60: Cryopreserved umbilical cord as a meningeal patch for in-utero spina bifida repair in sheep model

Abstract

Disruption of meningeal layers and spinal cord tethering is universally seen at the in-utero spina bifida repair site causing long-term functional deficits and the need for multiple de-tethering surgeries. The cryopreserved human umbilical cord (HUC) provides a watertight barrier at the spina bifida repair site. In this study, we tested the potential role of HUC patch to regenerate the arachnoid layer, reduce histological tethering and improve neurological function in a spina bifida sheep model. In timed-pregnant sheep, spina bifida defect (L2-L6 levels) was created in the fetuses at gestational day (GD) 75. On GD 96, the arachnoid layer was removed and the repair was performed. The fetuses were randomly assigned to two groups for the arachnoid layer closure: Conventional Repair (CR) using myofascial closure vs. HUC as a meningeal patch (n=4 each). Subsequently, the primary closure of the skin layer was performed. The lambs were delivered at GD 140 by C-section. The lambs without spina bifida creation served as controls (n=4). The clinical assessment for spinal cord function was performed using the Texas Spinal Cord Injury Scale (TSCIS) scale. The spinal cord was assessed at three levels (L3-L5) for spinal cord tethering, defined as lack of cerebrospinal fluid space (CFS) between pia layer and overlying tissue using Masson’s Trichrome and Picrosirius Red stain under circular polarized microscopy. Arachnoid layer regeneration was assessed using specific immunofluorescence markers for the arachnoid layer: Retinaldehydye dehydrogenase -2 (RALDH-2), and Pan-keratin staining. The TSCIS scores were lower in the conventional repair compared to HUC- meningeal patch, and the controls (Figure 1A). The loss of spinal cord function in the CR was mainly due to ataxia and loss of proprioception. The histology at the repair site showed higher spinal cord tethering in the CR lambs compared to HUC lambs at all levels of the repair site (Figure 1B and Figure 2). In the CR, there was an abrupt ending to the arachnoid layer at the tethering site (Figure 2- yellow arrow) whereas, in the HUC repair the arachnoid layer was continuous (green arrow) with underlying CFS. HUC as a meningeal patch allows regeneration of the arachnoid layer and prevents spinal cord tethering, and improves spinal cord function. Further exploration of mechanisms and application in human studies are needed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)