Surfactant Protein D Inhibits Interleukin-12p40 Production by Macrophages Through the SIRPα/ROCK/ERK Signaling Pathway.

OBJECTIVE: Interleukin (IL)-12 has a pivotal profibrotic role in the development of idiopathic pulmonary fibrosis (IPF). Medical research trials based on IPF registry databases have actively recruited patients. Surfactant protein D (SP-D) is a useful biomarker in patients with IPF. SP-D binds to signal regulatory protein α (SIRPα), which acts as an inhibitory receptor, and this SP-D/SIRPα interaction may have an anti-inflammatory effect. Accordingly, the inhibitory effect of SP-D on IL-12p40 production by lipopolysaccharide (LPS)-stimulated macrophages was investigated. MATERIALS AND METHODS: Human granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated macrophages (day 9 of culture) was used to investigate IL-12p40 production after stimulation with SP-D. RESULTS: GM-CSF was found to upregulate SIRPα expression by macrophages. PD98059 (an extracellular signal-regulated kinase [ERK] inhibitor) blunted induction of SIRPα expression by GM-CSF. SP-D significantly attenuated IL-12p40 production by macrophages after stimulation with LPS. Silencing of SIRPα/ß/γ significantly reversed this inhibitory effect of SP-D. In contrast, neither SB023580 (a p38α/ß MAPK inhibitor) nor BIRB796 (a p38γ/δ MAPK inhibitor) attenuated the inhibitory effect of SP-D on LPS-stimulated production of IL-12p40. Silencing of SHP also had no influence on this effect of SP-D. Interestingly, a Rho-associated protein kinase (ROCK) inhibitor (Y-27632) abolished the inhibition of LPS-stimulated IL-12p40 production by SP-D, whereas silencing of ERK 2 significantly blunted this effect of Y-27632. CONCLUSIONS: These findings suggest that SP-D inhibits LPS-stimulated production of IL-12p40 via the SIRPα/ROCK/ERK signaling pathway.

Roles of myeloperoxidase and GAPDH in interferon-gamma production of GM-CSF-dependent macrophages.

Interferon (IFN)-gamma is highly expressed in atherosclerotic lesions and may have an important role in atherogenesis. Myeloperoxidase (MPO), the most abundant protein in neutrophils, is a marker of plaque vulnerability and a possible bridge between inflammation and cardiovascular disease. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has also been implicated in the pathogenesis of atherosclerosis. The present study investigated the role of neutrophil activation in atherosclerosis. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IFN-gamma protein by GM-CSF-dependent-macrophages was investigated by enzyme-linked immunosorbent assay after stimulation with MPO. GM-CSF enhanced macrophage expression of the mannose receptor (CD206), which is involved in MPO uptake. MPO increased IFN-gamma production by GM-CSF-dependent macrophages in a concentration-dependent manner. Pretreatment of macrophages with small interfering RNA (siRNA) for CD206 or extracellular signal-regulated kinase (ERK)-2 attenuated IFN-gamma production, while siRNA for ERK-1 did not. GAPDH is known to bind to adenylate/uridylate (AU)-rich elements of RNA and may influence IFN-gamma protein expression by binding to the AU-rich element of IFN-gamma mRNA. Interestingly, pretreatment with siRNA for GAPDH significantly reduced IFN-gamma production by macrophages, while it did not affect TF protein expression. In conclusion, MPO upregulates IFN-gamma production by GM-CSF-dependent-macrophages via the CD206/ERK-2 signaling pathway, while silencing GAPDH reduces IFN-gamma production.

Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/ß-arrestin 2/Rho A signaling pathway.

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, ß-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/ß-arrestin 2/Rho A signaling pathway.

Human Neutrophil Elastase Induce Interleukin-10 Expression in Peripheral Blood Mononuclear Cells through Protein Kinase C Theta/Delta and Phospholipase Pathways.

OBJECTIVE: Neutrophils have an important role in the rapid innate immune response, and the release or active secretion of elastase from neutrophils is linked to various inflammatory responses. Purpose of this study was to determine how the human neutrophil elastase affects the interleukin-10 (IL-10) response in peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS: In this prospective study, changes in IL-10 messenger RNA (mRNA) and protein expression levels in monocytes derived from human PBMCs were investigated after stimulation with human neutrophil elastase (HNE). A set of inhibitors was used for examining the pathways for IL-10 production induced by HNE. RESULTS: Reverse transcription polymerase chain reaction (RT-PCR) showed that stimulation with HNE upregulated IL-10 mRNA expression by monocytes, while the enzyme-linked immunosorbent assay (ELISA) revealed an increase of IL-10 protein level in the culture medium. A phospholipase C inhibitor (U73122) partially blunt- ed the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expression was significantly reduced by a protein kinase C (PKC) inhibitor (Rottlerin). A calcium chelator (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester: TMB-8) inhibited the response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment with a broad-spectrum PKC inhibitor (Ro-318425) partly blocked the response to HNE. Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNA expression. CONCLUSION: These results indicate that HNE mainly upregulates IL-10 mRNA ex- pression and protein production in moncytes via a novel PKC theta/delta, although partially via the conventional PKC pathway.

A protease-activated receptor 2 agonist (AC-264613) suppresses interferon regulatory factor 5 and decreases interleukin-12p40 production by lipopolysaccharide-stimulated macrophages: Role of p53.

The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)-12 by macrophages. IRF5 is also a central mediator of toll-like receptor signaling and is a direct target of p53. Activation of protease-activated receptor 2 (PAR-2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR-2 agonists on expression of IRF5 and IL-12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration-dependent manner. HNE also caused a concentration-dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of ß-arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR-2 agonist, AC-264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC-264613 did not promote TLR4 transactivation. In conclusion, the PAR-2 agonist AC-264613 attenuated IRF5-associated IL-12p40 production by macrophages.

Chemokine profiles of human visceral adipocytes from cryopreserved preadipocytes: Neutrophil activation and induction of nuclear factor-kappa B repressing factor.

AIMS: In obesity, infiltration of adipose tissue by proinflammatory immune cells causes chronic low-grade inflammation. We investigated the chemokine profiles of human visceral adipocytes by the reverse transcription polymerase chain reaction and the effect of human neutrophil elastase (HNE) on monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels. MAIN METHODS: Human adipocytes were obtained from cryopreserved omental preadipocytes of subjects with a body mass index (BMI) <30kg/m(2) or >30kg/m(2) and were cultured to assess chemokine production. KEY FINDINGS: Chemokine responses associated with obesity-related inflammation were well preserved in cultured human adipocytes derived from cryopreserved preadipocytes. Visceral adipocytes from subjects with a BMI >30kg/m(2) expressed mRNA for MCP-1, regulated on activation, normal T cell expressed and secreted (RANTES), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), interleukin-8 (IL-8), lymphotactin-ß, and fractalkine. Although visceral adipocytes from subjects with a BMI <30kg/m(2) also expressed MCP-1, RANTES, ENA-78, and IL-8 mRNA, neither lymphotactin-ß nor fraktalkine mRNA was detected. Interestingly, expression of MCP-1 mRNA was decreased significantly after exposure to HNE (85×10(3)µM/L), suggesting the induction of nuclear factor-kappa B repressing factor. SIGNIFICANCE: Adipocytes from subjects with a BMI >30kg/m(2) or <30kg/m(2) have different chemokine profiles. Only adipocytes from subjects with a BMI >30kg/m(2) express lymphotactin-ß and fractalkine mRNA. Differential chemokine profiles of visceral adipocytes contribute to infiltration of adipose tissue by adaptive immune cells. Neutrophil activation is involved in induction of nuclear factor-kappa B repressing factor, resulting in regulation of immune cell trafficking.

Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. AIM: This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. METHODS: Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. DISCUSSION: Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation.

Mechanism of tissue factor production by monocytes stimulated with neutrophil elastase.

BACKGROUND: Monocytes and neutrophils are activated during disseminated intravascular coagulation. Tissue factor, the main initiator of coagulation, is expressed by monocytes, while elastase is released by neutrophils. AIMS: This study investigated tissue factor production by peripheral monocytes after stimulation with human neutrophil elastase. METHODS: Tissue factor mRNA levels were investigated by the reverse transcriptase-polymerase chain reaction and tissue factor protein production was assessed by western blotting when monocytes were exposed to neutrophil elastase with or without preincubation using various inhibitors. RESULTS: Neutrophil elastase upregulated tissue factor mRNA and protein levels in monocytes. Both U73122 (phospholipase C inhibitor) and TMB-8 (intracellular calcium antagonist) prevented the upregulation of tissue factor mRNA. SB203580 (p38 mitogen-activated protein kinase inhibitor) suppressed this response, but PD98059 (extracellular signal-regulated kinase inhibitor) did not. Ro-318425 (ATP-competitive and selective protein kinase C (PKC) inhibitor) and Go 6976 (inhibitor of conventional PKCs and PKCµ) blocked the upregulation of tissue factor mRNA expression. Go 6983 (broad-spectrum PKC inhibitor) and CGP 4125 (staurosporine analog) partially attenuated it, as did a PKC theta/delta inhibitor. CONCLUSIONS: Neutrophil elastase mainly enhances tissue factor production by monocytes via the phospholipase C/conventional PKC/p38 MAPK pathway, although a novel PKC is also involved.

The suppressive effects of dietary protein restriction on the progression of renal impairment in OLETF rats.

BACKGROUND: The suppressive effect of dietary protein restriction on the progression of diabetic nephropathy remains controversial. We investigated the effects of protein and energy restrictions on both albuminuria and morphology using diabetic-prone Otsuka Long-Evans Tokushima fatty (OLETF) rats. METHODS: In this study, male OLETF rats were divided into two groups according to their energy intake. They were then further divided into three subgroups based on their amount of dietary protein, which ranged between 10% and 30% of their total intake. Urinary albumin excretion (UAE) was used as a marker of renal impairment, and body weight fasting (F) and postchallenge (P), blood glucose (BG) levels, and systolic blood pressure (SBP) were all measured during various experimental periods up to 28 weeks of age. RESULTS: The OLETF rats fed with the high-calorie diet started to gain weight at 12 weeks, and their FBG and PBG were elevated at 22 weeks, while SBP did not differ between the two groups. In addition, UAE increased significantly in the rats fed with the high-calorie diet. However, the increasing rates of UAE with age were higher in the rats with a higher protein diet within the same energy groups. UAE correlated well with the amounts of dietary energy and protein at 16 and 28 weeks of age, while it correlated with both the FBG and PBG at only 28 weeks of age. A linear regression analysis, using the data obtained at 28 weeks, showed that the amount of protein intake and FBG explained 63.4% and 23.9% of the variation in UAE, respectively. Histological studies revealed that protein and energy restriction markedly reduced the sclerotic changes of the glomeruli. CONCLUSION: Dietary protein restriction starting very early in the life of OLETF rats, in combination with energy restriction, clearly suppressed UAE and the typical morphological changes that otherwise occurred at around 16 weeks of age. This method also seemed to be more effective than energy restriction alone in slowing down any increase in UAE. The influence of BG levels on UAE was lower at an early age, while it became an increasingly important factor at later ages in the experimental rat model.

Skull metastasis from clear cell chondrosarcoma.

A 67-year-old man presented with left lower cranial nerve paresis and dysfunction of the left cerebellar hemisphere 4 years after amputation of the left lower leg because of clear cell chondrosarcoma (CCC). Neuroimaging studies showed an osteolytic extradural mass with homogeneous enhancement in the left posterior fossa. Bone scintigraphy disclosed a single high-uptake lesion at the same site. The tumor was removed totally via a left suboccipital craniotomy. Histological examination found mainly clear cells arranged in a microlobular pattern separated by thin fibrovascular stroma. The nuclei were regular with few mitotic figures. Immunohistochemical staining showed the tumor cells reacted intensely for both S-100 protein and vimentin. Osteoclast-like multinucleated giant cells were found at the periphery of the lobules. The primary tumor showed the same findings and the metastatic tumor manifested no malignant change. The histological diagnosis was metastatic CCC. CCC is a very rare neoplasm with slow growth and low-grade malignancy. Distant metastasis is rare but can occur in the skull base bone despite radical resection of the primary tumor. Osteolytic findings of homogeneous enhancement on magnetic resonance imaging and a high uptake on bone scintigraphy are indicative of metastatic tumor from previous CCC.

The influence of sex and the presence of giant cells on postoperative long-term survival in adult patients with supratentorial glioblastoma multiforme.

OBJECT: Glioblastoma multiforme (GBM) remains incurable by conventional treatments, although some patients experience long-term survival. A younger age, a higher Karnofsky Performance Scale (KPS) score, more aggressive treatment, and long progression-free intervals have been reported to be positively associated with long-term postoperative patient survival. The aim of this retrospective study was the identification of additional favorable prognostic factors affecting long-term survival in surgically treated adult patients with supratentorial GBM. METHODS: Of 113 adult patients newly diagnosed with histologically verified supratentorial GBM who were enrolled in Phase III trials during the period between 1987 and 1998, six (5.3%) who survived for longer than 5 years were defined as long-term survivors, whereas the remaining 107 patients served as controls. All six were women and were compared with the controls; they were younger (mean age 44.2 years, range 31-60 years), and their preoperative KPS scores were higher (mean 85, range 60-100). Four of the six patients underwent gross-total resection. In five patients (83.3%) the progression-free interval was longer than 5 years and in three a histopathological diagnosis of giant cell GBM was made. This diagnosis was not made in the other 107 patients. CONCLUSIONS: Among adult patients with supratentorial GBM, female sex and histopathological characteristics consistent with giant cell GBM may be predictive of a better survival rate, as may traditional factors (that is, younger age, good KPS score, more aggressive resection, and a long progression-free interval).

Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

Myofibroblastoma in the suprasellar region. Case report.

Myofibroblastoma is a rare type of benign mesenchymal tumor; only two cases of intracranial myofibroblastoma have been reported in the literature. The authors report on the case of a 34-year-old woman with a myofibroblastoma in the suprasellar region who presented with the complaint of sudden onset of headache followed within 2 weeks by progressively worsening visual disturbance. Computerized tomography scanning demonstrated a mixed low- and high-density mass in the suprasellar region and contrast-enhanced magnetic resonance imaging revealed the mass to be of mixed intensity with heterogeneous enhancement. The tumor was subtotally removed via a right frontobasal translamina-terminalis approach and her vision improved immediately. Histologically, the tumor was characterized by alternating areas of spindle-shaped and round cells that were separated by collagen fibers. The diagnosis of myofibroblastoma was based on the tumor's intense immunoreactivity for alpha-smooth-muscle actin and the ultrastructural identification of myofibroblasts. The tumor was thought to have originated from the meninges in the suprasellar region.

A mixture of ethylene vinyl alcohol copolymer and ethanol yielding a nonadhesive liquid embolic agent to treat cerebral arteriovenous malformations: initial clinical experience.

OBJECT: The authors report their clinical experience with their new nonadhesive liquid embolic agent, an ethylene vinyl alcohol copolymer (EVAL)/ethanol mixture, to treat arteriovenous malformations (AVMs). METHODS: Between June 1995 and April 2001, 57 patients with confirmed AVMs underwent embolization of their lesions with the EVAL/ethanol mixture. In 87 procedures consisting of one to three stages, the authors embolized 185 feeding arteries to occlude as much of the AVM as possible. Repeated injections under fluoroscopic control could be performed smoothly without encountering cementing of the catheter to the vessel wall. Among the 87 embolizations undertaken in 57 patients, seven procedures (8%) in six patients produced new postembolization symptoms. Resolution of these symptoms occurred within hours or days after four of the seven procedures; permanent neurological deficits remained after the other three procedures (3.4%). Of the 57 patients, three underwent postembolization radiosurgery, and 54 underwent radical treatment with microsurgical extirpation. Histopathological examination of the 54 specimens disclosed mild inflammation within the embolized lumen without inflammatory reactions in the media or adventitia. Follow-up angiograms obtained 3 years after radiosurgery was administered showed that in all three patients treated in this fashion the nidus had completely disappeared. CONCLUSIONS: The EVAL/ethanol mixture is handled easily and appears to be an effective and safe agent for preoperative embolization of AVMs.

A nonadhesive liquid embolic agent composed of ethylene vinyl alcohol copolymer and ethanol mixture for the treatment of cerebral arteriovenous malformations: experimental study.

OBJECT: The authors have developed a mixture of ethylene vinyl alcohol copolymer (EVAL) and iopamidol, which is dissolved in ethanol, as an alternative solvent to provide a safe means of embolizing arteriovenous malformations (AVMs). METHODS: A two-stage delivery technique is required to prevent premature precipitation in the catheter when using this material: the catheter is first infused with 30% ethanol and this is followed by the delivery of the EVAL-ethanol mixture. Acute angiographic changes were analyzed after superselective delivery of dimethyl sulfoxide (DMSO) and 30% ethanol into the renal artery of rabbits. Histological changes following the embolization of the renal artery achieved using the EVAL-ethanol mixture were recorded at 1 hour and at 2 and 16 weeks after the procedure. Although DMSO always produced severe, rapidly progressive vasospasm in the renal artery during a 1- to 60-minute postinfusion, 30% ethanol did not. Microscopically, the lumens of embolized vessels examined 1 hour after embolization with EVAL-ethanol appeared to be filled with EVAL sponges, leaving almost no open spaces. The space between the EVAL sponges and the inner surface of the vessels was filled with fresh thrombus. In the vessel walls of specimens examined 2 weeks after embolization there was no or a slight inflammatory reaction. Scattered in the EVAL sponges were almost equal numbers of neutrophilic granulocytes and mononuclear cells, indicative of a mild inflammatory response. In specimens examined 16 weeks postembolization, the changes noted at 2 weeks were intensified. There was no definite histopathological evidence of mural hemorrhage, perivascular extravasation of the mixture, or perivascular hemorrhage in any specimen that was examined. CONCLUSIONS: Although the degree of permanence of this embolization material is yet unknown, the mixture was easy to handle, and appeared safe and effective for AVM embolization. Its nonadhesive characteristic and its ability to be infused by repeated injections make it an attractive alternative to currently available materials. The good results obtained in this study led us to undertake a clinical trial, the results of which are contained in a companion article in this issue of the Journal of Neurosurgery.