Primary Investigator University of Massachusetts Amherst, United States
Introduction:: Approximately 17,000 people sustain a spinal cord injury (SCI) in the U.S. each year, and over a quarter million Americans currently live with paralysis due to SCI. Injury severity and functional deficits due to SCI correlate with the extent of fluid accumulation (i.e., edema) occurring immediately after injury. Previous studies showed fluid pressure around the injured spinal cord (supraspinal) remains elevated for at least three days and contributes to a phase of tissue damage known as secondary injury. While neural cells will more directly interface with fluid within the spinal cord (interstitial), it is currently unknown how SCI affects interstitial fluid pressure and if interstitial forces also contribute to secondary injury. We use a combination of in silico and in vivo models to address these questions. Understanding the contributions of fluid forces and flows after SCI may enable strategies to limit tissue damage and functional deficits after SCI.
Materials and Methods:: We developed an in silico model to simulate interstitial flow after SCI using COMSOL Multiphysics and the porous medium module. We obtained the flow parameters of white matter from literature and set the initial and boundary conditions based on literature or experimentally determined values. The simulation of white matter and grey matter was conducted separately because the flow calculated in the grey matter would mask the flow in white matter due the difference in diffusion rate. For in vivo validation, Sprague-Dawley rats (8-10 weeks) were anesthetized and subjected to a cervical C4 hemi-contusion using an Infinite Horizons spinal cord impactor. Intraspinal pressure was measured at 1 hour, 3 days, and 7 days after injury using a catheter-based pressure sensor from ADinstrument. To label flow pathways in vivo, Evans Blue dye was injected in the tail vein 24 hours prior to tissue harvest at 3 days and 7 days post injury (dpi). In a separate cohort, we used the technique of convection enhanced delivery (CED) to exogenously enhanced interstitial flow after SCI. The spinal cord was re-exposed 7 days after injury, and a blunt-end 27-gauge catheter was used to deliver 5 µL of sterile saline at 1 µL/min, or three times the physiological rate of interstitial flow. The tissue was harvested three days later, cryosectioned at 20 microns, and stained with Luxol Fast Blue for stereological lesion volume quantification.
Results, Conclusions, and Discussions:: Results and Discussion
We find interstitial pressure increases from negative three mmHg to positive eight mmHg at 1 hour post injury, further increases by 3 days post injury, and plateaus near one mmHg after 7 days. This is similar to how the supraspinal pressure increases to 8 mmHg for the first hour post injury. The supraspinal pressure peaks at 12 mmHg for 3 dpi, then plateaus close to zero by 7 dpi. We used reported and measured pressure values to develop a 3D in silico model to interrogate pressure effects on interstitial fluid velocity after hemi-contusion SCI. For an idealized spherical injury, COMSOL simulations predict heightened flow out of the cavity and towards the center of the uninjured white matter, with higher flow velocities at 3 dpi versus at 7 dpi.(fig. 1a.) To validate the model, we used Evans Blue dye to label in vivo fluid flow pathways after SCI.(fig. 1b.) We show that, while the lesion size might be smaller at 3 days post injury, there is more dye transport out of the injury cavity and into adjacent uninjured tissue compared to the 7 days post injury timepoint. These results align with the pressure measurements and the model simulation results. Finally, we find adding flow increases lesion size after SCI, determined by volume of demyelination by Luxol Fast Blue staining.
Conclusion
Our results show traumatic injury causes an immediate and drastic increase in interstitial pressure in the spinal cord, which only resolves by 7 days after injury. COMSOL simulations suggest this increase in interstitial pressure drives elevated interstitial flow velocity into the tissue around the original lesion, which was confirmed by Evans Blue dye extravasation in vivo. Using convection enhanced delivery, we show that a significant increase in interstitial flow increases secondary spinal cord injury and the resulting lesion size. In the future, we can build on this knowledge to explore the mechanisms of how increased flow causes cellular death and potentially identify therapeutic targets for neuroprotection.
Acknowledgements (Optional): :
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