Ovine Hemisection Model of Spinal Cord Injury.

INTRODUCTION: We are developing ovine models of spinal cord injury to test novel neuromodulation-based methods on spasticity. The hemisection has been reported in a number of large animal studies. Our aim is to duplicate a hemisection injury in the sheep. Our effort is explored here. Methods and Results: Three sheep underwent hemi-sectioning of the spinal cord. Quantitative gait analysis was completed both pre- and post-injury. While measurable differences in most of the 20 gait metrics were observed, relatively few were above the predicted thresholds based on error levels expected from the data. Variations in severity of injury across the three sheep were observed. Conclusions: The hemisection ovine model of spinal cord injury shows promise as a large-animal platform for developing new therapies for treating spinal cord injuries. While variability in injury severity was observed across animals, as has been observed with weight drop-based SCI models, the hemi-section approach has the advantages of procedural ease and reduced technical complexity.

The Hemisection Approach in Large Animal Models of Spinal Cord Injury: Overview of Methods and Applications.

Introduction: Translating basic science research into a safe and effective therapy for spinal cord injury (SCI) requires suitable large animal models for testing both implantable devices and biologic approaches to better approximate human anatomy and function. Hemisection lesions, routinely used for investigational purposes in small animals, are less frequently described in large animals that might be appropriate for translational studies. Size constraints of small animals (mice and rats) limits the predictability of the findings when scaled up. Our goal is to review the status of hemisection SCI in large animals across species and time to prepare for the testing of a novel intradural spinal cord stimulation device for control of spasticity in an ovine model. Methods and Results: We surveyed the literature on hemisection in quadrupeds and nonhuman primates, and catalogued the species, protocols and outcomes of the experimental work in this field. Feline, lapine, canine, simian, porcine, ovine and bovine models were the primary focal points. There is a consistent body of literature reporting use of the hemisection approach in large animals, but with differences in surgical technique depending on the goals and nature of the individual studies. While the injuries are not always consistent, the experimental variability is generally lower than that of the contusion-based approach. In general, as the body size of the animal increases, animal care requirements and the associated costs follow. In most cases, this is inversely correlated with the number of animals used in hemisection models. Conclusions: The hemisection approach to modeling SCI is straightforward compared with other methods such as the contusive impact and enables the transection of isolated ascending and descending tracts and segment specific cell bodies. This has certain advantages in models investigating post-injury axonal regrowth. However, this approach is not generally in line with the patho-physiologies encountered in SCI patients. Even so, the ability to achieve more control over the level of injury makes it a useful adjunct to contusive and ischemic approaches, and suggests a useful role in future translational studies.

Treadmill measures of ambulation rates in ovine models of spinal cord injury and neuropathic pain.

Our laboratories are developing treadmill-based gait analysis employing sheep to investigate potential efficacy of intra-dural spinal cord stimulation in the treatment of spinal cord injury and neuropathic pain. As part of efforts to establish the performance characteristics of the experimental arrangement, this study measured the treadmill speed via a tachometer, video belt-marker timing and ambulation-rate observations of the sheep. The data reveal a 0.1-0.3% residual drift in the baseline (unloaded) treadmill speed which increases with loading, but all three approaches agree on final speed to within 1.7%, at belt speeds of ≈ 4 km/h. Using the tachometer as the standard, the estimated upper limit on uncertainty in the video belt-marker approach is ± 0.18 km h(-1) and the measured uncertainty is ± 0.15 km h(-1). Employment of the latter method in determining timing differences between contralateral hoof strikes by the sheep suggests its utility in assessing severity of SCI and responses to therapeutic interventions.

Direct vision in minimally invasive epicardial procedures: preliminary tests of prototype instrumentation.

This study investigated the use of direct visualization to enhance minimally invasive epicardial procedures. A commercially available miniature camera was placed in a prototype subxiphoid introducer needle and bench top, in vitro and in vivo tests of system performance were made during simulated and actual attempts at pericardial access and cardio-endoscopy. This system had an unshielded field of view of 100° and a resolution of 220 × 224 pixels. When a sleeve used to maintain depth of field was slid past the distal tip of the camera probe, the field of view would decrease by ≈15° per millimetre of sleeve extension, but without loss of image quality. While tests during in vivo subxiphoid access in a porcine model revealed that the pericardial membrane was difficult to localize, the results also showed excellent resolution of the coronary arteries on the epicardial surface. These findings and potential improvements are discussed in detail.

An in vivo rat model for visualizing glioma tumor cell invasion using stable persistent expression of the green fluorescent protein.

In our studies examining mechanisms of brain tumor cell migration/invasion into host tissue, the RT2 rat glioma cell line was stably transfected utilizing a green fluorescent protein (GFP) DNA construct. Stable transfected RT2 cells demonstrate high GFP expression at initial passages, maintain expression over 30 passages and have similar in vitro and in vivo growth patterns relative to controls. In rat brain tissue, individual tumor cells can be detected without any post-treatment. Using flow cytometry and cell sorting, we are able to retrieve and culture GFP positive cells from tumor core as well as adjacent tissue and contralateral hemisphere of tumor-bearing animals.

Nonlinear magnetic stereotaxis: three-dimensional, in vivo remote magnetic manipulation of a small object in canine brain.

In a series of in vivo experiments on five adult canines, a small cylindrical permanent magnet (approximately 5-mm diameter x 5 mm long) was magnetically moved under fluoroscopic guidance from an occipital-lobe burr hole to a predetermined destination within the brain and then removed. On three of the animals, dorsal and temporal skull markers were used to establish a coordinate system against which the motions of the seed were referenced. These procedures were sufficiently accurate to permit the guided motion of the seed along nonlinear paths within the brain, including traversal of the midline through the corpus callosum. For removal, the seed could be steered either to a frontal lobe location for extraction through an auxiliary burr hole, or back to the same burr hole through which it had been inserted. This article discusses the way in which stereotactic motions were obtained, the performance limits of the instrumentation and the precision of motion achieved.

Thermodynamics of movable inductively heated seeds for the treatment of brain tumors.

A thermodynamic study is presented of temperature distributions created by an inductively heated 6-mm-diam Ni sphere imbedded in vivo and in vitro into porcine brain tissue. This study was performed in support of the development of a system that creates localized heat-induced lesions in deep-seated brain tumors. In this system, a magnetic "seed" will be remotely repositioned within the brain by an externally produced magnetic field. Convective effects of a hot moving seed will produce a different thermodynamic situation than that arising from an array of static implants. In this work, a study is presented of part of the expected change, in which a static sphere is heated to high temperature. Measurements were made of the temporal and spatial dependence of the temperature rise in the vicinity of the heated sphere, in vivo in four animals and in one that was euthanized immediately prior to experimentation. These results are used for parameter estimation with a theoretical model based on a point source solution to a form of the thermal diffusion equation, i.e., the "bioheat transfer equation." With this model thermal distributions from a power source of arbitrary geometry can be found using appropriate integration methods, and the method has widespread applicability. Estimates of blood flow rates, tissue thermal conductivity, and seed power absorption were found using the parameter estimation algorithm. The estimated blood perfusion exhibits a step increase following the first heating in multiple heating experiments. Thermal conductivity estimated using data from the nonperfused (in vitro) animal is 0.6 W/m degrees C. Seed power absorption is estimated correspondingly to be 0.9 W, a result confirmed independently with calorimetry. Statistical uncertainty is established for the radial decrease of the tissue temperature rise created by this method. This result allows estimation of a cell death boundary uncertainty of 0.6 mm, caused by fluctuations in power delivered to the seed, uncertainty in the temperature probe placements, and thermal properties such as blood perfusion and tissue thermal conductivity.

Preliminary experimental investigation of in vivo magnetic manipulation: results and potential application in hyperthermia.

The first in vivo experiments in support of a new technique for delivering stereotaxic hyperthermia have been conducted at the Experimental Surgery Facility of the University of Virginia's Medical Center. We call this technique the "Video Tumor Fighter." In each of twelve trials a single, small permanent magnet or train of small permanent magnets was implanted on the brain surface of adult canine models. In three of the trials, this "seed" (typically 6-mm diameter X 6-mm long) was moved by magnetic manipulation to different locations within the brain. In two other trials, the seed moved along the interface between the brain and the inner vault of the skull. The noncontact magnetic manipulation was accomplished by coupling the permanently magnetized seed to the large dc magnetic field gradient created by a water-cooled coil surrounding the animal's head. The seed's motions were monitored with x-ray fluoroscopy; its rate of movement was found to be approximately 0.8 mm s-1. The forces required to produce these motions were on the order of 0.07 N. We document here the instrumentation used in these trials, describe the experimental procedures employed, and discuss the technical aspects of the results.

Magnetic stereotaxis: a technique to deliver stereotactic hyperthermia.

Advances in imaging techniques and computer software over the past decade now define brain abnormalities such as tumors in precise, three-dimensional images. We have taken advantage of these technological improvements in designing a system capable of performing magnetic manipulation of an object in a nonlinear trajectory and able to deliver hyperthermia to highly specific targets within the brain. This device relies on external magnets to pull a small metal pellet (thermoceptor) through the brain, and on biplane fluoroscopy to localize the thermoceptor with respect to previously obtained magnetic resonance images. A radiofrequency tuned circuit serves as the hyperthermia applicator and selectively heats the thermoceptor. This paper describes experiments conducted in a series of dogs showing that all three components of the system (magnetic drive, stereotactic real time imaging, and hyperthermia) can be achieved. Integration of the system was accomplished in one animal. These encouraging results need further detailed substantiation in each of the components, yet demonstrate the feasibility of such a device.

Antiproliferative effect of c-myc antisense phosphorothioate oligodeoxynucleotides in malignant glioma cells.

OBJECTIVE: To improve the prognosis for primary malignant tumors of the central nervous system, new therapeutic strategies are needed. Antisense oligodeoxynucleotides (ODNs) offer the potential to block the expression of specific genes within cells. The proto-oncogene c-myc has long been implicated in the control of normal cell growth and its deregulation in the development of neoplasia. We therefore reasoned that a strategy using ODNs complementary to c-myc messenger ribonucleic acid would be a potent inhibitor of glioma cell proliferation. METHODS: A variety of antisense, sense, and scrambled (15-mer) phosphorothioate ODNs targeted to rat and human c-myc messenger ribonucleic acid were synthesized and added to the media of cultured RT-2 cells (a rat glioblastoma cell line). Cell growth was assessed by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide dye assay 1 to 5 days after adding the ODNs. c-Myc protein expression was analyzed by Western blot analysis. The stability of the ODNs was confirmed by gel electrophoresis. RESULTS: Compared with cultures containing standard media, two of three antisense ODNs significantly inhibited the growth of glioma cells, whereas sense and scrambled sequence ODNs did not significantly affect cell growth at the concentrations tested. A human c-myc antisense sequence, which differed from the rat sequence by one base substitution, also had an inhibitory effect on RT-2 cells. Western blot analysis demonstrated that expression of immunoreactive c-Myc protein was also greatly reduced in the rat antisense ODN-treated cells (and not in sense-, scrambled-, or control-treated cells). The degree of reduction of c-Myc protein expression correlated well with the decrease in cell growth observed with several antisense ODNs. Phosphorothioate ODNs were stable in cell culture media for at least 5 days. CONCLUSION: These results suggest that c-Myc plays a critical role in glioma cell proliferation and demonstrate that antisense ODNs can suppress proto-oncogene expression and inhibit the proliferation of glioma cells. Our results indicate that the antiproliferative activity of these ODNs was mediated predominantly through sequence-specific antisense mechanisms, but that sequence-specific nonantisense effects may also contribute to the strongest effects demonstrated. These findings support a potential role for antisense strategies designed to inhibit c-myc expression in the treatment of malignant gliomas.

Magnetic movement of a brain thermoceptor.

Hyperthermia has significant potential as an adjuvant form of brain tumor therapy. Current intracranial hyperthermia methods, however, are limited in their ability to control spatiotemporal thermal distribution. A stereotaxic magnetic movement system that may be capable of heating discrete regions of brain to a preselected temperature is described. With this system, a ferromagnetic object (referred to as a thermoceptor) is directed through the brain by an external drive magnet. Real time thermoceptor position is monitored with biplanar fluoroscopy and superimposed on a preoperative magnetic resonance imaging scan using a computer. Once in position, the thermoceptor can be inductively heated by externally generated radiofrequency signals. Experiments on the magnetic drive and imaging aspects of this system have been conducted in vitro and in vivo. Mechanical studies of cadaver dog brains revealed that a mean force of 0.07 +/- 0.03 N was required to move a 3-mm diameter sphere through brain at a speed of less than 1 cm/15 s. A cranial phantom with mechanical properties similar to brain was constructed of gelatin and Plexiglas. With the use of a "neck loop" design drive magnet with a maximum magnetic field strength of 0.10 T, a 3 x 3 mm cylindrical neodymium iron boron thermoceptor was smoothly directed through the phantom in two dimensions. Additional experiments were conducted with a larger drive magnet in five anesthetized dogs. Neodymium iron boron and samarium cobalt thermoceptors of various shapes and sizes were placed into the cerebral cortex through a burr hole, then directed with the drive magnet. Fluoroscopy was used to follow the thermoceptor movements.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and stability of antisense phosphorothioate oligonucleotides in rodent brain following direct intraparenchymal controlled-rate infusion.

OBJECT: High-flow microinfusion is a novel technique for delivery of compounds directly into brain parenchyma, bypassing the blood-brain barrier. The feasibility of this technique has been demonstrated with low-molecular-weight compounds, macromolecular dyes, and proteins. Delivery of antisense oligonucleotides into brain parenchyma represents an additional potential application of this technique not previously described. In this report the authors sought to examine the distribution and disposition of phosphorothioate oligodeoxynucleotide (PS-ODN) for this reason. METHODS: An 18-mer 35S-PS-ODN (Mr approximately 6000) was infused over 1 hour into the caudate putamen of Fischer 344 rats. At 1, 6, 12, 24, and 48 hours after beginning the infusion, the brains were extracted and analyzed using quantitative autoradiographic techniques. Cerebrospinal fluid (CSF) was also aspirated from the cisterna magna and was analyzed to determine the radioactivity and stability of the 35S-PS-ODN. At 1 hour, the infused ODN was uniformly distributed in brain tissue, with a maximum average concentration of 4806.5 +/- 210.5 nCi/g. This represents a tissue concentration of 19.2 +/- 0.84 microM. Extensive spread into surrounding parenchyma was observed over the ensuing 47 hours. The 35S-PS-ODN radioactivity peaked in the CSF at the end of the 1-hour infusion, containing 1% (50 +/- 20 nCi) of the infused radioactivity. Activity then decayed exponentially over 11 hours, but stabilized at a lower CSF content of 0.2% (1 +/- 0.1 nCi) thereafter. The volume of distribution was 105 +/- 7.9 mm3 at 1 hour, representing a volume of distribution/volume of infusion ratio of 5.2. The volume of distribution increased to 443 +/- 62.3 mm3 at the end of 48 hours, whereas the average minimum tissue concentration decreased from 15.2 microM to 3.2 microM. Undegraded 18-mer was observed throughout the 48-hour period by means of 20% polyacrylamide/7 M urea gel electrophoresis. The animals tolerated the infusion without evidence of toxicity and minimal structural changes in tissue were observed on histological investigation. CONCLUSIONS: The authors found that PS-ODNs can be safely delivered in high concentrations to wide areas of rat brain by using high-flow microinfusion and are stable even after 48 hours in situ.

Utilization of type I collagen gel, demineralized bone matrix, and bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion.

Autologous bone grafts are currently considered "gold standard" material for achieving long-term spinal arthrodesis. The present study was performed to determine whether demineralized bone matrix (DBM), type I collagen gels, or bone morphogenetic protein-2 (BMP-2) can improve autologous bone spinal fusions. Using a unilateral decompression-contralateral fusion technique in dogs, each of these materials was added to an autologous bone graft. Volumetric analysis, histological analysis, and biomechanical testing were performed to assess the effectiveness of each material. The DBM had an inhibitory effect on solid bone fusion of the spine, whereas the type I collagen gels improved the bony interface between the graft and the host spine. The BMP-2 strongly enhanced the amount of bone deposition at the fusion site and increased the number of intervertebral levels that were solidly fused. This study strongly supports the use of BMP-2 as an additive to autologous bone grafts in spine stabilization.

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

OBJECT: The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. METHODS: Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). CONCLUSIONS: The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Determination of intracranial tumor volumes in a rodent brain using magnetic resonance imaging, Evans blue, and histology: a comparative study.

The measurement of tumor volumes is a practical and objective method of assessing the efficacy of a therapeutic agent. However, the relative accuracy of different methods of assessing tumor volume has been unclear. Using T1-weighted, gadolinium-enhanced magnetic resonance Imaging (T1-MRI), Evans Blue infusion and histology we measured intracranial tumor volumes in a rodent brain tumor model (RT2) at days 10, 16 and 18 after implantation of cells in the caudate putamen. There is a good correlation between tumor volumes comparing T1-MRI and Evans Blue (r2 = 0.99), T1-MRI and Histology (r2 = 0.98) and histology and Evans Blue (r2 = 0.93). Each of these methods is reliable in estimating tumor volumes in laboratory animals. There was significant uptake of gadolinium and Evans Blue in the tumor suggesting a wide disruption of the blood-brain barrier.

Quantitative three-dimensional analysis and diffusion modeling of oligonucleotide concentrations after direct intraparenchymal brain infusion.

We compared quantitative experimental results on the diffusion of 35S-labeled phosphorothioate oligonucleotide (PS-ODN) after intraparenchymal infusion in rat brain, with the distributions predicted by Fick's second law of diffusion. Fischer 344 rats underwent identical intracerebral infusions of 36S-PS-ODN. After 0, 5, 11, 23, and 47 h, groups of animals were sacrificed and sequential brain cryosections subjected to autoradiography. The resulting experimental data were compared to the predicted distributions, for estimation of the apparent free diffusion coefficient, D*. Volumes of distribution and total content of 36 S-PS-ODN in the parenchyma were also computed, to monitor loss of total material. The values for D* were within the expected range for the 21-mer PS-ODN used, but a progressive decrease in D* over time was noted. The model requires D* to remain constant and, thus, does not adequately explain the spread of 35S-PS-ODN following infusion. The progressive slowing of spread over time suggests that at later time points, 35S-PS-ODN may be fixed by tissue binding or cellular uptake in the brain. Loss of material via vascular and CSF clearance may also contribute to the lack of fit. Our results highlight issues to be addressed in the modeling and experimental design of the intraparenchymal infusion process.

An illuminating retractor for intracranial microneurosurgery.

During certain microneurosurgical procedures, the illumination provided by current coaxial microscope light sources is suboptimal. One potential solution is the surgical light blade (SLB), a malleable retractor incorporating a flat fiberoptic bundle along its surface. The SLB provides intense illumination within the surgical field without decreasing the amount of limited operating space. A prototype of the SLB was tested in six patients. Results suggest that with further development the SLB could become useful for intracranial microneurosurgery.

Goniometric motion controller for the superconducting coil in a magnetic stereotaxis system.

For the purpose of positioning the 80 kg, 2 T superconducting coil/cryostat used in the developmental version of a magnetic stereotaxis system, a four degree-of-freedom goniometer has been designed, built, and tested. Computer-controlled, stepping motor actuators enable movement of the coil via either joystick, keyboard, or translator-module keypad commands. An integral arrangement of counterweights and counterbalances minimizes the overall weight and size of the goniometer, while maintaining static and dynamic stability during operation. As much of the structure as possible has been made of nonmagnetic materials (mostly aluminum) to minimize distortion of the superconducting coil's field. In this paper, we present the design principles for the goniometer, describe the essential features of its construction, and discuss its performance characteristics and limitations. We also discuss a strategy for performing precision magnetic stereotaxis procedures with an arrangement of static superconducting coils.