Introduction: Mechanical pressure on dural or fascial wound edges exerted by cerebrospinal fluid (CSF) is thought to impair proper apposition of the wound borders and therefore prevent healing. Interestingly, it has been observed that the surgical tissues exposed to CSF do not bleed, exhibit smooth edges, and have no evidence of granulation tissue formation. This raises the question of whether the constituents of CSF themselves impair normal wound healing. However, the biochemical interaction of CSF with the healing process has not been investigated. Here, we hypothesize that CSF exhibits anti-angiogenic properties and therefore inhibits the healing process.
Methods: We used an in vitro model, in which human umbilical vein endothelial cells grow in a three-dimensional scaffold. Normally these cells form capillary-like structures named cords. We exposed the model to varying concentrations of CSF vs. Dulbecco’s Phosphate-Buffered Saline (DPBS), used as control. We then quantified cord length. To rule out potential direct cellular toxicity or a general effect on cell migration we performed a “scratch test” on human fibroblasts exposed to the same CSF or DPBS concentrations.
Results: In all samples (n=5) of CSF at 50, 75 and 100% Volume/Volume significantly diminished cell migration and subsequent formation of capillary-like structures compared to DPBS (used to control for dilutional effect) with a dose dependent tendency. Cell migration remained unchanged in the fibroblast culture, suggesting that CSF specifically inhibits capillary formation.
Conclusions: CSF inhibited cord formation with a dose-dependent tendency, implying that it contains signaling molecules that preclude angiogenesis. This effect was cell specific and not observed with fibroblasts. These experiments suggest that CSF, rather than acting as an inert bystander, may actively impair healing by inhibiting capillary formation. This could impact the understanding of postoperative leaks as well as the potential physiologic role of cerebrospinal fluid in precluding neo-vascular formation in the subarachnoid space.
Patient Care: The possibility that CSF inhibits new blood vessel formation has not, to our knowledge, been explored elsewhere. These findings could introduce new insights in the pathogenesis of leaks but also in other phenomena such as tumor spread within the central nervous system and brain embryology, since these both rely heavily on angiogenesis. This investigation will lead to a wider study of the roles of CSF in pathological states and therefore potential new treatments for CSF leaks.
Learning Objectives: By the conclusion of this session, participants should be able to
1. Describe the importance of increasing the current knowledge on the pathogenesis of CSF leaks
2. Discuss, in small groups, the relevance of identifying anti-angiogenic properties of the cerebrospinal fluid in brain development, pathology, and surgical technique.
3. Identify how addressing the pathogenesis of CSF leaks can lead to novel treatment modalities.
References: 1. Goldschmidt E, Hem S, Ajler P, et al. A new model for dura mater healing: human dural fibroblast culture. Neurol Res. 2013;35(3):300-307. doi:10.1179/1743132812Y.0000000136.
2. Goldschmidt E, Ielpi M, Loresi M, et al. Assessing the role of selected growth factors and cytostatic agents in an in vitro model of human dura mater healing. Neurol Res. 2014;36(12):1040-1046. doi:10.1179/1743132814Y.0000000429.
3. Stoop MP, Coulier L, Rosenling T, et al. Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples. Mol Cell Proteomics. 2010;9(9):2063-2075. doi:10.1074/mcp.M900877-MCP200.
4. Guldbrandsen A, Vethe H, Farag Y, et al. In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR). Mol Cell Proteomics. 2014;13(11):3152-3163. doi:10.1074/mcp.M114.038554.
5. Schutzer SE, Liu T, Natelson BH, et al. Establishing the Proteome of Normal Human Cerebrospinal Fluid. Gendelman HE, ed. PLoS One. 2010;5(6):e10980. doi:10.1371/journal.pone.0010980.
6. Cappabianca P, Iaconetta G, Califano L, eds. Cranial, Craniofacial and Skull Base Surgery. Milano: Springer Milan; 2010. doi:10.1007/978-88-470-1167-0.
7. Eloy JA, Choudhry OJ, Shukla PA, Kuperan AB, Friedel ME, Liu JK. Nasoseptal flap repair after endoscopic transsellar versus expanded endonasal approaches: Is there an increased risk of postoperative cerebrospinal fluid leak? Laryngoscope. 2012;122(6):1219-1225. doi:10.1002/lary.23285.
8. Jahangiri A, Wagner J, Han SW, et al. Morbidity of repeat transsphenoidal surgery assessed in more than 1000 operations. J Neurosurg. 2014;121(1):67-74. doi:10.3171/2014.3.JNS131532.
9. Kassam AB, Thomas A, Carrau RL, et al. Endoscopic reconstruction of the cranial base using a pedicled nasoseptal flap. Neurosurgery. 2008;63(1 SUPPL.):44-53. doi:10.1227/01.NEU.0000297074.13423.F5.
10. Zanation AM, Carrau RL, Snyderman CH, et al. Nasoseptal flap reconstruction of high flow intraoperative cerebral spinal fluid leaks during endoscopic skull base surgery. Am J Rhinol Allergy. 2009;23(5):518-521. doi:10.2500/ajra.2009.23.3378.
11. Hadad G, Bassagasteguy L, Carrau RL, et al. A novel reconstructive technique after endoscopic expanded endonasal approaches: vascular pedicle nasoseptal flap. Laryngoscope. 2006;116(10):1882-1886. doi:10.1097/01.mlg.0000234933.37779.e4.
12. Yip J, Macdonald KI, Lee J, et al. The inferior turbinate flap in skull base reconstruction. J Otolaryngol - Head Neck Surg. 2013;42(1):6. doi:10.1186/1916-0216-42-6.
13. Choby G, Pinheiro-Neto C, de Almeida J, et al. Extended Inferior Turbinate Flap for Endoscopic Reconstruction of Skull Base Defects. J Neurol Surg Part B Skull Base. 2014;75(4):225-230. doi:10.1055/s-0033-1358791.
14. Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014;6(265):265sr6. doi:10.1126/scitranslmed.3009337.
15. Park-Windhol C, Ng YS, Yang J, Primo V, Saint-Geniez M, D’Amore PA. Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling. Sci Rep. 2017;7(1):17138. doi:10.1038/s41598-017-16852-x.
16. Lee S-I, Lee E-S, El-Fiqi A, Lee S-Y, Eun-Cheol Kim, Kim H-W. Stimulation of Odontogenesis and Angiogenesis via Bioactive Nanocomposite Calcium Phosphate Cements Through Integrin and VEGF Signaling Pathways. J Biomed Nanotechnol. 2016;12(5):1048-1062. http://www.ncbi.nlm.nih.gov/pubmed/27305825. Accessed December 15, 2017.
17. Lazarovici P, Lahiani A, Gincberg G, et al. Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay. In: Methods in Molecular Biology (Clifton, N.J.). Vol 1727. ; 2018:239-250. doi:10.1007/978-1-4939-7571-6_18.
18. Liu X, Gao Q, Zhao N, et al. Sohlh1 suppresses glioblastoma cell proliferation, migration and invasion by inhibition of Wnt/ß-catenin signaling. Mol Carcinog. December 2017. doi:10.1002/mc.22774.
19. Zielke C, Lu Y, Poinsot R, Nilsson L. Interaction between cereal ß-glucan and proteins in solution and at interfaces. Colloids Surfaces B Biointerfaces. 2017;162:256-264. doi:10.1016/j.colsurfb.2017.11.059.