Cerebrospinal Fluid (CSF) Can Inhibit Wound Healing by Inhibiting Angiogenesis

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 (HuVECS) grow in a three-dimensional scaffold. Normally, these cells form capillary-like structures named cords. We exposed the model to varying concentrations of CSF versus Dulbecco’s Phosphate-Buffered Saline (DPBS) as a control. We then quantified cord length. To rule out a potential general effect on cell migration we performed a “scratch test” on human fibroblasts exposed to the same CSF or DPBS concentrations. We also tested if CSF had an effect on the migration velocity of endothelial cells, fibroblasts or smooth muscle cells in two-dimensional cultures.

Results: In all samples (n = 5) of CSF at 50, 75 and 100% significantly diminished cell migration and subsequent formation of capillary-like structures compared with DPBS with a dose-dependent tendency. Specifically, when HuVECS were exposed to normal human CSF they exhibited a reduction in cell migration and cord formation (7,677.2 ± 2,139.7; 7,428.4 ± 2,532; and 5,908 ± 1,488.7 pixels for CSF at 50, 75, and 100%, respectively) (n = 5) (p < 0.05). DPBS, used as control in the same concentrations to address the potential dilutional effects on the media, had no effect in the cord length and was no different than cells in normal full media (9,788.9 ± 1,276; 11.254.3 ± 1,568.2; and 8,712.6± 1,377.1 pixels for DPBS at 50, 75, and 100%, respectively, p > 0.1). Cell migration remained unchanged in the fibroblast scratch test and migration velocity was not altered by CSF in a two-dimensional culture of either fibroblasts, smooth muscle, or endothelial cells, suggesting that CSF specifically inhibits capillary formation.

Conclusion: CSF inhibited cord formation with a dose-dependent tendency, implying that it contains signaling molecules that preclude angiogenesis. This effect was cell and process specific since it was not observed with fibroblasts, smooth muscle cells or even two dimensional endothelial cell cultures. 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 neovascular formation in the subarachnoid space.

Fig. 1 Human endothelial cells (HuVEC) cord formation, ramification, and connectivity are inhibited in the presence of 50, 75, and 100% CSF compared with control-treated cultures. Scale bar = 200 mm.

Fig. 2 Quantification of cell migration as measured by cord length. Each point indicates the total length of the cords for each sample. The “y” axis depicts the total cord length in pixels.