Alexandre Rasouli

Laser-mediated cartilage reshaping with feedback-controlled cryogen spray cooling: Biophysical properties and viability

Recent studies have indicated that chondrocyte viability decreases with prolonged or repeated laser irradiation. To optimize laser‐mediated cartilage reshaping, the heating process must be finely controlled. In this study, we use high‐power Nd:YAG laser irradiation (λ = 1.32 μm) combined with cryogen spray cooling (CSC) in an attempt to reshape porcine septal cartilage while enhancing chondrocyte viability.

Transplantation of preconditioned Schwann cells following hemisection spinal cord injury.

Study Design. Chronically compressed sciatic nerve segments were transplanted to hemisected spinal cord injured rats. Histologic evaluation and behavior functional outcomes were tested after 6 weeks following surgery. Objective. To evaluate the outcome of preconditioned peripheral nerves as a permissive environment in axonal regeneration of the injured spinal cord. Summary of Background Data. Schwann cells have been used to facilitate a permissive environment for the injured spinal cord to regenerate. Previous experiments have shown compressive mechanical stress to be important in stimulating the regenerative behavior of Schwann cells. Transplantation of highly permissive Schwann cell-enriched peripheral nerve grafts may enhance regeneration in spinal cord injury. Methods. Adult Sprague-Dawley rats (n = 24) were used to create a hemisection injury of the spinal cord. At 1-week postinjury creation, the spinal cords were reexposed for all animals. Peripheral nerve grafts were obtained from rat sciatic nerve, either untreated or subjected to mechanical compression for 2 weeks with nonconstrictive tubing. Transplantation of grafts was performed after a resection of the glial scar. Functional outcome was measured using the Basso, Beattie, Bresnahan Locomotor Rating Score and footprint analysis. Tract tracing of descending and ascending spinal cord tracts was performed at 6 weeks after surgery for histologic evaluation of axonal regeneration. Results. Preconditioned transplants had significantly higher Basso, Beattie, Bresnahan Scores versus hemisection alone in the late postoperative period (P < 0.05). They also had significantly less foot exorotation and base of support when compared to nonconditioned transplants. Histologic analysis showed increased regeneration at lesional sites for preconditioned transplants versus control group (P < 0.05). Conclusions. Functional recovery after hemisection injury improved significantly in the late postoperative period with transplantation of preconditioned peripheral nerve. Preconditioned grafts also exhibit sustained axonal regeneration at and past the lesional site in histologic analysis. Further investigation with later time points is warranted.

Transplantation of preconditioned schwann cells in peripheral nerve grafts after contusion in the adult spinal cord. Improvement of recovery in a rat model.

BACKGROUND Recovery after injury to the peripheral nervous system is based on the pro-regenerative relationship between axons and the extracellular matrix, a relationship established by Schwann cells. As mechanical conditioning of Schwann cells has been shown to stimulate their regenerative behavior, we sought to determine whether transplantation of these cells to the central nervous system (i.e., the spinal cord), with its limited regenerative capacity after injury, would improve axonal regeneration and functional recovery. METHODS A moderate contusion injury of the spinal cord was created with a force-directed impactor in forty-eight adult Sprague-Dawley rats, and, at one week postinjury, the spinal cords were reexposed in all animals. In twenty-four of these animals, peripheral nerve grafts with Schwann cells that had been obtained from the sciatic nerves of donor animals, and had been either untreated or subjected to mechanical conditioning, were transplanted to the contused area of the cords following resection of the glial scar. Another group of animals was treated with glial scar excision only, and a fourth group had the contusion injury but neither glial excision nor transplantation. Scores according to the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale were assigned preoperatively and weekly thereafter. Tract tracing of descending and ascending spinal cord tracts was performed at six weeks postoperatively for quantitative histological evaluation of axonal regeneration. RESULTS While the recovery following glial scar excision without peripheral nerve transplantation was significantly worse than the recovery in the other groups, both transplantation groups had significantly higher BBB scores than the controls (no transplantation) in the early postoperative period (p < 0.05). Moreover, histological analysis showed markedly increased axonal regeneration at the lesional sites in the animals treated with the mechanically conditioned grafts than in the other groups (p < 0.05). CONCLUSIONS Functional recovery after spinal cord contusion improved following glial scar excision with transplantation of Schwann cells in peripheral nerve grafts to the contusion areas. Although recovery did not differ significantly between the transplantation groups, only the preconditioned grafts led to axonal regeneration at and past the lesional site. These grafts may further enhance functional recovery as the descending tracts eventually reach their target end-organs.

Resection of Glial Scar Following Spinal Cord Injury

While many studies have focused on modulating the immune response and enhancing axonal regeneration after spinal cord injury (SCI), there is limited work being performed on evaluating the role of glial scar in SCI. We sought to evaluate the effects of glial scar resection in contusion models and dorsal hemisection models of SCI. At 1‐week postinjury, 2 mm of glial scar was excised from specimens in one of the two groups from each injury model. Functional outcome was measured weekly using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale along with histologic evaluation of spinal cord tracts to determine axonal regeneration. Within the dorsal hemisection model, there was no significant difference in recovery for animals that underwent glial scar excision versus animals that did not have scar excision (p = 0.61). Animals subjected to the contusion model, however, demonstrated lower BBB scores in the glial resection group during the earlier postoperative periods (<4 weeks; p < 0.05). Histological analysis revealed no axons within the glial resection contusion model, and moderate axonal growth within the nonresection contusion group and both hemisection groups (p > 0.05 for differences among the three groups). While glial scar may serve to stabilize the preserved axonal tracts and thereby permit modest recovery in a contusion model of SCI, it may be of less importance with a dorsal hemisection model. These experiments highlight that basic biologic processes following SCI may vary tremendously based on the injury mechanism and that the role of glial scar in spinal cord regeneration must be elucidated.

Use of flow cytometry to assess chondrocyte viability after laser reshaping of cartilage

Lasers have been shown to cause permanent shape change in cartilage via photothermally induced mechanical stress relaxation. While the biophysical properties of cartilage during laser irradiation have been studied, tissue viability following laser irradiation has not been fully characterized. In this study, cell viability staining and flow cytometry were used to determine chondrocyte viability following photothermal stress relaxation. Porcine septal cartilage slabs (10 X 25 X 1.5 mm) were irradiated with light from a Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 s), determined from measurements of diffuse reflectance, which correlate with mechanical stress relaxation. Irradiated samples were then divided into two groups analyzed immediately and at five days following laser exposure (the latter group was maintained in culture). Chondrocytes were isolated following serial enzymatic digestion with hyaluronidase, protease, and collagenase II for a total of 17 hours. Chondrocytes were then stained using SYTOR/DEAD RedTM (Molecular Probes; Eugene, OR) wherein live cells stained green (530 nm) and dead cells stained red (630 nm) when excited at 488 nm. A flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ) was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post irradiation debris in each treatment population. Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after irradiation, were viable as determined using flow cytometry while non- irradiated controls were 100 percent viable. Specimens irradiated two or three times with the laser demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 percent following two laser exposures, and 16 percent after three laser exposures. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28 to 88 percent, with the least amount of deterioration in untreated and singly irradiated samples. Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. The flow cytometric approach to viability is accurate, fast, and can handle large sample numbers and sizes. Most importantly, since the method reveals that a single laser exposure of 6.7 s (sufficient for sustained shape change) causes less than 40 percent acute reduction in viability, photothermal reshaping of cartilage may be further researched as a clinical alternative to conventional techniques.

Femoral Neurogram Before Transpsoas Spinal Access at L4-5 Intervertebral Disk Space: A Proposed Screening Tool.

Study Design: Observational study. Objective: To illustrate the variability of the course of the femoral nerve across the L4–5 disk space, and to present a novel application of transforaminal epidural steroid injections (TFESI) in the visualization of femoral nerve roots. Summary of Background Data: A concern regarding the lateral retroperitoneal transpsoas approach is the proximity of the lumbar plexus. Current techniques of assessing the proximity of neural tissue to the L4–5 disk space have limited capabilities. Methods: A total of 100 patients were selected for L4–5 TFESI (L4 selective nerve root blocks) because of lumbar radiculopathy. L4 neurograms were obtained while performing L4–5 TFESI under flouroscopic guidance, using a retroneural technique. The course of the L4 root/femoral nerve was then evaluated under fluoroscopy in the anteroposterior and lateral planes. Images were then reviewed by a radiologist, physiatrist, and 2 orthopedic spine surgeons. Results: Fluoroscopic evaluation revealed that the pattern of location of the femoral nerve was highly variable. In males, it was located 4.7% in zone 2, 32.5% in zone 3, 53.5% in zone 4, and 9.3% in zone P. In female patients, it was located 7.0% in zone 2, 14% in zone 3, 54.4% in zone 4, and 24.6% in zone P. Conclusions: An L4 neurogram will provide an accurate trajectory of L4 root/femoral nerve as it crosses the L4–5 intervertebral disk space. An accurate assessment is essential to help minimize the increasing frequency of thigh pain, paresthesias, and weakness associated with the lateral access to the L4–5 intervertebral disk space. Femoral nerves that fall within zones 2 and 3 will require more manipulation during retraction and may be better suited with a different surgical approach.

5:3763. Novel transplantation of preconditioned Schwann cells following spinal cord contusion injury

CONTUSION INJURY *Rasouli, A; *Bhatia, N; *Suryadevara, S; Cahill, K; +*Gupta, R +*University of California, Irvine ranjang@uci.edu INTRODUCTION A pivotal difference between the two branches of the nervous system is that the peripheral nervous system (PNS) may successfully regenerate while the central nervous system may only do so in a limited capacity. This recovery in the PNS is based on the proregenerative relationship between axons and the extracellular matrix, a relationship established by Schwann cells. Compressive mechanical stress is important in stimulating the regenerative behavior of Schwann cells (1,2). The objective of this study was to determine whether transplantation of preconditioned peripheral nerves to the injured spinal cord improves functional recovery in a spinal cord contusion injury model (3,4). METHODS Forty adult Sprague-Dawley rats were used to create a moderate spinal cord injury contusion model. The PSI Infinite Horizon Impactor (IH; Precision Systems & Instrumentation, Lexington, KY) was used to create a contusion injury by uniformly delivering 175 kdyn to the exposed spinal cord. At 1 week post-injury creation, the spinal cords were re-exposed for all four groups. Peripheral nerve grafts were obtained from rat sciatic nerve, either untreated or subjected to mechanical compression for two weeks with inert, non-constrictive tubing. Transplantation of grafts to the contused area of cord was performed following resection of the glial scar. A subset of the animals underwent sham transplantation. Another group of animals underwent a sham operation in which the cord was exposed and 2 mm of friable glial scar excised, but no peripheral nerve was transplanted. Functional outcome was measured using the Basso, Beattie, Bresnahan (BBB) Locomoter Rating Scale. BBB scores were obtained preoperatively and weekly thereafter. Stereotactic injections of biotinylated dextran amineBDA (Molecular Probes, Eugene, Oregon) were performed via a craniotomy over the rat motor cortex to verify axonal regeneration in the descending tracts of the spinal cord. Ascending spinal cord tracts were evaluated in the remaining rats with intraneural injection of BDA to the L4 dorsal root ganglion. After tract tracing, the animals were allowed to survive for two additional weeks, following which their intact spinal cords were harvested and post-fixed in 4% paraformaldehyde, rinsed in Na2HPO4, equilibrated in 30% sucrose buffer, and embeded in TissueTek® (VWR International, West Chester, PA). The frozen tissue blocks were then sectioned to produce longitudinal sections of the lesion site, and cross-sections of the rostral and caudal segments. Visualizing the BDA-labeled axons was possible through histochemical means using avidin and biotinylated horseradish peroxidase (Vectastain ABC Kit, Vector Labs, Burlingame, CA) followed by diaminobenzidine (DAB) staining, allowing for dark staining of axons along with light staining of the gray matter. RESULTS Both transplanted groups had significantly higher BBB scores versus the untransplanted controls in the early postoperative period (postoperative weeks <4) (p<0.05). Although mechanical compression of the transplanted peripheral nerve did not have a significant effect on the functional outcome at these early time points, histological analysis showed increased axonal sprouting at lesional sites for the transplanted groups versus untransplanted groups (Fig. 1-3). Glial scar excision without peripheral nerve transplantation produced a significantly worse recovery in the early postoperative period versus nerve transplantation and versus sham operation without scar excision (p<0.05). CONCLUSIONS Functional recovery after a SCI contusion injury was improved following glial scar excision with transplantation of peripheral nerves to areas of spinal cord contusion. The amount of lesional-site axonal regeneration, verified by histological analysis, was higher around transplanted grafts. Although mechanical pre-conditioning of the transplanted nerve did not have a significant clinical effect on functional recovery at this early stage, there does appear to be an increase in axonal sprouting at the lesional site with these experimental groups. Thus, these grafts may improve axonal regeneration and functional outcome with further study required at later time points. Glial scar excision without nerve transplantation resulted in impaired functional and axonal recovery and should not be done alone after contusion injury. Peripheral nerve transplantation significantly improved functional and axonal recovery following contusion spinal cord injury in rats and and warrants further investigation for potential clinical applications.

Cartilage reshaping: an overview of the state of the art

The laser irradiation of cartilage results in a plastic deformation of the tissue allowing for the creation of new stable shapes. During photothermal stimulation, mechanically deformed cartilage undergoes a temperature dependent phase transition, which results in accelerated stress relaxation of the tissue matrix. Cartilage specimens thus reshaped can be used to recreate the underlying framework of structures in the head and neck. Optimization of this process has required an understanding of the biophysical processes accompanying reshaping and also determination of the laser dosimetry parameters, which maintain graft viability. Extensive in vitro, ex-vivo, and in vivo animal investigations, as well as human trials, have been conducted. This technology is now in use to correct septal deviations in an office-based setting. While the emphasis of clinical investigation has focused on septoplasty procedures, laser mediated cartilage reshaping may have application in surgical procedures involving the trachea, laryngeal framework, external ear, and nasal tip. Future directions for research and device design are discussed.

Two-photon excitation laser scanning microscopy of porcine nasal septal cartilage following Nd:YAG laser-mediated stress relaxation

Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within porcine nasal septal cartilage tissue over a 4-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation (lambda equals 1.32 micrometer) using parameters that result in mechanical stress relaxation (6.0 W, 5.4 mm spot diameter). TPM excitation (780 nm) result in induction of fluorescence from endogenous agents such as NADH, NADPH, and flavoproteins in the 400 - 500 nm spectral region. During laser irradiation diffuse reflectance (from a probe HeNe laser, (lambda) equals 632.8 nm), surface temperature, and stress relaxation were measured dynamically. Each specimen received one, two, or three sequential laser exposures (average irradiation times of 5, 6, and 8 seconds). The cartilage reached a peak surface temperature of about 70 degrees Celsius during irradiation. Cartilage denatured in 50% EtOH (20 minutes) was used as a positive control. TPM was performed using a mode-locked 780 nm Titanium:Sapphire (Ti:Al203) beam with a, 63X, 1.2 N.A. water immersion objective (working distance of 200 mm) to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns (lateral resolution equals 35 micrometer X 35 micrometer). Images were obtained immediately following laser exposure, and also after 4 days in culture. In both cases, the irradiated and non-irradiated specimens do not show any discernible difference in general shape or auto fluorescence. In contrast, positive controls (immersed in 50% ethanol), show markedly increased fluorescence relative to both the native and irradiated specimens, in the cytoplasmic region.