雷珠单抗联合Ahmed青光眼阀植入术对新生血管性青光眼患者眼动脉血流动力学和血清VEGF、PDGF的影响

摘要 目的：观察雷珠单抗联合Ahmed青光眼阀植入术治疗新生血管性青光眼（NVG）的疗效及对眼动脉血流动力学和血清血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）的影响。方法：选择2018年4月~2021年2月期间华中科技大学同济医学院附属协和医院眼科收治的NVG患者60例94眼,采用随机数字表法将患者分为对照组和研究组,分别为30例48眼和30例46眼。对照组患者予以Ahmed青光眼阀植入术,研究组眼内注射雷珠单抗一周后再行Ahmed青光眼阀植入术,观察治疗效果,对比两组眼动脉血流动力学和血清VEGF、PDGF,观察虹膜与前房角新生血管消退率及并发症发生率。结果：研究组的疗效明显优于对照组（P<0.05）。研究组治疗后3个月血流阻力系数（RI）低于对照组,收缩期峰值流速（PSV）、舒张末期流速（EDV）高于对照组（P<0.05）。治疗后3个月两组VEGF、PDGF均下降,且研究组低于对照组（P<0.05）。治疗后3个月两组眼压下降,视力升高,且研究组的改善程度优于对照组（P<0.05）,研究组虹膜与前房角新生血管消退率高于对照组（P<0.05）。研究组的并发症发生率少于对照组（P<0.05）。结论：NVG采用Ahmed青光眼阀植入术联合雷珠单抗治疗,可有效恢复视力、眼压,具有更好的疗效,这可能与联合治疗可改善眼动脉血流动力学及血清VEGF、PDGF水平有关。     

Computational Modeling of Fluid Flow and Intra-Ocular Pressure following Glaucoma Surgery

Background

Glaucoma surgery is the most effective means for lowering intraocular pressure by providing a new route for fluid to exit the eye. This new pathway is through the sclera of the eye into sub-conjunctival tissue, where a fluid filled bleb typically forms under the conjunctiva. The long-term success of the procedure relies on the capacity of the sub-conjunctival tissue to absorb the excess fluid presented to it, without generating excessive scar tissue during tissue remodeling that will shut-down fluid flow. The role of inflammatory factors that promote scarring are well researched yet little is known regarding the impact of physical forces on the healing response.

Methodology

To help elucidate the interplay of physical factors controlling the distribution and absorption of aqueous humor in sub-conjunctival tissue, and tissue remodeling, we have developed a computational model of fluid production in the eye and removal via the trabecular/uveoscleral pathways and the surgical pathway. This surgical pathway is then linked to a porous media computational model of a fluid bleb positioned within the sub-conjunctival tissue. The computational analysis is centered on typical functioning bleb geometry found in a human eye following glaucoma surgery. A parametric study is conducted of changes in fluid absorption by the sub-conjunctival blood vessels, changes in hydraulic conductivity due to scarring, and changes in bleb size and shape, and eye outflow facility.

Conclusions

This study is motivated by the fact that some blebs are known to have ‘successful’ characteristics that are generally described by clinicians as being low, diffuse and large without the formation of a distinct sub-conjunctival encapsulation. The model predictions are shown to accord with clinical observations in a number of key ways, specifically the variation of intra-ocular pressure with bleb size and shape and the correspondence between sites of predicted maximum interstitial fluid pressure and key features observed in blebs, which gives validity to the model described here. This model should contribute to a more complete explanation of the physical processes behind successful bleb characteristics and provide a new basis for clinically grading blebs.     

Clinical Outcomes of FP-7/8 Ahmed Glaucoma Valves in the Management of Refractory Glaucoma in the Mainland Chinese Population

BackgroundTo evaluate the efficacy and safety of the Ahmed glaucoma valve (AGV) and the risk factors associated with AGV implantation failure in a population of Chinese patients with refractory glaucoma.MethodIn total, 79 eyes with refractory glaucoma from 79 patients treated in our institution from November 2007 to November 2010 were enrolled in this retrospective study. The demographic data, preoperative and postoperative intraocular pressures (IOPs), best corrected visual acuity (BCVA), number of anti-glaucoma medications used, completed and qualified surgery success rates and postoperative complications were recorded to evaluate the outcomes of AGV implantation. Factors that were associated with implant failure were determined using Cox proportional hazard regression model analysis and multiple linear regression analysis.ConclusionAGV implantation was safe and effective for the management of refractory glaucoma. Patients with a greater number of previous surgeries were more likely to experience surgical failure, and patients with previous trabeculectomy were more likely to use multiple anti-glaucoma drugs to control postoperative IOP.     

Validation of the glaucoma filtration surgical mouse model for antifibrotic drug evaluation

Glaucoma is a progressive optic neuropathy, which, if left untreated, leads to blindness. The most common and most modifiable risk factor in glaucoma is elevated intraocular pressure (IOP), which can be managed surgically by filtration surgery. The postoperative subconjunctival scarring response, however, remains the major obstacle to achieving long-term surgical success. Antiproliferatives such as mitomycin C are commonly used to prevent postoperative scarring. Efficacy of these agents has been tested extensively on monkey and rabbit models of glaucoma filtration surgery. As these models have inherent limitations, we have developed a model of glaucoma filtration surgery in the mouse. We show, for the first time, that the mouse model typically scarred within 14 d, but when augmented with mitomycin C, more animals maintained lower intraocular pressures for a longer period of time concomitant with prolonged bleb survival to beyond 28 d. The morphology of the blebs following mitomycin C treatment also resembled well-documented clinical observations, thus confirming the validity and clinical relevance of this model. We demonstrate that the antiscarring response to mitomycin C is likely to be due to its effects on conjunctival fibroblast proliferation, apoptosis and collagen deposition and the suppression of inflammation. Indeed, we verified some of these properties on mouse conjunctival fibroblasts cultured in vitro. These data support the suitability of this mouse model for studying the wound healing response in glaucoma filtration surgery, and as a potentially useful tool for the in vivo evaluation of antifibrotic therapeutics in the eye.     

Long-Term Outcomes and Complications of Trabeculectomy for Secondary Glaucoma in Patients with Familial Amyloidotic Polyneuropathy

Objective

Secondary glaucoma is a serious complication in patients with transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP). We assessed the long-term outcomes and complications of trabeculectomy with mitomycin C (MMC) for secondary glaucoma associated with FAP.

Methods

Medical case records of Kumamoto University Hospital were retrospectively reviewed. Twenty-one eyes of 13 patients (10 with FAP ATTR Val30Met; 3 with FAP ATTR Tyr114Cys) underwent trabeculectomy with MMC and follow-up of at least 2 years. The primary outcome measure was Kaplan-Meier survival, with failure of this treatment being defined as an intraocular pressure (IOP) of ≤5 mm Hg or ≥22 mm Hg on two consecutive visits or as additional operations needed to reduce IOP. Secondary outcome measures included complications, bleb characteristics, and additional postoperative interventions required.

Results

The mean postoperative follow-up period was 5.7 years (range, 2.2–12.7 years). Kaplan-Meier analysis indicated probabilities of success of 0.76, 0.67, and 0.53 at 1, 2, and 3 years after operation, respectively. Significant complications included ocular decompression retinopathy in 7 eyes (33%) and bleb encapsulation in 10 eyes (48%). Twelve eyes (57%) needed additional surgery, such as bleb revision or trabeculectomy with MMC, to reduce IOP.

Conclusions

Trabeculectomy with MMC may not be optimal for patients with FAP-related glaucoma and may have several significant complications.     

Dry Eye Disease in Patients with Functioning Filtering Blebs after Trabeculectomy

Purpose

The aim of this study was to analyze dry eye disease (DED) in patients with functioning filtering blebs and to explore the relationship between the morphology of filtering blebs and ocular surface instability.

Methods

This was a cross-sectional, case-comparison study. Seventy glaucomatous patients (70 eyes) with functioning blebs who had undergone trabeculectomy more than 6 months prior (study group) and 35 control subjects (35 eyes) (control group) were included. All subjects completed an ocular symptom questionnaire that referred to the Shihpai Eye Study. Evaluation of meibomian gland obstruction, a tear film break-up time test (TFBUT), fluorescein corneal staining and a Schirmer’s tear test were then performed. Filtering bleb morphology was analyzed using Wuerzburg bleb classification scoring criteria in the study group. The presence of DED was defined as the concomitant presence of TFBUT <10 seconds and the presence of superficial punctate keratitis.

Results

The patients with functioning blebs presented higher corneal staining scores (P = 0.012) and lower TFBUT values (P = 0.043) than the control group. DED was present in 28/70 patients in the study group and 6/35 patients in the control group (P = 0.018). More patients in the study group complained of dryness (P = 0.001), a gritty or sandy sensation (P < 0.001) and redness (P = 0.048). In the study group, the patients with DED were significantly different from the patients without DED in both TFBUT (P < 0.001) and corneal staining (P < 0.001). More patients in the DED group were likely to report dryness (P = 0.013) and watery or teary eyes (P = 0.012). The differences in meibomian gland obstruction scores between the study and the control group, the DED and the non-DED group were not significant (P = 0.105 and P = 0.077, respectively). The values for microcysts and bleb heights were significantly higher in the DED group (P = 0.040 and P = 0.011, respectively). A Spearman’s rank correlation showed that microcysts were positively correlated with corneal staining (r = 0.270, P = 0.024). Bleb height was negatively correlated with TFBUT (r = -0.299, P = 0.012) and positively correlated with corneal staining (r = 0.275, P = 0.021). The relationships between DED and microcysts and between DED and bleb height were significant (r = 0.247, P = 0.039 and r = 0.307, P = 0.010, respectively).

Conclusion

DED is relatively common in patients with functioning filtering blebs following trabeculectomy. In DED patients, dryness and watery are common symptoms. Microcysts and bleb height are related to ocular surface instability and DED.     

Electron Microscope Studies of Nuclear Extrusions in Pancreatic Acinar Cells of the Rat

This paper describes "blebs" protruding from the surface of the nucleus into the cytoplasm. The "blebs" are separated from the cytoplasm by 2 membranes which are continuous with the outer and inner nuclear membranes. The "blebs" contain 3 structurally distinct substances. Two of these substances (β and γ substances) are similar to extranucleolar karyoplasm and nucleolar material. The other substance (α substance) is present in every "bleb," but it cannot be readily compared to a recognizable nuclear structure. Cytoplasmic vesicles are described that are apparently different from the Golgi vesicles or the vesicular component of the ergastoplasm. It is suggested that these vesicles may be of nuclear "bleb" origin. A dark karyoplasmic zone extending from the region of the nucleolus into the nuclear "bleb" is shown. This zone may be similar in some respects to the preformed pathway ("Leitbahn") described by Altmann (3) and Hertl (28) and could reflect movement of nuclear material from the nucleolar region into the cytoplasm. The "blebs" are thought to be homologous to structures described by many light microscopists, but they are considerably larger than the nuclear "blebs" described previously by electron microscopists.     

Tegafur Substitution for 5-Fu in Combination with Actinomycin D to Treat Gestational Trophoblastic Neoplasm

Although 5-fluorouracil (5-Fu) combination chemotherapy provides a satisfactory therapeutic response in patients with gestational trophoblastic neoplasms (GTNs), it has severe side effects. The current study analyzed the therapeutic effects and side effects of tegafur plus actinomycin D (Act-D) vs. 5-Fu plus Act-D for the treatment of GTNs based on controlled historical records. A total of 427 GTN cases that received tegafur and Act-D combination chemotherapy at the Second Xiangya Hospital of XiangYa Medical School between August 2003 and July 2013 were analyzed based on historical data. A total of 393 GTN cases that received 5-Fu plus Act-D between August 1993 and July 2003 at the same hospital were also analyzed, which constituted the control group. The therapeutic effects, toxicity and side effects after chemotherapy were compared between the groups. The overall response rate was 90.63% in the tegafur+Act-D group (tegafur group) and 92.37% in the 5-Fu+Act-D group (5-Fu group); these rates were not significantly different (P > 0.05). However, the incidence rates of myelosuppression (white blood cell decline), gastrointestinal reactions (nausea, vomiting, dental ulcer, and diarrhea), skin lesions and phlebitis were lower in the tegafur group than in the 5-Fu group (P < 0.05). The results of this study may provide useful data for the clinical application of tegafur in GTN treatment.     

Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.     

New Continuous Air Pumping Technique to Improve Clinical Outcomes of Descemet-Stripping Automated Endothelial Keratoplasty in Asian Patients with Previous Ahmed Glaucoma Valve Implantation

Background

To evaluate the outcomes of Descemet-stripping automated endothelial keratoplasty (DSAEK) with the use of continuous air pumping technique in Asian eyes with previous Ahmed glaucoma valve implantation.

Methods

The DSAEK procedure was modified in that complete air retention of the anterior chamber was maintained for 10 min using continuous air pumping at 30 mm Hg. The primary outcome measurement was graft survival, and postoperative clinical features including, rate of graft detachment, endothelial cell count, intraocular pressure (IOP), surgical time and cup/disc ratio were also recorded.

Results

A total of 13 eyes of 13 patients which underwent modified DSAEK and 6 eyes of 6 patients which underwent conventional DSAEK were included. There was a significant difference in graft survival curves between two groups (P = 0.029); the 1-year graft survival rates were estimated as 100% and 66.7% for patients with modified DSAEK and those with traditional DSAEK, respectively. The rate of graft detachment were 0% and 33.3% for the modified DSAEK and conventional DSAEK groups, respectively (P = 0.088). The significantly lowered surgical time for air tamponade was noted in the modified DSAEK group compared to that in the conventional DSAEK group [median (IQR): 10.0 (10.0, 10.0) min vs. 24.5 (22.0, 27.0) min; P<0.001] Postoperatively, patients in the modified DSAEK group had significantly lower IOP as compared to the conventional DSAEK group [12.0 (11.0, 15.0) mm Hg vs. 16.0 (15.0, 18.0) mm Hg; P = 0.047]. Modified DSAEK patients had higher endothelial cell counts as compared to conventional DSAEK patients [2148.0 (1964.0, 2218.0) vs. 1529.0 (713.0, 2014.0)], but the difference did not reach statistical significance (P = 0.072).

Conclusions

New continuous air pumping technique in DSAEK can be performed safely and effectively in patients with prior GDDs placement who have corneal failure.     

Prevention of ocular scarring post glaucoma filtration surgery using the inflammatory cell and platelet binding modulator saratin in a rabbit model

Clinical Relevance

Late complications can occur with use of current antimetabolites to prevent scarring following glaucoma filtration surgery (GFS). Safer, more targeted, anti-fibrosis agents are sought.

Objectives

The protein saratin has been shown to exhibit anti-fibrotic and anti-thrombotic properties in response to injury, but had not been used for glaucoma surgery. The goal of this study was to compare the efficacy of saratin with that of the widely accepted mitomycin-C (MMC) in prolonging bleb survival following GFS in the rabbit model. Two saratin delivery routes were compared; a single intraoperative topical application versus a combination of intraoperative topical application with two additional postoperative injections.

Methods

Twenty-four New Zealand White rabbits underwent GFS and received either intraoperative topical saratin, intraoperative topical saratin plus two injections on post-operative days 4 and 8, balanced saline solution (BSS), or MMC. The bleb tissues and their elevation durations were compared based on clinical and histological findings.

Results

Rabbits receiving topical+injections of saratin had a mean bleb survival of 33.6±8.5 days, significantly higher than the negative BSS controls, which averaged 17.4±6.0 days (p = 0.018). No improvement over BSS was seen for rabbits receiving topical saratin only (15.5±4.8 days, p = 0.749). Rabbits receiving saratin did not develop bleb avascularity and thinning associated with MMC treatment and there were no apparent clinical signs of toxicity.

Conclusions

Treatment with a single intraoperative topical application plus two additional postoperative injections significantly prolonged bleb elevation comparable to MMC, but without toxicity; however, topical application alone was ineffective.     

Sequential Therapy with Saratin,Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model

To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control—mean survival time of 19.7 ±2.7 days—(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)—mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.     

Analytical model for managing hypotony after implantation surgery of a glaucoma drainage device

The main aim of glaucoma treatment is to reduce the intraocular pressure (IOP). One of the most common surgical treatments of glaucoma is the implantation of a glaucoma drainage device to drain the aqueous humor from the anterior chamber to a filtration bleb, where the aqueous humor is absorbed. In some cases, the excess of drainage causes ocular hypotony, which constitutes a sight-threatening complication. To prevent hypotony after this intervention, surgeons frequently introduce a suture into the device tube, which increases the hydraulic resistance of the tube and, therefore, the IOP. This study aims to provide an analytical model to correct hypotony following implantation surgery of a glaucoma drainage device, which may help glaucoma surgeons decide on hypotony treatment. The results indicate that the IOP after implanting a cylindrical tube around 300 μm in diameter is essentially the same as that built up in the filtering bleb and can hardly be controlled by introducing a straight suture unless the suture diameter is slightly lower than that of the tube. On the contrary, when the tube diameter is smaller than, for example, 100 μm, significant reductions of the IOP can be obtained by introducing a thin suture into the tube.

     

Comparison of Ahmed Glaucoma Valve Implantation and Trabeculectomy for Glaucoma: A Systematic Review and Meta-Analysis

ObjectiveTo compare the efficacy and safety of Ahmed glaucoma valve implantation (AGV) with trabeculectomy in the management of glaucoma patients.MethodsA comprehensive literature search (PubMed, Embase, Google, and the Cochrane library) was performed, including a systematic review with meta-analysis of controlled clinical trials comparing AGV versus trabeculectomy. Efficacy estimates were the weighted mean differences (WMDs) for the percentage intraocular pressure reduction (IOPR %) from baseline to end-point, the reduction in glaucoma medications, and the odds ratios (ORs) for complete and qualified success rates. Safety estimates were the relative risks (RRs) for adverse events. All outcomes were reported with a 95% confidence interval (CI). Statistical analysis was performed using the RevMan 5.0 software.ResultsSix controlled clinical trials were included in this meta-analysis. There was no significant difference between the AGV and trabeculectomy in the IOPR% (WMD = -3.04, 95% CI: -8.36- 2.26; P = 0.26). The pooled ORs comparing AGV with trabeculectomy were 0.46 (0.22, 0.99) for the complete success rate (P = 0.05) and 0.97 (0.78–1.20) for the quantified success rate (P = 0.76). No significant difference in the reduction in glaucoma medicines was observed (WMD = 0.24; 95% CI: -0.27–0.76; P = 0.35). AGV was found to be associated with a significantly lower frequency of all adverse events (RR = 0.71; 95%CI: 1.14–0.97; p = 0.001) than trabeculectomy, while the most common complications did not differ significantly (all p> 0.05).ConclusionAGV was equivalent to trabeculectomy in reducing the IOP, the number of glaucoma medications, success rates, and rates of the most common complications. However, AGV was associated with a significantly lower frequency of overall adverse events.     

Postoperative Changes in Aqueous Monocyte Chemotactic Protein-1 Levels and Bleb Morphology after Trabeculectomy vs. Ex-PRESS Shunt Surgery

Purpose

To evaluate the postoperative changes in blebs and levels of aqueous monocyte chemotactic protein-1 (MCP-1) after trabeculectomy vs. Ex-PRESS tube shunt surgery.

Methods

Rabbits were subjected to trabeculectomy or Ex-PRESS tube shunt surgery and observed for up to 3 months. Intraocular pressure (IOP) was measured using a rebound tonometer. The MCP-1 level was measured by enzyme-linked immunosorbent assay (ELISA). Bleb morphology was evaluated using photos and anterior-segment optical coherence tomography (OCT).

Results

There were no differences in bleb appearance or IOP at any time between the groups. Bleb wall density in the anterior-segment OCT image was significantly lower 1 week after surgery in the Ex-PRESS group than the trabeculectomy group. The MCP-1 level in control eyes was 304.1 ± 45.2 pg/mL. In the trabeculectomy group, the mean aqueous MCP-1 level was 1444.9, 1914.3, 1899.8, 516.4, 398.3, 427.3, 609.5, 1612.7, 386.2, and 167.9 pg/mL at 3, 6, and 12 h, and 1, 2, 5, 7, 14, 30, and 90 days after surgery, respectively. In the Ex-PRESS group, the corresponding values were 1744.0, 1372.0, 932.5, 711.7, 396.1, 487.3, 799.5, 1327.9, 293.6, and 184.0 pg/mL. There were no significant differences in the aqueous MCP-1 level between the groups at any time point.

Conclusion

The postoperative changes were similar in the Ex-PRESS and trabeculectomy groups, except for bleb wall density in the anterior-segment OCT image. The postoperative aqueous MCP-1 level had bimodal peaks in both groups.     

Intracameral bevacizumab and mitomycin C Trabeculectomy for eyes with neovascular glaucoma: a case series

The purpose of this study was to describe the surgical outcomes and safety of intracameral bevacizumab during trabeculectomy in eyes with neovascular glaucoma. Pilot study included four eyes (four patients) with refractory neovascular glaucoma submitted to fornix-based trabeculectomy with adjunctive use of bevacizumab in the anterior chamber during the procedure. Patients were previously treated with panretinal photocoagulation as standard therapy. Variables evaluated were intraocular pressure, bleb appearance, iris neovascularization, intraoperative/postoperative complications, and visual outcomes. No intraoperative complication was observed. The mean follow-up period was 12.75 (range, 12–15 months). All eyes showed significant intraocular pressure control postoperatively. Iris neovascularization reduced significantly within 1 month after surgery. Mild anterior chamber inflammation was observed during follow-up in all eyes. No significant postoperative complication was observed, and no patient presented visual acuity deterioration. Intracameral bevacizumab may be used as an adjunctive therapy during trabeculectomy in eyes with neovascular glaucoma.     

Bleb formation during anoxia is not a prerequisite for eventual cell death in renal tubule cells

Membrane deformations, generally termed blebs, are a hallmark of cells subjected to noxious stimuli or injurious conditions. Although not lethal itself, bleb formation has been used as an indicator of the stage of cell injury. Whether or not bleb formation is an integral part of the sequence of events that lead to irreversible injury, or occurs in all cells within a group subjected to the same conditions is not known. We demonstrate here that renal epithelial cells in culture produce blebs when subjected to sustained periods of anoxia and substrate deprivation. However, the occurrence of blebs does not correlate with eventual cell death. The data suggest that bleb formation may be associated with prolonged survival.     

A Model of Blebbing in Mitotic Tissue Culture Cells

A preliminary study of blebbing in tissue cultures has been made. The tubal epithelium of fetal mouse oviduct was cultured at 37°C in Rose chambers. A cinematographic record was obtained of phase microscope observations of mitotic cells. Measurements of the size of both cells and blebs were made on the film using a “traveling” microscope. The duration and the rise and decay times of blebs were determined simply by counting frames on the film. Detailed observations are reported on blebbing in four cells undergoing mitosis. The results indicate that the frequency of blebbing as well as the duration of individual blebs exhibits a maximum during telophase. A model is proposed to account for blebbing in mitotic cells. The model attributes to local regions of the cell membrane the property of constant tension independent of stretch over some restricted range of stretch. This property implies that the cell membrane is locally unstable. Further assumptions stated explicitly in the model are that (i) cell division occurs at constant volume, (ii) the cell membrane stretches during cleavage, (iii) there is a positive pressure drop across the cell membrane. Evidence is cited in support of these assumptions as well as independent evidence that the cell membrane may be locally unstable. A physical model is described which would be expected to exhibit blebbing given the above assumptions.     

Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.