1438 Br J Ophthalmol 1998;82:1438–1440 Ultrasound enhanced thrombolysis in experimental retinal vein occlusion in the rabbit Jörgen Larsson, Jonas Carlson, S Bertil Olsson Abstract such as myocardial infarction, which is life Aims—To investigate if it was possible to threatening, this incidence of haemorrhage is lower the dose of streptokinase and main- acceptable, but in a patient with a retinal vein tain an eVective thrombolysis by adding occlusion it is hard to accept life threatening pulsed low energy ultrasound. side eVects. Methods—53 retinal veins in 27 rabbits Dye enhanced photothrombosis is a method were occluded by rose bengal enhanced where a dye that absorbs maximally at a laser treatment. Six rabbits were treated specific wavelength is injected intravenously with streptokinase (50 000 IU/kg), 10 rab- immediately before laser treatment in order to bits were treated with a low dose of strep- enhance the absorption of the laser light and tokinase (25 000 IU/kg), and 11 rabbits thus making it possible to use less laser energy. were treated with a low dose of streptoki- This method easily produces thrombi in the nase (25 000 IU/kg) and pulsed ultrasound vessels.18–20 during 1 hour. Fluorescein angiography Based on earlier in vitro experiences21 22 we was performed immediately before the wanted to investigate whether it was possible to thrombolytic treatment and after 12 lower the dose of streptokinase by adding hours. pulsed low energy ultrasound towards a Results—In the group treated with strep- thrombus in the eye. We investigated this in a tokinase (50 000 IU/kg) all vessels were model of experimental retinal vein occlusion in open. In the group that was given strep- the rabbit eye. tokinase (25 000 IU/kg), 21% of the vessels were open. In the group that was treated with streptokinase (25 000 IU/kg) and Methods ultrasound, 64% of the vessels were open. Twenty seven albino New Zealand rabbits were The diVerence between groups 2 and 3 is used (27 eyes, 53 vessels), each weighing 3–3.5 statistically significant (p= 0.011) kg. The rabbits were anaesthetised with a mix- Conclusion—Adding pulsed low energy ture of Ketalar and Rompun. Their pupils were ultrasound makes it possible to lower the dilated with 1% cyclopentolate eyedrops. The dose of streptokinase while maintaining a animals were treated according to the guide- good thrombolytic eVect. lines and requirements of the government (Br J Ophthalmol 1998;82:1438–1440) committee on animal experimentation ethics of the University of Lund and the ARVO conven- tion for animal experimentation ethics. Retinal vein occlusion is the second most com- Occlusion of the retinal veins was performed mon vascular disease in the human eye. The by dye enhanced photothrombosis. We used most severe form of retinal vein occlusion is the same method to induce thrombi in the reti- central retinal vein occlusion (CRVO). There nal veins as has been described previously by are two forms of CRVO, the ischaemic and the Oncel et al.23 Rose bengal solution, 50 mg/kg non-ischaemic, both leading to loss of visual (50 mg/ml in sterile water), was injected in an acuity. About 20–30% of the CRVOs are ear vein immediately before the laser treat- ischaemic and the majority of them develop ment. Argon green laser treatments were secondary glaucoma and rubeosis.1 Many performed with a power of 0.14 mW, a spot diVerent treatments have been tried such as size of 100 µm, and a duration of 0.3 seconds. Department of haemodilution,2–8 thrombolytic treatment,9–12 Each vessel was treated at a distance of a half to Ophthalmology, Lund 13 University Hospital, formation of chorioretinal anastomoses, and one disc diameter from the optic disc, until the Sweden diVerent drugs14 15 that influence the microcir- blood flow was completely stopped in the vein. J Larsson culation. For diVerent reasons, none of these About 5–20 laser eVects were needed for each treatments is widely used today, either because vessel. Department of of severe side eVects or they are considered to The rabbits were randomised into three Cardiology, Lund University Hospital, be too complicated, such as haemodilution. groups—group 1 (six rabbits, six eyes, 12 Sweden Thrombolytic treatment, with streptokinase vessels) was treated with an infusion in an ear J Carlson and tissue plasminogen activator, is one of the vein of 50 000 IU/kg streptokinase for 1 hour; S B Olsson most promising methods of treating a throm- group 2 (10 rabbits, 10 eyes, 19 vessels) was bus in the eye since it dissolves the clot, allow- treated with an infusion in an ear vein of Correspondence to: Jörgen Larsson, Department ing the circulation to be re-established within a 25 000 IU/kg streptokinase for 1 hour; group 3 of Ophthalmology, Lund few hours. The side eVects of thrombolytic (11 rabbits, 11 eyes, 22 vessels) was treated University Hospital, 221 85 treatment are haemorrhages and are a great with an infusion in an ear vein of 25 000 IU/kg Lund Sweden. problem. The incidence of severe haemorrhage streptokinase for 1 hour and was simultane- Accepted for publication when giving streptokinase to treat a myocardial ously given pulsed ultrasound directed towards 23 July 1998 infarction is around 0.5–1.0%.16 17 In a disease the occluded vessels. Ultrasound enhanced thrombolysis 1439 FLUORESCEIN ANGIOGRAPHY Thirty minutes after the laser treatment, fluorescein angiography was performed in order to confirm that the vessels were com- pletely occluded. Sodium fluorescein at a con- centration of 25% (250 mg/ml in sterile water) was injected in the ear vein. Fluorescein angio- graphy was performed using the scanning laser ophthalmoscope (Rodenstock, Heidelberg, Germany). Vessels that were only partially occluded were excluded from the study. Twelve hours after the treatment, fluorescein angio- graphy was performed again and the patency of the vessels was evaluated. The evaluation of the patency was performed by two of the authors (JL and JC) in a masked mode. All the pictures used for the evaluation were take 20–30 seconds after the injection of fluorescein. ULTRASOUND EXPOSURE Unfocused piezoelectric transducers (Ceram AB, Lund, Sweden) with a resonance fre- quency of 1.0 MHz and diameter of 20 mm were used. The transducers were excited by an electronic system consisting of a function gen- erator (HP 3314A, Hewlett-Packard, Washing- Figure 1 Theses fluorescein angiograms show an eye from group 3 that had been treated with both streptokinase and ton, USA) and a RF power amplifier (ENI ultrasound. (A) The angiogram 30 minutes after the laser 240L, ENI, Rochester, New York, USA). The treatment shows that the vein is totally occluded peripheral output of the function generator was manually to the thrombus. There is fluorescein in that part of the vein that is central to the thrombus, because of small veins that set to produce the desired ultrasound charac- empty in the central part of the vein. (B) The angiogram teristics, as described below. 12 hours after the treatment shows that the vessel is open. The transducers were calibrated by measur- Leakage from the retina of the fluorescein is noted around ing the total radiation pressure on an ultra- the place where the laser treatment had been performed. sound absorbing material (Ceram AB), using open were considered as closed. Leakage of the an electronic balance (Precisa 400M, PAG fluorescein was noted around the laser treat- Oerlikon AG, Zurich, Switzerland). In the ment site. experiment series, up to three transducers were In group 1 receiving streptokinase (50 000 coupled in parallel. Therefore, measurements IU/kg), all the vessels that had been occluded were made to verify that the ultrasound output were open. In group 2, which had only been was the same regardless of the number of treated with streptokinase (25 000 IU/kg), transducers coupled to the amplifier. 4/19 (21%) of the vessels were open, and in To allow good acoustic coupling between the group 3, which was treated with streptokinase transducer and the eye, ultrasound transmis- (25 000 IU/kg) and ultrasound, 14/22 (64%) sion gel was used (Aquasonic 100, Parker of the vessels were open (Fig 1). Laboratories Inc, NJ, USA). Owing to interfer- We used Fisher’s exact test to assess statisti- ence phenomena, the ultrasound beam is not cal significance. There is a statistical significant uniformly distributed close to the surface of diVerence between groups 1 and 2 (p<0.0001; the transducer. To avoid this aVecting the two sided), between groups 1 and 3 (p=0.03; experiment, the ultrasound transmission gel two sided), and between groups 2 and 3 was put between the cornea and the trans- (p=0.011; two sided). ducer. The distance was typically 10 mm between the transducer and the cornea, giving a total distance between the transducer and the Discussion vessel of about 25 mm. Thrombolytic treatment with streptokinase or Pulsed ultrasound of 1 MHz frequency was tissue plasminogen activator of retinal vein used: 100 pulses of 1 W/cm2 was sent every occlusions in humans has been performed millisecond (10% duty cycle), resulting in a before with fairly good results.9–12 24–26 The spatial average, temporal average intensity of thrombolytic treatment is not widely used 0.1 W/cm2. today in retinal vein occlusion, because of Pulsed low energy ultrasound was given dur- severe side eVects such as haemorrhage. If it ing the same period of time that streptokinase were possible to lower the dose of the was administered—that is, for 1 hour. thrombolytic drug and thus lower the fre- The rabbits were killed immediately after the quency of the side eVects, and still maintain second fluorescein angiography by an intra- good thrombolytic eVect by adding therapeutic venous injection of pentobarbitone.