- Email: [email protected]
Intravitreal fibrinolysis in experimental vitreous haemorrhage
Ex~I. Eye Res. (1976) 22, 181-188
Intravitreal Fibrinolysis in Experimental Vitreous Haemorrhage .J. Y. FORRESTICR: W. EDGAR: C’. R. M. PRENTICE, C'. D. FORBES AND J. WILLIAMSOS
(Receiwd
9 September
1975. Lon.dw)
The fibrinolytic system within experimentally induced vitreons haemorrhages was studied in rabbits. The presence of crosslinked fibrin was noted for up to five weeks following induction of the haemorrhage, while soluble fibrin degradation products were detected in low concentration for two months. Plasminogen activator activity of the vitreous did not decline following vitreous haemorrhage. In addition haemolysis appeared to occur simrlltaneously with fibrinolysis. It is suggested that fibrinolysis is important in the clearing of blood from t,he vitreous? and may be the initiating st,ep.
1. Introduction The pathophysiology of vitreous haemorrhage resolution has attracted the attention of many workers in the experimental field (for review. seeBenson and Spalter, 1971). Several aspects have been studied including haemolysis (Greer. Benson and Spalter, 1968), phagocytosis (Horven, 1964a), membrane formation (Lam, Ashrafzadeh and Lee. 1972; Swann. Chesney, Constable, Colman. Caulfield and Harper, 1974) ant1 the ocular t,oxicity of intravitreal blood (Regnault. 1970). Scant attention has been paid to the fibrinolytic mechanismswithin a vitreous haemorrhage. It is known that, vitreous has a procoagulant effect on blood, (Regnault. 1969) and histologicall! fibrin has been observed within experimental vitreous clots (Schimek and Steff~~nsen. 1955; Horven: 1964b). In a study of experimental vitreous haemorrhage report,ed bv Regnault (1970) where lzjl labelled human fibrinogen was injected into rabbit vitreons. the half-life of the fibrinogen was found to be four days with no trace of radioactivity after 20 days. On this basis, it was suggestedthat spontaneous lvsis of fibrin occurs as the initial step in the resolution of vitreous haemorrhage. * In a previous study (Forrester, PrenCce, Williamson and Forbes. 1974)we reported t’he presence of t,issueplasminogen activator activity within normal vitreous. and proposedthat intravitreal fibrinolysis occurred as the result of plasminogenactivat)ion by this enz”ymewithin the vitreous clot. The object of the experiments reported her{> was to record the progressive removal of fibrin from an intravitreal haemorrhage and to look for rlvidcnce of intra,vit,real fihrinolysis in the form of soluble fibrin breakdown pr”‘lll’t~s.
2. Materials and Methods .4 n Aal models 411 experiments were performed on white New Zealand rabbits weighing 3-4 kg and anaesthetized with intravenous pentobarbital (20 mg/kg wt). Local anaesthetic (Ametho(Gne drops lo/o) was instilled into the conjunctival sac and the lids were retracted with a speculum. The right globe of each animal was softened by withdrawing 0.2 ml aqueous from the anterior chamber using a 25-gauge needle gently inserted through the limbue. The eye was immobilized by fixing the superior rectue muscle and O-2 ml of whole blood 1Sl
182
FORRESTER
et al.
was withdrawn from the marginal ear vein into a plastic syringe. Under ophthalrnoscopit control the blood was injected immediately into the centre of the vitreous gel through the pars plana in the anterosuperior quadrant of the globe. A short pause was taken before the needle was sharply withdrawn, without tracking of blood under the conjunctiva. Chloramphenicol 0.5% and Atropine 1.0% drops were instilled into the lower fornix at the end of the operation. The resolution of the vitreous haemorrhage was monitored weekly by ophthalmoscop,v for a period of eight months following injection. Animals were sacrificed at, various stages and the globes were enucleated immediately post mortem, and transported on ice to the laboratory. Preparation of vitreorss extract The eyes were placed in polythene bags and immersed in a solution of CO, snow in acetone (--4O--60°C) for 60 sec. They were then sectioned with a skin-graft (Thiersch) knife and the frozen vitreous clot was removed with the aid of the dissecting microscope, thawed and centrifuged at x 200 g for 5 min. 10 ~1 of Trasylol (10 000 u/ml) (Bayer, Ltd.. Germany) and 10 N.I.H. u of bovine thrombin (100 u/ml) (Parke Davis & Co., Detroit, Mich.) were added to the supernates, which were recentrifuged at x 1750 g for 15 min and stored at -20°C. The residual solid vitreous clot was washed for 24 hr in 085% saline. The normal vitreous of the left eye was removed in similar fashion and served as a control. F&in
analysis
The detection and quantitation of fibrin within the vitreous clot was carried out bj polyacrylamide gel electrophoresis in sodium dodecyl sulphate using 7.5% acrylamide gel, as described by Weber and Osborne (1969). Control samples of rabbit fibrinogen, rabbit fibrin, normal rabbit vitreous and plasmin-incubated vitreous haemorrhage were also run. The quantity of crosslinked fibrin was estimated by densitometric scanning of the gels at 600 nm using a Gilford spectrophotometer with scanning attachment, comparing the y dimer peak heights with known concentrations of crosslinked rabbit fibrin. Detection of Fibrin Degradation Products (F.D.P.s) The presence of F.D.P.s within the supernate from the vitreous clot was estimated in all samples by the radial immunodiffusion (RI) technique [Mancini, Carbonara and Hergmans (1965)J. Anti-rabbit fibrinogen serum (Hoeehst Pharmaceuticals Ltd., Germany) was incorporated into the 176 agar support at a concentration of 0.4?$,. Rabbit plasma in dilutions of 1:50, 1 :lOOand 1:300 served as the standard. Plasma fibrinogen was measured by the method of Swain and Feders (1967)in blood collected in 4.896 citrate (8:l). Some samples were tested also by the tanned red cell haemagglutination inhibition immunoassay (TRCHII) (Merskey, Kleiner and Johnston, 1966) using anti-rabbit fibrinogen serum and clumping test sheep erythrocytes sensitized with rabbit plasma. The staphylococcnl (S.C.T.) (Hawiger, Niewiarowski, Guerewich and Thomas, 1970) was used to verif! quantitation of FDP concentration by the RI technique.
Plasminogen activator. activity The fibrin plate technique (Nilsson and Olow, 1962) was used to measure the plasminopen activator activity of the supernate from the vitreous haemorrhage, while the normal vitreous from the fellow eye served as the control.
3. Results ophthulmoscopy The changes
occurring in a resolving experimental vitreous haemorrhage have been documented by several workers (Schimek and Steffensen, 1955; Regnault, 1970).
INTRAVITREAL
FIBRIh-OLYSIS
183
Following injection, a circumscribed clot occupied the central vitreous for 24-48 hr and then from the third day the clot was redistributed to occupy the whole vitreous. A moderate proteinaceous iritis was noted at this stage. Numerous strands gradually developed throughout the blood clot but no fragmentation of the opaque haemorrhage occurred until the fifth to sixth week post-injection. Central white membranes were noted coming from the injection site in some animals. Between the sixth week and the thirteenth week increasing fundal detail was observed through gaps in t,he vit,reous opacities. These black and grey-white membranes were gradually removed until by three months the vitreous was clear, apart from a few floaters and minimal vit,reous haze. Very little solid material was obtained from the vitreous after 10 \\WkS. Polyucrylumicle
gel electrophoresis
Rabbit cross-linked fibrin [Fig. l(a)], obtained by incubating rabbit plasma with bovine thrombin in the presenceof Ca2+ions for 2 hr at 37”C, produced two prominent, banclscorresponding to the /3 chain and the yy dimer of the fibrin polymer (Lorand. 1972). The bands representing the c( and y monomers indicated that the fibrin was not fully cross-linked. Normal rabbit vitreous [Fig. 2(c)] contained no proteins with molecular weights corresponding to the fibrin chains, but several other soluble protein bands were present (Laurent. Laurent and Howe, 1962). Solubilized extracts from the solid vitreous clots of varying duration produced bands corresponding to the /3 monomer and yy dimer chains, indicating the presenceof strongly cross-linked fibrin within the vitreous for up to five weeks following induction of the haemorrhage [Fig. l(b) to (f)]. Ko fib rm was detected after this time [Fig. l(g) and(h)]. Several other lower molecular weight proteins were noted with two prominent bands appearing at molecular weights of 32 000 and 16 000 daltons (Fig. 1. x and 1. y). The lower band (Fig. 1, y) was thought to represent a monomer and the upper band a dimer of the constituent chains of the haemoglobulin molecule. released by haemolysis of the intact red cell within t.he solid clot by the extraction procedure with 8 M-urea, since any free haemoglobin would have been removed during the preliminary wash. Accordingly rabbit red cells wrerehaemolysed in distilled water, and the supernatant passedthrough a column of Sepharose-4B. The eluate corresponding to the haemoglobin peak (mol. wt 50 000.-100000) was then run on SDS-polyacrylamide gel electrophoresis as described. Two broad bands were observed in identical positions t,o Fig. 1. x and y. It is not understood why the globin molecule is not fully depolymerized by the 2-mercaptoethanol. but it may be related to the fact that the chain of rabbit globin is not a single polypeptide but a mixture of closely related proteins (Huisman and Schroeder. 1971). Further confirmation of the presenceof fibrin within the vit’reous clots was obtained by the disappearance of the fi and yy bands following incubation of the clot with plasmin (Fig. 2). The quantity of fibrin estimated by gel scan spectrophotometry ih shown in Table I. Removal of the fibrin occurred progressively during the first six weeks of resolution. E’.D.P.
estima~tiol~
Precipitation rings in the. Mancini plates surrounding the wells with supernatant vitreous were observed for up to six to eight weeks following injection of blood. Inconsistent results were obtained after this time, there generally being no F.D.P.s after two months. The concentration of F.D.P.s was measured by the diameter of
x-
Y-
FIG. 1. Polyacrylamide gel electrophorcsis. (a) prominent /3 and yy rlimer IM& II, control rruss-linkrtl rabbit fibrin. (b)-(f) /3 and yy bands present in vitreous haemorrhage extracts. (g) and (h) no fibrin ban&s present in vitreous haemorrhage extracts after 5 weeks. (h) -R days. (0) 1 week. (d) 2 lveeks. (e) 4 wwks. (f) 5 weeks. (g) 6 weeks. (h) 2 months.
FIG. 2. l’olyacrylamide and yy fibrin chains. vitreous control.
gel electrophoresis: (b) same haemorrhage
(a) vitreous haemorrhage rstract showing wcll.d~4inwl p extract following incubation with plasmin. (v) normal
IXTRAVITREAL TABLE
Vitreous
1x5
FIHRINOLVSIS 1
haewtorrhagejlwi~~~
comentration
E’ihrin n&ml
Rshhit no.
vitreous
Duration haemorrhitge
2 2 4 4 N 8 2 2 I 1 5 .5 6 2 2
21 10 20 Ii 1s 15 16 13 14 12 4 11 5 6
aayc days tlays tiays tlays days wreks weks month month wwks weeks weeks months months
TSHLE
Rabbit
Test
Control
1 2 3 4
0.40 0.41 0.43 0.00 0.41 0.41 0.40 0.m
0.37 0.39 0.46 0.42 0.41 O-RX 0.35 0.35
5
6 7 8
F.D.1’. Test
in
pg/ml (~‘ontrol
IO IX 8 8 5 .i
Xl Sil Xii Nil Sil
10
Xl Sil
14 s 8
8 47 Xl Xl I0
111
Duration of haemorrhage
vitreous
2 days
2 1 1 2 2 Y 8
weeks mont,h month months months months months
Sil
Xl
Sil Nil Nil Xii Xii Sil
186
FORRESTER
et al.
the precipitation ring against known concentrations of fibrinogen, and throughout the period of resolution of the haemorrhage, the level was low (Table II). Results obtained by the three methods (R.I., T.R.C.H.I.I. and S.C.T.) were within a comyarable range. No F.D.Ps were found in normal rabbit vitreous control. Plusminogen activator activity Fibrinolytic activity of the vitreous was similar following vitreous haemorrhage to that of normal control samples (Table III). Values are expressed in equivalent mean values of urokinase (Forrester et al., 1974). 4. Discussion These experiments have shown that it takes five weeks for fibrin to be removed from rabbit vitreous following the intravitreal injection of 0.2 ml autogenous whole blood. This time sequence is slow in comparison with other extravascular deposits of fibrin (Kwann and Astrup, 1963) and there are several possible reasons for t,his. Current theories of thrombolysis (Sherry, 1968) suggest that the rat,e of fibrin digestion is dependent on the plasminogen activator concentrations in the surrounding medium. Since the level of activator activity within the vitreous is known to be low (Forrester et al., 1974), a simple mass effect of this nature might explain the slow rate of intravitreal fibrinolysis. Further, if it is possible to extrapolate to the human situation. the notoriously poor resolution of recurrent intravitreal haemorrhage is probabl! not related to consumption of activator activity, since this appears to be similar before and after vitreous haemorrhage (Table III). A second explanation for the delayed clearance of blood from the vitreous is suggested by the type of fibrin within a vitreous clot. Lorand and Jacobsen (1962) have shown that cross-linked fibrin imore resistant to digestion by plasminogen than fibrin monomer. The experiments, reported here using whole blood show that not only was fibrin formed within rahbit vitreous clots, but that the fibrin was fully cross-linked, since there was no evidence of the M or y monomer on S.D.S. polyacrylamide gel electrophoresis (Fig. 1). lntravitreal fibrinolysis would therefore proceed at a slow rate. Whatever the reasons for slow intravitreal fibrin digestion, it is clearly imporbant to relate fibrinolysis to the whole of vitreous haemorrhage resolution. Most workers accept that there are at least three mechanisms involved in the clearance of blood from the vitreous; haemolysis, fibrinolysis and phagocytosis. Horven (196413) has shown by autoradiography that red cells cannot, he reabsorbed intact from the vitreous into the general circulation, but that they must first be degraded and their breakdown products reabsorbed. Greer et al. (1968) using 51Cr tagged red cell,* confirmed that haemolysis is a rate-limiting step in vitreous haemorrhage resolution. The presence of F.D.P.s within the soluble component of the vitreous (Table IIj indicates that intravitreal fibrinolysis is occurring and furthermore our data suggest that haemolysis occurs simultaneously (Fig. 1). since the quantity of haemoglobin remaining in the samples after the free haemoglobin has been removed bv washing. becomes progressively less. The role of phagocytosis is more difficult to define. Stimulation of macrophagr activity has been induced by short bouts of hyperpyexia in rats (Von Sallman. 1950). by immune uveitis with bovine serum albumin and by bacterial endophthalmitis in rabbits (Benson et al. 1969) and in all cases led to accelerated clearance of vitreous haemorrhage. Depression of the inflammatory response with systemic ACTH in
INTRAVITREAL
FIBRINOLYSIS
187
rabbits retarded the rate of vitreous haemorrhage resolution (Schimek and Steffensen, 1955) and at the same time? a reduction in the vitreous macrophage count was observed. It was therefore suggested that phagocytosis was the important step in vitreous haemorrhage resolution. On the other hand, Benson et al. (1969) found systemic cortisone acetate had no effect on vitreous haemorrhage resolution in rabbits at a dose of 1.3 mg kg-l day-l for 14 days. In addition. although Maberly and Chisholm (1970) confirmed the accelerated clearance of vitreous blood due to inflammation, in this case induced by the intravitreal injection of an inactivated fibrinolytic enzyme, these authors also observed that the active enzyme led to even more rapid clearance, suggesting that the clot lysis was due to the fibrinolytic effect of the enzyme rather than to the induced phagocytic response alone. From our observations, fibrinolysis has generally been completed when the first visible signs of clot fragmentation occur within the vitreous at five weeks (Fig. 1). This suggests that fibrinolysis may be the important ratelimiting factor. A further complication arises, however, in trying to separate fibrinolysis and phagocytosis as individual stages in vitreous haemorrhage resolution. It is known that lysosomes of macrophages possess fibrinolytic activity (Lack and Ali, 1964) and there is no evidence from these experiments to suggest that intravitreal fihrinolysis is due to vitreous activator activity rather than macrophage activity. The distinction may be artificial, but, whatever the mechanism, fibrinolysis is an integral part of vitreous haemorrhage resolution.
REFERESCES Benson, W. F., Wirostko. E. and Spalter. H. F. (1969). The effects of inflammation on experimentally induced vitreous hsemorrhage. Arch. Ophthalmol. 82,822-6. Benson, W. E. and Spalter, H. F. (1971). Vitreous haemorrhage: a review of experimental and clinical investigation. Surv. Ophthalmol. 15, 2977311. Forrestcr. J. V., Prentice. C. R. M., Williamson, J. and Forbes, C. D. (1974). Fibrinolytic activit,y of the vitreous body. Incest. Ophthdmol. 13, 875-9. t:recr. D. F., Benson, W. E. and Spalter, H. F. (1968). A study of simulated vitreous haemorrhage using labelled blood. Arch. Ophthalmol. 79, 755-8. Hawiger, J., Niewiarowski, S., Guerewich, V. and Thomas. D. P. (1970). Measurement of fibrinogen and fibrin degradation products in serum by t,he staphylococcal clumping test. J. Lab. Clin. Med. 75, 93-108. Horven, I. (1964a). A radioautographic study of erythroryte resorption from the anterior chamber of the human eye. Acta. Ophthdmol. 42,600-k .Horven, I. (1964b). A radioautographic study of the crythrocyte phagoryting cells in the rabbit eye. Acta. Ophthulmol. 42, 800-11. Huisman, T. H. J. and Schroeder, W. A. (1971). ,Vew dspects in the Btructure, Function and Synthesis of Haemoglobins. P. 40. Butterworth. London. Kwaan, H. C. and Astrup, T. (1963). Localization of fibrinolytic activity in the eye. Arch. Pathol. 76, 595-9. Lack. C. H. and Ali, S. Y. (1964). Tissue activator of plasminogen. Natwe (London) 201, 1030. Lam. K. W., Ashrafzadeh, M. T. and Lee, G. B. (1972). Vitreous membranes: induction in rabbits by intravitreous leukocyte injection. Arch. Ophthulmol. 88,655-8. Laurent, 0. B. G., Laurent, T. C. and Howe, A. F. (1962). Chromatography of soluble proteins from the bovine vitreous body on DEAE-cellulose. Exp. Eye Res. 1, 276-85. Lorxnd. L. (1972). Fibrinoligase: the fibrin-stabilizing factor system of blood plasma. Ann. N. Y. dead. Sri. 202, B-30. Lorand. L. and Jacobsen, A. (1962). Accelerated lysis of blood clots. Nature (London) 195, 911. Mahrrly, A. L. and Chisholm, L. D. J. (1970). The effect of a fibrinolytic agent, in vitreous haemorrhage in rabbits. Can. J. Ophthnlmol. 4, X-64.
188
FOKRES
TEK
et, al.
Mancini, G., Carbonara, A. 0. and Hergmans, J. I?. (1965). Immunochemiral quant,itation of antigens by single radial immunodiffusion. Immune-chemistry 2, 235-54. estimation of split products Merskey, C., Kleiner. G. J. and Johnston. A. J. (1966). Q uantit,at,ive of fibrinogen in human serum, relation to diagnosis and t,reatment. Blood 28, l-18. Nillson, I. M. and Olow, B. (1962). Fibrinolysis induced by streptokinase in man. Scfn. (!hir. Scar&. 123, 247. Regnault, F. R. (1969). Action du vitr& sur la czoagulation. t’nthol. Biol. 17, 31-i. Regnault, I?. R. (1970). Vitreous haemorrhage, an experimental study. Arch. Ophth/llwol. 83, 458-74. E. H. (1955). Vitreous hazc~morrhagc absorption. .~wI,,.. d. Schimek, R. j\. and Steffensen, Ophth&mol. 39, 677-83. Sherry, S. (1968). Fibrinolysis. Ann. Rev. Xed. 19, 27-i. Swain, m’. R. and Feders, M. B. (1967). Fibrinogen assay. (‘lin. (‘hw,)~. 13, 1 lY?-Ci. Swaan, 1). a., Chesney, C. MCI., Constabk, I. J.. Colman. It. \\‘., (‘aulfield, .J. 1~. and Harpy, E. (1974). The role of vitreous collagen in plat,elet a,,ovregation in rifro and in. I+O. .I. Lrrh. Gin. Med. 84, 264-74. Von Sallman, L. (1950). Experimental studies on the vitreous. I I. Experiments on dissppr>lrau(.e of red blood cells from the vitreous. Arch. Ophthalmol. 43, 638-52. Weber, K. and Osborne, M. (1969). The reliability of molecular weight determinations by (lodrcyl-sulphate polyacrylamide gel ele&ophoresis. J. Biol. Chem. 244, 44OCi- 12.
Intravitreal Fibrinolysis in Experimental Vitreous Haemorrhage .J. Y. FORRESTICR: W. EDGAR: C’. R. M. PRENTICE, C'. D. FORBES AND J. WILLIAMSOS
(Receiwd
9 September
1975. Lon.dw)
The fibrinolytic system within experimentally induced vitreons haemorrhages was studied in rabbits. The presence of crosslinked fibrin was noted for up to five weeks following induction of the haemorrhage, while soluble fibrin degradation products were detected in low concentration for two months. Plasminogen activator activity of the vitreous did not decline following vitreous haemorrhage. In addition haemolysis appeared to occur simrlltaneously with fibrinolysis. It is suggested that fibrinolysis is important in the clearing of blood from t,he vitreous? and may be the initiating st,ep.
1. Introduction The pathophysiology of vitreous haemorrhage resolution has attracted the attention of many workers in the experimental field (for review. seeBenson and Spalter, 1971). Several aspects have been studied including haemolysis (Greer. Benson and Spalter, 1968), phagocytosis (Horven, 1964a), membrane formation (Lam, Ashrafzadeh and Lee. 1972; Swann. Chesney, Constable, Colman. Caulfield and Harper, 1974) ant1 the ocular t,oxicity of intravitreal blood (Regnault. 1970). Scant attention has been paid to the fibrinolytic mechanismswithin a vitreous haemorrhage. It is known that, vitreous has a procoagulant effect on blood, (Regnault. 1969) and histologicall! fibrin has been observed within experimental vitreous clots (Schimek and Steff~~nsen. 1955; Horven: 1964b). In a study of experimental vitreous haemorrhage report,ed bv Regnault (1970) where lzjl labelled human fibrinogen was injected into rabbit vitreons. the half-life of the fibrinogen was found to be four days with no trace of radioactivity after 20 days. On this basis, it was suggestedthat spontaneous lvsis of fibrin occurs as the initial step in the resolution of vitreous haemorrhage. * In a previous study (Forrester, PrenCce, Williamson and Forbes. 1974)we reported t’he presence of t,issueplasminogen activator activity within normal vitreous. and proposedthat intravitreal fibrinolysis occurred as the result of plasminogenactivat)ion by this enz”ymewithin the vitreous clot. The object of the experiments reported her{> was to record the progressive removal of fibrin from an intravitreal haemorrhage and to look for rlvidcnce of intra,vit,real fihrinolysis in the form of soluble fibrin breakdown pr”‘lll’t~s.
2. Materials and Methods .4 n Aal models 411 experiments were performed on white New Zealand rabbits weighing 3-4 kg and anaesthetized with intravenous pentobarbital (20 mg/kg wt). Local anaesthetic (Ametho(Gne drops lo/o) was instilled into the conjunctival sac and the lids were retracted with a speculum. The right globe of each animal was softened by withdrawing 0.2 ml aqueous from the anterior chamber using a 25-gauge needle gently inserted through the limbue. The eye was immobilized by fixing the superior rectue muscle and O-2 ml of whole blood 1Sl
182
FORRESTER
et al.
was withdrawn from the marginal ear vein into a plastic syringe. Under ophthalrnoscopit control the blood was injected immediately into the centre of the vitreous gel through the pars plana in the anterosuperior quadrant of the globe. A short pause was taken before the needle was sharply withdrawn, without tracking of blood under the conjunctiva. Chloramphenicol 0.5% and Atropine 1.0% drops were instilled into the lower fornix at the end of the operation. The resolution of the vitreous haemorrhage was monitored weekly by ophthalmoscop,v for a period of eight months following injection. Animals were sacrificed at, various stages and the globes were enucleated immediately post mortem, and transported on ice to the laboratory. Preparation of vitreorss extract The eyes were placed in polythene bags and immersed in a solution of CO, snow in acetone (--4O--60°C) for 60 sec. They were then sectioned with a skin-graft (Thiersch) knife and the frozen vitreous clot was removed with the aid of the dissecting microscope, thawed and centrifuged at x 200 g for 5 min. 10 ~1 of Trasylol (10 000 u/ml) (Bayer, Ltd.. Germany) and 10 N.I.H. u of bovine thrombin (100 u/ml) (Parke Davis & Co., Detroit, Mich.) were added to the supernates, which were recentrifuged at x 1750 g for 15 min and stored at -20°C. The residual solid vitreous clot was washed for 24 hr in 085% saline. The normal vitreous of the left eye was removed in similar fashion and served as a control. F&in
analysis
The detection and quantitation of fibrin within the vitreous clot was carried out bj polyacrylamide gel electrophoresis in sodium dodecyl sulphate using 7.5% acrylamide gel, as described by Weber and Osborne (1969). Control samples of rabbit fibrinogen, rabbit fibrin, normal rabbit vitreous and plasmin-incubated vitreous haemorrhage were also run. The quantity of crosslinked fibrin was estimated by densitometric scanning of the gels at 600 nm using a Gilford spectrophotometer with scanning attachment, comparing the y dimer peak heights with known concentrations of crosslinked rabbit fibrin. Detection of Fibrin Degradation Products (F.D.P.s) The presence of F.D.P.s within the supernate from the vitreous clot was estimated in all samples by the radial immunodiffusion (RI) technique [Mancini, Carbonara and Hergmans (1965)J. Anti-rabbit fibrinogen serum (Hoeehst Pharmaceuticals Ltd., Germany) was incorporated into the 176 agar support at a concentration of 0.4?$,. Rabbit plasma in dilutions of 1:50, 1 :lOOand 1:300 served as the standard. Plasma fibrinogen was measured by the method of Swain and Feders (1967)in blood collected in 4.896 citrate (8:l). Some samples were tested also by the tanned red cell haemagglutination inhibition immunoassay (TRCHII) (Merskey, Kleiner and Johnston, 1966) using anti-rabbit fibrinogen serum and clumping test sheep erythrocytes sensitized with rabbit plasma. The staphylococcnl (S.C.T.) (Hawiger, Niewiarowski, Guerewich and Thomas, 1970) was used to verif! quantitation of FDP concentration by the RI technique.
Plasminogen activator. activity The fibrin plate technique (Nilsson and Olow, 1962) was used to measure the plasminopen activator activity of the supernate from the vitreous haemorrhage, while the normal vitreous from the fellow eye served as the control.
3. Results ophthulmoscopy The changes
occurring in a resolving experimental vitreous haemorrhage have been documented by several workers (Schimek and Steffensen, 1955; Regnault, 1970).
INTRAVITREAL
FIBRIh-OLYSIS
183
Following injection, a circumscribed clot occupied the central vitreous for 24-48 hr and then from the third day the clot was redistributed to occupy the whole vitreous. A moderate proteinaceous iritis was noted at this stage. Numerous strands gradually developed throughout the blood clot but no fragmentation of the opaque haemorrhage occurred until the fifth to sixth week post-injection. Central white membranes were noted coming from the injection site in some animals. Between the sixth week and the thirteenth week increasing fundal detail was observed through gaps in t,he vit,reous opacities. These black and grey-white membranes were gradually removed until by three months the vitreous was clear, apart from a few floaters and minimal vit,reous haze. Very little solid material was obtained from the vitreous after 10 \\WkS. Polyucrylumicle
gel electrophoresis
Rabbit cross-linked fibrin [Fig. l(a)], obtained by incubating rabbit plasma with bovine thrombin in the presenceof Ca2+ions for 2 hr at 37”C, produced two prominent, banclscorresponding to the /3 chain and the yy dimer of the fibrin polymer (Lorand. 1972). The bands representing the c( and y monomers indicated that the fibrin was not fully cross-linked. Normal rabbit vitreous [Fig. 2(c)] contained no proteins with molecular weights corresponding to the fibrin chains, but several other soluble protein bands were present (Laurent. Laurent and Howe, 1962). Solubilized extracts from the solid vitreous clots of varying duration produced bands corresponding to the /3 monomer and yy dimer chains, indicating the presenceof strongly cross-linked fibrin within the vitreous for up to five weeks following induction of the haemorrhage [Fig. l(b) to (f)]. Ko fib rm was detected after this time [Fig. l(g) and(h)]. Several other lower molecular weight proteins were noted with two prominent bands appearing at molecular weights of 32 000 and 16 000 daltons (Fig. 1. x and 1. y). The lower band (Fig. 1, y) was thought to represent a monomer and the upper band a dimer of the constituent chains of the haemoglobulin molecule. released by haemolysis of the intact red cell within t.he solid clot by the extraction procedure with 8 M-urea, since any free haemoglobin would have been removed during the preliminary wash. Accordingly rabbit red cells wrerehaemolysed in distilled water, and the supernatant passedthrough a column of Sepharose-4B. The eluate corresponding to the haemoglobin peak (mol. wt 50 000.-100000) was then run on SDS-polyacrylamide gel electrophoresis as described. Two broad bands were observed in identical positions t,o Fig. 1. x and y. It is not understood why the globin molecule is not fully depolymerized by the 2-mercaptoethanol. but it may be related to the fact that the chain of rabbit globin is not a single polypeptide but a mixture of closely related proteins (Huisman and Schroeder. 1971). Further confirmation of the presenceof fibrin within the vit’reous clots was obtained by the disappearance of the fi and yy bands following incubation of the clot with plasmin (Fig. 2). The quantity of fibrin estimated by gel scan spectrophotometry ih shown in Table I. Removal of the fibrin occurred progressively during the first six weeks of resolution. E’.D.P.
estima~tiol~
Precipitation rings in the. Mancini plates surrounding the wells with supernatant vitreous were observed for up to six to eight weeks following injection of blood. Inconsistent results were obtained after this time, there generally being no F.D.P.s after two months. The concentration of F.D.P.s was measured by the diameter of
x-
Y-
FIG. 1. Polyacrylamide gel electrophorcsis. (a) prominent /3 and yy rlimer IM& II, control rruss-linkrtl rabbit fibrin. (b)-(f) /3 and yy bands present in vitreous haemorrhage extracts. (g) and (h) no fibrin ban&s present in vitreous haemorrhage extracts after 5 weeks. (h) -R days. (0) 1 week. (d) 2 lveeks. (e) 4 wwks. (f) 5 weeks. (g) 6 weeks. (h) 2 months.
FIG. 2. l’olyacrylamide and yy fibrin chains. vitreous control.
gel electrophoresis: (b) same haemorrhage
(a) vitreous haemorrhage rstract showing wcll.d~4inwl p extract following incubation with plasmin. (v) normal
IXTRAVITREAL TABLE
Vitreous
1x5
FIHRINOLVSIS 1
haewtorrhagejlwi~~~
comentration
E’ihrin n&ml
Rshhit no.
vitreous
Duration haemorrhitge
2 2 4 4 N 8 2 2 I 1 5 .5 6 2 2
21 10 20 Ii 1s 15 16 13 14 12 4 11 5 6
aayc days tlays tiays tlays days wreks weks month month wwks weeks weeks months months
TSHLE
Rabbit
Test
Control
1 2 3 4
0.40 0.41 0.43 0.00 0.41 0.41 0.40 0.m
0.37 0.39 0.46 0.42 0.41 O-RX 0.35 0.35
5
6 7 8
F.D.1’. Test
in
pg/ml (~‘ontrol
IO IX 8 8 5 .i
Xl Sil Xii Nil Sil
10
Xl Sil
14 s 8
8 47 Xl Xl I0
111
Duration of haemorrhage
vitreous
2 days
2 1 1 2 2 Y 8
weeks mont,h month months months months months
Sil
Xl
Sil Nil Nil Xii Xii Sil
186
FORRESTER
et al.
the precipitation ring against known concentrations of fibrinogen, and throughout the period of resolution of the haemorrhage, the level was low (Table II). Results obtained by the three methods (R.I., T.R.C.H.I.I. and S.C.T.) were within a comyarable range. No F.D.Ps were found in normal rabbit vitreous control. Plusminogen activator activity Fibrinolytic activity of the vitreous was similar following vitreous haemorrhage to that of normal control samples (Table III). Values are expressed in equivalent mean values of urokinase (Forrester et al., 1974). 4. Discussion These experiments have shown that it takes five weeks for fibrin to be removed from rabbit vitreous following the intravitreal injection of 0.2 ml autogenous whole blood. This time sequence is slow in comparison with other extravascular deposits of fibrin (Kwann and Astrup, 1963) and there are several possible reasons for t,his. Current theories of thrombolysis (Sherry, 1968) suggest that the rat,e of fibrin digestion is dependent on the plasminogen activator concentrations in the surrounding medium. Since the level of activator activity within the vitreous is known to be low (Forrester et al., 1974), a simple mass effect of this nature might explain the slow rate of intravitreal fibrinolysis. Further, if it is possible to extrapolate to the human situation. the notoriously poor resolution of recurrent intravitreal haemorrhage is probabl! not related to consumption of activator activity, since this appears to be similar before and after vitreous haemorrhage (Table III). A second explanation for the delayed clearance of blood from the vitreous is suggested by the type of fibrin within a vitreous clot. Lorand and Jacobsen (1962) have shown that cross-linked fibrin imore resistant to digestion by plasminogen than fibrin monomer. The experiments, reported here using whole blood show that not only was fibrin formed within rahbit vitreous clots, but that the fibrin was fully cross-linked, since there was no evidence of the M or y monomer on S.D.S. polyacrylamide gel electrophoresis (Fig. 1). lntravitreal fibrinolysis would therefore proceed at a slow rate. Whatever the reasons for slow intravitreal fibrin digestion, it is clearly imporbant to relate fibrinolysis to the whole of vitreous haemorrhage resolution. Most workers accept that there are at least three mechanisms involved in the clearance of blood from the vitreous; haemolysis, fibrinolysis and phagocytosis. Horven (196413) has shown by autoradiography that red cells cannot, he reabsorbed intact from the vitreous into the general circulation, but that they must first be degraded and their breakdown products reabsorbed. Greer et al. (1968) using 51Cr tagged red cell,* confirmed that haemolysis is a rate-limiting step in vitreous haemorrhage resolution. The presence of F.D.P.s within the soluble component of the vitreous (Table IIj indicates that intravitreal fibrinolysis is occurring and furthermore our data suggest that haemolysis occurs simultaneously (Fig. 1). since the quantity of haemoglobin remaining in the samples after the free haemoglobin has been removed bv washing. becomes progressively less. The role of phagocytosis is more difficult to define. Stimulation of macrophagr activity has been induced by short bouts of hyperpyexia in rats (Von Sallman. 1950). by immune uveitis with bovine serum albumin and by bacterial endophthalmitis in rabbits (Benson et al. 1969) and in all cases led to accelerated clearance of vitreous haemorrhage. Depression of the inflammatory response with systemic ACTH in
INTRAVITREAL
FIBRINOLYSIS
187
rabbits retarded the rate of vitreous haemorrhage resolution (Schimek and Steffensen, 1955) and at the same time? a reduction in the vitreous macrophage count was observed. It was therefore suggested that phagocytosis was the important step in vitreous haemorrhage resolution. On the other hand, Benson et al. (1969) found systemic cortisone acetate had no effect on vitreous haemorrhage resolution in rabbits at a dose of 1.3 mg kg-l day-l for 14 days. In addition. although Maberly and Chisholm (1970) confirmed the accelerated clearance of vitreous blood due to inflammation, in this case induced by the intravitreal injection of an inactivated fibrinolytic enzyme, these authors also observed that the active enzyme led to even more rapid clearance, suggesting that the clot lysis was due to the fibrinolytic effect of the enzyme rather than to the induced phagocytic response alone. From our observations, fibrinolysis has generally been completed when the first visible signs of clot fragmentation occur within the vitreous at five weeks (Fig. 1). This suggests that fibrinolysis may be the important ratelimiting factor. A further complication arises, however, in trying to separate fibrinolysis and phagocytosis as individual stages in vitreous haemorrhage resolution. It is known that lysosomes of macrophages possess fibrinolytic activity (Lack and Ali, 1964) and there is no evidence from these experiments to suggest that intravitreal fihrinolysis is due to vitreous activator activity rather than macrophage activity. The distinction may be artificial, but, whatever the mechanism, fibrinolysis is an integral part of vitreous haemorrhage resolution.
REFERESCES Benson, W. F., Wirostko. E. and Spalter. H. F. (1969). The effects of inflammation on experimentally induced vitreous hsemorrhage. Arch. Ophthalmol. 82,822-6. Benson, W. E. and Spalter, H. F. (1971). Vitreous haemorrhage: a review of experimental and clinical investigation. Surv. Ophthalmol. 15, 2977311. Forrestcr. J. V., Prentice. C. R. M., Williamson, J. and Forbes, C. D. (1974). Fibrinolytic activit,y of the vitreous body. Incest. Ophthdmol. 13, 875-9. t:recr. D. F., Benson, W. E. and Spalter, H. F. (1968). A study of simulated vitreous haemorrhage using labelled blood. Arch. Ophthalmol. 79, 755-8. Hawiger, J., Niewiarowski, S., Guerewich, V. and Thomas. D. P. (1970). Measurement of fibrinogen and fibrin degradation products in serum by t,he staphylococcal clumping test. J. Lab. Clin. Med. 75, 93-108. Horven, I. (1964a). A radioautographic study of erythroryte resorption from the anterior chamber of the human eye. Acta. Ophthdmol. 42,600-k .Horven, I. (1964b). A radioautographic study of the crythrocyte phagoryting cells in the rabbit eye. Acta. Ophthulmol. 42, 800-11. Huisman, T. H. J. and Schroeder, W. A. (1971). ,Vew dspects in the Btructure, Function and Synthesis of Haemoglobins. P. 40. Butterworth. London. Kwaan, H. C. and Astrup, T. (1963). Localization of fibrinolytic activity in the eye. Arch. Pathol. 76, 595-9. Lack. C. H. and Ali, S. Y. (1964). Tissue activator of plasminogen. Natwe (London) 201, 1030. Lam. K. W., Ashrafzadeh, M. T. and Lee, G. B. (1972). Vitreous membranes: induction in rabbits by intravitreous leukocyte injection. Arch. Ophthulmol. 88,655-8. Laurent, 0. B. G., Laurent, T. C. and Howe, A. F. (1962). Chromatography of soluble proteins from the bovine vitreous body on DEAE-cellulose. Exp. Eye Res. 1, 276-85. Lorxnd. L. (1972). Fibrinoligase: the fibrin-stabilizing factor system of blood plasma. Ann. N. Y. dead. Sri. 202, B-30. Lorand. L. and Jacobsen, A. (1962). Accelerated lysis of blood clots. Nature (London) 195, 911. Mahrrly, A. L. and Chisholm, L. D. J. (1970). The effect of a fibrinolytic agent, in vitreous haemorrhage in rabbits. Can. J. Ophthnlmol. 4, X-64.
188
FOKRES
TEK
et, al.
Mancini, G., Carbonara, A. 0. and Hergmans, J. I?. (1965). Immunochemiral quant,itation of antigens by single radial immunodiffusion. Immune-chemistry 2, 235-54. estimation of split products Merskey, C., Kleiner. G. J. and Johnston. A. J. (1966). Q uantit,at,ive of fibrinogen in human serum, relation to diagnosis and t,reatment. Blood 28, l-18. Nillson, I. M. and Olow, B. (1962). Fibrinolysis induced by streptokinase in man. Scfn. (!hir. Scar&. 123, 247. Regnault, F. R. (1969). Action du vitr& sur la czoagulation. t’nthol. Biol. 17, 31-i. Regnault, I?. R. (1970). Vitreous haemorrhage, an experimental study. Arch. Ophth/llwol. 83, 458-74. E. H. (1955). Vitreous hazc~morrhagc absorption. .~wI,,.. d. Schimek, R. j\. and Steffensen, Ophth&mol. 39, 677-83. Sherry, S. (1968). Fibrinolysis. Ann. Rev. Xed. 19, 27-i. Swain, m’. R. and Feders, M. B. (1967). Fibrinogen assay. (‘lin. (‘hw,)~. 13, 1 lY?-Ci. Swaan, 1). a., Chesney, C. MCI., Constabk, I. J.. Colman. It. \\‘., (‘aulfield, .J. 1~. and Harpy, E. (1974). The role of vitreous collagen in plat,elet a,,ovregation in rifro and in. I+O. .I. Lrrh. Gin. Med. 84, 264-74. Von Sallman, L. (1950). Experimental studies on the vitreous. I I. Experiments on dissppr>lrau(.e of red blood cells from the vitreous. Arch. Ophthalmol. 43, 638-52. Weber, K. and Osborne, M. (1969). The reliability of molecular weight determinations by (lodrcyl-sulphate polyacrylamide gel ele&ophoresis. J. Biol. Chem. 244, 44OCi- 12.