Evaluation of fibrin sealants for central nervous system sealing in the mongrel dog durotomy model.

BACKGROUND: Watertight repair of the dura is imperative after neurosurgical procedures involving the brain or spinal cord because inadequately treated leakage of cerebrospinal fluid (CSF) from punctured dura can have serious consequences such as meningitis, arachnoiditis, or epidural abscess. OBJECTIVE: To assess the efficacy of Evicel Fibrin Sealant (Human) to prevent CSF leakage using a 2.0-cm durotomy mongrel dog repair model and to compare the tissue response with Tisseel (a fibrin sealant) and Duraseal (a synthetic polyethylene glycol [PEG] hydrogel sealant). METHODS: The canine durotomy repair model was used. This well-characterized model assesses the ability of sealants to achieve intraoperative watertight seals of the dura mater, as well as long-term safety and efficacy. This study included 27 mongrel dogs and had a 28-day duration. RESULTS: The 3 sealants were 100% effective in preventing CSF leakage intraoperatively at 15 mm Hg. The 2 fibrin sealants were 100% effective in postoperative sealing; the PEG hydrogel was not. Microscopically, the tissue changes induced by Evicel at the durotomy site were similar in nature except for foamy macrophages seen only with the PEG hydrogel. The extent and severity of adhesions at 28 days were less with the fibrin sealants than with the PEG hydrogel. CONCLUSION: Evicel, a fibrin sealant, was safe and effective in achieving and maintaining a watertight seal of the dura. The performance of the fibrin sealants was similar to that of the synthetic PEG hydrogel sealant with the exception of a Duraseal seal, which leaked.

Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells.

OBJECTIVE: A proapoptotic BH3-only protein BIM (BCL-2 interacting mediator of cell death) can link cytokine receptor signaling with the apoptotic machinery in hematopoietic cells. We investigated here the role of BIM in erythropoietin (EPO)-mediated survival in erythroid cells. MATERIALS AND METHODS: We downregulated BIM in EPO-dependent HCD57 erythroid cells with short hairpin RNA (shRNA), and used real-time polymerase chain reaction, Western blots, and flow cytometry to characterize BIM expression and apoptosis. Hematologic analyses of BIM-deficient (Bim(-/-)) mice were conducted. RESULTS: BIM expression increases in primary murine erythroid cells and HCD57 cells deprived of EPO. Whereas Bim mRNA increased less than twofold, BIM protein increased more than 10-fold after EPO withdrawal, suggesting posttranscriptional regulation of BIM. EPO treatment resulted in rapid phosphorylation of BIM at Serine 65 and phosphorylation correlated with degradation of BIM. Inhibition of extracellular signal-regulated kinase (ERK) by a MEK/ERK inhibitor, U0126, blocked both phosphorylation and degradation of BIM, resulting in apoptosis. Treatment with a proteasome inhibitor, MG-132, also blocked degradation of phosphorylated BIM. Downregulation of BIM with the shRNA resulted in HCD57 cells more resistant to apoptosis induced by either EPO withdrawal or ERK inhibition. Although we observed no significant changes in the number of erythrocytes or reticulocytes in the circulation of Bim(-/-) mice, erythroid progenitors from bone marrow in Bim(-/-) mice were reduced in number and more resistant to apoptosis induced by U0126 MEK/ERK inhibitor. CONCLUSION: EPO protects erythroid cells from apoptosis in part through ERK-mediated phosphorylation followed by proteasomal degradation of BIM.

Role of JunB in erythroid differentiation.

The role of junB as a regulator of erythroid cell survival, proliferation, and differentiation was tested by controlled expression of JunB in the erythropoietin (EPO)-dependent erythroleukemia cell line HCD57. JunB induced erythroid differentiation as evidenced by increased expression of the erythroid-specific proteins beta-globin, spectrin-alpha, and TER-119. Expression of JunB for at least 48 h was required for the differentiated phenotype to emerge. Differentiation was accompanied by a slower rate of proliferation and an increase in the expression of the cell cycle inhibitory protein p27. p27 protein expression increased due to reduced turnover without changes in transcription, indicating global changes in cell physiology following JunB induction. JunB expression was also studied in mouse and human primary erythroid cells. JunB expression increased immediately in both primary mouse cells and HCD57 cells treated with EPO and quickly returned to base-line levels, followed by a secondary rise in JunB in primary erythroid cells, but not in HCD57 cells, 36-48 h later. This result suggested that the initial EPO-dependent JunB induction was not sufficient to induce differentiation, but that the late EPO-independent JunB expression in primary erythroid cells was necessary for differentiation. This study suggests that JunB is an important regulator of erythroid differentiation.