Expression of Caspase Signaling Components in the Outer Membranes of Chronic Subdural Hematomas

Abstract

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.

Introduction

Chronic subdural hematoma (CSDH) occurs frequently in elderly patients after mild head injury. The accumulation of liquefied blood in the subdural space induces slowly progressing symptoms such as headache, hypesthesia, and hemiparesis. Surgical intervention under local anesthesia is accepted as one of the best treatments, even in elderly patients. Spontaneous resolution of CSDH has been reported in some cases, however.^1
–3 We have also encountered cases in which septal membranes within a CSDH still existed after trepanation surgery, especially in trabecular CSDH. Even in such cases, the CSDH outer membrane, including the septum, gradually resolved with no further surgical treatment (Supplementary Fig. 1; see online supplementary material at ftp.liebertpub.com).

CSDH is believed to be an angiogenic and inflammatory disease. The expression of vascular endothelial growth factor (VEGF) is increased in CSDH fluid compared with serum.^4
–6 In previous studies, we showed that VEGF activates the mitogen-activated protein kinase (MAPK) signaling pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and nuclear factor-kB (NF-kB) in endothelial cells of vessels,^7

–10 suggesting that these molecules are involved closely in CSDH angiogenesis. Moreover, expression of the inflammatory cytokine interleukin-6 (IL-6) is increased in CSDH fluids compared with serum,⁵ and IL-6 activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway in endothelial cells and fibroblasts in CSDH outer membranes.^11,12 Transforming growth factor β (TGF-β), which is enriched in CSDH fluids, activates the Smad signaling pathway in fibroblasts and induces their growth.¹³ These signaling pathways may play an important role in the enlargement of the outer membrane. Mechanisms that drive the resolution of CSDH outer membranes have not been fully clarified, however.

Apoptosis is a selective process of programmed cell death that is essential to the homeostasis of organisms. Fas, a cell surface protein with a relative molecular weight of 45 kDa, transduces apoptotic signals in cells.^14,15 Fas-associated death domain (FADD) functions as an important adaptor in coupling death signaling from membrane receptors, such as those in the Fas and tumor necrosis factor (TNF) families.¹⁶ Overexpression of FADD in MCF7 breast carcinoma cells and BJAB cells induces apoptosis.¹⁶ Caspase-8 binds to the death effector domain of FADD, and on overexpression, it induces apoptosis.¹⁷ Caspase-8 subsequently activates downstream effector caspases, such as caspase-3, resulting in the cleavage of proteins involved in the execution of apoptosis.

The TNF receptor type 1 (TNFR1)-associated death domain (TRADD) interacts specifically with the death domain of TNFR1 and can activate caspase-8.¹⁸ Overexpression of TRADD leads to two major TNF-induced responses: apoptosis and activation of NF-kB.¹⁸ Receptor-interacting protein (RIP) contains a death domain response element that enables interaction with the death domain receptor Fas¹⁹ and recruitment to TNFR1 by TRADD.²⁰ Overexpression of RIP has a role in apoptosis and NF-kB activation.^19,20

Caspase-9 is also an important member of the cysteine aspartic acid protease family and further activates caspase-3 and caspase-7 to initiate caspase signaling.²¹ Caspase-7 is an effector molecule responsible for cleaving downstream substrates such as poly-(adenosine diphosphate [ADP]-ribose) polymerase (PARP).²² Caspase-3 is a critical effector of apoptosis and is responsible for the proteolytic cleavage of key proteins such as PARP and deoxyribonucleic acid (DNA) fragmentation factor 45 (DFF45).²³ Caspase signaling thus plays important roles in cell death (Fig. 1A), which ensures normal tissue turnover.

FIG. 1.

Caspase signaling pathway (A). Western blotting showing protein expression patterns in the outer membranes of chronic subdural hematomas from eight patients (B). The membranes were homogenized in homogenization buffer and subjected to Western blotting with anti-Fas (α-Fas); anti-Fas-associated death domain (α-FADD); anti-tumor necrosis factor receptor type 1-associated death domain (α-TRADD); anti-receptor-interacting protein (α-RIP); anti-caspase-3, 8, 9, and 7 (α-Caspase-3, 8, 9, and 7); anti-poly-(ADP-ribose) polymerase (α-PARP); anti-DNA fragmentation factor 45 (α-DFF45), and anti-β-actin (α-β-actin) antibodies. Notably, all molecules involved in the caspase signaling pathway were detected in nearly all cases. Positive CNT: positive control using Jurkat cells.

The present study explored the mechanism that enables resolution of CSDH outer membranes with a focus on caspase signaling. To accomplish this, we performed immunoblotting and immunohistochemical analyses of CSDH outer membranes.

Methods

Patients

Eight patients (six men and two women; 59–79 years; mean age, 68 years) with CSDH confirmed by computed tomography (CT) or magnetic resonance imaging (MRI) were enrolled in this study. All patients underwent a burr hole drainage surgical procedure under local anesthesia at Aichi Medical University Hospital. The Ethics Committee of Aichi Medical University approved this clinical study.

Materials

All chemicals were obtained from Sigma Chemicals (St. Louis, MO) unless specified otherwise.

Western blotting analysis

Outer membranes from CSDHs were obtained during trepanation and then homogenized in 80 μL of homogenization buffer containing 50 mM Tris base/HCl (pH 7.5), 0.1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetate, 0.2 mM ethylene glycol bis(aminoethyl ether)tetraacetate, 0.2 mM phenylmethylsulfonyl fluoride, 1.25 μg/mL of pepstatin A, 0.2 μg/mL of aprotinin, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, and 1% Nonidet P-40. The homogenates were centrifuged at 12,000 × g for 10 min at 4°C. The protein concentrations of the supernatants were determined using a Bradford assay with bovine serum albumin as the standard. The crude samples (25 μg of protein each) were separated using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

The proteins were transferred to polyvinylidene difluoride membranes and incubated with primary polyclonal antibodies against β-actin (Sigma), PARP (Cell Signaling Technology, Danvers, MA), caspase-3 (Cell Signaling Technology), caspase-8 (Cell Signaling Technology), and caspase-9 (Cell Signaling Technology), each at a 1:750 dilution, as well as monoclonal antibodies against caspase-7 (1:750 dilution; BD Bioscience, San Jose, CA), DFF45 (1:500 dilution; BD Bioscience), RIP (1:750 dilution; BD Bioscience), TRADD (1:250 dilution; BD Bioscience), FADD (1:250 dilution; BD Bioscience), and Fas (1:750 dilution BD Bioscience) overnight at 4°C. After being washed, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Sigma) at a 1:3000 dilution for 30 min at room temperature. The reactions were developed with ECL Plus (GE Healthcare, Buckinghamshire, UK). Untreated Jurkat cells were lysed in Chaps cell extract buffer, and a cytoplasmic fraction (Cell Signaling Technology) served as the positive control.

Histological examinations

To study the cellular localization of caspase-3, PARP, and cleaved caspase-3, immunohistochemical staining was performed on samples from three patients at room temperature using the avidin-biotinylated peroxidase complex (ABC) technique. To preserve the outer membranes of the CSDH samples, they were incubated in 10 mL of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h. Serial axial cryostat sections (10 μm) were placed on slides for staining. Nonspecific immunoreactivity was blocked by incubation with goat serum for 30 min.

The samples were treated with primary antibodies against caspase-3 (Cell Signaling Technology) at a dilution of 1:300, PARP (Cell Signaling Technology) at a dilution of 1:100, and cleaved caspase-3 (Cell Signaling Technology) at a dilution of 1:100 overnight at 4°C. After being washed, the samples were incubated with biotinylated anti-rabbit immune globulin G (IgG) for 1 h and then ABC for 1 h. Sera for the blocking step, biotinylated antibodies, and ABC were purchased from Vector Laboratories (Burlingame, CA). The reaction products were developed by incubating the sections in 0.05% 3,3'-diaminobenzidine tetrachloride and 0.01% H₂O₂ in 50 mM Tris-HCl (pH 7.5) for 10 min.

Results

Clinical data

Clinical data are presented in Table 1. All patients had a history of mild head injury without any hemostatic disorder, and no patients had received antiplatelet or anticoagulation therapy. Some patients had hypertension, but none were prescribed β-blockers. The Glasgow Outcome Scale showed good recovery to previous levels of daily living activity in all patients except one who had dementia.

Table 1.

Clinical Data for the Eight Patients with Chronic Subdural Hematoma

Case	Age, sex	Mechanism of injury	Trauma (weeks ago)	GCS	Symptoms	Comorbidity	Medication	GOS
1	75, M	Traffic accident	10	15	Hemiparesis	DM	Sitagliptin	GR
2	66, M	Mild head trauma	6	15	Headache, aphasia	None	None	GR
3	59, M	Fall	7	15	Headache	None	None	GR
4	68, F	Mild head trauma	8	14	Hemiparesis	Dementia	Donepezil	MD
5	74, M	Fall	6	15	Headache	Gastric cancer	None	GR
6	79, F	Mild head trauma	9	14	Hemiparesis	HL, HT	Pitavastatin, amlodipine	GR
7	63, M	Fall	8	14	Hemiparesis	DM, HT	Vildagliptin, olmesartan	GR
8	60, M	Fall	7	14	Hemiparesis	HT	Azilsartan	GR

GCS, Glasgow Coma Scale at admission; GOS, Glasgow Outcome Scale three months after operation; DM, diabetes mellitus; GR, good recovery; MD, moderate disability; HL, hyperlipidemia; HT, hypertension.

Western blotting analysis of caspase signaling

Nearly constant β-actin levels were detected in all samples, which suggested that equal amounts of protein were applied to the SDS gels. Fas, caspase-3, 7, 8, and 9, and DFF45 were detected in nearly all samples (Fig. 1B). The FADD, TRADD, RIP, and PARP were detected; however, their signals were weaker in some cases (Fig. 1B). Western blotting analysis using positive controls, however, revealed that these molecules were correctly detected (Fig. 1B). All molecules of the caspase signaling pathway—that is, all the components that relay signals from the cell surface into the nucleus—were detected in CSDH outer membranes (Fig. 1A).

Histological observations

Hematoxylin and eosin staining of CSDH membranes showed fibroblasts located between collagenous fibers (Fig. 2A) that expressed caspase-3, PARP, and cleaved caspase-3 (Fig. 2B, D, and F, respectively). Caspase-3 expression was located in the cytoplasms of the fibroblasts (Fig. 2C), whereas PARP and cleaved caspase-3 expression were found in the nuclei of the fibroblasts (Fig. 2E and G, respectively). Caspase-3 was observed in the cytoplasms of endothelial cells (Fig. 2H), whereas PARP was observed in nuclei (Fig. 2I). For the negative controls without primary antibodies, the fibroblasts and the endothelial cells were consistently negative for the markers listed above (J and K arrowheads, respectively).

FIG. 2.

Hematoxylin and eosin staining demonstrating the presence of fibroblasts, collagenous fibers, inflammatory cells, and vessels (A). Ten-micrometer slices were immunostained with polyclonal antibodies against caspase-3 (B, C, H), poly-(ADP-ribose) polymerase (PARP, D, E, I) and cleaved caspase-3 (F, G) using the avidin-biotinylated peroxidase complex method. The areas within the rectangle, labeled in B, D, and F, are shown at higher magnification in panels C, E, and G, respectively. Note that caspase-3 is expressed in the cytoplasms of fibroblasts and endothelial cells (C and H, respectively), whereas PARP is expressed in the nuclei of these cells (E and I, respectively). Some fibroblasts showed especially high expression of cleaved caspase-3 in their nuclei (G). Immunostaining without primary antibodies is presented in fibroblasts and endothelial cells (J, K arrowheads, respectively). Scale bars = 50 μm (A, B, D, F, J).

Discussion

In this study, we used Western blotting analysis to evaluate the expression of caspase signaling molecules in CSDH outer membranes. Immunohistochemical analysis showed that caspase-3 was expressed mainly in the cytoplasms of fibroblasts and endothelial cells, whereas PARP was expressed in the nuclei of these cells. Further, some fibroblasts expressed cleaved caspase-3 within their nuclei.

The radiological characteristics of spontaneous resolution of CSDH have been investigated previously, as well as the mechanisms driving this resolution. The use of corticosteroids inhibits inflammation and neomembrane formation, promoting the resolution of CSDH.^24,25 Hyperosmotic agents are also a useful nonsurgical treatment method.²⁶ The modified smooth-muscle cells in the CSDH outer membrane may also play a role in the resolution of CSDH, because they produce collagen and reinforce the membrane, which reduces its fragility and makes it resistant to the rupture of microcapillaries.²⁷ Ventricular dilatation, which increases the counterpressure against CSDHs and low- or isodensity hematomas, is an important CT finding in cases of spontaneous resolution.¹ In addition, asymptomatic CSDHs localized in the frontal region with small mass signs can be expected to disappear spontaneously.² Although the above-referenced retrospective studies described the CT findings that characterize spontaneous resolution cases, the molecular mechanism enabling CSDH resolution remains unclear.

Apoptosis is involved in both pathological conditions and aging. In the present work, we detected all molecules of the caspase signaling pathway (i.e., from Fas located at the cell surface to PARP and DEF45 located in the nucleus) in CSDH outer membranes. Our immunohistochemical results revealed that caspase-3 is expressed in the cytoplasms of fibroblasts, and after cleavage, it translocates into the nucleus, where PARP is located. Likewise, in endothelial cells, caspase-3 is expressed in the cytoplasm and PARP the nucleus. PARP is one of the main cleavage targets of caspase-3, and cleaved PARP facilitates cellular disassembly and ensures the irreversibility of apoptosis.²⁸ DFF45 is a subunit of a heterodimeric DNase complex that is critical for inducing DNA fragmentation. DFF45 is cleaved by caspase-3 and/or caspase-7, resulting in nuclear DNA degradation by DFF40, which leads to apoptosis.^29,30 These findings suggest that caspase signaling is activated within CSDH outer membranes and has a role in the spontaneous resolution of CSDH. It should be noted, however, that the expression of death factors such as Fas ligand and TNF is highly important in terms of caspase signaling activation, and further studies are necessary to explore the precise mechanism activating this pathway.

Levels of growth factors and inflammatory cytokines are increased in CSDH fluid compared with serum. The VEGF plays an important role in angiogenesis through MAPK. In CSDH outer membranes, the extracellular signal-regulated dual kinase (MEK)/extracellular signal-regulated kinase (ERK), PI3K/Akt, and NF-kB signaling pathways are particularly important to the role of VEGF.^7

–10 The VEGF prevents apoptosis induced by high glucose or serum starvation in human endothelial cells by activating ERK.^31,32 The VEGF also inhibits staurosporine-induced apoptosis in human endothelial cells via the PI3K and ERK signaling pathways.³³ Tissue plasminogen activator (tPA) is also present at greater levels in CSDH fluid in serum, which causes local hyperfibrinolysis in CSDH fluid.⁴ Further, high tPA concentrations in CSDH fluid are associated with a high probability of recurrence.⁴ The tPA is a potent survival factor that prevents renal interstitial fibroblasts from undergoing apoptosis through the ERK pathway.³⁴ Moreover, the inflammatory cytokine IL-6 activates the JAK/STAT signaling pathway in endothelial cells and fibroblasts, resulting in the growth of CSDH outer membranes.^11,12 We have reported recently that JAK/STAT signaling is precisely regulated by soluble IL-6 receptor and soluble gp130 in CSDH fluid and by suppressor of cytokine signaling 3 (SOCS3) and protein inhibitor of activated STAT3 (PIAS3) in fibroblasts, which controls CSDH growth.³⁵ The SOCS3 promotes apoptosis by both aggravating inflammation and inhibiting JAK/STAT3 signaling activity in adipocytes.³⁶ Moreover, specific inhibition of STAT3 induces the onset of apoptosis in prostate cancer lines.³⁷ Considering these previous studies and our data, these growth factors and inflammatory cytokine are activated more strongly than the caspase signaling pathway in cases that require surgical treatment. Surgical treatment might decrease the levels of these factors in CSDH fluid, which activates caspase signaling and may play a role in the resolution of CSDH after the surgical procedure. Further, from the perspective of translational medicine, these molecules may be novel therapeutic targets for intractable CSDH.

This study has several limitations. First, our findings are purely observational. We measured the expression of caspase signaling molecules in CSDH outer membranes; however, we did not perform any animal experiments to corroborate our findings. Additional definitive experiments are necessary to determine whether these molecules are cleaved in CSDH outer membranes after trepanation. Second, no control cases, such as chronic subdural effusion, were included for comparing the expression levels of these molecules. Moreover, the mechanism of caspase signaling activation in cases of spontaneous resolution of CSDH without operation must still be elucidated.

Conclusion

This study was the first to examine the expression of caspase signaling pathway molecules in CSDH outer membranes. The results showed that cleaved caspase-3 is expressed in fibroblasts. In addition, the caspase signaling pathway may regulate precisely the growth and mediate the resolution of CSDH outer membranes, although additional molecules may also be involved in this resolution. Additional in vivo studies or clinical trials targeting the caspase signaling pathway are necessary to understand the potential of targeting its components to treat patients with recurrent and intractable CSDH.

Footnotes

Acknowledgments

This work was supported in part by a Japanese Grant-in-Aid for Scientific Research (C), Grant Number 26462174 and 17K10853 (K.O.), and by a grant (K.O.) from the General Insurance Association of Japan. We thank Masahiro Kokubo for his technical assistance.

Author Disclosure Statement

No competing financial interests exist.

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