**Effects of Spine Surgery on Paraspinal Musculature
R.G. Fessler, MD, PhD; F.D. Brown, MD; E.D. Wirth, MD, PhD
Lumbar stenosis is one of the most common diseases of the spine in the geriatric population and has become the leading indication for spinal surgery in patients over the age of 65. Rates of surgery for those over 65 in the United States have increased from 7.8 to 61 procedures per 100,000 people in the past 13 years. The current standard surgical treatment is a wide midline decompressive laminectomy, which entails dissection and retraction of the paraspinal muscles, followed by extensive resection of the posterior spinal elements. The wide exposure an tissue resection can result in significant operative blood loss, and is likely responsible for postoperative pain and paraspinal muscle weakness. The disadvantages in functional outcomes associated with this procedure have led surgeons to develop new minimally invasive techniques to minimize the weaknesses associated with the open procedure while achieving similar outcomes.
Minimally invasive surgery is thought to decrease muscle injury be retracting rather than resecting the paraspinal muscles. Our preliminary investigations into the decompression of a stenotic spinal canal to date suggest that the microendoscopic decompression of stenosis (MEDS) technique substantially decreases blood loss and postoperative pain resulting in better patient outcome making the MEDS procedure a viable alternative to the standard open laminectomy. However, it is not known whether minimally invasive surgery preserves muscle strength and what effect the preservation of strength has on patient long-term functional outcome. The reason for this is because the effects of neither the open nor the MEDS procedure on paraspinal muscle strength have been measured. Accordingly, our specific aims will address 1) the change in lumbar muscle strength following open or MEDS surgery and 2) the relationship between patient outcome measures and postoperative paraspinal muscle strength in a controlled, prospective study.
Completion of the specific aims established in this proposal will test the hypothesis that the MEDS procedure better preserves paraspinal muscle strength following surgery relative to the standard open procedure resulting in improved long-term patient outcome. The results of this study will provide objective validation of minimally invasive surgical approaches and help to increase the use of this technique.
** A Mouse Model of Intervertebral Disc Degeneration
X. Li, MD, PhD; G. Balian, PhD; M. Cui; C.T. Laurencin; B.L. Leo
The research project is going on very well. It is progressed in two parts: 1. In vitro data report on the Spine Journal; 2. In vivo study on Ad-GDF-5 treatment for disc degeneration induced by needle-puncture.
FINDINGS OR CONCLUSIONS TO DATE
1. In vitro data have been reported on the Spine Journal.
2. In vivo study on Ad-GDF-5 treatment for disc degeneration induced by needle-puncture.
Animals Groups
A total of 40 Balb/c mice (8 weeks old) were used in this research. All of the animal work procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia. Mice were separated into 4 groups with 10 mice in one group. The time point was set to 1, 2, 4 and 8 weeks after operation. One group of animal was sacrificed at each timepoint.
Surgical procedures and the in vivo transduction
The animals were under general anesthesia. Using aseptic technique, the spine was exposed through an anterior midline transperitoneal approach. The intestinal contents were carefully removed from the abdominal cavity and packed in a saline soaked gauze pad and protected during the surgical procedure. After separating the hind peritoneum and psoas major muscles, the L4, L5 and L6 vertebral bodies were identified. The IVD of L3/4, L4/5 and L6/S were randomly assigned to receive 5µl Ad-GDF5 (1.8×1010/ml) or Ad-Luc (2×1011/ml) solution injection through a 30 gage needle or left intact.After the injection, the incision was closed in a layered fashion and the animals were allowed to act freely in the cage.
In vivo bioluminescence imaging
Four of the animals received noninvasive, real-time in vivo imaging of bioluminescent assay at the first and third day after the injection followed with once a week until to the 8th week postoperation. The imaging system included a cryogenically cooled In Vivo Imaging System (IVIS) (Xenogen Corp., Alameda, CA) coupled to a data-acquisition computer running Living Image software (Xenogen Corp.). The animals were anesthetized by put into a cabinet filled with 4% isofluorane/oxygen mixture. During imaging, 2% isoflurane anesthesia was maintained using a nose cone delivery system. N-Luciferin (potassium salt; Xenogen Corp.) was injected intraperitoneally at a dose of 150 mg/kg body weight. A gray scale body surface image overlaid with pseudocolor image was obtained prior or posts the injection of luciferin by every 2 minues in the posteroanerior position. The spatial distribution and quantity of photon counts emitted by luciferase from transduced cells within the animal were represented by colored pixels produced by the computer. A defined round region of interest (ROI) including the peak photon count was made with 1.5-mm diameter at the same position of the same animal. The data of different time from same animal were normalized by the data of first day.
Radiographic analyses
The same four mice were used to receive radiographic study at different time point. Animals were general anesthetized with 2% isofluorane/oxygen mixture. The data were acquired using the small animal Digital X-ray scanner located at the University of Virginia. The component of this scanner consisted of a Source Ray SB-1k-800 x-ray source (50 micron focal spot) and a Hamamatsu C7940DP-03 flat panel image sensor (50 micron detector element size). The distance from the radio source to the sensor is 234mm with the animal put between them and 166mm from the source. The digital lateral radiographies were obtained preoperation and at 1, 2, 4, 8 weeks postoperation. The heights of vertebral bodies and IVDs were measured with National Institutes of Health imaging software (Image J, NIH) and were expressed as Disc Height Index (DHI) using the method developed by Lu et al. The average measurement of anterior, middle and posterior height of IVD was divided by the average height of adjacent vertebral body. The change of DHI was expressed as percentage DHI and normalized by the DHI of preoperation.
Magnetic Resonance Imaging
Two mice received MRI scan at different time point until 8 weeks after operation. By using 2% isofluorane/oxygen mixture, the animals were anesthetized and placed on the tray of magnetic resonance image scanner on dorsal position. Imaging was obtained by using a Varian Inova 4.7 Telsa (T) MR (Varian Inc., Palo Alto, CA) scanner with a quadrature birdcage radiofrequency coil. T2-weighted sections in the sagittal plane were obtained as two-dimensional multislice. The fast spin-echo sequence with time to repetition of 4000 milliseconds and time to echo of 75 milliseconds was performed as a resolution of 60 × 60 × 1000 µm with a slice thickness of 1 mm and an area of 3 × 3 cm.
Biochemical assay
In each group, 5 mice were used for the biochemical assay. Animals were sacrificed by CO2 inhalation and the discs were harvested immediately after death at 1, 2, 4 and 8 weeks post operation. After removing the endplate, the discs were digested in 200 µL of sterile papain solution (125 g/mL in 1× PBE, pH 6.5) at 60°C for 24 hours. Deoxyribonucleic acid (DNA) content was determined by Hoechst 33258 dye assay with calf thymus DNA as a standard. Glycosaminoglycan (GAG) of the disc was detected by the dimethylmethylene blue (DMMB) colorimetric assay with chondroitin sulfate as a standard. The data of Ad-Luc and Ad-GDF5 injection IVD was normalized by the data of the intact IVD in each animal.
Histologic Evaluations
Five mice of each gourp were sacrificed at 1, 2, 4 and 8 weeks after the injection. The specimens of lumbar spine column used for histologic evaluation were harvested with surrounding tissue and fixed in 4% paraformaldehyde in PBS solution for 48 hours. And then, the specimens were decalcified by 0.25M ethylenediamine tetraacetic acid (EDTA) in phosphate buffered solution (PBS) for 2 weeks. After embedded in paraffin, the specimens were sectioned midsagittally at 8µm and stained with either hematoxylin and eosin, or type II collagen immunostaining. Immunostaining for type II collagen was performed using a type II Collagen Staining Kit (Chondrex, Redmond, Washington) according to the manufacturer protocol.
RESULT
In vivo bioluminescence imaging
We tracked luciferase expression in animals transduced with Ad-Luc in situ by using LivingImage software and IGOR analysis software to define a region of interest (ROI). The fluorescence appeared at the first day after Ad-Luc injection. In most of the animals it disappeared at 6th weeks post operation, but some of mice could keep it until the termination of 8th week. The image also showed the vast majority of luciferase activity was found only at the injection site and was not distributed throughout the animal. (Figure 1) After normalized by the data of the first day, the semi-quantitative analysis of the peak amplitudes of fluorescence from 4 mice demonstrated the activity of luciferase declined over time since the third day. After the first week, the intensity of fluorescence decreased nearly to the half of the initial expression, but it takes 2 weeks more to fall off to the 1/4 of original intensity. Even after 28 days, the expression of luciferase was more than 10% of the maximum expression.
Radiographic analyses
The diversity of IVD was expressed with disc height index (DHI). At each time point, DHI of each IVD was normalized by the data collected before operation and showed as percent disc height index (%DHI) (Figure 2). The intact IVD was used as control, and there is no difference among the different time points. At the first week after the injection, both of IVDs received Ad-Luc or Ad-GDF5 showed a similar decrease of the %DHI compared with intact IVD (P<0.05). %DHI of the IVDs received Ad-Luc keep on diminishing as time pass over. Although decreased at the first week time point, the %DHI of the IVDs received Ad-GDF5 transduction recovered at 2 weeks after injection and keep the height throughout the research. From the 2 weeks time point, there is no statistic difference of %DHI between intact and the Ad-GDF5 injected IVD, but they both higher than the %DHI of Ad-Luc injection IVD at 2 weeks (P<0.05) and become more serious at 4 and 8 weeks time points (P<0.01) (Figure 3).
Magnetic Resonance Imaging
The animals were scanned by MRI at 2, 4, 6 and 8 weeks post operation. The T2-weighted signal of both injected IVD groups showed obviously lower intensity at 2 and 4 weeks time points. But the signal of the IVD received Ad-GDF5 injection begin to recover since 6 weeks postinjection and displayed a higher signal at 8 weeks time point. Compare with the normal IVD and the IVD received Ad-GDF5 injection, the IVD received Ad-Luc didn’t show obviously recover of the signal intensity (Figure 4).
Biochemical assay
The average of DNA content in Ad-Luc injection group decreased at 4 and 8 weeks time points, although the difference didn’t show the statistical significance. The possible reason is the number of specimens maybe was not enough to show the statistical significance. There was not obvious variation of the DNA content of intact IVD and IVD received Ad-GDF5 injection groups at different time points. The GAG content of Ad-Luc injected IVD level decreased at the 2nd weeks postoperation (P<0.05) (Figure 5).
Histology image
The specimens were harvested at 1, 2, 4 and 8 weeks after the injection. By hematoxylin and eosin staining, both kinds of the IVDs received injection showed the loss of nucleus pulposus at the first week post operation. The chondroid cells began to proliferate from anulus fibrosus towards the center of the IVD. In Ad-GDF5 groups, the proliferation of the cells was more obvious than Ad-Luc groups. The preponderance of the cells growth in Ad-GDF5 groups could be found throughout the whole research period. At the 8 weeks time point, the IVD height of the Ad-Luc groups was obviously lower than Ad-GDF5 groups. Although there was not typical nucleus pulposus appeared, the Ad-Luc injected IVD was filled with more fiber and less cells than Ad-GDF5 groups (Figure 6). The collagen II immunostaining was processed on the 8 weeks time point specimens, and the adjacent intact IVD was used as positive control. The staining reaction of extracellular matrix in the center of IVD in Ad-GDF5 groups was stronger than Ad-Luc groups (Figure 7).
** Prospective Cohort Study of the COMT val 158 met Genotype and Outcome from Surgery for Degenerative Disc Disease
D.H. Kim, MD; L.G. Jenis, MD; R.J. Banco, MD; M. Max, PhD
The purpose of this study is to determine the clinical relevance of the catechol-O-methyltransferase (COMT) gene polymorphism as it applies to clinical outcome following spinal surgery for degenerative disc disease. Inheritance of specific allelic variations of the COMT gene has been demonstrated to play a significant role in human perception and emotional response to standardized pain stimuli.
The study hypothesis suggests that analysis of COMT genotype for an individual with severely symptomatic degenerative disc disease will reveal that individual’s predisposition to experience heightened emotional response to pain and may identify that patient as a relatively poorer candidate for surgical intervention. COMT genotype analysis is a simple, inexpensive, and standardized procedure requiring a small venous blood sample. Potential clinical gains of such a new informative diagnostic test would include valuable information for patient counseling regarding natural history and response to therapy, a reduction in the rate of unsuccessful surgery, and reduction in overall morbidity in terms of treatment of degenerative disc disease. Reduced and, presumably, more targeted surgical rates would translate immediately into substantial cost savings in terms of more rational distribution of medical resources and avoidance of a potentially large number of failed back surgery cases.
The proposed study is designed as a prospective cohort study of patients undergoing spinal surgery for lumbar degenerative disc disease. All patients will have a blood sample drawn at the time of surgery for genotypic analysis of the COMT gene. All clinical personnel having direct patient contact will be blinded to the results of this analysis. Standard validated outcome measures will be administered preoperatively and at regular follow-up intervals through two years following surgery. Preoperative and postoperative measures will include a visual analogue scale (VAS) for low back pain, an Oswestry low back pain disability questionnaire, and a short-form 36 (SF-36) generic health instrument. A pre-study power analysis set at >80% suggests that a sample size of 100 should provide sufficient power. The study will conclude two years following surgical treatment of the last patient enrolled in the study. At that point, genotype data will be unblended and subjected to statistical analysis.
ENROLLMENT:
The target of goal of 100 eligible subjects has been reached for this study. In a Continuing Review Report submitted to the site’s IRB in March 2007, the study was closed to enrollment.
A total of 108 subjects have signed informed consent forms for this study. Of these subjects, 8 have been withdrawn from the study. Six were considered ineligible, 1 delayed surgery outside the study window and 1 cancelled surgery.
Of the 100 active and eligible subjects, 59% are male and 41% are female. Two percent of the subjects are black and 98% of the subjects are white.
TREATMENT:
All active subjects underwent surgical treatment for a primary diagnosis of lumbar degenerative disc disease between 11/18/04 and 1/1/07.
CLINICAL OUTCOMES:
After signing an informed consent, all patients were required to complete pre-operative questionnaires. These questionnaires include the SF-36, Oswestry Disability Index, Visual Analog Scales, and questions pertaining to work and smoking status. Patients are also required to complete similar questionnaires post-operatively at 6 weeks, and 3, 6, and 12 mos. These post-operative questionnaires include patient satisfaction portions addressing both their in-house stay and office visit.
BLOODWORK:
Blood samples were obtained from all 100 active subjects in this study. These samples were sent to BioServe Biotechnologies Ltd, where DNA extraction was performed. One hundred DNA samples were sent from BioServe to an NIH laboratory under the direction of Dr. Mitchell Max. Upon receipt, one sample was found to be desiccated and was not able to be analyzed. A DNA analysis which included genotyping of the COMT and GCH1 genes was performed on all 99 viable DNA samples.
FINDINGS TO DATE:
The results of the genotyping have been received. Further statistical analysis has yet to be performed.
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research