Histological analysis of retinas sampled during translocation surgery: a comparison with normal and transplantation retinas.

AIMS: To carry out a histopathological analysis of retinal specimens of patients undergoing translocation surgery for age-related macular degeneration (ARMD). METHODS: A histopathological analysis, using confocal microscopy, was performed on six retinal specimens. Results were compared with those from two further retinal specimens, collected during RPE transplantation, to control for the effects of vitrectomy and ARMD. In addition, a third control specimen from a cadaver with no history of ophthalmic disease was also analysed. RESULTS: In the translocation specimens, rods and cones were relatively well preserved but showed reduced density and outer segment length. In four specimens, there were focal areas of rod opsin redistribution to the inner segment, but this was not observed in the controls. Staining with calbindin was decreased in cones compared with controls but normal in horizontal and amacrine cells. Rod bipolar cells were mildly disorganised, and in one there was evidence of neurite sprouting. Glial fibrillar acidic protein was raised in both translocation and transplantation retinae but not in the cadaver control. CONCLUSIONS: In this study, there was little evidence of cellular injury following iatrogenic detachment; however, the rate of PVR following translocation surgery infers that cellular events set in motion may continue despite early reattachment.

[Possible role of alkylphosphocholines in retinal reattachment surgery]. / Mögliche rolle von alkylphosphocholinen bei der ablatio-chirurgie.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a major complication after retinal detachment surgery, but there is no established pharmacotherapy available to control the cell biology of the disease. The aim of this study was to investigate the role of alkylphosphocholines [APCs; erucylphosphocholine (ErPC) was used in this study], novel pharmacologic substances with antiproliferative properties, on intraretinal proliferation initiated by experimental retinal detachment in a well-established in vivo model. METHODS: Retinal detachments were created in adult pigmented rabbits. ErPC was injected intravitreally on either day 1 or day 2 after detachment. Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) was injected on day 3. Following fixation, retinas were triple-labelled with anti-BrdU (proliferation marker), Isolectin B4 (retinal microglia marker), and anti-vimentin (retinal Mueller glia cell marker). The number of anti-BrdU-labelled cells per millimeter of retina was determined from sections imaged by laser scanning confocal microscopy. Toxicity was assessed by light and electron microscopy. RESULTS: A single intravitreal injection of ErPC had a significant effect on reducing the number of proliferating non-neural retinal cells on day 3 after experimental retinal detachment in the rabbit. Injection of ErPC on day 1 was more effective than when given on day 2. No evidence of toxicity was observed in the retina on day 3 for any of the conditions. CONCLUSIONS: APCs are novel pharmacologic substances that significantly inhibited intraretinal proliferation after experimental retinal detachment in this in vivo model. They could be considered as an adjunct therapy at the time of retinal reattachment surgery to potentially prevent proliferative vitreoretinal diseases such as PVR. However, long-term toxicity studies must be performed before APCs can be considered for clinical application.

Proliferative vitreoretinopathy-developments in adjunctive treatment and retinal pathology.

Proliferative vitreoretinopathy (PVR) remains a difficult management problem despite advances in vitreoretinal surgery. There is still a significant incidence of PVR in rhegmatogenous retinal detachment and other forms of retinal disease. Surgery for PVR now has a high anatomical success rate although visual results are often disappointing. The use of adjunctive treatments to prevent cellular proliferation holds promise for the prevention of PVR or recurrences after surgery. Control of proliferation and strategies aimed at improving visual outcome are important areas of future research in PVR and other forms of retinal disease. Studies of the intraretinal and peri-retinal pathology of PVR have demonstrated characteristic changes which may have a significant influence on visual outcome and surgical management.

Animal models of retinal detachment and reattachment: identifying cellular events that may affect visual recovery.

Retinal detachment continues to be a significant cause of visual impairment, either through the direct effects of macular detachment or through secondary complications such as subretinal fibrosis or proliferative vitreoretinopathy. Animal models can provide us with an understanding of the cellular mechanisms at work that account for the retinopathy induced by detachment and for the generation of secondary effects. As we understand the mechanisms involved, animal models can also provide us with opportunities to test therapeutic agents that may reduce the damaging effects of detachment or improve the outcome of reattachment surgery. They may also reveal information of use to understanding other causes of blindness rooted in retinal defects or injuries. Understanding the effects of detachment (and reattachment) are likely to become even more important as surgeons gain skills in subretinal surgical techniques and macular translocation, both of which will generate short-lived detachments. Here we discuss the fundamental events that occur after detachment, present changes associated with reattachment, and discuss retinal changes that may affect the return of vision.

FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Müller and RPE cells.

PURPOSE: To identify changes in cellular signaling pathways and AP-1 expression in retina and retinal pigmented epithelium (RPE) after experimental retinal detachment (RD). METHODS: Cat and rabbit neural retinas were separated from the RPE in vivo for 5 minutes to 28 days. Tissues were removed and processed for Western blotting, immunohistochemistry, in situ hybridization, and immunoprecipitation experiments. RESULTS: An ordered sequence of events occurs after RD: (1) fibroblast growth factor (FGF) receptor 1 (FGFR1, flg) is phosphorylated in the retina within 15 minutes and dephosphorylated 2 hours after RD; (2) The extracellular signal-regulated kinase (ERK) is phosphorylated in both Müller and RPE cells within 15 minutes and remains so for several days; (3) De novo expression of c-fos mRNA coincides with increased c-Fos and c-Jun immunoreactivity in both Müller and RPE cells; (4) CREB is phosphorylated in a subpopulation of photoreceptors; and (5) STAT3 and NF-kappaB are activated in inner nuclear layer cells by 1 day of RD. CONCLUSIONS: These data suggest that nonneuronal cells (RPE and Müller cells) respond to RD very rapidly by stimulating ERK signaling and AP-1 transcription factor expression. Furthermore, these data suggest that basic fibroblast growth factor (FGF-2, bFGF) is involved in initiating the retina's earliest responses to RD. The events described here precede changes in gene expression and morphology that can have serious effects on visual outcome in humans treated for retinal detachment or other retinal injuries.

Doxazosin in the treatment of benign prostatic hyperplasia in normotensive patients: a multicenter study.

A 16-week, double-blind, placebo controlled, dose titration study was done on 100 normotensive patients age 45 years or older to determine the efficacy and safety of doxazosin, a selective alpha 1-adrenoceptor antagonist, in the treatment of benign prostatic hyperplasia (BPH). Of the 41 efficacy evaluable patients 88% underwent dose titration to a maximum of 8 mg. doxazosin once daily. Maximum and average urinary flow rates increased significantly above baseline with doxazosin (2.9 ml. per second and 1.4 ml. per second, respectively) compared with placebo (0.7 ml. per second and 0.3 ml. per second, respectively). A significant effect on maximum flow rate was noted as early as week 2 of double-blind treatment at the initial efficacy evaluation. Doxazosin was superior to placebo in patient and investigator assessments of total, obstructive and irritative BPH symptoms. The onset of efficacy for total patient-assessed symptoms was significant for doxazosin compared to placebo 4 weeks after the start of the treatment regimen. Statistically significant decreases in mean blood pressure of 4 to 6 mm. Hg were noted with doxazosin compared with placebo. Adverse events, primarily mild to moderate in severity, were reported in 44% of patients given doxazosin and 30% of those given placebo. Our results strongly demonstrate that doxazosin is significantly superior to placebo in the treatment of BPH in normotensive patients, with the patient experiencing significant relief early after initiation of therapy.

Use of the MIB-1 antibody for detecting proliferating cells in the retina.

PURPOSE: To study intraretinal proliferation as a response to experimental retinal detachment using an antibody that recognizes the nuclear specific antigen Ki-67 in proliferating cells. METHODS: Experimental retinal detachments were produced in cats (1, 3, 7, and 28 days) and rabbits (1, 3, and 7 days). The animals were killed and the eyes were fixed and embedded in paraffin. Histologic sections were processed for immunohistochemistry using the MIB-1 antibody to detect the Ki-67 protein. Labeled cells were identified, and the proliferative response was quantified. RESULTS: In normal cat retina, approximately 0.05 cells per millimeter of retina are labeled. In cat retina detached for 1, 3, 7, or 28 days, the number of cells labeled by MIB-1 is 0.06, 5.03, 1.38, and 0.23 cells per millimeter of retina, respectively. MIB-1 labeling yields an approximate fivefold increase over the number of proliferating cells detected in retinal sections using 3H-thymidine autoradiography. Detachment of the rabbit retina elicits a similar response as measured by MIB-1 immunohistochemistry. CONCLUSIONS: In contrast to 3H-thymidine, which labels cells in S-phase only, the MIB-1 antibody labels proliferating cells regardless of their location within the cell cycle. MIB-1 labeling, therefore, is a more accurate means of evaluating cellular proliferation in the retina and elsewhere in the central nervous system, and it is a relatively simple way of evaluating the effects of agents that may affect this response.

Role of tumor necrosis factor in flavone acetic acid-induced tumor vasculature shutdown.

Flavone acetic acid (FAA), a novel investigational antitumor agent, has been shown to cause early vascular shutdown in several experimental murine tumors, and this phenomenon is believed to be crucial to FAA's antitumor effects. However, the basis of this FAA-induced tumor vascular shutdown is unknown. In this study a radioactive tracer-clearance technique has been used as an objective indication of tumor blood flow to show that i.p. administered FAA induces a progressive and sustained reduction in blood flow in a colon 26 tumor growing s.c. in syngeneic mice. As early as 1 h after administration, there was a significant increase in the t1/2 clearance value for intratumorally injected 133Xe, reaching a peak at 3 h (117.3 +/- 36.4 versus 7.8 +/- 0.85 min for controls). Significant inhibition of blood flow was still apparent 48 h after a single injection of drug. This FAA-induced vascular shutdown was virtually abolished in tumor-bearing mice pretreated with an antiserum against tumor necrosis factor, while no such effect was observed in controls pretreated with nonimmune serum (t1/2 of 10.8 +/- 1.2 versus 65.6 +/- 8.0 min for controls). Furthermore, in vitro FAA was seen to induce tumor necrosis factor secretion from murine peritoneal cells and splenocytes. These studies suggest that FAA-induced tumor vascular shutdown in the colon 26 tumor is mediated by tumor necrosis factor.

Interleukin-1 potentiates bradykinin- and TNF alpha-induced PGE2 release.

The ability of interleukin-1 (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF alpha) and bradykinin to cause prostaglandin E2 (PGE2) release from human synovial cells was examined. IL-1 alpha and IL-beta proved equipotent in their effect, and were up to four orders of magnitude more potent than TNF alpha after incubation for 24 h. Bradykinin proved the weakest of all the agonists examined. When the cells were pretreated with IL-1 alpha or IL-1 beta for 24 h, their ability to release PGE2 in response to a short incubation (1 h) with bradykinin, TNF alpha or a second dose of IL-1 was potentiated. In addition, TNF alpha and bradykinin were shown to increase the level of free arachidonic acid (AA) in the cells. Furthermore, a similar potentiation in the response of pretreated cells was observed with exogenous AA. It is already known that pretreatment with IL-1 for 24 h results in an induction of cyclo-oxygenase (CO). It seems likely, therefore, that activation of phospholipase A2 which occurs during a short incubation with IL-1, TNF alpha or bradykinin releases substrate, AA, which is more rapidly converted to PGE2 by cells in which CO has been induced. The result of these events might indicate a sustained release of PGE2 at sites of inflammation where such mediators are released.

PMN stimulation by factors from IL-1-treated human synovial cell cultures.

Following exposure of cultured human synovial cells to human recombinant interleukin 1 alpha (IL-1 alpha), we demonstrate the appearance of factors in the supernatant which stimulate human polymorphonuclear leukocyte (PMN) locomotion and elevate intracellular free calcium ([Ca++]i). The production of these factors can be abolished by actinomycin D or dexamethasone but not by cyclo-oxygenase or lipoxygenase inhibitors. In vivo, the supernatant induces a rapid accumulation of PMNs in rabbit skin following intradermal injection. These activities were not due to IL-1 itself, tumour necrosis factor (TNF alpha) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Such factors may play an important role in inflammatory responses involving IL-1.

Increased vascular permeability and polymorphonuclear leucocyte accumulation in vivo in response to recombinant cytokines and supernatant from cultures of human synovial cells treated with interleukin 1.

The inflammatory effects of intradermal injections of the human recombinant cytokines interleukin 1 (IL-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF alpha) have been assessed in rabbit skin, and compared with the effects of a novel polymorphonuclear leucocyte (PMN)-stimulating activity (PSA) produced by IL-1-treated human synovial cell cultures. IL-1 (84 fmol) and GM-CSF (10 pmol) caused increases in vascular permeability with a delayed onset, as assessed by the dermal accumulation of intravenously-administered 125I-human serum albumin. These cytokines also stimulated extravascular accumulation of PMNs. In contrast, PSA-containing supernatant caused a more rapid and prolonged increase in vascular permeability and PMN accumulation. TNF alpha (84 fmol) was unable to stimulate either of these responses. The increases in vascular permeability and PMN accumulation following IL-1 administration in vivo may be a consequence of the local generation of PMN-stimulating activity by connective tissue cells, such as the activity produced by IL-1-treated synovial cell cultures that we have described.

Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique.

A new quantitative assay for measuring angiogenesis in a s.c. located sponge implant in rats is described. Using this model, which detects neovascularization by measuring alterations in 133Xe clearance, it has been shown that the known angiogenic factors, transforming growth factor alpha and tumor necrosis factor alpha, cause maximum vascularization of the sponge to occur by Day 11 postimplantation compared with Days 15 to 17 in control animals. The monokine interleukin 1 alpha is shown to be strongly angiogenic, suggesting that more than one macrophage-derived cytokine may be the active mediator in macrophage-induced angiogenesis. Extracellular matrix proteins appear to play a role in regulating the angiogenic response such that presoaking sponges in laminin (40 micrograms/ml) or fibrinogen (500 micrograms/ml) solutions induced a significant reduction in the time taken to achieve maximum 133Xe clearance values; no such enhancement of neovascularization was observed when sponges were presoaked in type IV collagen (100 micrograms/ml) solution. The assay described here, which is reproducible, objective, and quantitative, should be of considerable use in elucidating the molecular basis of angiogenesis regulation.

Neutrophil stimulation by recombinant cytokines and a factor produced by IL-1-treated human synovial cell cultures.

Neutrophil accumulation and activation are early events in the inflammatory response in vivo. Using human recombinant forms of the putative inflammatory mediators interleukin-1 (IL-1) and tumour necrosis factor (TNF alpha) we were unable to detect direct effects on human neutrophil locomotion or intracellular free calcium concentration ([Ca2+]i) in vitro. Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to stimulate significant locomotion, but was unable to elevate neutrophil [Ca2+]i. In contrast, supernatant from cultured human synovial cells that had been treated with human recombinant IL-1 alpha (28 pM) released a factor that stimulated both neutrophil locomotion and elevated neutrophil [Ca2+]i. Our studies demonstrate that the production of this factor is time-dependent, requiring exposure of the synovial cells to IL-1 for more than 4 hr, is not influenced by cyclo-oxygenase or lipo-oxygenase inhibition, but can be abolished by dexamethasone (100 nM) or actinomycin D (0.8 microM). The factor has a molecular weight above 10,000 and does not cross-react with anti-C5a antisera. IL-1 beta and TNF alpha were also able to stimulate its production. Our findings suggest that the neutrophil accumulation that is known to occur in response to IL-1 in vivo may be a consequence of the local production of such a factor.

Nonoperative versus operative treatment of obstructive jaundice in pancreatic cancer: cost and survival analysis.

Pancreatic carcinoma is complicated by malignant obstructive jaundice in 40-70% of cases. Patients frequently are old, debilitated, unresectable, and faced with a dismal prognosis. Invasive endoscopic and radiologic procedures in the inoperable patient can provide palliation without the need for surgery, in most cases. Few studies have compared nonoperative palliation with conventional surgical biliary enteric bypass procedures. In a retrospective study of patients with pancreatic carcinoma, we found no difference between operative and nonoperative treatment in survival, total hospitalization, or morbidity and mortality. Cost analysis revealed significant savings with nonoperative treatment.

Infected pancreatic fluid collections: percutaneous catheter drainage.

Thirty-eight infected pancreatic fluid collections in 23 patients with acute or chronic pancreatitis were drained percutaneously following initial diagnosis with computed tomography and fine-needle aspiration. Fifteen (65.2%) patients were cured completely without surgery. Eight (34.8%) patients required some type of surgery despite successful treatment of the fluid collection, and in two (6.5%) the collection recurred after catheter removal. Complications occurred in three (13%) patients, but only one complication (4%), empyema, was a direct result of catheter drainage. Catheter drainage time averaged 29 days for 16 patients with isolated collections and 96 days and 104 days for patients with collections with pancreatic duct fistulas (nine patients) or gastrointestinal fistulas (14 patients), respectively. This study confirms that infected pancreatic fluid collections can be safely and effectively treated with percutaneous catheter techniques in most patients.

Antiproliferative and anticontractile effects of liposome encapsulated fluoroorotate.

Incorporation of fluoroorotate into liposomes increases its growth inhibitory potency for rabbit dermal fibroblasts 30-fold. The optimal lipid composition of the liposomes is dipalmitoylphosphatidylglycerol:cholesterol (67:33). Liposomes prepared by reverse phase evaporation without extrusion are the optimal liposomes for delivery. Fluoroorotate, like other RNA directed fluoropyrimidines, inhibits the contractility of rabbit dermal fibroblasts. The effect is greatest when the cells are exposed to the drug for the 48-72 hr immediately prior to measurement of cell contractility. Encapsulation of fluoroorotate increases its anticontractile potency 10-fold. The anticontractile effects of both free and encapsulated fluoroorotate on the cells last at least 12 days. Leakage studies suggest that the loss of drug from the liposomes under storage conditions will be quite low. Leakage studies also confirm that serum will accelerate the loss of drug from the liposomes and that sonicated liposomes leak much more rapidly than larger liposomes. However, the large difference between egg phosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes for drug delivery is not explained by leakage studies. These results suggest that encapsulated fluoroorotate may be a useful adjunct to surgery for treatment of proliferative vitreoretinopathy.

The roles of prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation in man and the rat.

The effects of aspirin, carboxyheptylimidazole (CHI) and creatine phosphate/creatine phosphokinase (CP/CPK) on platelet aggregation and thromboxane B2 (TxB2) formation induced by collagen have been examined in vitro. Platelets from two species, man and the rat, have been used. In man, aspirin and CHI abolished TxB2 production but only partially inhibited aggregation. CP/CPK partially inhibited aggregation and TxB2 formation. In the rat, aspirin and CHI abolished TxB2 formation but had no effect on aggregation. CP/CPK completely inhibited aggregation and partially inhibited TxB2 generation. In man, collagen-induced aggregation is largely dependent on ADP and to a lesser extent on arachidonate metabolites whereas, in the rat, ADP alone mediates aggregation induced by this agonist. The results with CP/CPK suggest that TxB2 formation is dependent either on the prior release of platelet ADP or on aggregation itself rather than being responsible for the aggregation response.

Mechanism of cyclosporin A-induced inhibition of prostacyclin synthesis by macrophages.

In vitro studies of PG production over a 24 h period by adherent rat peritoneal macrophages activated by serum-opsonized zymosan revealed that CSA (0.3-10 micrograms/ml) caused a dose-related inhibition of PGI2 (assayed as 6-oxo-PGF1 alpha) formation. Indomethacin (IND, 0.01-10 micrograms/ml) and dexamethasone (DEX, 0.01-10 micrograms/ml) also inhibited the PG production in a dose-related manner. When arachidonic acid (10 micrograms/ml) was added together with the inhibitors, there was no change in the level of PGI2 produced by IND-treated cells whilst the PGI2 levels of DEX- and CSA-treated cells were elevated to the control level. Therefore CSA like DEX does not inhibit cyclo-oxygenase activity. However unlike DEX, CSA (1-30 micrograms/ml) caused inhibition of phospholipase A2 (PLA2) activity when assayed on the hydrolysis of a synthetic substrate by pancreatic PLA2 in a cell-free system. The direct inhibition of PLA2 might well be a manifestation of the fundamental activity of CSA on immunocompetent cells.

Sex and hormonal influences on platelet sensitivity and coagulation in the rat.

Platelet sensitivity to adenosine di-phosphate (ADP), thrombin, collagen, arachidonic acid and prostaglandin I2 (PGI2) and the activity of the coagulation system as measured by the activated partial thromboplastin time, prothrombin time, Russell's viper venom time and plasma fibrinogen have been examined in male and female rats, female rats during the oestrous cycle and female rats treated with oestrogen and a progestogen. Male rat platelets were less sensitive to thrombin and more sensitive to inhibition by PGI2 than those from females and fibrinogen levels in male rat plasma were approximately twice those seen in females. During the oestrous cycle, platelets were more sensitive to ADP and less sensitive to thrombin at dioestrus. Following 6 weeks treatment with 17 beta-oestradiol or ethynyl oestradiol, both platelet aggregation and release of granular ATP induced by collagen were significantly reduced. Platelet sensitivity to other agents, ADP, arachidonic acid, thrombin and PGI2 was, however, unchanged following oestrogen treatment. Activation of factor X by Russell's viper venom was accelerated in rats treated with ethynyl oestradiol, although this enhancement was not reflected in the overall clotting times.