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Chronic subdural hematoma (CSDH) is fundamentally treatable, although it sometimes recurs. We observed, however, several cases of spontaneous resolution of CSDH outer membranes, even in a trabecular type of CSDH, after a trepanation surgical procedure. In this study, we examined the expression of molecules of the autophagy signaling pathway in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation were included in this study. By Western blot analysis, we examined the expression of mammalian target of rapamycin (mTOR); GβL; UNC-51-like kinase-1 (ULK1); Beclin-1; autophagy-related genes (Atg) 3, 5, 7, 12, 13, and 16L1β,α; the autophagy marker Light Chain3A/B (LC3A/B); and β-actin, which constitute the autophagy signaling pathway. The expression levels of Beclin-1, Atg12, and LC3A/B were also examined by immunohistochemistry. Almost all of these molecules could be detected in all samples. Beclin-1, Atg12, and LC3A/B were found to be localized in the endothelial cells of vessels and fibroblasts in CSDH. We detected molecules of the autophagy signaling pathway in CSDH outer membranes. Autophagy contributes to the tissue homeostatic process, maintaining cellular integrity by clearing debris. Our data suggest that autophagy might play an important role in the spontaneous resolution of CSDH. Therefore, these molecules may be novel therapeutic targets for the treatment of those with CSDH.

Eosinophils induce inflammation by releasing cytokines and cytotoxic granule proteins. Infiltration of eosinophilic granulocytes occurs in the outer membrane of chronic subdural hematomas (CSDHs). Eosinophils play an important role in the growth of CSDHs. However, the manner in which eosinophils accumulate within CSDH fluid remains undetermined. In the current study, we assessed the expression of eosinophil chemoattractants in CSDH fluids according to growth stage of CSDHs and examined the correlation between the two. CSDH fluids were obtained from 38 patients during trepanation surgery. Ecalectin, eotaxin-3, interleukin-5 (IL-5), and eosinophil-derived neurotoxin (EDN) concentrations were measured using enzyme-linked immunosorbent assay kits. For use as controls, serum samples were collected from 5 healthy adults, and cerebrospinal fluid (CSF) samples were collected from 5 adults with unruptured aneurysms. The percentage of eosinophils (%eosinophil) in CSDH fluids was calculated using Giemsa staining. Concentrations of ecalectin, eotaxin-3, IL-5, and EDN were nearly equivalent in serum and CSF samples; however, their concentrations were high in CSDH fluids. In particular, ecalectin and EDN levels in CSDH fluids were significantly higher than those in serum and CSF. Levels of eotaxin-3, IL-5, EDN, and %eosinophil were significantly higher in laminar type of CSDH, whereas that of ecalectin was not. The correlations between eotaxin-3 and IL-5, IL-5 and EDN, and EDN and %eosinophil were statistically significant (p 0.01). Our data suggest that eotaxin-3 is a chemoattractant of eosinophils. IL-5 induces the activation of eosinophils subsequent to degranulation of EDN into CSDH fluids. These factors may serve as novel therapeutic targets for managing CSDH.

We previously demonstrated that the inflammatory cytokine interleukin-6 (IL-6) activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway in fibroblasts within the outer membranes of chronic subdural hematomas (CSDHs), and the activation of this pathway may induce CSDH outer membrane growth. The inhibitory system for this signal transduction pathway is unknown. CSDH fluids were obtained from 10 patients during trepanation surgery as the case group, and cerebrospinal fluid (CSF) samples were obtained from seven patients suffering from subarachnoid hemorrhage (SAH) on Day 1 as the control group. The concentrations of IL-6, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) in CSDH fluid and CSF were measured using enzyme immunoassay kits. The co-localization of IL-6 and sgp130 in CSDH fluid was examined by immunoprecipitation. The expression levels of STAT3, JAK2, suppressor of cytokine signaling 3 (SOCS3), and protein inhibitor of activated Stat3 (PIAS3) in the outer membranes of CSDHs were examined by immunostaining. Soluble IL-6R and sgp130 concentrations in CSDH fluid were significantly higher than those in CSF after SAH. Sgp130 and IL-6 were co-immunoprecipitated from CSDH fluid. Immunostaining revealed STAT3, JAK2, SOCS3, and PIAS3 expression in fibroblasts located in the outer membranes of CSDHs. Soluble gp130 binds to IL-6/sIL-6R and acts as an antagonist of the JAK/STAT signaling pathway. SOCS3 also binds to JAK and inhibits its signaling pathway. In addition, PIAS3 regulates STAT3 activation. These factors might down-regulate the IL-6/JAK/STAT signaling pathway in fibroblasts within CSDH outer membranes. Therefore, these molecules may be novel therapeutic targets for the inhibition of CSDH growth.

Growth factors and inflammatory cytokines activate the mitogen-activated protein kinase (MAPK) cascade. Previous studies have shown that chronic subdural hematoma (CSDH) fluid contains these factors and cytokines. In this study, expression of three major MAPK cascade transmitters in the outer membrane of CSDH was assessed. Eleven patients whose outer membrane and CSDH fluid were successfully obtained during trepanation surgery were included in this study. Expression of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, and actin was examined by Western blot and immunostaining. We examined whether CSDH fluid could activate MAPKs in cultured endothelial cells (ECs) or fibroblasts in vitro. Western blot analysis showed that p-ERK was present in all samples, whereas p-p38 and p-JNK were detected, but not in all cases. Immunostaining showed that all three p-MAPKs were expressed in vascular endothelium. However, only p-ERK was expressed in fibroblasts. Expression of p-extracellular signal-regulated kinase kinase (MEK) and p-ERK in ECs and fibroblasts was significantly induced immediately after treatment with CSDH fluid, whereas p-p38 and p-JNK expression was significantly induced in ECs 60 min after treatment, but not in fibroblasts. Activation of MEK was significantly inhibited by treatment with antibodies directed against interleukin-6 and vascular endothelial growth factor in ECs, but not in fibroblasts. Inflammatory cytokines and growth factors in CSDH fluids might activate major MAPKs in ECs, which might be associated with neovascularization in the outer membrane of CSDH. These MAPK pathways could become novel targets for treatment of CSDHs.

Chronic subdural hematoma (CSDH) is fundamentally treatable through surgery, although CSDH recurs in some cases. We have observed several cases of spontaneous resolution of CSDH outer membranes, including in trabecular CSDH, after trepanation surgery. In this study, we examined the expression of molecules involved in caspase signaling in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation surgery were included in this study. The expression of Fas; Fas-associated death domain (FADD); tumor necrosis factor receptor type 1-associated death domain (TRADD); receptor-interacting protein (RIP); caspases 3, 7, 8, and 9; poly-(ADP-ribose) polymerase (PARP); DNA fragmentation factor 45 (DFF45) and β-actin was examined by Western blot analysis. The expression levels of PARP, caspase-3, and cleaved caspase-3 were also examined by immunohistochemistry. Fas; FADD; TRADD; RIP; caspases 3, 7, 8, and 9; PARP, and DFF45 were detected in nearly all samples. Caspase-3 and PARP were localized in the endothelial cells of vessels and in fibroblasts in CSDH outer membranes. In addition, cleaved caspase-3 was detected in fibroblasts. We detected molecules of the caspase signaling pathway in CSDH outer membranes. In particular, cleaved caspase-3 was detected, which suggests that apoptosis may occur within these membranes. Thus, during the growth of CSDH outer membranes, the caspase signaling pathway may be restrained. Once the pathway is activated, gradual resolution of CSDH outer membranes may occur. Therefore, these molecules may be novel therapeutic targets for intractable CSDH.

Chronic subdural hematoma (CSDH) is considered to be an inflammatory disease. Eosinophils are frequently expressed in the outer membrane of CSDH and are major sources of transforming growth factor beta (TGF-β). The mothers against decapentaplegic (Smad)-signaling pathway, which is activated by TGF-β, has been shown to be involved with fibrosis. In the present study, we compared the concentrations of eotaxin-3, eosinophil-specific chemoattractant, and TGF-β between CSDH fluid and cerebrospinal fluid (CSF) from control patients. We also explored the expression of the Smad-signaling pathway in the outer membrane of CSDH. Eight patients whose outer membrane and 12 whose CSDH fluid were successfully obtained during trepanation surgery were included in the study. Concentrations of eotaxin-3 and TGF-β were measured by enzyme immunoassay kits. Expression levels of Smad2, phosphorylated Smad3, Smad3, Smad4, and actin were examined by Western blot analysis. In addition, expression of Smad3 was also examined by immunohistochemistry. Concentrations of eotaxin-3 and TGF-β in CSDH fluid were significantly higher than those in CSF. Smad2, Smad3, phosphorylated Smad3, and Smad4 were detected in all cases. Smad3 was shown to be present in fibroblasts. These findings indicate that eotaxin-3 is expressed in CSDH fluid, inducing eosinophils into the outer membrane and resulting in elevation of TGF-β with the Smad pathway activated by TGF-β. These data suggest a potential mechanism for CSDH formation and growth.