1Department of Neurosurgery, General Hospital of Tianjin Medical University, Tianjin, China. 2Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin Neurological Institute, Tianjin, China. 3Department of Neurosurgery, The First Central Hospital of Tianjin, Tianjin, China. 4Department of Neurology, Tianjin Huanhu Hospital, Tianjin, China. 5Department of Neurology, Tangdu Hospital, Baqiao, Shanxi, China. 6Department of Neurosurgery, The First Central Hospital of Baoding City, Lianchi, Baoding, China. 7Department of Neurology, Henry Ford Hospital, Detroit, MI 48202, USA
Chronic subdural hematoma (CSDH) is a neurological disorder with a substantial recurrence rate. Atorvastatin is an effective drug for treating hyperlipidemia and known to improve neurological outcome after intracerebral hemorrhage. Previous studies have reported that atorvastatin treatment promotes hematoma absorption in CSDH, while the underlying mechanisms remain unclear. In this study, we investigated whether the anti-inflammatory effects of atorvastatin mediate absorption of CSDH. 144 male, Wistar rats (6 months old) were randomly divided into the following groups: 1) sham surgery control, 2) treatment: CSDH + atorvastatin, and 3) vehicle control: CSDH + saline. Atorvastatin or saline was orally administered daily for 19 days after CSDH procedure. A T2WI MRI was used to evaluate CSDH volume changes during the time course of the study. Flow cytometry and immunohistochemical staining were used to measure the number of regulatory T cells (Treg). ELISA was used to measure cytokine level in the hematoma border. Neurological function and cognitive outcome were evaluated using Foot-Fault test and Morris Water Maze test, respectively. When compared to saline treatment, atorvastatin treatment accelerated the absorption of CSDH as indicated by decreased hematoma volume in T2WI MRI data on 14th and 21st day after CSDH (P<0.05). Atorvastatin treatment significantly increased the number of Treg in circulation and hematoma border from 3rd to 21st day after CSDH. Atorvastatin treatment significantly decreased the levels of interleukins (IL-6 and IL-8) and tumor necrosis factor-α (TNF-α), but increased IL-10 level in the hematoma border. Atorvastatin treatment also improved neurological function and cognitive outcome compared to vehicle treated group. Atorvastatin induced anti-inflammatory responses and increased Treg in circulation and brain which may contribute to the accelerated CSDH absorption in rats.
Figure 1. Measurement of hematoma volume at various time points after CSDH using MRI. T2WI sequence MRI scanning shows that atorvastatin treatment decreases hematoma volume from 14 to 21 days after CSDH. (A-C) Representative MRI images of the CSDH saline-treated rats and (D-F) CSDH-atorvastatin treated rats. (G) Hematoma volume quantitative data.
Figure 2. Atorvastatin treatment increases Treg expression in brain tissue of CSDH rats. FoxP3 immunohistochemical staining was used to detect Treg expression in brain tissue. FoxP3+ cells are primarily detected in the vicinity of the intracranial hematoma lesions, and atorvastatin promotes its expression. The dura membrane and its hematoma on days 3, 14 and 21 after CSDH are represented in panels A-C for non-treated CSDH rats, and in panels D-F for atorvastatin treated CSDH rats with quantitative data presented in panel G.
Figure 3. Atorvastatin treatment increases Treg expression in peripheral blood of CSDH rats as measured by flow cytometry. A) Representative dot plots showing the gating strategy. B) Representative dot plots of Treg cells in the sham control and CSDH-non-treated rats. Panel C) Representative dot plots of Treg cells in Atorvastatin treated rats. Quantitative data for Treg in peripheral blood is presented (D).
Figure 4. Higher circulating Treg correlates with lower hematoma volume in CSDH rats. Linear correlation analysis indicates that lower hematoma volume after CSDH correlates with Treg increase in peripheral blood (r2= 0.448, P<0.05, panel A), and brain (r2= 0.727, P<0.05, panel B).
Figure 5. Atorvastatin treatment increases IL-10, and decreases IL-6, IL-8, and TNF-α expression in brain tissue of CSDH rats. Atorvastatin treatment significantly decreases expression of (A) TNF-α, (B) IL-6 and (C) IL-8 while increasing (D) IL-10 expression in the dura and hematoma boundary tissue compared to saline-treated CSDH rats. #P<0.05 for CSDH saline treated group vs. sham control group; *P<0.05 for CSDH atorvastatin treated group vs. CSDH saline treated group.
Figure 6. Atorvastatin treatment improves neurological and cognitive outcome in CSDH rats. A) CSDH induces significant neurological impairment compared to sham control rats, and Atorvastatin treatment improves neurological function as indicated by foot-fault test. B) CSDH induces significant cognitive impairment compared to sham control rats, and Atorvastatin treatment improves spatial learning and memory as indicated by Morris water maze test. #P<0.05, CSDH non-treated group vs. sham control group and *P<0.05, CSDH Atorvastatin treated group vs. CSDH-non-treated group.
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