Exp. Eye Res. (1992) 55, 831-838

Pentoxifylline Modulates Deformability, F-actin Superoxide Anion Production of Polymorphonuclear from Diabetic Cats PETER

L.SONKIN”,

SHARON

F. FREEDMAN”, DAVID NEEDHAM”, DIANE L. HATCHELL”“’

Content, and Leukocytes K. MURALI

K. RAO”

AND

Departments of a Ophthalmology and Cell Biology, b Mechanical Engineering and Materials Science, Duke University, and the c Geriatric Research, Education and Clinical Center, Durham Veterans Administration Medical Center, Durham, NC, U.S.A. (Received Rockville

29 November

7991 and accepted in revised form 8 April

1992)

Capillary occlusion is an early event in the development of diabetic retinopathy, and white blood cells have recently been shown to be involved. We have shown previously that pentoxifylline improves deformability and decreasesF-actin content of unstimulated polymorphonuclear leukocytes from normal human subjects. The purpose of this study was to determine if pentoxifylline would improve three properties of unstimulated polymorphonuclear leukocytes from diabetic cats. The measured parameters were mechanical (whole cell deformability), structural (F-actin content) and biochemical (rate of superoxide anion production). Chronic hyperglycemia was induced in three cats by partial pancreatectomy. and they were kept in poor glycemic control for at least 6 months prior to the study. Polymorphonuclear leukocytes were isolated and the entry time of individual passive cells was measured during aspiration into a 4-pm micropipette under constant suction pressure ( - 15 cmH,O). Deformability was defined as the inverse of the entry time. F-a&in content of passive cells was measured by NBDphallacidin labeling followed by flow cytometry. The rate of superoxide anion production was measured spectrophotometrically by superoxide dismutase-inhibitable cytochrome c reduction. Following incubation for 15 min with 0.1, 1.0 and 10.0 mM pentoxifylline, the average entry time of passive polymorphonuclear leukocytes was reduced from control by 11 f 5 % (P = 0.04 S), 17 + 6 % (P = 0.00 7), and 36 + 5 % (P < OOOl), respectively. The F-actin content decreasedby Ox, 4+0.6x (P < O.OOl), and 10 f 3 % (P < 0001 ), respectively. Superoxide anion production by resting polymorphonuclear leukocytes was reduced from control by 10 + 7.6% (P > 0.05. not significant). 74 f 7.6% (P < 0.001). and 100% (P < O.OOl), respectively. Improved polymorphonuclear leukocyte deformability in the presence of pentoxifylline correlated to some extent with the decrease in F-a&in content, and may result from a disruption of cytoplasmic structures that rely on F-a&n for their integrity. The promotion of the passive state by pentoxifylline was further evidenced by the inhibition of superoxide anion production. These three effects may combine to reduce the risk of intravascular injury by polymorphonuclear leukocytes. Although achievable plasma levels of pentoxifylline are less than the concentrations used in this study, therapy would result in continuous exposure of polymorphonuclear leukocytes to the drug and its metabolites, and may therefore have the potential to be clinically effective in prevention of capillary occlusion and diabetic retinopathy. Key words: pentoxifylline ; diabetic cat ; polymorphonuclear leukocyte : deformability, F-a&in : superoxide anion : retinopathy ; capillary occlusion.

1. Introduction Capillary occlusion is the hallmark of diabetic retinopathy and any pathogenic mechanism proposed has to explain capillary closure (Kohner, 1989). Although numerous theories have been tested previously, only recently has a role for leukocytes been proposed (SchrGder, Palinski and Schmid-Schanbein, 1991; Sinclair, 1991). In the first study, monocytes and granulocytes were shown to cause capillary obstruction in a rat model of diabetic retinopathy. Local leukocyte accumulation strongly correlated with other * For correspondenceand reprint requestsat: Departmentof Ophthalmologyand Cell Biology. Box 3802, Duke University Eye Center.Durham.NC 27710. U.S.A. 00144835/92/120831+08

$08.00/O

retinal vascular pathology, such as endothelial cell damage, non-perfusion areas, and extravascular leukocytes. In the second study, using the blue field entoptic simulation technique, patients with diabetes perceived decreased leukocyte density and increased speed of leukocytes circulating in their own macular capillaries versus age-matched controls. The authors report that these results indicate that in diabetes, capillary occlusion, either transient or permanent, may focally occur within the retina associated with vasodilation in the adjacent microvasculature because of the relative tissue hypoxia. Several aspects of polymorphonuclear (PMN) function are known to be altered in diabetes, and these changes may promote and contribute to microvascular injury and early diabetic retinopathy. For example. 0 1992 Academic Press Limited

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there are reports of decreasedfilterability of leukocytes and PMNs isolated from diabetic patients as compared with normal controls (Ernst and Matrai, 1986 ; Vermes et al., 198 7 : DeVaro et al., 1988). Somestudies report an increased superoxide anion (O,-) production by resting PMNs (Shah, Wallin and Eilen, 1983 : WieruszWysocka et al., 19 8 7) and stimulated PMNs (Baranao et al., 198 7) from diabetic humans as compared with normal controls. Also. we have shown that stimulated PMNs from diabetic cats have an increased rate of 0, production compared with those from normal cats (Freedman and Hatchell, 1992). Increased PMN O,production may increase the potential for vascular endothelial damage and subsequent vascular occlusion. Pentoxifylline is a methylxanthine derivative used to treat chronic occlusive arterial disease and intermittent claudication (Schubotz, 19 76 : Aviado and Porter, 1984 ; RGssnerand Miiller, 198 7). Pentoxifylline increases arterial blood flow in the leg and improves exercise tolerance (Schwartz et al., 1989). The direct effects of pentoxifylline on normal blood include increased filtration rate of whole blood, increased red cell deformability, and decreasedred cell and platelet aggregation (Ehrly, 19 76 ; Schubotz and Miihlfellner, 19 77 ; Angelkort, Maurin and Boateng, I9 79 ; Gaillard et al., 19 79 ; Manrique and Manrique, 1987). One study has suggested a beneficial effect of pentoxifylline on diabetic retinopathy ; improvement was documented in both the microvasculature. (i.e. retinopathy and nephropathy), and the macrovasculature (i.e. ischemic heart disease and peripheral occlusive arterial disease), after a long-term (4 year) clinical trial with pentoxifylline (Ferrari et al., 198 7). We have shown previously that pentoxifylline in vitro increases the deformability and decreasesactivation (pseudopod formation) of PMNs from normal human subjects (Armstrong et al., 1990; Needham et al., 1989). This increased deformability and reduced activation were correlated with the results of other studies that reported a decreasein F-actin content in pentoxifylline-treated PMNs from normal subjects (Rao et al., 1988). Pentoxifylline also reduces the response of PMNs to specific stimulators ; for example, it inhibits PMN priming by platelet activating factor (Hammerschmidt et al., 1988), phagocytosis of latex particles, and phorbol ester-stimulated production of 0,~ (Bessleret al., 1986). The purpose of our study was to determine the effectsof the drug pentoxifylline on three properties of PMNs from diabetic cats that were kept purposely hyperglycemic for at least 6 months. The resistance of passive cells to flow deformation (deformability) was determined by measuring the time required for cells to completely enter a suction micropipette. F-actin content of cells was measured by NBD-phallacidin labeling and flow cytometry. Superoxide anion production was measured by superoxide dismutaseinhibitable cytochrome c reduction.

ET AL.

2. Materials and Methods

Partial pancreatectomies were performed on three adult male cats (4.5-55 kg) to induce diabetes (Reiser et al.. 1987). Morning blood glucose levels were maintained between 19.6 and 25.2 m&l by single morning injections of protamine zinc insulin. The cats were kept hyperglycemic for at least 6 months prior to study. The average morning pre-insulin glucose level for the three diabetic cats was 22.5 + 1.5 mM, compared to 4.03 I 1.2 mM for normal cats in our laboratory. PMN Isolation

Whole venous blood anticoagulated wit,h EDTA was drawn from the cats following I.M. injection of ketamine (7 5 mg kg-‘), prior to morning insulin injection. The blood was diluted 1: 1 with calcium and magnesium-free Hank’s Balanced Salt Solution (HBSS- pH 7.4, osmolarity 300f3) and PMNs were isolated using a Ficoll-Hypaque (Sigma Chemical Co., St Louis, MO, U.S.A.) double-density centrifugation method (English and Andersen, 1974). The plasma/ HBSS- layer ( 50% plasma) was collected and saved for later use. The cells were washed in HBSS- and resuspended in either 50% plasma for determination of deformability, complete Hank’s Balanced Salt Solution (HBSS+ ) for determination of F-a&in content, or Hepes-buffered phenol red-free HBSS+ for determination of O,- production. Wright’s stain was used to identify cell types. Cell viability was greater than 99% as judged by trypan blue exclusion. Determination of PMN Deformability

Pentoxifylline (Sigma Chemical Co.) was prepared as a 100 mM stock solution using HBSS- and sterile filtered. Further dilution yielded pentoxifylline solutions of 10 and 1 mM. The 50% plasma was aliquoted into four samples and the appropriate pentoxifylline solution was added to yield plasma/pentoxifyIline solutions with drug concentrations of 10, 1 and 0.1 mM. HBSS- was added to the fourth plasma aliquot (0 mM pentoxifylline) to be used as control. Isolated PMNs were resuspended in 50% plasma and aliquoted into four 900-,ul samples, and kept on ice until use. The aspiration apparatus consisted of a glass micropipette mounted in a micromanipulator attached to a Zeiss inverted microscope (Sung et al., 1982). Pipettes were pulled with a vertical pipette puller and cut with a heated glass bead to achieve an inside tip diameter of 4.0 pm. The microscope employed a x 40 objective and videocassette recorder. The cell suspensions were placed in a 16 x 24 x 2 mm glass chamber, which was open on one edge and had a disposable coverslip for its floor. The aspiration

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FIG. 2. PMN pseudopod formation. Activated PMN approaching the pipette tip with pseudopod positioned perpendicularly to the pipette. PMNs with visible pseudopods were excluded from data analysis.

FIG.1. PMNmicropipetteaspiration.A, PMNapproaching pipette tip. B, Contact frame, definedas the initial time of contact betweenthe PMN and pipettetip. C and D, Further progressionof PMN entry into the pipette. E, Entry frame, definedasthe time at which the entire PMN has enteredthe pipette.Entry time is defined as the elapsed time from contact frame to entry frame.

pressure was continuously monitored with an in-line manometer. The pentoxifylline concentration sequence was randomized. The sample chamber was filled with plasma/pentoxifylline solution, and was placed on the stage of the inverted microscope. The micropipette was placed carefully into the chamber 10 min prior to PMN addition to allow plasma proteins to coat the chamber and micropipette. During this time, an aliquot of PMN suspension was removed from the ice and 100 ,~l of the appropriate pentoxifylline solution was added to yield the same pentoxifylline concentration as the plasma coating the chamber. After 10 min. approximately one-half of the plasma/

pentoxifylline in the chamber was removed, and PMN/pentoxifylline suspension was added to the chamber. The cells were allowed to settle to the bottom of the chamber for 5 min, yielding a total incubation time (in the pentoxifylline) of 15 min. Once the PMNs settIed, the micropipette was located and placed into the field of view, and then focused at x 1000 magnification on the bottom surface of the chamber. Suction was maintained at - 15 cmH,O, the videorecorder was started, and PMNs were individually aspirated with the frame counter running. After 50 cells were aspirated, the sample was scanned at a lower magnification ( x 400) to allow for approximation of the percent of activated PMNs (as determined by the presence of visible pseudopods). The above stepswere done at room temperature, and were repeated for each pentoxifylline dose. The videotape was later analysed by a trained, masked observer to determine the entry times of the PMNs into the micropipette (Fig. 1). Only cells identified as PMNs were anaiysed (Armstrong et al., 1990). Wright’s stain revealed cell suspensions consisting of 903) 1.5% PMNs and 9.7+ 1.5% eosinophils, and this corresponded well with the two cell types identified on the videoscreen. PMNs exhibiting visible pseudopods (Fig. 2). and those attached to platelets or other cells were excluded. PMNs were used within 4 hr of venipuncture and samples were excluded from the study if greater than 2 5 % of the PMNs were activated. Determination of PMN F-actin Content

For this assay, PMNs were resuspended in HBSS+ following hypotonic lysis to remove contaminating erythrocytes. NBD-phallacidin labeling followed by flow cytometry was used to quantitate the F-actin content of PMNs (Rao et al., 1988). Briefly, 100 ~1 of the PMN suspension (lo-20 x lo6 cells ml-l) was incubated with 0, 0.1. 1, or 10 mM PTX in HBSS+ in a 37*C water bath for 10 min. Cells were then fixed

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and stained in a single step by adding 100 /cl of a staining cocktail consisting of paraformaldehyde. lysolecithin and NBD-phallacidin in phosphatebuffered saline (PBS)for 60 min at room temperature. The cells were pelleted, washed twice, and resuspended in PBS for flow cytometric analysis. Flow cytometry of PMN suspensions revealed two distinct cell populations. To distinguish PMNs from eosinophils, the cell suspension was passedthrough a flow cytometer equipped with a cell sorter, and the two populations were separated. One group was greater than 99% PMNs by Wright’s stain, and represented 88% of those leukocytes sorted. Wright’s stain of the other population revealed a predominance of eosinophils. Only the population of PMNs was analysed for determination of F-actin content. In each sample. the fluorescence of 5000 PMNs was determined by the flow cytometer.

Determination of PMN SuperoxideAnion Production Assay for superoxide anion production followed previously described procedures (Freedman and Hatchell, 1992). Briefly, PMNs were resuspended in Hepes-buffered phenol red-free HBSS+ (Hepes/ HBSS+, pH 7.4, 5.6 mM glucose) to 2-8 x lo6 cells ml-‘, and kept on ice prior to assay. Hypotonic lysis was not routinely performed, unless RBC contamination in PMN preparations exceeded 50%; control experiments show that neither addition of excess RBCs nor hypotonic lysis affected O,- production by PMNs in these assays. Cell counts were normalized to obtain equal numbers of leukocytes in each experiment. Eosinophil content of leukocyte preparations averaged 11) 7 % and was in the normal range for healthy adult cats (Duncan and Prasse, 1988). Superoxide anion production was measured spectrophotometrically by superoxide dismutaseinhibitable cytochrome c reduction (Cohen and Chovaniec, 1978). Assayswere performed in duplicate at pH 7.4, 37°C. in disposable plastic cuvettes. Standard assayscontained 50 PM ferricytochrome c (type III, Sigma Chemical Co.) and 0.5 x lo6 PMNs ml-’ in 2.5 ml Hepes/HBSS+ . After pre-incubation of cells with 0. 0.1. 1, and 10 mM pentoxifylline for 10 min at 37°C absorbance at 550 nm (A,,,) was followed at 5-min intervals for 40 min to calculate unstimulated O,- production. Control assayswere performed in the presence of superoxide dismutase (SOD, 40 kg ml-‘, Diagnostic Data. Inc., Mountainview, CA, lJ.S.A.). which completely abolished cytochrome c reduction. For measurement of stimulated PMN O,- production, PMNs were pre-incubated with pentoxifylline as above, then assayed in the presence of 10 ng ml-’ phorbol myristate acetate (PMA, Sigma Chemical Co). Following addition of this maximally stimulating dose of PMA. A 550gradually increased to a limiting linear rate, which was taken to be the maximum rate of O,-

ET AL.

production. For selected assays, PMA was added to stimulate PMNs after the lo-min pentoxifylline incubation. Statistical Analyses

The unit of analysis for determination of deformability was entry time (in seconds)per cell. Entry time was determined for 40-50 PMNs at each concentration of pentoxifylline. A two-way ANOVA was used to determine whether entry time differed according to pentoxifylline concentration, with cat and concentration as the predictors. Pairwise comparisons with the control dose are reported using unadjusted P values and confidence intervals. Thesewere calculated on an entry time scale and converted into percent of control values. Analysis of F-actin data was performed as above, with the exception that the F-actin content of 5000 PMNs was determined at each concentration of pentoxifylline. Flow cytometry produced summary statistics ; therefore, the method of unweighted means was used to implement the two-way ANOVA. O,- production was quantified as the amount of O,produced per minute per lo6 cells in each of two replicates at each concentration of pentoxifylline. As above. a two-way ANOVA was used to determine if O,- production differed according to pentoxifylline concentration. All values are reported as rneank~.~.~., with II values as described above. 3. Results PMN Defurmability

Incubation with pentoxifylline significantly decreasedthe entry time (improved the deformability) of

716

704 Cat ID#

650

FIG. 3. The effectsof pentoxifylline (PTX)on PMN entry time. Mean entry times as a function of pentoxifylhe concentration. Values are mean &standard error for n =

40-50 cellsper cat/concentration combination.( mM PTX: (&I) 1 mM PTX: (m) 10 mM PTX.

PTX: (0) 0.1

PENTOXIFYLLINE

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PMNs

835

PMN Superoxide Anion Production

V”

716 cot

704 ID#

650

FIG. 4. The effects of pentoxifylline on PMN F-actin content. Mean fluorescence (F-actin content) as a function of pentoxifylline concentration. Values are mean + standard error for n = 5000 cells per cat/concentration combination. Key as for Fig. 3.

Incubation with 1 mM and 10 mM pentoxifylline significantly decreased the O,- production by unstimulated PMNs (Fig. 5). In the absence of pentoxifylline, unstimulated PMNs generated 0.20 _+0.06 nmol O,- min-’ lo-” cells. Incubation with 0.1, 1 and 10 mM pentoxifylline resulted in a decrease in O,production, relative to control, of 10 ) 7.6 % (P > 0.05), 74+7.6x (P < O.OOl), and 100% (P < O*OOl), respectively. When stimulated with 10 ng ml-’ PMA in the absence of pentoxifylline, PMNs had a maximum rate of O,- production of 2.6 + 0.5 3 nmol min-’ 1Om6cells. Interestingly, pentoxifylline was less effective at inhibiting O,- production by maximally stimulated PMNs, compared to passive PMNs. Incubation with pentoxifylline at 0.1 and 1 mM did not alter PMA-stimulated O,- production, even after a 40-min incubation period ; however, 10 mM pentoxifylline did inhibit PMN O,production, relative to control, by 91+ 4% (P < O.OOl), and had its effect within seconds of addition to PMNs (data not shown). 4. Discussion

PTX concentration

(mtd

FIG. 5. The effects of pentoxifylline on resting PMN superoxide anion production. Mean O,- production of unstimulated PMNs as a function of pentoxifylline concentration. Values are meanf standard error for n = 2 replicates per cat/concentration combination. (0) cat #716: (m) cat #704; (0) cat #650.

passive PMNs (Fig. 3). Incubation with 0.1, 1 and 10 mM pentoxifylline resulted in a decrease of the mean entry time, relative to control, by 11 f 5 % (P= 0.045), 1746% (P= 0.007), and 36+5x (P < O.OOl), respectively.

PMN F-actin Content Incubation with 1 mM and 10 mM pentoxifylline significantly decreased the mean F-actin content of PMNs (Fig. 4). Incubation with 0.1, 1 and 10 mM pentoxifylline resulted in a decrease of the average fluorescence, relative to control, by OS, 4 f 0.6 % (P < 0.001). and 10 f 3 % (P < O.OOl), respectively.

Although the role of PMNs in diabetic retinopathy has not been extensively studied, there is now direct evidence that WBCs play a role in retinal capillary occlusion (Schroder et al., 1991). Comparing alloxaninduced diabetic rats to controls, Schrijder et al. (199 1) reported the following diabetes-related findings: an increased percentage of activated WBCs in the circulating blood; many retinal capillary occlusions by leukocytes, with endothelial cell damage, extravascular macrophage accumulation, and tissue damage: and areas of capillary dropout and neovascularization in the retina coinciding with sites of extravascular leukocytes. PMNs play a major role in tissue ischemia and in determining survival after hemorrhagic shock and reperfusion injury (Barroso-Aranda and SchmidSchonbein, 1990). The reasons appear to hinge on the large resistance of PMNs to mechanical deformation and flow and on the acutely sensitive response of PMNs to priming and activating stimuli that result in the release of free radicals and other toxins and transform the passive cell into a more rigid and and Schmidadherent state (Barroso-Aranda Schonbein, 1990; Frank, 1990). The grossly altered mechanical properties of ‘active ’ cells compared to ‘passive ’ cells appear to be largely a result of the polymerization of F-actin in the cortical region of the cell during chemotaxis and the formation of phagocytic pseudopodia. The cell membrane also becomes inherently ‘ sticky ’ because of the presence of adhesion proteins and shows strong tendencies to adhere to the vascular endothelium. Thus, the slow passage of cells through capillaries under reduced perfusion pressure

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is important in microvascular occlusion, especially if the cells are inadvertantly stimulated to activate in the circulation. A recent report indicates that PMNs in venous blood from patients ’ legs are less deformable and more activated after intermittent claudication than when the same patients are at rest (Neumann et al., 1990). This finding supports the hypothesis that PMN deformability and activation may play a role in microvascular injury and occlusion. The present study suggests that enhanced blood flow in the presence of pentoxifylline may be due to an improvement in the deformability and a decrease in the activation of PMNs (the latter evidenced by decreased O,- production). Incubation with pentoxifylline for 15 min significantly reduced entry time (improved deformability) of passive (spherical) PMNs from diabetic cats at all concentrations tested (0.1, 1 and 10 mM). F-actin content and O,- production were significantly reduced by pentoxifylline at 1 and 10 mM. Although achievable plasma levels for pentoxifylline (standard therapy of 1200 mg day-‘) are lower than 0 1 mM, PMN exposure to the drug in vivo would be continuous. and active metabolites of pentoxifylline would also be present (Ward and Clissold, 198 7). Clinical studies will be necessary to determine if our in vitro findings can be confirmed in vivo. Pentoxifylline has been used clinically in chronic occlusive arterial disease and intermittent claudication (Schubotz, 1976; Ehrly, 1982; Porter et al., 1982; Aviado and Porter, 1984 : Riissner and Miiller, 198 7), ischemic heart disease (Insel, Halle and Mirvis, 1988), and cerebrovascular insufficiency (Herskovits et al., 19 8 5 ; Schultheiss et al., 19 8 7 ; Hartmann and Tsuda, 1988) because of its ability to improve arterial blood flow. In vitro exposure to pentoxifylline increases the filterability of whole blood, increases RBC filterability, decreases platelet aggregation, and increases PMN filterability (Ehrly, 1976; Schubotz et al., 1977; Angelkort et al., 1979 ; Schmalzer and Chien, 1984 ; Matrai and Ernst, 1985 ; Schwartz et al., 1989). In similar studies using micropipette aspiration, we showed that pentoxifylline improves the deformability of spherical ‘passive ’ PMNs and decreases the percentage of activated (pseudopod-forming) PMNs from normal humans (Armstrong et al., 1990; Needham et al., 1989). These changes may partially account for the improved arterial bood flow seen in patients treated with pentoxifylline (Schwartz et al., 1989). Deformability is dependent upon, in addition to other things, the shape of the cell. In fact, filtration studies have shown that stimulation of PMNs, which results in pseudopod formation (as well as other changes), severely decreases the filterability of these cells (Nees and Schonharting, 19 8 7). Pentoxifylline therefore yields a dual benefit in that it improves the deformability of passive PMNs and decreases the fraction of PMNs with pseudopods. Pentoxifylline, in vitro, also inhibits O,- production of stimulated PMNs from normal humans (Currie et al., 1990: Ham-

ET AL,

merschmidt et al., 1988; Bessler et al.. 19X6: Hand et al.. 1989). By decreasing O,- production. pentoxifylline may reduce the risk of PMN-induced vascular endothelial damage and capillary occlusion. The mechanism of action of pentoxifylline is unknown. Cell filterability and aggregation are cytoskeleton-dependent functions, and alterations in the actin state might affect cell deformability and the dynamics of blood circulation (Rao et al., 1988). Pentoxifylline decreases the F-a&in content in vitro of PMNs from normal humans (Rao et al., 1988). The present study confirms this effect on PMNs from diabetic cats, and demonstrates improved deformability in the presence of pentoxifylline in cells from the same diabetic cats. Since F-a&in is a critical component of cellular structure, the ability of pentoxifylline to decrease the F-actin content probably plays a major role in the improved deformability of PMNs. Pentoxifylline, by virtue of its observed effects on PMNs from normal humans and diabetic cats, may prove useful in both prevention and early treatment of the microvascular complications of diabetes, including retinopathy. Acknowledgements This study was supported by Veterans Administration Medical ResearchFunds (Dr Hatchell) ; National EyeInstitute Research Grant EY02903 (Dr Hatchell) and Core Grant EY05722 : a generous gift from Dr and Mrs Harris Vernick. Student Research Fellowships SF9001 7 and SF9001 7C (P. Sonkin ; awarded in memory of JamesThurber, by Fight for Sight, Inc., Research Division of the National Society to Prevent Blindness) ; grant PD90036 (Dr Freedman; awarded in memory of Mary E. and Alexander P. Hirsch, by Fight for Sight, Inc., Research Division of the National Society to Prevent Blindness). and Research to Prevent Blindness, Inc. We are grateful to Dr Gregory Samsa for statistical assistance, and to Camille Barden and Jan Davis for animal care and data analysis. References Angelkort. B., Maurin, N. and Boateng, K. (1979). Influence of pentoxifylline on erythrocyte deformability in peripheral occlusive arterial disease.Curr. Med. Res.Opin. 6. 255-8.

Armstrong, M., Needham, D., Hatchell, D. L. and Nunn, R. S. (1990). Effect of pentoxifylline on the flow of polymorphonuclear leukocytes through a model capillary. Angiology 41; 253-62. Aviado. D. M. and Porter, J. M. (1984). Pentoxifylline. A new drug for the treatment of intermittent claudication. Mechanism of action. pharmacokinetics, clinical efficacy and adverse effects. Pharmacotherapy 4, 297-307. Baranao, R.. Garberi, J. C., Tesone. P. A. and Rumi, L. S. (198 7). Evaluation of neutrophil activity and circulating immune complexeslevels in diabetic patients. Harm. Metab. Res. 19, 371-4. Barroso-Aranda. J. and Schmid-SchGnbein, G. W. (1990). Pentoxifylline pretreatment decreasesthe pool of circulating activated neutrophils, in-vivo adhesion to endothelium, and improves survival from hemorrhagic shock. Bioheology27. 401-18.

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