results.

RESULTS--Gender and race differences were observed for both subjective and employment outcomes.

IMPLICATIONS--This research has been helpful in identifying the roles of gender and race in adaptation after SCI.

FUTURE PLANS--A longitudinal follow-up is planned within the next two years.

RECENT PUBLICATIONS FROM THIS RESEARCH

B. Treatment and Rehabilitation

[306] FES ON SPINAL CORD INJURED PATIENTS: EFFECTS ON MUSCLE BLOOD FLOW AND METABOLISM

Erika AM Scremin, MD
VA Medical Center, West Los Angeles 90073

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B2002-RA)

  No report was received for this issue.

[307] FUNCTIONAL ELECTRICAL STIMULATION OF SPINAL CORD INJURED PATIENTS

A.M. Erika Scremin, MD; Karen L. Perell, PhD; Deborah L. Mutton, MA; Oscar U. Scremin, MD, PhD; Thomas J. Barstow, PhD; Livia Kurta, PT; Jerry Baranick, BS; Donna Leonard, BS; Barbara Wiseman, BS
Physical Medicine and Rehabilitation Service, VA Medical Center, West Los Angeles, CA, 90073; University of California, Los Angeles, 90024; Division of Respiratory and Critical Care Physiology and Medicine, Harbor-UCLA Medical Center, Torrance, CA 90509

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B603-RA)

PURPOSE--The purpose of this project is to study the effects of functional electrical stimulation-induced lower limb cycling (FESILEC) on complete, spastic SCI subjects and determine the therapeutic benefits and associated risks of this form of rehabilitation.

METHODOLOGY--Subjects participated in a 48-session training protocol on a computerized REGYS ergometer, powered by lower limb muscles and activated by cutaneous electrodes. A total of 37 subjects were screened, of which 25 (3 with quadriplegia and 22 with paraplegia) were admitted to the study. In addition, 17 nondisabled subjects were studied as controls for the muscle blood flow studies and 10 nondisabled and 6 flaccid SCI subjects were studied as controls for the spasticity studies.

RESULTS--Metabolic Studies. 1) Oxygen uptake (VO2) kinetics: the VO2 kinetics during arm ergometry and FESILEC were compared in nine subjects during a 10-min session of FESILEC before and after training. Exercise VO2 was the same for both arm and leg exercise; however, FESILEC exhibited slower VO2 kinetics and was accompanied by an attenuated increase in heart rate and a greater rise in blood lactate. The improvement in blood lactate levels with leg, but not arm exercise, in the absence of changes in exercise heart rate, suggests peripheral changes to the contracting leg muscles with FESILEC training. 2) Hybrid exercise training (arm ergometry combined with FESILEC): eight subjects completed a hybrid exercise training program immediately following the FESILEC training. Results showed that hybrid exercise training performed 2×wk provided sufficient exercise intensity to significantly improve aerobic capacity, achieved higher VO2 values during actual training sessions and increased caloric expenditure as compared with FESILEC training, alone. Long-term FESILEC and/or hybrid exercise may ultimately reduce the risk of cardiovascular disease in these patients.

  Muscle Blood Flow. Studies on H215O positron emission tomography (PET) muscle perfusion: the PET technique was used on 4 SCI subjects and 5 nonimpaired controls before and immediately after exercise and 20 min after recovery from a standardized exercise. This exercise was induced by FES in the SCI subjects and by voluntary contraction to perform a comparable amount of work in the nondisabled subjects. The purpose of this study was to compare the skeletal muscle blood flow parameters in both groups and to determine if the limited efficiency of FES-induced exercise in SCI subjects was due to a restriction of muscle perfusion. The kinetics of H215O by muscle was studied in 16 consecutive frames obtained simultaneously in 32 tomographic planes through the activated area. Registration of PET images with CT images allowed identification and measurement of the volume of areas activated by exercise in both groups of subjects. The preliminary results (more SCI subjects are being recruited) showed that the volume of muscle with enhanced blood flow was greater, and had less variability than that of nondisabled controls. The kinetics of H215O uptake by muscle were comparable in both groups, but an enhanced rate of tracer uptake was still evident 20 min after exercise in the SCI subjects while it had returned to baseline levels in the controls at this time. Taken together, these results suggest that there is no limitation to the maximal blood flow level attainable but a slower recovery of blood flow indicative of a greater O2 debt following FES stimulation when compared to nondisabled subjects performing the same work voluntarily.

  Muscle Mass. Final analysis of the computerized tomographic and magnetic resonance imaging studies before and after FESILEC training to assess muscle mass changes in thigh (stimulated muscles) and in the shank (nonstimulated muscles) is being performed in 15 subjects.

FUTURE PLANS--Our plans are to complete work in the areas of muscle blood flow and muscle mass.

RECENT PUBLICATIONS FROM THIS RESEARCH

[308] CLINICAL TRIAL OF ARTIFICIAL PERIPHERAL NERVE GRAFT

Eric E. Sabelman, PhD; Vincent R. Hentz, MD; Feng Zhang, MD; Paula Koran, BS
Rehabilitation R&D Center, VA Palo Alto Health Care System, Palo Alto, CA 94304; Dept. of Functional Restoration, Stanford University Medical School, Stanford CA 94305; email: sabelman@roses.stanford.edu

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(#B588-3RA)

PURPOSE--The recovering damaged nerve normally has a high population of Schwann cells that made up the myelin sheaths of axons prior to injury. These cells secrete growth factors and repair the extracellular matrix in preparation for the extension of regenerating axons into the damaged region. This project is based on replacing the Schwann cells in an otherwise acellular artificial nerve graft as a substitute for an autograft taken from elsewhere in the patient's body. The latest graft formulation is to be tested in lieu of an autograft in patients having trauma to the hand or arm, or to replace sural nerves removed for autografting.

METHODOLOGY--Preparation of the graft essentially consists of re-polymerization of solubilized collagen fibers with added cultured Schwann cells and insertion into a biodegradable conduit. Type I collagen is preferred because it is readily available, relatively inexpensive, and its properties are reasonably well understood. The matrix or conduit walls could include regeneration-promoting agents such as nerve growth factor. The conduit limits penetration of inflammatory cells into the region of axonal regrowth, as well as facilitating microsurgical reanastomosis with the proximal and distal ends of the nerve.

PROGRESS--In a series of animal implantations, a graft formulation has been achieved having the same functional recovery as an autograft. Based on this result, a proposal for a limited clinical trial (up to 10 patients per year) has been approved. A preclinical phase is underway, in which culture methods for adult human Schwann cells are being optimized, potentially immunoreactive components are being omitted from the fabrication process, long (30 mm) grafts are being tested for efficacy and durability in a rat model, and clinical sensorimotor measurements for functional recovery are being refined.

FUTURE PLANS--There is potential for tissue-engineered grafts to bridge traumatic defects in the central nervous system. Our laboratory is collaborating with Hines VA Rehabilitation R&D Center by fabricating grafts for testing Schwann cell-seeded implants in spinal cord injuries in rats.

RECENT PUBLICATIONS FROM THIS RESEARCH

[309] FUNCTIONAL RESTORATION OF GRASP IN QUADRIPLEGIA

V. Rodney Hentz, MD; Felix E. Zajac, PhD; Inder Perkash, MD; Charles Burgar, MD; Kevin McGill, PhD; Machiel Van der Loos, PhD; Francisco J. Valero-Cuevas, MS; Kai-Nan An, PhD;
Surgery Service, VA Palo Alto Health Care System, Palo Alto, CA 94304-1200; Department of Functional Restoration, School of Medicine, Stanford University, Palo Alto, CA; Department of Orthopaedics, Mayo Clinic, Rochester, MN 55905; email: zajac@roses.stanford.edu

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B898-2RA)

PURPOSE--Our overall objective is to improve functional grasping in quadriplegics by providing them, through surgical musculoskeletal intervention, with the biomechanical ability to grasp objects. Reconstructive surgeries, including tendon transfers, are commonly performed to restore partial grasping function. To predict functional outcome, the surgeon relies heavily on what the biomechanical properties of the hand and arm are before and are expected to be after surgery. However, surgeons are understandably reluctant to risk performing new techniques if they are unable to guarantee the best possible outcome to the patient. Uncertainty in the functional outcome of new surgical approaches hinders our ability to achieve improvements in the treatment of quadriplegics.

METHODOLOGY--A biomechanical model of the hand musculotendinoskeletal system will be developed to provide surgeons with an "in vitro" testbed for improving existing techniques or trying new ones and predicting functional outcomes. Such a model will allow surgeons to determine the precise musculotendinoskeletal parameters to which the functional outcome of a surgical or rehabilitation procedure is most sensitive. Thus, those aspects of the surgical procedure needing close clinical scrutiny can be identified. We will develop and test the validity of a computer-implemented musculotendinoskeletal model, first of the normal hand and next the quadriplegic hand, where the emphasis is on identifying the biomechanical factors bounding grasping. The ability of the index finger and the thumb to exert maximum grasping (pinch) forces will be the focus because of its importance to quadriplegic grasping (e.g., tip and key pinch) and because of the anatomic similarity between the index finger and the other fingers. Maximum pinch forces are emphasized because they specify the biomechanical limit of grasping performance.

PROGRESS--We have developed a computer model of the index finger. The index finger is modeled as a metacarpal and three phalanges articulated by pin joints, two at the metacarpophalangeal and one at each interphalangeal joint. All index finger musculotendons are included. Force generation of a muscle is assumed to depend on its excitation level (to be determined by the model) and on experimentally obtained musculotendon architectural parameters. The moment arm of each tendon at each spanned joint, as a function of joint angle, was found from a fresh cadaver.

RESULTS--We analyzed the model to find the maximum static index finger tip force biomechanically possible at any finger posture, the excitations required of muscles to generate the finger forces, and the sensitivity of force production to musculotendon parameters and the excitation pattern. The model predicts that the muscle excitation pattern producing maximum finger forces is quite robust to the direction of the finger tip force being generated (palmar vs. radial) and to musculotendinoskeletal parameters. However, the amount of force the finger can produce in a given direction depends mostly on a few muscle moment arms.

FUTURE PLANS--We will test the validity of the model by recording finger tip forces and EMGs with intramuscular electrodes from the muscles acting on the finger in subjects instructed to press as hard as possible against either a low or high frictional surface with the finger in different postures. We will also develop a musculotendinoskeletal model of the thumb. With a model of the finger and thumb, the complex biomechanical and neural interactions that make grasping possible can be analyzed.

[310] HIGH-FREQUENCY MAGNETIC STIMULATION OF THE BLADDER AND BOWEL

Vernon Wen-Hau Lin, MD; Inder Perkash, MD
VA Palo Alto Health Care System, Palo Alto, CA 94304; Stanford University, School of Medicine, Stanford, CA 94305

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B803-RA)

PURPOSE--The purpose of this research was to determine the effects of high-frequency magnetic stimulation (HFMS) of the sacral nerves on bladder and rectal pressures (BP and RP) in spinal cord injured (SCI) patients, and to optimize the magnetic stimulation parameters to obtain functional micturition and defecation.

METHODOLOGY--Two-day experiments were performed in eight chronic SCI patients, C4-T12. In day one (bladder day), each subject received a screening history and physical examination, bulbocavernosus reflex latency, as well as a full urodynamic study. This was followed by magnetic stimulation of the bladder using a Cadwell stimulator, with a 9-cm magnetic coil placed near the L2-L4 vertebrae while measuring BP and RP. The frequency and power output parameters were initially fixed at 20 Hz and 175 Joules/pulse while optimizing the focus of stimulation, and were later varied to generate the frequency and intensity profiles. The second day (bowel day) consisted of a full rectodynamic study, and magnetic stimulation of bowel. Functional micturition and defecation were also attempted at the end of each experimental day. Laboratory tests, including a set of Chem 20, CBC with differentials, PT, PTT, UA, and urine cultures, were performed both before and after the experiment.

PROGRESS--We have demonstrated that HFMS of the sacral nerves was effective in elevating the bladder and rectal pressures; 20 Hz frequency and 70 percent of maximal power provide adequate sacral nerve stimulation in most subjects. Functional magnetic micturition and defecation was achieved in more than 10 subjects.

RESULTS--Thirty SCI subjects with reflex bladders were recruited. The bladder capacity varied from 150 to greater than 600 ml. Peak voiding pressure varied from 24 to 94 cm H2O. And post void residual varied from 0 to 360 ml. Rectodynamic studies revealed an average rectal capacity of 310±30 ml, and peak defecating pressure of 85±13.3 cm H2O. HFMS of the bladder via sacral nerve stimulation using 20 Hz, 70 percent intensity and 2 second burst length resulted in an averaged increase in BP of 24.4±4.88 cm of H2O. Similarly the mean rise in RP was 22.8±6.05 cm of H2O. The frequency and intensity profile results demonstrated that 20 Hz frequency setting, and 175 Joules/pulse intensity offer adequate pressure changes in most subjects, although higher intensities and frequencies usually generate higher BP and RP. We observed micturition in 10 subjects and defecation in 2 individuals. Either suprapubic or lumbosacral stimulation would result in urination.

FUTURE PLANS--We plan to optimize the magnetic stimulation characteristics and anatomical approach to produce functional micturition and defecation, critically evaluate the relative response of HFMS data to existing information using the electrical field stimulation methodology, and determine whether bowel motility is modified by HFMS by evaluating colonic transit time.

RECENT PUBLICATIONS FROM THIS RESEARCH

[311] MANAGEMENT OF MUSCULOSKELETAL COMPLICATIONS OF SPINAL CORD INJURY

Leonard J. Deftos, MD; Jacqueline G. Parthemore, MD; Susan Szollar, MD; Kevin D. Gerhart, MD; Estralita Martin, PhD
San Diego VA Medical Center, San Diego, CA 92161; University of California, San Diego, CA 92161

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B576-3RZ)

PURPOSE--We seek to develop and apply newly discovered, bone cell-specific serum markers and new densitometry/imaging procedures for the skeleton to clinical studies of musculoskeletal assessment in patients with spinal cord injury (SCI) from trauma or illness.

METHODOLOGY--We have recently obtained a bone densitometer and have applied it to our studies of bone markers. The bone markers we have developed and used are immunochemically based. They are classical and novel immunoassys for the respective bone proteins and regulatory hormones under study.

PROGRESS--We have achieved our goals or made substantial progress toward them. We have developed new procedures for the measurements of bone alkaline phosphatase and bone gla protein. We have conducted studies in the following conditions:

  Spondylopathy. We analyzed AP radiographs of lumbar spine (obtained within 1 month of DEXA, dual energy X-ray absorptiometry) in 116 SCI patients for various aspects of spondylopathy, and matched the result to the each vertebral level (L1, 2, 3, and 4). There were 227 (49 percent) abnormal individual vertebrae. Significant elevation (15, 15, 18, and 20 percent; p<0.001-p<0.05) of bone mineral density (BMD; g/cm2) was observed at all levels, particularly at those abnormal without hardware compared to valid. The L4 was most severely affected.

  Heterotopic Ossification (HO) We analyzed plain radiographs of the hip (obtained within 1 month of DEXA) in 107 SCI patients for HO, and matched the result to the three regions of interest: the femoral neck, Ward's triangle, and the trochanter. Significant elevation of densitometric values (p<0.05) observed at all sites.

  Bone markers. We have purified BAcP from surgical specimens from human bone by tracking tartrate resistant acid phosphatase enzymatic activity (TRAP). Three basic steps were involved in our purification procedure: gel filtration on Sephadex, SDS-PAGE electrophoresis, and affinity chromatography. This preparation was then used to immunize mice for monoclonal antibody production. We have developed a panel of monoclonal antibodies and we are evaluating them for their specificity by immunohistology and Western analyses.

FUTURE PLANS--We hope to continue the application of the new procedures we have developed and are in the process of developing to continued studies of the SCI patient.

RECENT PUBLICATIONS FROM THIS RESEARCH

[312] VERTEBRAL FUSION BY NEW OSTEOGENIC AGENTS TO ACCELERATE REHABILITATION

Basil S. Strates, PhD; Michael MacMillan, MD
VA Medical Center, Gainesville, FL 32608; Department of Orthopaedic Surgery, University of Florida College of Medicine, Gainesville, FL 32610

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #A736-2RA)

PURPOSE--The purpose of this project is to stimulate inervertebral bone formation and increase the likelihood of spinal fusion using an osteogenic agent with a carrier. Our hypothesis is that pain relief and vertebral stability, allowing early rehabilitation, often depend on a solid fusion, which can be significantly stimulated by nanogram quantities of an osteogenic agent when delivered to the fusion site by a suitable carrier.

METHODOLOGY--Our rabbit vertebral fusion model utilizes a surgical procedure that minimizes the animal vs human anatomical differences. It involves T11-12 and T12-L1 (or L1-L2 and L2-L3) discectomies, creation of a cylindrical defect, and application of autologous bone (AB) comparative control or an osteogenic agent (DBM: demineralized bone matrix; BMP: bone morphogenetic protein or TGFb1: transforming growth factor b1) and their composites with a resorbable carbonated hydroxyapatite (MHA) carrier of crystal size and composition similar to the mineral of newly formed bone.

PROGRESS--Since the initiation of this project in 1993, we prepared ample quantities of the osteogenic agents DBM and BMP and of the biodegradable carrier microcrystals of hydroxyapatite (MHA) and tested the feasibility of: (a) using a modified lateral approach for intradiscal spinal fusion in the rabbit, similar to that used clinically by our orthopaedic-neurosurgical team; (b) adapting to the rabbit model our digitized imaging method for radiographic density measurements; and (c) stimulating fusion with a composite of a recombinant human TGFb1 (commercially available as rhTGFb1) with MHA and comparing it to AB. Adult male NZW rabbits were randomly assigned to six groups according to graft type (AB control, MHA carrier and rhTGFb1+MHA) and time allowed for fusion (6 and 12 weeks post-grafting). Grafts were placed in a 3.5 x 5 mm tunnel drilled into each disc and adjoining end plates without additional fixation. Spines of euthanized rabbits were cleaned of adhering soft tissues, sawed sagittally into two halves and x-rayed using high resolution mammography film. Radiographic density was quantitated by digitized imaging, and new bone formation and graft consolidation were examined histologically (H&E and Masson-trichrome stains). Fusion was evaluated by radiographic densitometry and biomechanical testing (rigidity by four-point bending) and statistical analyses by Student's t-test and ANOVA.

RESULTS--The bone-forming capacity (bone induction activity) of our preparations of the osteogenic agents DBM and BMP and the commercially obtained rhTGFb1, with and without the MHA carrier, was tested using our intramuscular implantation rabbit bioassay model. The total number of rabbits required for the adaptation of our clinical surgical procedure to the rabbit model and for the preparation of DBM and the extraction and purification of BMP and subsequent implantation exceeded 75. An initial study comparing AB chips to AB powder (T12 rib bone ground intraoperatively in liquid N22) at 6 and 12 weeks post-grafting in 18 rabbits found substantial fusion rate differences in favor of powdered AB at both time intervals. The roentgenographic density data showed statistically significant differences between all discs grafted with either chipped or powdered AB (p<0.01) compared to nongrafted controls. Histologically, cartilage and bone were seen 6 weeks after grafting of powdered AB with some consolidation on the vertebral bone, compared to the presence of unresorbed AB chips, cartilage, and bone with les consolidation in grafts of chipped AB. At 12 weeks post-grafting, new bone associated with consolidation was seen in most discs grafted with powdered AB. Moreover, evaluation of data at 6 and 12 weeks post-grafting in 72 rabbits, randomly assigned to animals grafted with AB (10 mg), MHA (3 mg), MHA (3 mg)+rhTGFb1 (100 ng) and non-grafted controls showed that in all cases intervertebral fusion was effected by endochondral bone formation. Radiographic density and rigidity data indicated that the composite rhTGFb1+MHA produced: (a) radiographically, a high rate of fusion as early as 6 weeks post-grafting and (b) biomechanically, fusion more rigid than AB and significantly better (p<0.02) than that by MHA alone 12 weeks after surgery.

IMPLICATIONS--Because of its fast resorption and effectiveness as an osteogenic agent and the relatively easy intraoperative preparation, AB powder has the potential of becoming clinically useful for stimulation of bone formation and early spinal fusion. Intradiscal grafting of small amounts of the growth factor rhTGFb1 with resorbable microcrystals of hydroxyapatite as carrier could be clinically preferable to AB for the stimulation of spinal fusion leading to earlier rehabilitation.

RECENT PUBLICATIONS FROM THIS RESEARCH

[313] SPINAL CORD INJURY-INDUCED BONE LOSS

Guy A. Howard, PhD; Bernard A. Roos, MD
Research Service 151, VA Medical Center, Miami, FL 33125; Department of Medicine, University of Miami School of Medicine, Miami, FL 33101; email: howard.guy_a@miami.va.gov

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B684-2RA)

PURPOSE--The central as well as the peripheral nervous system are altered after spinal trauma. We hypothesize that changes in neuropeptides and neurotransmitters in nerves supplying bone are involved in the osteopenia which develops as a result of spinal cord injury (SCI). We propose to understand how these changes affect bone metabolism after SCI, since a considerable number of veterans suffer SCI, are paralyzed, and are treated in the VA system. The significance of the research lies in the potential for discovering the mechanism(s) involved in the osteopenia following SCI. These results could lead to a therapy to prevent the loss of bone in newly injured veterans, or aid in the recovery of bone in those with chronic SCI. Such treatment would result in enhanced rehabilitation, and potentially increased independence in many veterans.

METHODOLOGY--The studies utilize a rat model, in which the bone loss is both dramatic and progressive over time. Histomorphometry, radioimmunoassays, and molecular biology techniques characterize bone loss following SCI, as well as changes in neuropeptide distribution and levels in bone and periosteum. We have focused on calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY), all known to be in nerves in bone and implicated as modulators of bone metabolism. Immunohistochemistry, receptor binding assays, and autoradiographic methods are used to evaluate receptor changes.

PROGRESS--We have defined the model and histomorphometrically evaluated the effects of SCI on the bone at various times post lesion, as the animals age. We have developed methods to isolate bone cells for in vitro evaluation from the bones of lesioned animals, as well as to evaluate the neuropeptide content and respective neuropeptide mRNAs in bone and periosteum. We have established a human bone cell model to evaluate the effect of neuropeptides on mRNA of proteins involved in cell-cell communication via gap junctions. We have shown gap junctions to be present and functionally regulated by neuropeptides (e.g., VIP and CGRP) in these bone cells.

RESULTS--Characterization of the effects of SCI on bone metabolism indicate that as the animals age they lose approximately 60 percent of their trabecular bone compared to nonlesioned animals. This loss results in loss of mechanical strength in the femurs of lesioned animals. Immunohistochemical and retrograde tracing studies of nerves in bone showed sensory nerves containing CGRP, VIP, and NPY are particularly dense in the periosteum and penetrate the bone surface. VIP, but not SP, is capable of acutely up-regulating the mRNA for the gap junction protein, connexin 43, in osteoblasts. CGRP is capable of regulating osteoblast function via potassium channels and intracellular calcium, but with moderate increases in cAMP, suggesting other second messengers for CGRP. We have cloned the gene for a receptor component protein of CGRP.

FUTURE PLANS--We will continue our studies with bone cells of lesioned and nonlesioned animals for neuropeptide effects on mRNA levels and functional status of cell-cell communication, as well as on mineralization of collagen matrix formed in vitro. We are beginning studies to identify post-SCI changes in bone cell receptors for these neuropeptides. We will continue our studies on the mechanism of CGRP modulation of osteoblasts. We will evaluate the role of the CGRP receptor component protein in bone metabolism in both an animal model and in human bone cells.

RECENT PUBLICATIONS FROM THIS RESEARCH

[314] PROPHYLACTIC MONITORING OF BLADDER PRESSURE AND VOLUME

James S. Walter, PhD; John S. Wheeler, Jr., MD; John Markley, MD; Rani Chintam, MD; Sherry Gruber, RN; Paul Zaszczurynski; Lisa M. Blacker
Hines VA Medical Center, Rehabilitation Research and Development Center, Hines, IL 60141; Loyola Medical Center, Department of Medicine, Maywood, IL 60153

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B883-RA)

PURPOSE--It is known that patients with spinal cord injury (SCI) and multiple sclerosis (MS) have a high incidence of high bladder pressures, urinary incontinence, and other urological pathologies. However, the use of the clinical urodynamic equipment to monitor bladder function is limited to the clinic and is, therefore, not likely to be utilized as frequently as is required to prevent urological pathologies. To aid in the early detection and possibly the prevention of these pathologies, we evaluated a simple home-use pressure gauge with tubes to measure bladder pressure along with urine volume recording as an adjunct to intermittent self-catheterization (IC) in SCI and MS patients.

METHODOLOGY--Our home-monitoring pressure gauges and clinical urodynamics equipment were calibrated with a standard water column. All pressure recording devices were adjusted to read the same pressure within 1 cm of water. Two different home pressure gauges were used. The first was a digital gauge, which had a zero adjustment, a highly accurate pressure reading and a small volume displacement for pressure recording. However, subjects found this device hard to read. The second home gauge was a mechanical bellows type with pressure indicated in cm H2O on the dial face, which the subjects found easier to read. We currently use this second gauge. Initial clinical urodynamic evaluation was conducted with simultaneous recording of pressure using the clinical equipment and our home monitor. Detrusor pressures were also determined, taking the pressure of the bladder when empty as a measure of abdominal pressure and subtracting this from the full bladder pressure.

  For home use, subjects were given instructions on connecting a sterile tube from the pressure gauge to their catheter for IC. They were asked to record their bladder pressure and volumes on a weekly basis, more often if they were having a urological problem. Subjects are monitoring their pressures for up to 1 year.

PRELIMINARY RESULTS--Five subjects have entered this study: four SCI and one with MS. In the urodynamic clinic, the home pressure gauge was found to record the same pressures obtained with standard clinical urodynamic equipment. Accurate detrusor pressures could also be obtained. The average age of the five subjects was 46.4±9.3 (SD). The SCI subjects were upper motor neuron lesioned. All subjects were on IC for bladder emptying, and home monitoring was conducted without adverse effects. Daily pressure recordings with home monitoring in four subjects showed little change over time and one subject is just beginning our study. Three of the five subjects recorded low pressures at volumes from 150 ml to 450 ml. Detrusor pressures were less than 23 cm H2O with a high pressure of 34 cm H2O recorded in one subject at the high bladder volume of 340 ml. The fourth subject regularly recorded high detrusor pressures from 30 to 44 cm H2O at large volumes up to 825 ml. This subject has refused to conduct more frequent IC to lower his filling volume before SCI but has not had upper urinary tract problems. Use of the home device is ongoing in two SCI subjects. Home monitoring was stopped within 6 months in the three others. Reasons for stopping related to the time available to subjects, and their feeling that they were not receiving benefits from continued monitoring. In summary, these findings indicate that the home monitoring of bladder pressure may be an important evaluation technique.

FUTURE PLANS--Because regular home monitoring of the detrusor pressure and volume may aid in the early detection or even prevention of urological pathologies, additional subjects are being recruited for this study.

[315] TREATMENT OF SCIATIC NERVE INJURY WITH GONADAL STEROIDS

Kathryn J. Jones, PhD; Talat Khan, PhD
Hines VA Medical Center, Rehabilitation Research and Development Center, Hines, IL 60141; Loyola Medical Center, Department of Medicine, Maywood, IL 60153

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B884-RA)

PURPOSE--Gonadal steroids are neurotrophic agents capable of modulating many aspects of neuronal growth and function, and thus have therapeutic potential in the treatment of nerve injuries involving lower motor neurons. The key question that will be addressed is the following: Will systemic administration of the gonadal steroid, testosterone propionate (TP), augment the regenerative properties of spinal motor neurons, analogous to the effects of the steroid on cranial motor neurons? The goal of this research is to determine if TP can be used as a therapeutic agent in spinal cord injury (SCI).

METHODOLOGY--The experimental approach that will be used will range from functional, for the determination of rehabilitation potential, to molecular, for the determination of mechanism. The long-term goal of this research is to determine the therapeutic potential of gonadal steroids in spinal motor neuron regeneration in animal models and subsequently extrapolate this information to human peripheral nerve and SCI situations. There are four sets of experiments to be conducted on the rat sciatic motor neuron. The sciatic nerve is axotomized at mid-thigh levels in castrated male rats, with half of the animals receiving subcutaneous implants of TP and the other half sham implanted. Functional assessment of lower limb movement and locomotor behaviors is being done. Molecular analysis of the effects of TP directly on injured sciatic motor neurons is being accomplished using in situ hybridization with specific probes.

PROGRESS--Currently, all the details of the behavioral and molecular analyses are being accumulated in pilot studies. The appropriate behavioral tests have been identified and the conditions for use of DNA probes in the in situ hybridization experiments established.

PRELIMINARY RESULTS--The surgical conditions for sciatic nerve injury have been accomplished. All probes to be used in the in situ hybridization experiments work in rat tissue and can be used in subsequent experiments.

FUTURE PLANS--With the pilot experiments accomplished, the behavioral analysis and regeneration rate studies will be done next. If TP improves functional recovery from paralysis induced by sciatic nerve damage, the mechanisms underlying this will then be determined.

[316] ACUTE EFFECTS OF SCI ON SPERM FUNCTION

Todd A. Linsenmeyer, MD; John Ottenweller, PhD
Kessler Institute For Rehabilitation, West Orange, NJ 07006; VA Medical Center, East Orange, NJ 07018-1095

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B887-RA)

PURPOSE--With advances in techniques of electroejaculation, semen may be obtained in approximately 90 percent of men with spinal cord injury (SCI). Unfortunately, semen parameters, particularly sperm motility, are usually of poor quality following SCI. An acute decline in semen quality has been documented to occur shortly after SCI both in humans and in an SCI animal model. Advances in assisted reproductive technologies such as in vitro fertilization have allowed some men to father children. However, these techniques are extremely expensive and often have limited availability. The purpose of this study is to identify the causes of the decline in sperm quality following SCI and to identify treatment strategies which may help to prevent this decline.

METHODOLOGY--We have studied both an SCI animal model (Sprague Dawley rat) and men with recent SCI. Animal studies involve mature male rats with a T9 transection and sham operated controls. Short- and long-term testicular blood flow studies using a doppler probe were performed in the rats. Drug studies investigating the possible impact of medications, specifically verpamil and L arginine, on preventing decline in sperm motility have also been evaluated in our animal model.

  Scrotal temperature studies in men with SCI have just begun. Since electroejaculation has been found to be unreliable in obtaining ejaculates in SCI rats, sperm is obtained by epididymal puncture. Possible use of a new SCI animal model (guinea pig), which will allow repeated electroejaculation rather than animal sacrifice at specific time intervals, has been evaluated.

PROGRESS--Testicular blood flow studies have revealed that there was a persistent decrease in testicular blood flow in the SCI animals compared to sham controls at 2 weeks and 4 weeks post SCI. However, at 2 months post SCI there was normal testicular blood flow. The decline in blood flow was found to correspond closely to a decline in sperm count and motility. This decline in semen quality shortly after SCI may be prevented by maintaining normal testicular blood flow after SCI. Our study has evaluated several medications to maintain testicular blood flow. Pilot studies have shown improvement in semen quality with L arginine but not verpamil when SCI animals were treated and compared to nontreated SCI animals. While there has been difficulty obtaining ejaculates using electroejaculation in the SCI rat model, there was a high success rate (70 percent) in obtaining ejaculates in the SCI guinea pig.

FUTURE PLANS--Work will continue to focus on medical interventions to prevent a decline in sperm function. Testicular blood flow studies using doppler and ultrasound in men with SCI will begin shortly. Studies are also going to begin to evaluate the impact of SCI on testicular temperature regulation, in both the animal model and in men. Further studies are planned to evaluate the guinea pig SCI animal model.

[317] PREVENTION AND TREATMENT OF SPINAL CORD ISCHEMIA AND PARAPLEGIA IN THORACOABDOMINAL ANEURYSM REPAIR: A PILOT STUDY

Bok Y. Lee, MD; Marcelo Da Silva, MD; Richard Zeman, PhD; Irfan Jameel, MD; Lee E. Ostrander, PhD; Burton Herz, MD
Surgical Service, VA Medical Center, Castle Point, NY 12511; BME Department, Rensselaer Polytechnic Institute, Troy, New York 12180; email: ostral@rpi.edu

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Pilot Project #B1661-PA)

PURPOSE--This pilot project examines the prevention of neuronal degeneration after ischemic injury to the spinal cord (SC) by treatment with clenbuterol. Paraplegia is an important devastating complication of SC ischemia during thoracoabdominal aortic aneurysm repair. Clenbuterol is a selective b2-agonist which has been shown to increase gene expression of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) in the central nervous system. NGF can oppose neuronal degeneration caused by Alzheimer's disease and promotes axonal regeneration and synaptic plasticity. bFGF antagonizes glutamate-induced increases in intracellular calcium that cause neurotoxicity. Since glutamate toxicity is thought to occur extensively during and after SC ischemia, clenbuterol treatment may prevent neuronal cell death and paralysis occurring within several days of ischemia. The phenomenon of neuronal preservation after ischemic injury was studied in the New Zealand rabbit, an excellent experimental model for reproducing ischemic paraplegia since these rabbits have a segmental distribution of SC blood supply.

METHODOLOGY--Thirty evaluable rabbits (15 control, 15 experimental) weighing 4 to 6 kg were premedicated with atropine sulfate (0.005 mg/kg) administered subcutaneously and anesthetized with i.m. ketamine hydrochloride (40 mg/kg) and xylazine (3 mg/kg). The experimental group was given clenbuterol (9 mg) in drinking water 24 hours before surgery. Intermittent intravenous readministration of one-quarter dose of the anesthetic agents maintained an adequate level of anesthesia and prevented the need for endotracheal intubation and mechanical ventilation. All rabbits were kept on 100 percent oxygen during the procedure. Preoperative chloramphenicol and heparin (70 units/kg) were given 5 min prior to surgery. The fur on the abdomen was clipped with electric shears and the skin prepared with Betadine solution. In the supine position, a midline incision of approximately 5 cm was made between xiphistemum and pubic symphysis. The abdominal aorta was identified. A flowmeter probe was placed 1 cm below the renal arteries and direct blood flow measurements recorded at the infrarenal aorta before and after aortic clamping. The degree of reproducible ischemic injury was graded by cross-clamping infrarenal aorta for 22 or 30 min. Using a vascular clamp, the abdominal aorta was clamped just distal to the renal arteries for the specified time interval. Confirmation of aortic occlusion was obtained by a zero reading of the flowmeter. Abdominal aortic blood flow was recorded at the time of declamping the aorta, until it approached baseline again. At the end of the procedure, the abdomen was closed in layers with absorbable fascial and nonabsorbable skin sutures. All rabbits were kept in close observation postoperatively and neurological assessment recorded. The anal and bladder sphincters functions were also assessed daily. The rabbits were euthanized at the end of 30 days and the SC histologically analyzed.

RESULTS--Assessment of degree of paralysis of the hind limb was recorded. All of the rabbits with 30-min cross-clamping of the aorta (2 control and 2 experimental) developed complete paraplegia. Of the 13 control group rabbits with 22-min cross-clamping 77 percent developed paraplegia, 9 with no movement and 1 with slight movement of the limb, and 23 percent did not develop paraplegia: 3 were able to stand but not walk normally and 2 recovered completely.

  Of the 13 experimental group rabbits on clenbuterol with 22-min aortic cross-clamping, 38 percent developed paraplegia (3 with no movement and 2 with slight movement of the limb); 62 percent did not develop paraplegia (2 were able to stand but not walk normally and 6 recovered completely). An interesting and fairly consistent laboratory observation was that the rabbits that did not develop paraplegia had minimal increase in aortic blood flow; whereas the rabbits that developed paraplegia had a significant increase following aortic declamping. Although the exact mechanism of paraplegia following intraoperative aortic clamping and declamping is unknown, our preliminary laboratory observations in the rabbit ischemic injury model suggests that the variations in aortic blood flow due to aortic clamping and declamping may play a role in the development of paraplegia. This may be due to ischemia and/or declamping hyperperfusion. Further aortic blood flow studies will help to clarify the causes of paraplegia following thoracoabdominal aortic repairs, and it may turn out that better control of the variation in aortic blood flow may help prevent paraplegia following aortic clamping and declamping.

FUTURE PLANS--Dosage and temporal administration of clenbuterol was empirical in our pilot study. We have designed a detailed protocol to evaluate optimal dose and temporal chronology of clenbuterol. Histological analysis for evidence of neural preservation in the experimental animal group versus the control group will also be evaluated in our future study.

[318] PERFORMANCE CAPACITY AND PHYSICAL STRAIN IN SUBJECTS WITH A SPINAL CORD INJURY

L.H.V. van der Woude; A. Dallmeijer; Rients H. Rozendal; A.P. Hollander; H. van As; E. Angenot; M.T. Hopman
Institute for Fundamental and Clinical Human Movement Sciences Vrije Universiteit, Faculty of Human Movement Sciences, 1081BT Amsterdam, The Netherlands; Rehabilitation Center Amsterdam, Amsterdam, The Netherlands; Department of Physiology, Catholic University Nijmegen, The Netherlands; email: L_H_V_van_der_Woude@FBW.VU.NL

Sponsor: Dutch Prevention Fund

PURPOSE--We study the evolution of physical capacity in association with physical strain over time. Parameters of performance capacity and physical strain in activities of daily living (ADL) are evaluated with repeated standardized wheelchair exercise tests as well as ADL tests in different groups of subjects with spinal cord injury (SCI). This involves both subjects with long-term SCI (both sedentary and physically active) as well as those in the course of rehabilitation. Thus, the effects of wheelchair use and a wheelchair-confined lifestyle on cardio-respiratory, musculo-skeletal, and health risk parameters are evaluated.

METHODOLOGY--A longitudinal study of male subjects with a longstanding spinal injury has been concluded, as has a study of subjects with a cervical SCI. Different subject groups are studied in both cross-sectional as well as longitudinal research designs in the course of the rehabilitation process. Maximum aerobic capacity, anaerobic sprint performance, and isometric strength are individually determined according to standardized procedures at fixed times during and/or after rehabilitation. The physical strain of daily life in rehabilitation, and more specifically in the therapy sessions, is evaluated with the Percentage Heart Rate Reserve (%HRR) with a simple SportTester PE4000. Risk factors for cardiovascular disease and musculo-skeletal problems are repeatedly determined with questionnaires, which are also used to study different physical and personal characteristics.

RESULTS--The results on intramurally treated SCI indicate an inverse association between physical strain in standardized ADL wheelchair tasks and indicators of maximum performance capacity, which is similar to results previously found in a group of males with a longstanding SCI. Initial results on the physical strain of wheelchair-specific therapy sessions and physical and vocational therapy showed a strong interindividual (lesion level dependent) variance as well as a strong intertherapy variance. Physical therapy appears the most straining and therefore seems the most effective in terms of training stimulus for the cardio-respiratory system. However, physical strain during rehabilitation does not seem to meet criteria for training as formulated by the American College of Sports Medicine. Intensity, duration, and frequency of physical activity should be tuned more carefully to the individual. Preliminary findings on risk indicators for cardio-vascular disease do not show an increased risk among the subjects with SCI. Sports activity has a reducing effect on such risk factors among subjects with longstanding SCI.

FUTURE PLANS--A further analysis of the rehabilitation process and its effects upon subjects with SCI will be conducted in a longitudinal perspective. The effects of therapy sessions and daily life in rehabilitation will be documented on a larger subject group, possibly within an experimental design. Additionally, the evolution of wheelchair propulsion technique will be studied over time as a mediating component of performance capacity. Thus, we hope to grasp the process of learning in this complex motor task. To increase the strength of the results, a multicenter approach is planned in which seven rehabilitation centers will cooperate to study a group of subjects with SCI during and after rehabilitation during a 3-year period. With four research groups, different aspects associated with the restoration of mobility will be evaluated.

RECENT PUBLICATIONS FROM THIS RESEARCH

[319] IMMUNE RESPONSES TO PNEUMOCOCCAL VACCINE IN SPINAL CORD INJURY

K. B. Waites, MD
University of Alabama at Birmingham, Department of Pathology, Birmingham, AL 35233; email: waites@sun.rehabm.uab.edu

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202-2646

PURPOSE--Pulmonary complications, with pneumonia being the most frequent, are a major cause of both morbidity and mortality in persons with spinal cord injury (SCI). Both bacterial and viral immunizations have been recommended to prevent infectious pulmonary complications in patients with neuromuscular disorders producing mechanical dysfunctions of the respiratory system. Although patients with SCI, particularly those with tetraplegia and high paraplegia, have been shown to be at increased risk for the development of serious pulmonary complications, including pneumonia, we are unaware of any studies documenting the efficacy of either bacterial or viral immunizations to reduce the incidence of pulmonary complications in the population with SCI. Objectives of this study are to document changes in immunologically related laboratory values of patients vaccinated at varying intervals after spinal cord injury; and to compare the incidence of pulmonary complications in unimmunized patients with SCI with the incidence in a series of patients with SCI vaccinated at varying times following injury.

METHODOLOGY--This study entails random assignment of SCI patients into one of four groups following their entry into the University of Alabama at Birmingham (UAB) Hospital care system. Groups 1 and 2 will receive the vaccine or placebo at 17 days (±24 hours) of injury. Groups 3 and 4 will receive the pneumococcal vaccine or placebo at 4-6 months postinjury. The groups for which a patient is eligible to be randomized as a subject (to receive vaccine) or control (to receive placebo) are determined according to the time at which the patient is admitted to the UAB Hospital or Spain Rehabilitation Center. Following enrollment, four blood samples are collected: the first at the time of vaccination or administration of placebo, the second 1 month later, the third 2 months later, and the fourth at 1 year following enrollment.

  Laboratory tests performed at each blood sampling interval include: antipneumococcal antibody titers to four major representative serotypes, quantitative immunoglobulins, complete blood count with differential leukocyte count, liver profile, total serum protein and albumin. Subjects and controls are monitored during their initial hospitalization for the occurrence of respiratory or other systemic complications of pneumococcal disease. Appropriate microbiological and/or immunological diagnostic procedures are implemented whenever possible to determine whether or not such complications are indeed due to infection with Streptococcus pneumoniae.

PROGRESS--Recent developments in the acute care of persons with SCI made it necessary to alter the study design and eliminate the group immunized immediately postinjury because of the high dose of methylprednisolone often given within 8 hours of injury. Steroid presence negates the immunogenicity of the pneumococcal vaccine unless at least 2 weeks elapse prior to immunization. Therefore, no groups will be vaccinated at 72 hours. Groups 1 and 2 will receive vaccine/placebo at 17 days and Groups 3 and 4 will receive vaccine/placebo at 6 months postinjury.

  Data collection instruments and accompanying syllabus have been completed and are in use. Subject identification, enrollment, administration of vaccine or placebo, follow-up, and collection of blood samples are underway. As of June 1996, 87 persons completed the study with a complete dataset and the study is complete.

FUTURE PLANS--Measurement of antibody levels will be completed by October 1996. All four samples from each person will be assayed at the same time. All laboratory data and pulmonary complications are being recorded and entered into the computer database. A final report on the results of this study will be completed by December 1996.

[320] ULTRASOUND FOR URINARY TRACT SURVEILLANCE OF PERSONS WITH SPINAL CORD INJURY

L. Keith Lloyd, MD
University of Alabama at Birmingham, Spain Rehabilitation Center, Birmingham, AL 35233; email: lloyd@sun.rehabm.uab.edu

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202-2646

PURPOSE--Patients with SCI require long-term surveillance to detect and treat urinary tract dysfunction. Because such dysfunction is often asymptomatic, continued screening of patients who appear to be doing well is important. Screening of the urinary tract requires an examination that is sensitive, specific, easily performed, well-tolerated by the patient, and cost effective. The renal ultrasound examination (RUSE) is less invasive than either excretory urography (EXU) or comprehensive renal scintigraphy (CRSP) and therefore might further increase the likelihood of patients returning for routine annual evaluations. The RUSE eliminates the risk of ionizing radiation, can be performed in considerably less time than CRSP, and costs substantially less to perform.

  Objectives of this project are: 1) to determine the sensitivity and specificity of the RUSE compared to CRSP for detecting upper urinary tract abnormalities of persons with SCI; 2) to determine the sensitivity and specificity of the RUSE compared to EXU for detecting upper urinary tract abnormalities of persons with SCI; and 3) to determine the role of the RUSE in the long-term urologic follow-up of persons with SCI.

METHODOLOGY--Standardized data collection instruments and syllabus have been developed. At this Center, CRSP is routinely performed on all patients with SCI who have neurogenic bladders prior to first definitive discharge and annually thereafter.

  The RUSE will be performed on a random sample of 10 percent of patients scheduled for routine CRSP. The RUSE will be performed using an ACCUSAN 128 Real Time ultrasound scanner utilizing 3.5 and 5.0 Mhz transducers. Renal size, parenchymal thickness, presence, size, and location of calculi; presence, size, location, and character of renal masses; presence and severity of hydronephrosis; size of ureters (normal or enlarged); bladder volume and anterior wall thickness; presence of other abnormalities; and the overall quality of the examination will be recorded. Overall, at least 100 patients will receive both the RUSE and CRSP within 4 weeks of each other during the 5-year project time frame. Most will receive both the RUSE and CRSP within 2 weeks of each other.

  EXU is routinely performed only once per person just prior to the first definitive discharge from the rehabilitation hospital. The RUSE will be performed on a random sample of 25 percent of persons scheduled for EXU. Overall, at least 100 persons will receive both the RUSE and EXU within 2 weeks of each other during the 5-year project time frame. Most will receive the RUSE and EXU on the same day.

FINAL RESULTS--A total of 72 patients were entered into the study, 66 males and 6 females. The mean age was 35 (16-64 years). The mean years from time of injury was 4.4: 35<1 yr; 15 1-5 yrs; 9 5-10 yrs; 13>10 yrs. There were 33 patients who received EXU/RUSE/CRSP all within a 2-week period. Other correlative study groups include: 63 patients who received EXU/RUSE within 2 weeks; 42 patients who received RUSE/CRSP within 2 weeks; 44 patients who received RUSE/CRSP within 4 weeks; 18 patients who received RUSE/ CRSP greater than 4 weeks. A comparison of RUSE to EXU in the detection of urinary tract calculi was performed, including the number and size of calculi. A comparison of RUSE and CRSP was performed using mean parenchymal thickness for "normal" RUSEs and established normal ERPF values for CRSP. A comparison of RUSE to EXU in the detection of hydronephrosis was performed. Seven cases with moderate/severe hydronephrosis by either RUSE or EXU were reviewed and compared to CRSP to evaluate RUSE versus EXU for detection of obstruction.

  Study limitations included logistical difficulty of scheduling EXUs. There was no established form for EXU. There were small numbers of patients with pathology.

IMPLICATIONS--RUSE and CRSP may replace EXU for routine urinary tract surveillance. RUSE is better accepted by patient and logistics are easier. RUSE is equivalent for detection of calculi though inaccurate for size and number. CRSP is more sensitive for renal function loss. EXU is useful for selected cases with calculi, obstruction. A baseline EXU is helpful.

[321] OBSTETRIC/GYNECOLOGIC COMPLICATIONS IN WOMEN WITH SPINAL CORD INJURY

Amie B. Jackson, MD
Department of Physical Medicine and Rehabilitation, University of Alabama at Birmingham, Birmingham, AL 35233; email: jackson@sun.rehabm.uab.edu

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202-2646

PURPOSE--Aside from the immediate and obvious consequences of spinal cord injury (SCI), many physiological systems ultimately are altered for varying periods. It is becoming increasingly evident that the reproductive axis is one such system. In addition to problems with menorrhea, galactorrhea, fertility, and sexual function in women after SCI, disordered hormone production could potentially result in systemic changes such as accelerated bone loss, accentuated catabolism and nitrogen imbalance, and possibly hypercholesterolemia and atherosclerosis. To date there is little information concerning these complications. The purpose of this research is to determine the changes in reproductive endocrine function both immediately following SCI and in the remainder of the first year postinjury. This research will also examine how these changes affect menstruation, ovulation, vaginal and cervical pathology, and sexual function. As it is believed that many complications of SCI, such as autonomic dysreflexia, muscle spasticity, and bladder management problems are influenced by fluctuations in the woman's hormonal cycles, these correlations will be examined as well. Possible factors that could be responsible for postinjury endocrine changes will be explored.

  Objectives of this study are 1) to document hormonal changes that influence ovulation and menstrual cycles, as well as cause complications such as hyperprolactinemia, with or without galactorrhea; 2) to document endocrine imbalances that may occur following SCI as a result of cardiovascular instability, chest trauma, nutritional or metabolic changes, or concomitant head injury; 3) to document the relationship between reproductive hormone levels following SCI and fertility, sexual well-being, and sexual activity; and 4) to determine the relationship between reproductive hormone levels following SCI and complications such as autonomic dysreflexia, increased muscle spasticity, and occurrence of bladder spasms.

METHODOLOGY--The basic design of this proposed project will be that of a prospective cohort study to assess the natural history of reproductive hormonal imbalances that cause changes in the menstrual cycle leading to sexual dysfunction and infertility in women during the first year after SCI.

  All women who agree to participate in the study will be interviewed initially by a designated nurse/clinician who will obtain a complete obstetrical and gynecological history. Other information collected during this interview will be first day of the last menstrual period, age, height, weight, etiology of injury, and loss of consciousness, or occurrence of closed heat trauma or chest trauma at the time of injury. Also during the initial evaluation, vaginal wall samples will be obtained by gently scraping the upper third of the lateral vaginal wall.

  To evaluate postinjury reproductive endocrine status, blood sampling for hormone assays will be performed once a week for 6 weeks. A single electrolyte profile, including serum sodium, chloride, potassium, blood urea nitrogen, albumin, creatinine, and glucose will be obtained on admission to the study to assess the metabolic status of each woman.

  Throughout the initial inpatient stay, careful documentation of the onset, duration and amount of flow of any menses will be recorded. Upon discharge, each woman will be given a menstrual calendar and instructed on recording this same information.

  Over the 3.5-year study, at least 28 women should be enrolled and 336 monthly calendars obtained.

PROGRESS--To date, entry into the study has been slow and determined by the low census of women with SCI who meet the study criteria. One patient has completed the study.

RESULTS--Data have not been analyzed. It is anticipated that the patient numbers will increase in the next few months as measures for early detection have been initiated.

[322] PREDICTION OF MORTALITY AFTER SPINAL CORD INJURY: A 20-YEAR PROSPECTIVE STUDY

J. Stuart Krause, PhD
Shepherd Center, Atlanta, GA 30309

Sponsor: National Center for Medical Rehabilitation Research, National Institute of Health, Bethesda, MD 20892; American Association of Spinal Cord Injury Psychologists and Social Workers; Minnesota Medical Foundation; National Institute for Handicapped Research

PURPOSE--The purpose of this study is to identify psychosocial, vocational, and medical risk factors related to mortality after spinal cord injury (SCI). The primary focus of this research is the utilization of prospective data on life adjustment to predict later mortality.

METHODOLOGY--Participants. All study participants were identified from outpatient files from the University of Minnesota Hospital. To be included in the study, they had to meet the following criteria: (a) had a traumatic SCI, (b) the injury was at least 2 years duration, and (c) were at least 18 years of age at the time of the study. Two study samples have been utilized. Sample 1 (n = 256) began participating in 1974; Sample 2 (n = 193) began participating in 1985.

  Instruments. The Life Situation Questionnaire (LSQ) was developed in 1974 to measure mostly objective information on a broad range of areas relevant to persons with SCI. Although the original form of the LSQ was rather limited in scope, subsequent revisions were made in 1985 and 1989 to expand the content areas. The resulting questionnaire includes eight outcome scales, five of which were developed via factor analysis of life satisfaction and problems items.

  Procedures. Prospective data on life adjustment were collected at three separate times: 1974, 1985, and 1989. A total of 256 individuals completed the LSQ in 1974. In 1985, 154 of these individuals completed a second LSQ. In addition, 193 new participants from Sample 2 completed LSQs in 1985 (a total of 347 participants in 1985). In 1989, 286 of the 347 participants again completed the LSQ, (Sample 1 = 135; Sample 2 = 151). The survival status of all former participants was ascertained in 1996.

PROGRESS--Survival status was ascertained during 1996 in order to use prospective data collected during the three previous study stages (1974, 1985, 1989) to predict 1996 survival status. Among the 256 participants from the 1974 data collection, 95 were deceased by 1996. Fifty-two of the 347 participants from the 1985 follow-up study and 27 of the 286 participants from the 1989 follow-up were deceased by 1996.

RESULTS--Consistent with previous findings, survival status was highly correlated with a positive overall adjustment pattern. Participants who were more active socially and vocationally and who had a higher overall level of subjective well-being were more likely to have survived their injuries until 1996. More detailed analyses are currently underway.

IMPLICATIONS--Findings from this study have been instrumental in identifying life adjustment patterns which place individuals at differential risk for early mortality after SCI.

FUTURE PLANS--Dissemination of the results of the current study will be carried out over the next 12 to 18 months. Further information is being collected on causes of death. The next major follow-up study is planned for the year 2000.

[323] MEASURING THE EFFECTS OF VESTIBULAR STIMULATION ON CHILDREN WITH CEREBRAL PALSY

James W. Fee, Jr., MS; Katherine Samworth, PT, BEE
RERC on Rehabilitation Robotics, Applied Science and Engineering Laboratories, University of Delaware/A.I. duPont Institute, Wilmington, DE 19899; email: fee@asel.udel.edu

Sponsor: National Institute on Disability and Rehabilitation Research, U.S. Department of Education, Washington, DC 20202; Nemours Foundation, A.I. duPont Institute, Wilmington, DE 19899

PURPOSE--The Vestibular Stimulation project seeks a correlation between whole body vertical oscillations and the reduction of spasticity in those with cerebral palsy.

METHODOLOGY--We have constructed an electro-mechanical system which can apply a predetermined vertical acceleration to a child and his/her normal seating system and tested it on 30 subjects. As a means of evaluating changes in spasticity, a leg drop pendulum test was performed on each subject, twice before (at 15-min intervals) and twice after 15 min of up and down motion. In addition, breathing capacity and postural stability tests were made before and after vertical oscillations. Parameters of the vertical oscillations were set at a frequency of 1.57 hertz and an amplitude of 8.82 cm. This is equivalent to about 0.79 g acceleration.

PROGRESS--We are presently studying 30 subjects, 6 to 21 years of age, with a primary diagnosis of spastic diplegia. At this writing 27 subjects have been tested, and while changes have not been as dramatic as those of the pilot study of 1993, they are being observed. It appears that whatever causes these changes seems to last about 15 min. One interesting aside that has resulted from this study is the observation that changes in spasticity in muscles which cause lateral motion during the leg drop pendulum tests are much more consistent, and much easier to detect than the major muscle groups of the limb. In the first 27 subjects, changes in quadricep and hamstring spasticity was detected in 11 of the 27 subjects. Changes in lateral motion, most likely due to changes in spasticity in the sartorius, were detected in 17 of the 27 subjects

FUTURE PLANS/IMPLICATIONS--Our next set of experiments will attempt to show functional improvement after vestibular stimulation. The 10 subjects showing the greatest amount of change in the present study will undergo a gait analysis performed before and after stimulation. If changes in gait are demonstrated, further investigations will be made with variations of frequency and amplitude of oscillation to determine optimum values.

RECENT PUBLICATIONS FROM THIS RESEARCH

[324] CHEMICAL TRIGGERS OF REFLEX DEFECATION IN SPINAL CORD INJURY: COMPARISONS OF EFFECTIVENESS

Steven A. Stiens, MD, MS; J. Glen House, MD; Lance L. Goetz, MD
SCI Unit, VA Puget Sound Health Care System, Seattle, WA 98108-1597; email: stiens@seattle.va.gov

Sponsor: None listed

PURPOSE--Neurogenic bowel dysfunction resulting from spinal cord injury (SCI) can produce constipation, incontinence, and inability to willfully defecate. Many persons with SCI rank bowel and bladder dysfunction among their major life-limiting problems. The upper motor neuron (UMN) bowel results from a lesion of the spinal cord above the conus medullaris. This condition typically presents with fecal distention of the colon, overactive segmental peristalsis, underactive propulsive peristalsis, and a hyperactive holding reflex with spastic anal constriction. These impairments of sphincter control, along with gross immobility from paralysis, interact to compound their functional significance.

  Rehabilitative interventions to regain personal control of fecal elimination emphasize the importance a carefully designed bowel program with regularly scheduled bowel care. A bowel program is a comprehensive individualized treatment plan focused on prevention of incontinence, effective efficient colonic evacuation, and prevention of complications of neurogenic bowel dysfunction. Bowel care, a subcomponent of the bowel program, is the individually developed and prescribed procedure carried out by the patient or attendant to periodically evacuate stool from the colon. The goals of bowel care are to facilitate normal defecation of the maximal stool volume in the least amount of time with avoidance of stool incontinence thereafter. The bowel care procedure typically consists of introduction of a colonic stimulant medication into the rectum and mechanical facilitation of reflex defecation with intermittent digital rectal stimulation. Unfortunately, some bowel care sessions can require up to 3 hours for completion, and still yield insufficient stool results. More than 20 percent of persons with SCI report difficulty with evacuation of their bowels.

  There are many medications on the market utilized as chemical stimulants to enhance reflex defecation. Bisacodyl is a common active ingredient. The effectiveness of hydrogenated vegetable oil-based bisacodyl (HVB) suppositories, polyethylene glycol-based bisacodyl (PGB) suppositories, and polyethylene glycol-based, glycerine, docusate sodium mini enemas (TVC) was compared in subjects with UMN SCI. Thereafter, general strategies for bowel programs and techniques of bowel care were published for consumers and practioners.

METHODOLOGY--The HVB suppositories contained 10 mg bisacodyl in a hydrogenated vegetable-oil base. The PGB suppositories contained 10 mg bisacodyl dissolved in a mixed polyethylene glycol polymer base of two molecular weights: E1450 and E400. TVC consists of a solution of polyethylene glycol, glycerine, and docusate sodium. Separate open label and randomized, prospective, double-blind studies comparing bowel care initiated by these agents were completed.

  The outcome parameters were as follows. The total bowel care period was divided into intervals by discrete timed events: First Flatus (end of the interval from insertion of the suppository until the first flatus is passed), Begin Stool Flow (beginning of the defecation interval), End Stool Flow (marks the end of the defecation interval), Time off toilet (marks the end of the interval from the last stool flow to transfer off toilet or completion of clean-up if in bed), Total Time (includes the time from insertion of the suppository to the last stool flow). Both duration and frequency of digital stimulations and manual evacuations were recorded. Stool results were recorded as: 0, 1 minimal, 2 small, 3 moderate, 4 large, 5 very large. All episodes of incontinence were recorded and defined as: any passage of substance through the anus (include stool, mucus, liquid, and so forth) at any time outside the designated bowel care session.

PROGRESS--Both studies revealed a significant decrease in total bowel care time using the PGB suppository with comparable stool volumes and numbers of bowel incontinence episodes. TVC mini enemas and PGB suppository initiated bowel care sessions were similar in all time intervals, stool production, incontinence, and the number of digital stimulations required.

  The mean bowel program interval that was most reduced when comparing the suppositories was the Time to Flatus (PGB 15 min, HVB 36 min) suggesting that the rate of dissolution of the base is directly related to the bioavailability of the bisacodyl and the subsequent peristaltic response of the colon.

FUTURE PLANS--We are currently developing automated quantitative instrumentation to record the progress of bowel care, with the intent to test other pharmacologic triggers of defecation and the effectiveness of various techniques of bowel care.

IMPLICATIONS --The findings to date suggest that the delivery of bisacodyl to the colonic mucosae in a polyethylene glycol base initiates defecation sooner and can reduce the average duration of bowel care by up to one half as compared with HVB.

RECENT PUBLICATIONS FROM THIS RESEARCH

C. Spinal Cord Regeneration

[325] ELECTRIC FIELDS AND CARBON FIBERS IN THE TREATMENT OF SPINAL CORD INJURY

Talat Khan, PhD; Joel B. Myklebust, PhD; Scott Sayers PhD; Robert Havey BS
Rehabilitation Research and Development Center, Hines VA Medical Center, Hines, IL 60141; Zablocki VA Medical Center, Milwaukee WI 53295

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B423-3RA)

PURPOSE--The purpose of this study was to evaluate the functional benefit of combining carbon filament implants with pulsed DC electrical stimulation following severe contusion injury to the spinal cord in cats.

METHODOLOGY--Seventeen cats were anesthetized and given a severe contusion injury to their spinal cord at the T8 level by dropping a 30 g weight from a height of 18 cm. A mid-dorsal myelotomy was performed 1-2 hours after the injury; by this time edema and hemorrhage develop extensively in the center of the injured spinal cord. Utilizing an operating microscope, the hemorrhagic and edematous tissue was removed by aspiration leaving a small cavity. A bundle of approximately 30,000 carbon filaments of 5 µm in diameter, cut to the appropriate length to completely fill the lesion cavity, was lowered into the injury site and anchored into place with a piece of dura film.

  The animals were divided into four groups. In Group One, cats sustained the injury, received a dorsal myelotomy, and subsequently received implantable stimulators 1 hour after injury. In Group Two, cats sustained the injury and then received a dorsal myelotomy and carbon filament implants 1 hour after injury. In Group Three, cats sustained the injury and then received a dorsal myelotomy and carbon filament implants, in addition to implantable electrical stimulators, 1 hour after injury. Group Four served as a control group in which cats sustained the injury and subsequently underwent a dorsal myelotomy 1 hour afterward.

  In groups One and Three, battery-powered stimulators, with 2 mm diameter platinum discselectrodes, were surgically implanted with the electrodes configured to produce current flow parallel to the long tracts of the spinal cord across the injury site. The stimulation parameters were 25 µA pulsed direct current, which result in a 100 Hz unipolar square wave with a 20 percent duty cycle.

  All animals received daily care in accord with AAALAC guidelines. Electrophysiological and behavioral tests were performed before injury and then bi-monthly after injury throughout the 6-month experimental period.

RESULTS--Various degrees of electrophysiological recovery have been observed in the animals that received implantable stimulators after severe contusion injury. However, the animals that received both carbon filament implants and electrical stimulation have consistently shown weight bearing and minimum ambulation, in addition to electrophysiological recovery. The control animals have all remained paraplegic at the end of the experimental period.

  These preliminary findings indicate that the application of an electrical field, in combination with a suitable substrate such as carbon filaments, provides a favorable environment at the lesion site that results in functional recovery.

FUTURE PLANS--We will continue to evaluate (electrophysiologically, behaviorally, and histologically) the beneficial effects of the use of carbon filament implants, combined with electrical stimulation of the injury site as a means of repairing the damaged spinal cord.

RECENT PUBLICATIONS FROM THIS RESEARCH

[326] ENHANCED CARBON FILAMENT PROSTHESES AS SUBSTRATES FOR REGROWTH OF INJURED SPINAL CORD: ELECTROPHYSIOLOGICAL RECOVERY

Talat Khan, PhD; Scott Sayers, PhD; Lian-Sheng Liu, MD
Rehabilitation Research and Development Center, Hines VA Medical Center, Hines, IL 60141

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B742-2RA)

PURPOSE--The purpose of this study was to determine whether culturing fetal spinal cord on carbon filaments, and then subsequently implanting these filaments into the injured spinal cord, would enhance funtional recovery with axonal regrowth across the lesion site.

METHODOLOGY--Adult rats were anesthetized, and subjected to a severe contusion injury at the T8 level. The rats were divided into five groups: Group One consisted of normal rats (n=7); Group Two consisted of rats that sustained contusion injuries and the lesion sites were subsequently filled with a bundle of approximately 10,000 carbon filaments of 5 µm in diameter, cultured with 15-day-old rat fetal spinal cord explants (n=5); Group Three consisted of rats that received fetal spinal cord tissue implants after contusion injury (n=4); Group Four consisted of rats that received carbon filament implants after contusion injury (n=4); and Group Five consisted of rats that sustained a contusion injury only (n=4). The implantation was performed one hour after injury. Both somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were recorded 10 weeks after injury. The MEPs were recorded from both the left and right tibialis anterior muscles. Animals received injections of 1 percent WGA-HRP in the motor cortex or lumbar spinal cord, or injection of Fast Blue in the lumbar spinal cord.

RESULTS--SSEPs and MEPs were recorded from all animal groups at the end of the 10-week survival period. The most significant electrophysiological recovery, as determined by SSEPs and MEPs, was seen in the group of animals which received carbon filament implants cultured with fetal spinal cord tissue, suggesting that this combination plays an important role in promoting electrophysiological recovery after injury. In addition, retrograde labelling showed labelled axons and cells across the lesion in the group which received carbon filament implants cultured with fetal spinal cord tissue, as compared to the other three injury groups.

FUTURE PLANS--These results of our preliminary study suggest that the transplantation of the combination of carbon filaments and fetal spinal cord tissue play an important role in promoting spinal cord recovery after injury, as demonstrated by increased axonal conduction of the motor and somatosensory tracts in the injured host spinal cord, and by retrograde labelling techniques. We will continue to evaluate the use of these implants for spinal cord repair after injury.

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[327] MOLECULAR MECHANISMS UNDERLYING REHABILITATION AFTER NEURONAL INJURY: A PILOT STUDY

Kathryn J. Jones, PhD
Hines VA Medical Center, Rehabilitation Research and Development Center, Hines, IL 60141; Loyola Medical Center, Department of Medicine, Maywood, IL 60153

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Pilot Project #B94-1743PA)

PURPOSE--Damage to the nervous system, either through trauma or disease, often results in extensive neurological disabilities that severely compromise the quality of life for the affected person and his/her family. While many rehabilitation approaches exist for improving outcomes, these generally depend upon stimulating uninjured brain regions to provide alternate routes for recovery. However, in order to obtain full recovery and complete rehabilitation from neurological disorders, including spinal cord injury (SCI), promoting survival and regrowth of directly damaged brain and spinal cord cells must ultimately be accomplished. Elucidating the molecular mechanisms underlying nervous system damage is therefore important in identifying and implementing rehabilitation strategies designed to promote full functional recovery. The purpose of the experiments in this proposal is to establish a new technological approach for determining the molecular basis for successful regeneration in the nervous system.

METHODOLOGY--The overall research plan that is being followed is: obtain tissue punches of injured and normal brain tissue, isolate and tag messenger RNA that is contained in these punches, and then hybridize the RNA to various DNAs encoding genes known to be important for regeneration. In this way, immediate early events that occur within damaged neurons can be identified and the sequence of changes important to successful regeneration established.

PROGRESS--This pilot project involved development of molecular methodology to amplify expressed genes from brain tissue of hamsters. The specific steps consist of: 1) rapid isolation of nondegraded mRNA from brain tissue; 2) making a full length antisense cDNA copy from the isolated mRNA templates, by using a special primer encoding a T4 polymerase promoter; 3) making a second full length cDNA strand complementary to the first cDNA strand; 4) using the copy of the T4 polymerase promoter encoded on the first cDNA strand to synthesize (and amplify) radioactive RNA copies of the original mRNAs present in the tissue; 5) transfection of bacteria with plasmids containing the known genes of interest, to produce cDNAs to affix to a nylon membrane; 6) attaching the cDNAs to the membrane; 7) testing the bound cDNAs by hybridization to known, synthesized, radiolabeled RNAs; 8) hybridizing the radiolabeled, amplified RNAs to the membrane; 9) washing and exposing the membrane to autoradiographic film to detect bound RNAs; 10) analyzing the autoradiographs to detect which RNAs are present.

PRELIMINARY RESULTS--The results to date are as follows: 1) isolation of undegraded RNA from tissue has been accomplished by two methods, using guanidinium thiocyanate or lithium dodecyl sulfate to inhibit degradative enzymes. To increase the efficiency of the process and minimize sample losses, magnetic beads bound to the cDNA primer have been synthesized, which allows efficient retrieval of cDNA from solutions. 2) Full length first strand cDNA copies of the mRNA templates have been verified, radiolabeling the synthesis, separating the cDNAs by size on an agarose gel, and detecting the radiolabeled species by autoradiography. 3) Combinations of T4, Klenow and DNA polymerase I enzymes, with 3 different buffer systems and various temperature conditions, have been tested to increase the length of the second cDNA strand product. To date, second strand synthesis incorporates only 15-64 percent as much radionucleotide as the first strand synthesis. Currently, Taq polymerase is under study as an alternative enzyme to increase efficiency of this step. 4) Amplification of short second cDNA strands has been demonstrated by incorporation of radiolabeled nucleotides and separation on agarose gels, as above. 5) Several cDNAs for initial testing purposes (c-fos, negative injury control; b-tubulin positive injury control; HSP, constitutively expressed, and a vector, negative control) have been synthesized. 6) Synthesized cDNAs were blotted onto nytran membranes and covalently cross-linked by UV radiation. 7) Integrity of the cDNA-bound nytran membrane was verified by binding of radiolabeled synthesized RNA encoding c-fos, washing, and film autoradiography. 8-10) Hybridization of radiolabeled, amplified RNAs to the cDNAs affixed on nytran membranes has met with limited success.

FUTURE PLANS--Steps 8-10 need validation and replication. Once this has been accomplished, we shall seek to apply this method to the nerve regeneration field in order to identify why some neurons die after injury and others survive and vigorously regenerate.

[328] TRANSPORT OF NGFs±MIF-1 INTO SPINAL CORD

Abba J. Kastin, MD
VA Medical Center, New Orleans, LA 70146

Sponsor: Department of Veterans Affairs, VA Rehabilitation Research and Development Service, Washington, DC 20420
(Project #B2004-RA)

PURPOSE--Severe damage to the spinal cord has devastating consequences because of the lack of regeneration of the severed neural pathways. Experimental evidence now exists that axonal regrowth can be stimulated so as to re-establish supraspinal connections of the isolated segments to provide function.

  In order to accomplish this regeneration of the spinal cord, an adequate supply of nerve growth factors (NGFs) of the neurotrophin family of small polypeptide proteins is required. Penetration of such substances from the circulation into the spinal cord would open up new approaches to treatment of injuries of the spinal cord.

  Until relatively recently, the possibility that NGFs administered peripherally could enter the spinal cord has not been taken seriously, probably because of erroneous dogmas concerning size of the compounds and the blood-brain- (spinal cord-) barrier (BBB). Similar misconceptions had previously occurred with peptides and cytokines like the interleukins that are the same size as the NGFs. Although the rate of entry of these substances into the spinal cord and brain is relatively small, for many compounds it is similar to that of substances like morphine and dopa that have always been accepted as readily penetrating the BBB.

METHODOLOGY--We are in the process of characterizing the entry of NGFs into the spinal cord and brain from blood by state-of-the-art methods. These include measurement of rates of entry (Kis), washout, and capillary depletion methods to exclude substantial sequestration by vascular endothelial cells, and high performance liquid chromatography (HPLC) to determine the intact nature of the entering NGFs. Nonspecific entry is being tested with albumin to rule out a disrupting effect of the administered NGF, as we previously have done for interleukin at the spinal cord.

PROGRESS--Our preliminary results are showing that NGFs can enter the spinal cord. The rates of entry differ among the various neurotrophins being tested (NGF, BDNF, NT3, and NT 4/5). They also differ between different areas of the spinal cord (cervical, thoracic, and lumbar) and the brain.

IMPLICATIONS--It is hoped that characterization of the entry of NGFs into the spinal cord from the periphery will lead to new approaches to the treatment of spinal cord injuries. This should provide stimulus to studies of the beneficial effects of NGFs in spinal cord injury.

[329] GENETICALLY ENGINEERED NEUROTROPHIN SECRETING SCHWANN CELLS FOR THE TREATMENT OF SPINAL CORD INJURY

Scott Sayers, PhD; Talat Khan, PhD
Rehabilitation Research and Development Center, Edward Hines Jr. VA Hospital, Hines, IL 60141

Sponsor: Hines VA Rehabilitation Research and Development Center, Hines, IL 60141 (Core Funds)

PURPOSE--Neurotrophins are proteins which are essential for the survival, target innervation, and function of different populations of neurons. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT-4/5) all belong to the same family. The neurotrophins have great potential as pharmacological agents. However, the use of neurotrophins for the treatment of spinal cord injury (SCI) has not been evaluated at present.

  Studies have suggested that regeneration can be encouraged in the damaged mammalian spinal cord by providing trophic factors, and by introducing substrates that provide a favorable attachment surface and provide directionality to regrowing axons. Recent studies have shown many populations of central nervous system neurons which are responsive to BDNF and NT-3, providing a rationale for the use of BDNF and NT-3 for encouraging the regrowth of motor and sensory fibers after SCI.

  Recently, through the use of genetic engineering technology, we have infected Schwann cells with a retrovirus-based vector containing the cDNA for either BDNF or NT-3. Once infected, these cells act as biological pumps that continuously secrete either BDNF or NT-3. These BDNF or NT-3 secreting Schwann cells, if implanted into the injured spinal cord, could then continuously deliver these growth factors and maintain an enriched environment for the injured spinal cord axons to regenerate. The primary purpose of the present study was to infect Schwann cells with a replicative incompetent retrovirus-based vector into which the cDNA for BDNF or NT-3 had been inserted.

METHODOLOGY--The cDNA for BDNF and NT-3 were each inserted into retroviral vectors, and the orientation of the cDNA with respect to the promoter was determined by restriction enzyme digestion and agrose gel electrophoresis. Retroviruses were generated from the plasmid forms of the retroviral vectors by transient transfection of PA 317 amphotropic retroviral packaging cells. The resulting BDNF and NT-3 retroviruses were used for infecting Schwann cells. Stable BDNF- and NT-3 secreting colonies were selected. Total RNA was prepared from the BDNF and NT-3 secreting Schwann cells, and poly (A+) RNA was isolated from the total RNA. The mRNA levels for these two neurotrophic factors was evaluated by Northern blots. The levels of BDNF and NT-3 secreted by the Schwann cells will be measured by using an ELISA for these two neurotrophic factors.

PROGRESS--At the present time, both the BDNF and NT-3 retroviral vectors have been constructed. Amphotropic retrovirus packaging cells have been transfected with BDNF and NT-3 retroviral vectors, and BDNF and NT-3 retrovirus have been harvested. Schwann cells have been infected with the BDNF and NT-3 retroviruses, and stable BDNF- and NT-3 secreting colonies have been selected. The Northern blots of the poly (A+) RNA prepared from the BDNF and NT-3 secreting Schwann cells showed the presence of the mRNA signal for either BDNF or NT-3 when the blots were hybridized with 32P-labeled riboprobes prepared to detect either BDNF or NT-3 mRNA. The levels of BDNF and NT-3 secreted by these Schwann cells are currently being measured using an ELISA for BDNF and NT-3.

FUTURE PLANS--The BDNF and NT-3 secreting Schwann cells will be evaluated in vitro for their effect on the growth of spinal cord axons by co-culturing them with rat fetal spinal cord explants. Subsequent studies will involve culturing these cells on carbon filaments and implanting them into the lesion sites of spinal cord contused rats to determine their effect on the regrowth of injured spinal cord axons in vivo.

  The use of genetically modified cells, which secrete BDNF or NT-3, have great potential as a means of gene therapy for the treatment of SCI. This technology, once evaluated in an animal model, can be directly applied to the treatment of human SCI.

RECENT PUBLICATIONS FROM THIS RESEARCH

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Last revised Wed 05/26/1999