Vitamin D-A New Perspective in Treatment of Cerebral Vasospasm

Sepide Kashefiolasl, Matthias S Leisegang, Valeska Helfinger, Christoph Schürmann, Beatrice Pflüger-Müller, Voahanginirina Randriamboavonjy, Andrea E Vasconez, Geert Carmeliet, Klaus Badenhoop, Gudrun Hintereder, Volker Seifert, Katrin Schröder, Juergen Konczalla, Ralf P Brandes, Sepide Kashefiolasl, Matthias S Leisegang, Valeska Helfinger, Christoph Schürmann, Beatrice Pflüger-Müller, Voahanginirina Randriamboavonjy, Andrea E Vasconez, Geert Carmeliet, Klaus Badenhoop, Gudrun Hintereder, Volker Seifert, Katrin Schröder, Juergen Konczalla, Ralf P Brandes

Abstract

Background: Cerebral vasospasm (CVS) is a frequent complication after subarachnoid hemorrhage (SAH), with no sufficient therapy and a complex pathophysiology.

Objective: To explore the vitamin D system as a potential treatment for CVS.

Methods: 25-vitamin D3 levels tested between 2007 and 2015 and data of SAH patients admitted during the months with a peak vs nadir of VitD3 values were analyzed, retrospectively. We prospectively correlated VitD3 and vasospasm/outcome data in SAH patients admitted in 2017. An experimental mice SAH model and cell culture model were used to investigate the effect of 1,25-dihydroxyvitamin D3 (1,25-VitD3). Additionally, the mediators acting in the VitD mechanism were researched and detected.

Results: Based on the retrospective analysis demonstrating an increased frequency of vasospasm in SAH patients during the low vitamin D period in winter, we started basic research experiments. Active 1,25-VitD3 hormone attenuated CVS, neurological deficit, and inflammation after intrathecal blood injection in mice. Deletion of the vitamin D receptor in the endothelium or in myeloid cells decreased the protective 1,25-VitD3 effect. Co-culture experiments of myeloid and endothelial cells with blood confirmed the anti-inflammatory 1,25-VitD3 effect but also revealed an induction of stroma-cell-derived factor 1α (SDF1α), vascular endothelial growth factor, and endothelial nitric oxide synthase by 1,25-VitD3. In mice, SDF1α mimicked the protective effect of 1,25-VitD3 against CVS. From bench to bedside, CVS severity was inversely correlated with vitamin D plasma level, prospectively. Patients with more severe CVS exhibited attenuated expression of SDF1α and 1,25-VitD3-responsive genes on circulating myeloid cells.

Conclusion: 1,25-VitD3 attenuates CVS after SAH by inducing SDF1α. However, VitD administration should be tested as optional treatment to prevent CVS.

Keywords: Cerebral vasospasm; Subarachnoid hemorrhage; Translational study; Vitamin D.

© Congress of Neurological Surgeons 2020.

Figures

FIGURE 1.
FIGURE 1.
1,25-VitD3 attenuates vascular dysfunction in a murine SAH model. A, Representative ink images of the cerebral vasculature and BA in magnification and statistics of vessel diameter of mice 12 h after intrathecal injection of blood (SAH, 60 μl) or saline (sham) with and without 1,25-VitD3 pretreatment (100 ng/kg/d i.p.; 5 d). n = 9. ANOVA, Fisher test, #P < .05 sham vs SAH. *P < .05 with vs without 1,25-VitD3. Mean with 95% (CI). Scale bars are 1000 and 200 μmeters, respectively. B, Endothelium-dependent relaxation of the isolated BA harvested from the animal groups indicated (n = 5). ANOVA for repeated measurements. *P < .05. Mean with 95% CI. C and D, Relative weight loss, ANOVA, Fisher test C, and neuroscore, ANOVA Mann-Whitney test D, n = 9, #P < .05 sham vs SAH. *P < .05 with vs without 1,25-VitD3. Shown are mean +/− 5/95% CI. E, Correlation between different CVS degrees defined as reduced diameter of BA in percent compared to the mean BA diameter in mice (<33%, ≥33%-66%, and > 66%) and worse outcome using the 14-point score by Chen et al (2001). n = 9. Multiple t-test. #P < .05 SAH with vs without 1,25-VitD3. Mean with 95% CI. F and G, quantitative reverse transcription PCR (RT-q PCR) polymerase chain reaction (PCR) of the genes indicated from the BA of mice 12 h after intrathecal injection of blood (SAH, 60 μl) or saline (sham) with and without Control 1,25-VitD3 pretreatment (100 ng/kg/d i.p.; 5 d). n = 9. ANOVA, Fisher test, #P < .05 sham vs SAH. *P < .05 with vs without 1,25-VitD3. Mean with 95% CI.
FIGURE 2.
FIGURE 2.
The protective effect of 1,25-VitD3 requires the vitamin D receptor in LysM-positive cells and in the endothelium. A-E, Statistics of vessel diameter A, weight loss, and neuroscores B of the VDR flox-flox mouse lines indicated 12 h after intrathecal injection of blood (SAH, 60 μl) and after 1,25-VitD3 pre-treatment (100 ng/kg/d i.p.; 5 d). n = 3 (cre0/0) and n = 5 (cre+/0). ANOVA, #P < .05 cre0/0 vs cre0/+, shown are mean +/− 5/95% CI. C-E, qRT-PRT of the genes indicated from the BA of the mice lines indicated 12 h after intrathecal injection of blood (SAH, 60 μl) with 1,25-VitD3 pretreatment (100 ng/kg/d i.p.; 5 d). ANOVA, Fisher test, #P < .05 cre0/0 vs cre0/+, shown are mean +/− 5/95% CI.
FIGURE 3.
FIGURE 3.
Macrophages induce protective genes in the endothelium in response to 1,25-VitD3. Relative expression changes as determined by RT-qPCR of the genes indicated from the cells denoted. Cells were kept in mono- or co-culture for 48 h in the presence or absence of blood (10% blood in endothelial cell basal medium (EBM)) and presence or absence of 1,25VitD pretreatment (100 nmol/L in EBM; Control: dimethyl sulfoxide (DMSO)), n = 3. ANOVA, Fisher test, #P < ,05 with vs without blood. *P < ,05 with vs without 1,25-VitD3. Shown are Mean +/− 5/95% CI.
FIGURE 4.
FIGURE 4.
SDF1α prevents SAH-induced vascular dysfunction and mediates the effects of vitamin D. A, Statistics of vessel diameter of mice 12 h after intrathecal injection of blood (SAH, 60 μl) with and without 1,25-VitD3 pretreatment (100 ng/kg/d i.p.; 5 d). In subgroups, the CXCR4 receptor antagonist AMD3100 (5 mg/kg, i.p.) or SDF1α (250 ng intrathecal) were administered at time of blood injection (n = 3). ANOVA, Fisher test, B, Relative weight loss, ANOVA, Fisher Test, and neuroscore, ANOVA Mann-Whitney test. C and D, RT-qPCR of the genes indicated from the BA of mice 12 h after intrathecal injection. ANOVA, Fisher test. #P < .05 SAH vs SAH + 1,25-VitD3 or SAH + SDF1α. *P < .05 SAH + 1,25-VitD3 vs SAH + 1,25-VitD3 + AMD3100. Shown are mean +/− 95% CI.
FIGURE 5.
FIGURE 5.
Prospective study cohort: vitamin D is associated with favorable outcome in SAH patients. A, Spearman correlation of plasma vitamin D level on time of admission and degree of CVS. Patients (n = 23) were divided into 4 groups: no CVS (0% decrease of vessel diameter) (n = 5), mild CVS (<33% decrease of vessel diameter) (n = 5), moderate CVS (33-66% decrease of vessel diameter) (n = 7), and severe CVS (>66% decrease of vessel diameter) (n = 6). B, Spearman correlation of plasma vitamin D level on time of admission and clinical outcome level described as mRS 0 to 6 in 23 patients. P < .0001 is significant. C-H, RT-qPCR of the genes indicated from CD14-positive peripheral blood mononuclear cells isolated before (pre-CVS) during (CVS) and after (post) the CVS period. C-E, Pooled data of all patients. n = 16, ANOVA, *P < .05. Mean with 95% CI. F-H, Comparison of gene expression at time of CVS between patients with mild or no CVS (n = 8) to those with moderate and severe CVS (n = 8). ANOVA, *P < .05. Mean with 95% CI.

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