Branch Retinal Vein Occlusion: Pathogenesis, Visual Prognosis, and Treatment Modalities

In branch retinal vein occlusion (BRVO), abnormal arteriovenous crossing with vein compression, degenerative changes of the vessel wall and abnormal hematological factors constitute the primary mechanism of vessel occlusion. In general, BRVO has a good prognosis: 50–60% of eyes are reported to have a final visual acuity (VA) of 20/40 or better even without treatment. One important prognostic factor for final VA appears to be the initial VA. Grid laser photocoagulation is an established treatment for macular edema in a particular group of patients with BRVO, while promising results for this condition are shown by intravitreal application of steroids or new vascular endothelial growth factor inhibitors. Vitrectomy with or without arteriovenous sheathotomy combined with removal of the internal limiting membrane may improve vision in eyes with macular edema which are unresponsive to or ineligible for laser treatment.
BACKGROUND
Method of Literature Search
Eligible studies were identified through a comprehensive literature search of electronic databases (Medline, 1966–September 2007 and Science Direct, all years). Additional articles were selected from review of the reference lists of the articles generated from the above search. The following keywords and combinations of these words were used in compiling the search: branch retinal vein occlusion, retinal circulatory disorders, pathogenesis, hematological disorders, risk factors, therapy methods, visual prognosis. In total, 150 of these were used for this mini-review.
Epidemiology, Classification
Retinal vein occlusion (RVO) is the second most common retinal vascular disorder after diabetic retinopathy and is a significant cause of visual handicap. Its prevalence has been shown to vary from 0.7% to 1.6%.1,2 In a population-based study,3 an overall incidence of symptomatic RVO was found in 0.21% of patients aged 40 or older. Hayreh et al.4 investigated the demographic characteristics of various types of RVO in 1108 patients (1229 eyes). In this study, a male:female ratio of 1.2:1 was noted in a group of patients with RVO. Of the two main types of RVO, central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO), the latter is more common. A further group is hemi-vein occlusion, a distinct clinical entity presenting as occlusion of only one trunk of the central retinal vein in the area of the anterior part of the optic nerve.4 Hayreh et al.4 postulated that its pathogenesis is closely related to CRVO.
The first case of BRVO was reported by Leber in 1877.5 Some studies showed a higher proportion of BRVO patients older than 65 at the onset of the disease compared to CRVO,4,6 but others found no significance of age in the distribution of CRVO and BRVO.7,8 BRVO is divided into two distinct entities: major BRVO, when one of the major branch retinal veins is occluded, and macular BRVO, when one of the macular venules is occluded. In 66% of eyes with BRVO, there is occlusion of the major branch in the superotemporal quadrant followed by 22–43% of eyes with occlusion of the major branch in the inferotemporal quadrant.9 Owing to absent subjective BRVO symptoms in nasal quadrants, the diagnosis of occlusion in this localization is mostly accidental and therefore rare.10 Very often BRVO in nasal quadrants is diagnosed only when its complication as bleeding from neovascularizations into the vitreous cavity occurs. The cumulative probability of developing a second episode of occlusion in the other eye within 4 years is about 7% in patients with BRVO.4
Pathogenesis
The pathogenesis of RVO is multifactorial while BRVO may be due to a combination of three primary mechanisms: compression of the vein at the arteriovenous (A/V) crossing, degenerative changes of the vessel wall, and abnormal hematological factors. In the following sections these factors are discussed.
Arteriovenous Crossing
Koyanagi in 192811 first reported the association between BRVO and A/V crossing, and now it is established that mechanical narrowing of the venous lumen at these intersections plays a role in the pathogenesis of BRVO. Anatomic features of A/V crossings and secondary effects of arteriolar sclerosis may explain the apparent vulnerability of the crossing site to venous occlusion. In the majority of A/V crossings, the thin-walled vein lies between the more rigid thick-walled artery and the highly cellular retina. The sharing by artery and vein of the common adventitial sheath and the narrowing of the venous lumen that normally occurs at the A/V crossing provide the setting for BRVO.12 The risk of occlusion may be accentuated when arteriolar sclerosis results in increased rigidity of the crossing artery. Duker and Brown13 provided further support for a mechanical basis of BRVO development when they examined the relative anatomic position of the crossing artery and vein at the site of occlusion in 26 eyes with BRVO. They found in all 26 eyes the artery anterior to the vein (towards the vitreous cavity). Zhao et al.12 evaluated the anatomic position of the crossing vessels in 106 eyes with BRVO and found the artery anterior to the vein at the obstructed site in 99% of affected eyes. However, other mentioned risk factors must play a role, too, because in approximately 60% of normal A/V crossings without BRVO the artery lies anterior to vein.12
Degenerative Changes of Vessel Wall
A number of studies have investigated the histological changes of vessel wall at the A/V crossing.14,15 An investigation by Jefferies et al.14 showed that the expected venous compression at the crossing in histological view does not exist. He described the bending of the vein into the nerve fiber layer at this point without its compression. Histological investigation of the venous lumen at the A/V crossing in patients with a number of months to several years duration of BRVO showed organized thrombus with varied extent of recanalization in this part. Seitz15 described the clinical histological correlation in one eye with BRVO of a few hours after onset. There was no blood thrombus obliterating the venous lumen at the A/V crossing and even the fundoscopic examination showed strong dilated and tortuous vein distal to the crossing. In the area of the A/V crossing, alteration of the endothelium and intima media was present. Seitz suggests that the trophic changes of venous endothelium and intima media, as they follow the compression from overlaying artery, is the root of the pathogenesis of BRVO.15 The formation of the thrombus follows as a secondary process. The findings of Frangieh et al.16 support this hypothesis; 90% of the patients in their study had evidence of intima media layer hypertrophy, and all had evidence of intravenous thrombosis.
Systemic hypertension, diabetes mellitus, atherosclerosis, and smoking are reported to be more common in patients with RVO.1,2,10 Sclerosis of the retinal artery which is associated with these systemic disorders may result in further compression of the vein, when the increased rigidity of arterial wall and contraction of the adventitial sheath shared by artery and vein occur. Mechanical obstruction of the vein through the rigid artery in the A/V crossing may result in turbulent blood flow producing damage to venous endothelium and intima media and the sequence of events leading to occlusion of the vein.12,14 The turbulent blood flow was confirmed by Christoffersen and Larsen in an investigation which analyzed the fluorescein angiograms of 250 patients with BRVO.17
Hematological Disorders
Some studies have revealed an association between BRVO and hyperviscosity due to high hemotocrit.18,19 Higher blood viscosity increases under conditions of low blood flow and erythrocyte aggregation.18 Viscosity is mainly dependent upon the hematocrit (the greater the number of erythrocytes, the larger they aggregate) and plasma fibrinogen (required for aggregation to occur).20 Another discussed hematological disorder in the pathogenesis of BRVO is dysregulation of the thrombosis-fibrinolysis balance.21 The coagulation cascade including different blood factors results in the production of thrombin which converts circulating fibrinogen to fibrin. The coagulation sequence is held in check and inhibited by specific anticoagulants including protein C, protein S, and antithrombin. Table 1 shows the major disorders studied in patients with RVO. The results of published studies, however, are inconsistent, and the role of coagulation factors in the development of RVO remains unclear.
TABLE 1
Most discussed coagulation and anticoagulation disorders in the etiology of BRVO
Resistance to activated protein C (especially factor V Leiden mutation)
Protein C or protein S deficiency
Deficiency of antithrombin III
Genetic mutation in the prothrombin (factor II) gene
Anti-phospholipid antibodies
Hyperhomocysteinemia
Resistance to Activated Protein C and Deficiency of Protein C or Protein S
Protein C is serine proteinase whose activated form is a potent inhibitor of coagulation factors V and VIII.22 Factors V and VIII are a part of the coagulation cascade leading to conversion of fibrinogen to fibrin. Patients with protein C deficiency frequently manifest superficial and deep venous thrombosis and pulmonary embolism. Protein S and phospholipids are co-factors in the inactivation of factors V and VIII by activated protein C.22 An absolute deficiency of protein C or S is relatively rare. Tekeli23 and several other authors have reported normal levels in patients with RVO.24–26 The concept of resistance to activated protein C (so-called APC resistance) was first introduced by Dahlbäck et al. in 1993.27 APC resistance was subsequently shown to be a risk factor for venous thrombosis.28 More than 90% of patients with APC resistance have been shown to have a single point mutation in factor V gene.29 This mutation hinders the degradation of factor V normally occurring through protein C. Several investigators have reported an increased frequency of APC resistance in a cohort of patients with RVO,30–34 but this association has not been confirmed in other studies.35,36 Moreover, some results are inconclusive due to small patient samples or the lack of control groups. The meta-analysis of Janssen et al.21 showed the odds ratios for factor V Leiden mutation in patients with RVO 1.5 (95% CI 0.8–3.2). Despite the evidence of the significance of Leiden mutation, the effect of this hematological disorder in the etiology of RVO is only marginal.21
Deficiency of Antithrombin and Mutation in the Prothrombin Gene
In recent studies of patients with RVO, no significant association with a deficiency of antithrombin or with prothrombin mutation was found.21,26,34,37–40
Anti-Phospholipid Antibodies and Hyperhomocysteinemia
Antiphospholipid antibodies (APA) consist of a heterogeneous group of immunoglobulins, mainly anti-cardiolipin antibodies (ACA) and lupus anticoagulants (LA). Circulating APA leads to a hypercoagulable state and recurrent thrombosis through thrombocyte activation and inhibition of the natural anticoagulant pathways by binding of membrane phospholipids. Both the presence of LA and increased level of ACA are associated with a 3- to 10-fold increased risk of venous thrombosis.41
An elevated level of the amino acid, homocysteine is now generally accepted to be a risk factor for systemic vascular disease.42 Homocysteine appears to have a deleterious effect on vascular endothelium and may induce increased platelet aggregation and thrombosis. Levels of homocysteine may be increased by dietary habits, prescription medicines, or enzymatic mutations affecting homocysteine metabolism.43 The results of meta-analyses confirm total homocysteine to be an independent risk factor for RVO.21,44 Loewenstein et al.45 investigated the prevalence of genetic mutation in the enzyme methylentetrahydrofolate reductase (MTHFR) whose impaired activity may lead to hyperhomocysteinemia. The prevalence of this mutation was significantly higher in patients with RVO compared with the incidence of MTHFR in a control population. However, these results were not confirmed in other studies.46 The meta-analysis of Cahill et al.44 showed an association between retinal vascular occlusion and hyperhomocysteinemia but not with the mutation in the gene for MTHFR.
Pathogenesis of Macular Edema in BRVO
The development of macular edema (ME) followed by BRVO has been hypothesized to be caused by fluid flux from vessels to tissue according to Starling's law,47,48 which is based on the breakdown of the blood-retinal barrier (BRB) as a result of damage to the tight junctions of capillary endothelial cells,49 vitreoretinal adhesion,50 and secretion into the vitreous of vasopermeability factors produced in the retina.51,52 Observations by Noma et al.52 suggest that in patients with BRVO, vascular occlusion induces the expression of vascular endothelial growth factor (VEGF) and Interleukin-6 (IL-6), resulting in BRB breakdown and increased vascular permeability. Thus, VEGF and IL-6 may contribute to the development and progression of vasogenic ME in BRVO. ME is closely associated with retinal hypoxia, and the degree of hypoxia in the center of the macula corresponds to the decrease in visual acuity (VA). If marked hypoxia persists, irreversible structural changes in the macula occur, and the disturbed VA is almost always lasting. It is generally known that ME and intraretinal hemorrhage occurring in BRVO usually disappear within 6 to 12 months.53 In these cases, collateral systems often develop. The main purpose of the treatment is to decrease the duration of edema to prevent photoreceptor damage, if no spontaneous improvement occurs.
Clinical Signs and Diagnosis
In general, diagnosis of BRVO is not a problem owing to its classical features. Major BRVO can be asymptomatic or with visual blurring usually involving the sector of visual field corresponding to the area of the retina involved. In macular BRVO, there is always a central visual disturbance with normal peripheral vision. Acute BRVO presents characteristic clinical features with flame-shaped, dot and blot hemorrhage, soft and hard exudates, retinal edema, and dilated, tortuous vein in a segmental distribution. Signs of old occlusion are vascular sheathing and venous collaterals. The diagnosis is based on clinical examination under slit lamp and fundoscopy in artificial mydriasis. VA is of great importance for future visual prognosis. BRVO often leads to retinal non-perfusion zones in the occlusion area. Fluorescein angiography is particularly useful in determining the extent of ME and ischemia, although the ischemic areas are often obscured by the presence of intraretinal hemorrhage. Retinal neovascularization occurs in 36% of eyes with an area of non-perfusion greater than 5 disc diameter.54
RVO is associated with an increase in vascular causes of death (both cerebral and cardiac) in large prospective follow-up studies.55 In all patients with RVO, the systemic risk factors (hypertension, diabetes mellitus, blood lipid disorders) should be investigated and managed by appropriate specialists.
Natural Course and Visual Prognosis
The visual outcome following the natural course of BRVO is well documented.56–58 In general, BRVO has a good prognosis: 50–60% of eyes have been reported to have a final VA of 20/40 or better even without any treatment.56–59 The natural course of BRVO is determined by the site and degree of occlusion, the integrity of arterial perfusion to the affected sector, and the efficiency of the developing collateral circulation.59 Chronic ME and bleeding into the vitreous from neovascularizations account most frequently for a poor final VA.54,58,60 Retinal neovascularization and persistent ME develop in 25% and 60% of eyes, respectively.57,61 Gutman et al.60 found that in the natural course of BRVO, only 14% of eyes with chronic ME retained a VA of 20/40 or better, while 86% had a final VA of 20/50 or worse. He concluded that chronic ME has a poor prognosis in terms of final VA.60 Schilling et al.62 observed a worse visual prognosis in cases of ischemic ME compared to perfused ME. However, findings by Finkelstein63 showed that 91% of 23 eyes with macular ischemia recovered vision within one year with a VA of 20/40 or better. The conflicting reports and small number of studied eyes make it difficult to reach definitive conclusions on visual prognosis in patients with BRVO.
VA is a very sensitive indicator of the oxygen situation in the macula. For this reason, pre-treatment VA may be an important prognostic factor. Six studies analyzing the relation between initial and final VA were found.53,56,58,64–66 Five were used in an analysis of the data of eyes with unsatisfactory final VA (20/200 or worse) in relation to initial VA (Table 2). There were 2 groups; the first consisted of eyes with an initial VA of 20/50 or better and the second group of eyes with an initial VA of 20/200 or worse. In the second group were found a considerably higher percentage of eyes with a final VA of 20/200 or worse, regardless whether the eyes had undergone laser treatment or not. Since there were differently divided subgroups for final VA, the study of Subramanian et al.65 was not included in our analysis. Magargal et al.58 investigated the visual prognosis in 246 eyes with BRVO divided into two groups: with and without laser treatment. The obtained analysis illustrates that in the group of eyes with an initial VA 20/50 or better, no eye (not receiving laser treatment) and only 13% eyes (had undergone laser treatment) had a final VA of 20/200 or worse, whereas in the group of eyes with an initial VA 20/200 or worse, 83% of eyes (not receiving laser treatment) and 50% of eyes (had undergone laser treatment) had this unsatisfactory final VA. In an analogous way, the data for the final VA 20/50 or better in relation to the initial VA were analyzed (Table 3). We can see that in the group of eyes with an initial VA 20/50 or better, 89% of eyes (not receiving laser treatment), and 75% of eyes (had undergone laser treatment) retained this good VA, whereas in the group of eyes with an initial VA 20/200 or worse, only 14% of eyes (not receiving laser treatment) and only 22% of eyes (had undergone laser treatment) had a final VA 20/50 or better.58 Similar data are reported in the other studies (Tables 2 and 3). A chi-squared test with Yates correction was used to analyze the data. In 4 cases, in Table 2, and in 5 cases, in Table 3, respectively, the results were statistically significant (p < 0.05). Our analysis shows that in eyes with an initial VA 20/50 or better, the visual prognosis is good even without treatment. It could also be concluded that the cases of BRVO with an initial VA of 20/200 or worse have a statistically significantly poorer visual prognosis than those with an initial VA of 20/50 or better. Subramanian et al.65 showed that in patients with BRVO who underwent laser treatment of ME, the level of preoperative VA can be a useful predictor of visual outcome.
TABLE 2
Final visual acuity of 20/200 or worse in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05)
Initial visual acuity 20/50 or better
Initial visual acuity 20/200 or worse
Chi-squared test p < 0.05
Natural course—without laser treatment
Gutman56
5% (1/20)
50% (6/12)
Significant
Magargal58
0% (0/35)
83% (24/29)
Significant
Had undergone laser treatment
Wetzig53
25% (2/8)
67% (10/15)
No
Jalkh64
0% (0/9)
33% (3/12)
No
Magargal58
13% (5/40)
50% (32/64)
Significant
Lang66
8% (1/13)
50% (8/16)
Significant
TABLE 3
Final visual acuity 20/50 or better in relation to initial visual acuity. Chi-squared test with Yates correction (p < 0.05)
Initial visual acuity 20/50 or better
Initial visual acuity 20/200 or worse
Chi-squared test p < 0.05
Natural course—without laser treatment
Gutman56
90% (18/20)
33% (4/12)
Significant
Magargal58
89% (31/35)
14% (4/29)
Significant
Had undergone laser treatment
Wetzig53
63% (5/8)
20% (3/15)
No
Jalkh64
56% (5/9)
9% (1/12)
Significant
Magargal58
75% (30/40)
22% (14/64)
Significant
Lang66
77% (10/13)
13% (2/16)
Significant
Treatment
Current treatment options focus on the sequelae of the occluded venous branch, such as ME, retinal neovascularization, vitreous hemorrhage, and traction retinal detachment. There have been a number of treatment modalities advocated for the management of BRVO (Table 4). Many studies that examine interventions for BRVO suffer from methodological limitations, including insufficient power resulting from small sample sizes, short follow-up periods, absence of a control group or inappropriate control group (absence of placebo or best practice intervention as control groups), and lack of distinction between clinical entities. A number of such investigations have therefore produced conflicting data. Hence, the results of randomized clinical trials are the most important. The complex pathogenesis of this disease requires investigation and treatment of all risk factors (hypertension, diabetes mellitus, blood lipid disorders, hematological disorders).
TABLE 4
Treatment modalities for BRVO
Anti-aggregative therapy and firbrinolysis
Isovolemic hemodilution
Laser treatment
Intravitreal and periocular application of steroids
Intravitreal injection of VEGF inhibitors
Sheathotomy and vitrectomy
Anti-Aggregative Therapy and Fibrinolysis
Systemic treatment with oral acetylsalicylic acid, subcutaneous heparin, or intravenous thrombolysis have not been shown to be effective treatments for CRVO, while for BRVO no randomized clinical trials have been published as of the date of this review. Thrombolysis using administration of tissue plasminogen activator intravitreally or directly into the retinal vein (mostly upper temporal branch close to the optic disc) has been demonstrated to improve VA in patients with CRVO,67 but the results were based only on noncomparative interventional series. There is no general current acceptance of this treatment.
Houtsmuller et al.,68 in a double-blind study, examined the platelet aggregation inhibiting effect of ticlopidine in 54 patients with BRVO less than 3 weeks from the onset of symptoms. Compared with placebo therapy a significant improvement in VA was observed with ticlopidine therapy for six months. In the treated group, 69% of patients experienced an improvement in VA, whereas 52% of placebo group reported improvement.
Troxerutin has been suggested to inhibit erythrocyte and platelet aggregation and to improve erythrocyte deformability, thus reducing blood viscosity and the retinal microcirculation.69 A double-blind randomized study of 26 patients with BRVO compared troxerutin with placebo.69 At 4 months follow-up, more of the patients receiving troxerutin treatment had a mean VA of 20/40 or better than the control group, although this difference was not found to be statistically significant. After 4 months, all patients were treated with troxerutin for 2 years. At the completion of this follow-up period for those patients initially treated with troxerutin, a significant improvement in VA and improvement of ME was demonstrated. The limitation of this study is that there is no separation in the analysis of results for patients with BRVO and CRVO who were included in the study, too.
Both studies mentioned that investigated the medical treatment of BRVO are limited by a small sample size and short follow-up period (6 and 4 months).
Isovolaemic Hemodilution
Chen et al.20 demonstrated positive results for isovolemic hemodilution given up to 3 months after the on-set of the symptoms of BRVO in patients with a hematocrit of 35% or more. In this randomized controlled study, 18 patients were treated for 6 weeks with venesection and volume replacement using hydroxyethylstarch and compared to 16 untreated control patients. After a one year follow-up, the final VAs were 20/40 and 20/80 for treated and untreated patients, respectively (p = 0.03). Patients with ME and a VA 20/40 or worse underwent 3 months after including into the study macular grid laser photocoagulation (MLG). Sector photocoagulation was applied if ocular neovascularization developed or if, at 3 months, the fluorescein angiogram showed an area of capillary non-perfusion greater than 5 disc areas. 28% of the hemodiluted patients required MLG compared to 44% of the control group; this difference was not statistically significant (p = 0.2). Sector photocoagulation was required by 50% of both groups of patients.20
Hydroxyethylstarch has a capacity to expand the plasma volume by up to 172% of the volume infused and has a duration of action of approximately 36 hours.70 It is non-antigenic and has a low incidence of allergic reactions.71 Poupard et al.72 randomized 25 patients to either hemodilution with dextran for 21 days (n = 10), hemodilution combined with heparin for 21 days (n = 10), or heparin treatment for 21 days followed by anti-vitamin K drugs for a further 30 days (n = 5). The study showed that, for those receiving heparin followed by anti-vitamin K drugs, mean VA remained unchanged to baseline values by 60 days. For those treated with hemodilution and heparin, a statistically significant increase in VA was found by 60 days. For those treated with hemodilution alone, a significant improvement in VA was found by day 14. In a randomized study by Hansen et al.73 of 35 patients with BRVO, 18 patients were treated by hemodilution for a period of 5 to 6 weeks (targeted hematocrit 30–35%). A control group of 17 patients were only observed. At follow-up 12 months later, 25 patients had completed the therapy. Seven of the 13 who received hemodilution demonstrated a VA increase of 2 lines or more compared with none of the 12 patients who did not receive hemodilution (p < 0.005). Reported complications of hemodilution include headache, exertional dyspnea, tiredness, deep vein thrombosis, and hypotension. The treatment was noted to be generally well-tolerated even in elderly patients.20,73,74
The use of hemodilution to treat BRVO is currently not generally accepted. Interpretation of the above-mentioned studies is difficult because most of them incorporated other treatments in combination with the hemodilution. Further prospective randomized trials with adequate controls and sufficient follow-up are required for any definitive conclusions and recommendations.
Arteriovenous Crossing Sheathotomy and Vitrectomy
Osterloh and Charles75 first reported improvement in VA in patients with BRVO after treatment using the technique of surgical sheathotomy. The principle steps of this procedure are a pars plana vitrectomy followed by separation of the retinal artery from the vein by creating an incision in the adventitial sheath adjacent to the A/V crossing and then separation of the adhesions. Several studies have shown significantly better functional outcomes in patients treated by sheathotomy compared to controls (Table 5).75–96 Reported complications are few but include cataract, hemorrhage, retinal tears, postoperative gliosis, and retinal detachment.75–96 Garcia-Arumi76 described a combination of A/V sheathotomy and injection of thrombolytic into the occluded vein which resulted in thrombus release in 28% cases and significant correlation with early surgery and better final VA. The role of the sheathotomy alone in visual improvement is insufficiently clear. Some authors suggest that vitrectomy is the most important part of the sheathotomy surgery, leading to reduction of ME.77,80,96 Yamamoto et al.77 compared the effect of sheathotomy combined with vitrectomy to the effect of vitrectomy alone and found no advantage of sheathotomy. Eyes with pre-existing posterior vitreous detachment were not studied. For this reason, the benefit of vitrectomy of these eyes is unknown. Surgical detachment of posterior hyaloid could be more important than the sheathotomy itself .78 The vitreous is postulated to have a role in the pathogenesis of neovascularization and ME, which may complicate BRVO and its removal may help in the management of these sight threatening complications.78 Vitrectomy and removal of the posterior hyaloid with peeling of the internal limiting membrane (ILM) appears to improve oxygenation of the retina, which may lead to visual improvement.97,98 Peeling of the ILM improves the surgical outcome during A/V adventitial sheathotomy, too.84 To date, no randomized clinical trials on the surgical treatment of BRVO have been published. Any evidence supporting these procedures is based on non-ramdomized case series only.
TABLE 5
Summary of studies evaluating the treatment of macular edema in BRVO by sheathotomy (VA = visual acuity, ME = macular edema, ILM = internal limiting membrane)
Author
Study type
Patients
Follow-up (mean)
Outcomes
Comments
Osterloh and Charles75
Case report.
1 eye.
8 months.
VA improved from 20/200 to 20/25.
First report of sheathotomy.
Garcia-Arumi et al.76
Prospective interventional nonrandomized study.
40 eyes—all underwent vitrectomy, sheathotomy and injection of 25 mg of tissue plasminogen activator into occluded vein.
13 months.
Thrombus release in 11 eyes (27.5%)—correlated with early surgery. VA increased from 20/100 to 20/40 (p = 0.016).
Yamamoto et al.77
Retrospective interventional comparative case series.
20 eyes—sheathotomy 16 control eyes (posterior vitreous detachment via vitrectomy).
12 months.
VA: significantly better in both groups (p = 0.008 and p = 0.001, respectively). VA and foveal thickness were not significantly different between the groups.
Charbonnel et al.78
Prospective nonrandomized, interventional case series.
13 eyes—sheathotomy.
7 months.
Improvement in VA ≥ 2 ETDRS lines in 9 eyes (69%).
Absence of previous posterior vitreous detachment correlated with improvement in VA.
Sohn et al.79
Retrospective interventional case series.
22 eyes—sheathotomy + ILM peeling in all eyes.
3 months.
Improvement in VA (log MAR) from 0.79 ± 0.29 to 0.57 ± 0.33 (p < 0.01).
All eyes pretreated with grid laser or triamcinolone.
Kumagai et al.80
Prospective, randomized, comparative, interventional study.
Group 1: 18 eyes—sheathotomy. Group 2: 18 controls (vitrectomy without sheathotomy).
31 months.
VA (log MAR) in group 1: 0.52 → 0.08. In group 2: 0.53 → 0.014. Differences between group 1 and 2 was not significant.
Avci et al.81
Retrospective interventional comparative case series.
11 eyes—sheathotomy. 10 control eyes—grid laser photocoagulation.
9 months.
VA (log MAR): sheathotomy: 0.84 → 0.36. Grid laser: 1.06 → 0.82. Difference was significant.
Horio et al.82
Interventional case series.
7 eyes.
6 months.
Significant improvement in retinal blood flow (p < 0.01) and reduced macular thickness (p = 0.03).
Lakhanpal et al.83
Retrospective interventional case series.
12 eyes.
49.9 weeks.
VA (logMAR) improved from 1.00 ± 0.32 to 0.56 ± 0.28 (p = 0.0003).
25-gauge transvitreal limited arteriovenous crossing manipulation without vitrectomy.
Mester et al.84
Prospective interventional nonrandomized case-control study.
43 eyes—sheathotomy. 16 eyes additionally + ILM peeling. 25 control eyes.
6 weeks.
26 patients (60%) gained ≥ 2 lines of VA. Better result in patients with ILM peeling. ME and intraretinal hemorrhage resorbed in all patients.
All patients had isovolaemic hemodilution for 10 days.
Opremcak et al.85
Prospective interventional case series.
15 eyes.
6.5 years.
Snellen VA improved in 10 patients (67%) by an average of 4 lines vision (range 1–9 lines). In 3 patients resolution of ME but no improvement of VA.
Retinal vascular bleeding in 2 patients.
Asensio Sanchez et al.86
Prospective interventional nonrandomized study.
13 eyes—sheathotomy, 5 eyes underwent additionally ILM peeling.
12 months.
VA improved in 12 patients (92%). Better results in patients with ILM peeling.
Lerche et al.87
Prospective nonrandomized intervention case series.
12 eyes – sheathotomy.
3 months.
VA (logMAR) improved from 0.74 to 0.56.
Mason et al.88
Prospective, nonrandomized, comparative interventional study with concurrent control group.
20 eyes—sheathotomy. 20 control eyes (10 of them without intervention and another 10 underwent grid laser).
14 months (sheathotomy). 19 months (controls).
VA improvement: Sheathotomy: from 20/250 to 20/63. Controls: from 20/180 to 20/125 (p = 0.02). 45% of the surgical group had final VA ≥ 20/40 compared with 15% of the controls.
Data only for whole control group together.
Cahil et al.89
Retrospective non-controlled case series.
27 eyes—sheathotomy.
12 months.
Resolution of ME in 8 (29.6%) patients, reduction in 14 (51.8%) and persistence in 5 (18.5.%).
Becquet et al.90
Prospective nonrandomized interventional case series.
6 eyes (sheathotomy + ILM peeling. 6 controls (ILM peeling only).
6 months.
Significant improvement of VA in both groups. No difference in VA or foveolar thickness between the groups (p = 0.5; p = 0.6 respectively).
Martinez-Soroa et al.91
Retrospective interventional case series.
17 eyes—sheathotomy.
6 months.
Improvement in VA from 0.26 to 0.4. 53% patients improved ≥ 4 lines (Snellen).
Le Rouic92
Retrospective interventional case series.
3 eyes—sheathotomy.
10 months.
No improvement in VA observed.
All patients with initial VA < 20/40.
Dotrelova et al.93
Retrospective interventional case series.
3 eyes—sheathotomy.
12 months.
VA improved in 2 patients to 20/40, in 1 patient stabilized (20/180).
Shah et al.94
Retrospective interventional case series.
5 eyes—sheathotomy.
6.5 years.
VA preoperative in all patients ≤ 20/200. Improved in 4 eyes from 20/30 to 20/70. 1 eye with counting fingers remained unchanged.
Crafoord et al.95
Retrospective interventional case series.
12 eyes—sheathotomy.
20 months.
VA improved in 9 eyes (75%), in 1 eye (8.3%) remained unchanged and deteriored in 2 eyes (16.7%).
2 patients received additionally 25 mg triamcinolone acetonide at the end of the surgery.
Han et al.96
Retrospective interventional case series.
20 eyes—pars plana vitrectomy and dissection of the arteriovenous crossing without separation of the vessels.
10.5 months.
In 16 eyes (80%) improved VA ≥ 2 lines. Mean improvement of VA (logMAR) was = 0.44 ± 0.14 (p = 0.016).
Steroids
Intravitreal Corticosteroids
In several nonrandomized comparative studies, intravitreal corticosteroids were successfully used for the treatment of BRVO. Currently published randomized studies are very rare and limited by virtue of evaluating patients with ME of different etiology, making comparisons difficult. In various studied doses from 4 to 25 mg, triamcinolone acetonide (TA) has been reported to be effective99–117 (Table 6). In a randomized, interventional, three-arm clinical trial, Avitable et al.99 compared the results of treating diabetic patients and a small group of BVRO patients with cystoid ME by TA and MLG. From a total of 63 patients, 22 were treated by TA (4 mg), 21 underwent MLG, and in 20 patients these methods were combined (TA + MLG). The greatest improvement in VA was found in patients treated by TA combined with MLG. VA (log MAR) in this group increased significantly from 0.83 at baseline to 0.20 at the end of follow-up 9 months later (p = 0.003). In patients treated by TA, VA improved significantly, from 0.82 at baseline to 0.23 at 9 months after injection (p = 0.04). VA in the group of patients treated by MLG remained the same. The results of this study are limited, however, owing to the different ME etiologies in evaluated patients; only 6 patients had ME secondary to BRVO. Oh et al.100 used a retrospective interventional case series to compare VA after single TA injection (4 mg) in 10 patients with mean duration of ME ≤ 3 months after onset of BRVO versus 10 patients with ME > 3 months after onset. In patients with a disease duration ≤3 months, VA significantly improved from baseline over 6 months of follow-up. However in those with a duration of >3 months, improved VA, though apparent at one month, was not maintained at 3 or 6 months after TA injection. This study is limited by its retrospective design and short follow-up period. Ozkiris et al.101 evaluated the effect of TA injection on persistent ME in BRVO that failed to respond to previous laser photocoagulation. During a mean follow-up time of 6.2 months, best corrected VA (log MAR) improved significantly (p < 0.001) from 1.01 at baseline to 0.55 at one month after the injection. VA after 3 months was 0.56, and at the end of follow-up was 0.62. The authors concluded that intravitreal application of TA may be helpful in patients who do not respond to laser photocoagulation. However, in published studies, the resulting reduced macular thickness and improved VA, is only temporary and requires repeated treatment. One to four times re-application has been reported. Cekic et al.103 performed a retrospective chart review of 13 patients who underwent intravitreal injections with 4 mg TA. Six eyes received a single injection. Repeated injections were performed in 1 eye twice, 4 eyes three times, and 2 eyes four times. During a mean follow-up of 13 months, central foveal thickness decreased by more than 50%. Final VA improved in 7 eyes (range 2–6 Snellen lines), remained the same in 4 eyes (range 0–1 Snellen lines), and worsened in 2 eyes (range 1–4 Snellen lines) compared to baseline. Retinal thickness decreased in all cases, while vision improved in most cases. One of the most common side effects of TA was steroid-induced elevation of intraocular pressure.118 Other complications were infectious endophthalmitis, post-injection steroid-induced cataract, and retinal detachment.119,120 Reported risk of infectious endophthalmitis per injection range was from 0.1% to 1.6%.120 The most recent report by Bhavsar et al.121 found in two large studies-Diabetic Retinopathy Clinical Research Network (DRCR.net) and SCORE (Standard Care versus Corticosteroid for Retinal Vein Occlusion), an endophthalmitis prevalence of 0.05% (one case in the 2009 injections).
TABLE 6
Summary of studies evaluating the treatment of macular edema in BRVO by intravitreal application of triamcinolone acetonide (TA = triamcinolone acetonide, VA = visual acuity, ME = macular edema, MLG = macular laser grid photocoagulation)
Author
Study type
Patients
Follow-up (mean)
Outcomes
Comments
Avitabile et al.99
Randomized interventional, parallel, three-arm clinical trial.
Intravitreal TA (4mg): 22 eyes. MLG: 21 eyes. TA+MLG: 20 eyes.
9 months.
TA group: VA improved from 0.82 to 0.23 log MAR (p = 0.04). MLG-group: VA unchanged. TA+MLG group, VA improved from 0.83 to 0.20. logMAR (p = 0.003).
Different etiology of ME, only 6 eyes with BRVO.
Oh et al.100
Retrospective interventional comparative case series.
20 eyes with ME (4 mg TA) Disease duration: 10 eyes ≤ 3 months; 10 eyes > 3 months.
6 months.
Group ≤ 3 months: VA (logMAR) improved from 1.07to 0.63 in 1 month (p = 0.012) and to 0.34 in 6 months (p = 0.005).
Ozkiris et al.101
Retrospective, non-controlled case series.
19 treated eyes (8 mg TA).
6.2 months.
VA (logMAR) improved from 1.01 ± 0.16 to 0.62 ± 0.22. VA improved in 17 eyes and remained unchanged in 2 eyes.
Jonas et al.102
Prospective nonrandomized comparative study.
10 treated eyes (20 mg TA). 20 untreated controls.
TA patients: 10.1 months. Controls: 6 months.
TA patients: VA increased from 0.27 ± 0.11 to 0.45 ± 0.27 (p = 0.02). Controls: VA decreased significantly (p = 0.007).
VA increased higher in non-ischemic group. Significant increase of intraocular pressure in treated group.
Cekic et al.103
Retrospective non-controlled case series.
13 eyes (4 mg TA).
13 months.
VA: improved in 7 eyes, remained the same in 4 eyes, worsened in 2 eyes. Foveolar thickness decreased in 56% of patients (p < 0.001).
VA improvement significantly correlated with patient age (p = 0.026).
Lee et al.104
Retrospective, non-controlled case series.
6 eyes (4 mg TA).
149.5 days.
Improvement in VA ≥ 2 lines in 5 eyes (83.3%). VA from 20/166 to final 20/106.
3 eyes treated with re-application of TA.
Ozkiris et al.105
Retrospective interventional comparative case series.
15 eyes (8 mg TA). 19 eyes MLG.
6.3 months.
VA (logMAR) improved in TA group from 0.98 to 0.24 and in MLG group from 1.02 to 0.5 (in both groups p < 0.001). Improvement in TA group was significantly higher than in MLG (p < 0.001).
Yepremyan et al.106
Retrospective, non-controlled case series.
12 eyes (4 mg TA).
15.3 months.
VA improved >3 lines in 50% of eyes after 1 month and in 42% of eyes at last follow up.
8 eyes developed recurrent ME at an average of 5.5 months after initial TA injection.
Cheng et al.107
Prospective nonrandomized interventional comparative study.
16 eyes (4 mg TA). 11 controls (without TA).
103 days in TA-group. 94.5 days in controls.
VA (logMAR). In TA-group: improvement from 0.77 ± 0.43 to 0.44 ± 0.43 (p < 0.001). No significant change of VA in controls.
Significant reduction of ME in TA-group (P < 0.001).
Chen et al.108
Case report.
1 eye (4 mg TA).
3 months.
Improvement in VA from counting fingers to 20/80.
Eye with macular ischaemia.
Chen et al.109
Prospective interventional non-controlled case series.
18 eyes (4 mg TA).
All patients completed 9 months, 12 eyes completed 12 months.
VA (logMAR) improved from 0.81 ± 0.36 to 0.65 ± 0.3 (p = 0.03) after 1 months, no significant difference in VA after 3, 6, 9 and 12 months.
Eye with macular ischaemia. All eyes with macular ischaemia.
Tsujikawa et al.110
Prospective interventional non-controlled case series.
17 eyes (vitrectomy + 10 mg TA intravitreal), 12 eyes of them with recurrent ME received sub-tenon 20 mg TA.
12.1 months.
82% of eyes rapid resolution of ME within 2 months (p = 0.041). 12 eyes (70.5%) received sub-tenon TA because of recurrent ME. Final VA (logMAR) improved from 0.74 ± 0.40 to 0.40 ± 0.34 (p = 0.010).
14 eyes with vitrectomy underwent additional phacoemulsification with lens implantation.
Karacorlu et al.111
Prospective interventional non-controlled case series.
8 eyes (4 mg TA). All eyes with serous macular detachment.
6 months.
After TA regression of ME and serous macular detachment in all eyes. After 6 months recurrence in 2 eyes (25%) re-treatment occurred. Final VA improved in 7 eyes (87.5%).
Krepler et al.112
Prospective interventional non-controlled case series.
9 eyes (4 mg TA).
6 months.
Significant improvement in reading VA only after 1 month (p = 0.02). No significant improvement in VA for distance. No significant reduction in macular thickness.
5 eyes non-ischaemic BRVO. 4 eyes ischaemic BRVO.
Degenring et al.113
Case report.
2 patients: 1 eye BRVO 1 eye CRVO (25 mg TA).
5 weeks.
Patient with BRVO improved VA from 0.25 to 0.5. Patient with CRVO from 0.4 to 0.5.
Wakabayshi et al.114
Prospective interventional non-controlled case series.
5 eyes with CRVO. 11 eyes with BRVO. All eyes received sub-tenon injection of 20 mg TA.
7 months.
8 eyes (50%) improved VA and 2 eyes (12.5%) had worsening of VA at the time of final examination. Reduction of ME >30% of initial thickness in 13 eyes (81.3%). Because of recurrent ME – in 7 eyes repeated sub-tenon application of TA.
1 eye with BRVO pretreated with laser photocoagulation because of retinal ischemia.
Salinas-Alaman et al.115
Retrospective interventional case series.
5 eyes (4 mg TA).
6 months
Improvement of VA in 4 eyes. 1 eye underwent re-injection after 3 months because of recurrent ME.
Hirano et al.116
Retrospective interventional comparative case-control study.
8 eyes TA-injected group (simultaneous intravitreal and sub-tenon TA injection). 7 eyes vitrectomy with TA group (treated by vitrectomy and intravitreal or sub-tenon TA).
12 months.
VA improved significantly from baseline in both the TA-injected (p = 0.0069) and vitrectomy with TA groups (p = 0.0145). There was no significant difference in VA and macular thickness between the two groups.
Kuppermann et al.117
Randomized interventional clinical trial.
105 (20 with venous occlusion) eyes in each group: I. 700 μg Posurdex II. 350 μg Posurdex III. controls.
3 months.
Improvement of VA of ≥10 lines (ETDRS) 35% eyes in group 700 μg Posurdex, 24% in 350 μg Posurdex and 13% in control group (p < 0.001 versus 700 μg group; p = 0.04 versus 350 μg group).
Preliminary report 60 eyes with retinal venous occlusion include eyes with CRVO and BRVO.
Most published studies on intravitreal TA for BRVO, however, suffer from two serious flaws: either the designs are not randomized or they often do not clearly differentiate between nonischemic types and ischemic types of occlusion. To compare the effectiveness and safety of standard care versus TA injection in the treatment of ME in patients with CRVO and BRVO, the multicenter randomized study SCORE is ongoing (https://web.emmes.com/study/score). In each of the two disease areas, 630 participants will be randomized in a 1:1:1 ratio to one of three groups: standard care, intravitreal 4 mg of TA, or 1 mg of TA. The follow-up is planned for 3 years. The results are not published as yet. Biodegradable intravitreal implants may allow steroid delivery over a more sustained period, permitting longer duration of action. A multicenter randomized clinical trial which evaluates implantation of dexamethasone 350 μg or 700 μg (Posurdex) versus observation (no therapy) for ME secondary to a variety of retinal disorders (including BRVO) has been reported.117 The preliminary 90-day results of all 315 evaluated patients showed that an improvement in VA of 10 letters or more (in ETDRS) was achieved by a greater proportion of patients treated with dexamethasone 700 μg (35%) or 350 μg (24%), than untreated patients (13%; p < 0.001 versus 700 μg group; p = 0.04 versus 350 μg group). The results were similar for patients with diabetic retinopathy, retinal vein occlusion, or uveitis or Irvine-Gass syndrome. In total, 60 patients with BRVO were randomized 1:1:1 to receive 350 μg or 700 μg dexamethasone or observation (no therapy). In the case of RVO, the effect of the treatment was evaluated only in a common group (CRVO and BRVO patients together): an improvement in VA of 10 letters or more was achieved in 15% of untreated patients versus 31% of patients treated with dexamethasone 700 μg. The number of patients with an increase in intraocular pressure of more than 10 mmHg from baseline anytime during the study was 12% for 350 μg, 17% for 700 μg, and 3% for the untreated controls.117
Periocular Application of Triamcinolone Acetonide
Kawaji et al.122 evaluated in 20 patients the effectiveness and safety of trans-tenon retrobulbar injection of 40 mg of TA for ME associated with BRVO after vitrectomy. Improvement in VA was seen in 14 (70%) eyes. Hayashi et al.123 compared in a randomized clinical trial, the short-term effect of intravitreal versus retrobulbar injection of TA for the treatment of ME caused by BRVO. Sixty patients received either a single intravitreal injection (4 mg) or repeated retrobulbar injections (40 mg, three times) of TA. The first injection in the retrobulbar group was given approximately one week after focal laser photocoagulation. Foveal thickness, macular volume, and improvement in VA were significantly better after intravitreal injection than after repeated retrobulbar injections. The need for re-injections was significantly greater in the retrobulbar group than in the intravitreal group.
Intravitreal Injection of VEGF Inhibitors
VEGF inhibitors are a treatment option for ME associated with RVO that target the disease at the causal molecular level. Randomized studies evaluating the results of treatment of all available VEGF inhibitors (bevacizumab, ranibizumab, and pegaptanib) are ongoing. Case reports, small retrospective or prospective non-controlled studies of VEGF inhibitors in the treatment of ME and retinal neovascularizations secondary to BRVO, have been published.124–140
Rosenfeld et al.124 first reported improved VA and reduced ME measured by optical coherent tomography (OCT) following intravitreal injection of bevacizumab for recurrent ME secondary to CRVO in an eye previously treated by intravitreal TA injection. In a short-term study, Iturralde et al.125 treated 16 eyes of CRVO with ME that had failed intravitreal corticosteroid therapy, and nearly every eye showed some anatomic or VA improvement following bevacizumab injection. In various reports, doses from 1.25 to 2.5 mg bevacizumab have been intravitreally administrated.125–134 The most recently published studies evaluated the results in a group of patients with BRVO combined with patients with CRVO. In all of these studies, bevacizumab injection improved VA and reduced macular thickness measured by OCT within the first 3 to 9 weeks. Few studies are available for BRVO patients alone.126,127 Rabena et al.126 reported a significantly increased VA and reduced macular thickness after treatment with 1.25 mg bevacizumab in a retrospective study of 27 patients with BRVO. Recurrent ME was observed in 6 (22%) patients an average of 2.1 months after the initial injection. These patients were reinjected and all showed moderate to complete reduction in ME. The limitations of this retrospective study are short follow-up and lack of control group. Additionally, most of the eyes in the study were previously treated and thus failed standard treatment, and perhaps represent a group unlikely to benefit from any treatment. All published reports provide evidence that this treatment is well tolerated. The most common adverse events were conjunctival hyperemia and subconjunctival hemorrhage at the injection site. However, the duration of reduced ME after bevacizumab administration is currently unknown. Frequent repeated injections are required to prevent a rebound effect with no clearly defined endpoint.128
Campochiaro et al.129 presented preliminary results of a randomized study in the treatment of BRVO with intravitreal injection of ranibizumab at the 2007 Annual Meeting of Association on Research and Vision in Ophthalmology (ARVO). Patients with ME due to CRVO or BRVO were randomized 1:1 to receive 3 monthly injections of 0.5 or 0.3 mg of ranibizumab. Interim results without regard to treatment assignment, which is unknown, showed that 12 randomized patients with BRVO gained an improvement in VA (in ETDRS) from 21 to 37 letters and a reduction in ME from 508 to 208 μm after 3 months of treatment. The endpoint results are expected to clarify any differences between the treatment groups. Another indication for anti-VEGF drugs are retinal neovascularizations, rubeosis iridis, and neovascular glaucoma. Rapid regression of neovascularizations and compensation of intraocular pressure have been described in several studies.136–139 Intracameral application of bevacizumab as successful treatment of rubeosis iridis and neovascular glaucoma has also been reported.140
Prospective, controlled studies are mandatory to develop standardized treatment protocols that allow safe and effective application of anti-VEGF drugs.
Laser Treatment
Laser treatment is an established method for use in patients with BRVO. A large number of publications concerning the role of photocoagulation in the management of BRVO have appeared. Various laser techniques can be used: macula grid photocoagulation and the method of arterial crimping for treatment of ME, and peripheral scatter photocoagulation for treatment of retinal and/or disc neovascularization.
Macular Grid Laser Photocoagulation
The Branch vein occlusion study group remains the largest randomized prospective trial that has evaluated the efficacy of grid-pattern laser photocoagulation for the treatment of ME in BRVO.141 In this study, only eyes with recent BRVO, perfused ME, resolved foveal hemorrhage, VA 20/40 or worse, and no other ocular comorbidities were included. After a 3-year follow-up period, 65% of treated eyes gained improvement of 2 or more lines from baseline, as opposed to 37% of untreated eyes. The number of eyes that lost 2 or more lines was not significantly different in the two groups.141 Parodi et al. published two randomized controlled studies, in which no significant benefit of MLG on VA was found.142,143 MLG is recommended as an effective treatment to reduce the ME in BRVO after a period of 3 to 6 months after onset and following absorption of the majority of hemorrhage if VA is 20/40 or worse.61,62,141 If the fluorescein angiogram reveals macular nonperfusion, laser therapy is not warranted.141 Subramanian et al.65 recommended laser treatment in patients with poor VA (20/200 or worse) secondary to ME due to BRVO, before more aggressive approaches (as intravitreal TA). Argon MLG is usually used for this purpose. However, diode laser (810 nm) and krypton red laser (647 nm) also can be used.63,141,144
Scatter Photocoagulation
The randomized controlled study by Branch vein occlusion study group54 reported that peripheral scatter laser photocoagulation significantly reduced the development of retinal neovascularization and vitreous hemorrhage. This study also demonstrated that, if all eyes with large retinal nonperfusion were treated, 64% of these patients would never develop neovascularization. If only the eyes that develop neovascularization were treated, the events of vitreous hemorrhage would decrease from 61% to 29%. Since loss in the lower part of the visual field can produce marked disability and BRVO involving the superior retina is common, a significant worsening of visual fields with laser treatment becomes a very important, clinically relevant finding.145 Therefore, waiting is generally advocated until neovascularization actually develops before scatter photocoagulation is considered.54
Arteriolar Constriction
An alternative type of laser treatment involves arteriolar constriction (called also “crimping technique”) and may be considered in order to reduce the inflow into the affected area if the ME is excessive. This procedure was first described by L'Esperance146 in 1975. It may lead to a decrease in arterial pressure in the occluded region resulting in better drainage of the ME due to reduced blood inflow. The technique is employed by placing coagulations at approximate intervals of ½ disc diameter (using the green beam of argon laser) through the afferent arteriole in the region of venous blockage. In 1984 Jalkh et al.64 proposed their own modification of this method and published the results obtained in 41 eyes. In this study, arterial constriction was applied in the treatment of the chronic stage of BRVO. Rehak et al. published several studies describing the modified arteriolar constriction in patients with BRVO.147–149 This technique consists of the application of coalescent coagulation spots through the afferent arteriole that supports the occluded venous region. 83% of patients treated by this method within the first 2 months after the onset of occlusion achieved a final VA 20/40 or better.149 In a study by Erdol and Akyol,150 the improvement in VA was higher in a group of patients receiving the MLG combined with arteriolar constriction than in a group treated by MLG only. However, the difference in the resolution of ME between the groups was not statistically significant. The authors suggest that arteriolar constriction in addition to grid pattern laser photocoagulation is more effective for resolving ME in patients with BRVO.
CONCLUSIONS
The pathogenesis of BRVO is multifactorial. Its resulting visual loss is due primarily to ME, macular nonperfusion, and retinal neovascularization. A large number of treatments have been advocated in its management. Unfortunately, almost all of these lack sufficient evidence for their effectiveness. Randomized prospective trials are essential. The only one established treatment for ME is macular grid photocoagulation in patients with BRVO longer than 3 months and a VA of 20/40 or worse. Additionally, the initial VA may play a crucial role in the prognosis of BRVO and determinates the final VA.

branch retinal vein occlusion
pathogenesis
visual prognosis
treatment
risk factors


A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein–protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas.
Introduction
The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model organism for plant biology (Harris 2001; Gutman and Niyogi 2004; Pröschold et al. 2005). It represents one of the simplest photosynthetic eukaryotes, can be easily grown at large scale either photoautotrophically, mixotrophically or heterotrophically, and can be propagated sexually or asexually. Moreover, Chlamydomonas combines a powerful genetics with the availability of unique genetic and genomic resources: all three genomes are fully sequenced (nuclear, plastid, and mitochondrial; Merchant et al. 2007), large mutant collections have been established, and all three genomes are amenable to genetic manipulation by transformation (Hippler et al. 1998; Remacle et al. 2006). One of the few drawbacks of Chlamydomonas is that it has been notoriously difficult to express transgenes to reasonably high levels from the nuclear genome. Use of specialized promoters (Schroda et al. 2000; Fischer and Rochaix 2001) and adjustment of the transgene’s codon usage to that of the highly GC-rich nuclear genome of the alga (Fuhrmann et al. 1999, 2004) helped in some cases, but no general solution to the problem has been found to date. This is highly unfortunate, because a number of valuable tools available in higher plants currently cannot be used routinely in Chlamydomonas. These include all applications of in vivo reporters of gene expression, such as, promoter-YFP/GFP fusions for gene expression analyses and subcellular localization studies, as well as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) for monitoring protein–protein interactions.
Recently, codon-optimized reporter genes have been developed for nuclear and chloroplast expression in C. reinhardtii. Two such genes have been developed for nuclear expression: a gfp gene encoding the green fluorescent protein from the jellyfish Aequorea victoria (Fuhrmann et al. 1999) and a luciferase gene (R-Luc) from the sea pansy Renilla reniformis (Fuhrmann et al. 2004). Synthetic luciferase and gfp genes were also designed for chloroplast transformation and successfully used to measure plastid gene expression (Minko et al. 1999; Mayfield and Schultz 2004; Barnes et al. 2005). However, the use of these reporter genes for nuclear transformation is still far from being routine. While both reporters allowed detection or quantitation of the expression of some (fusion) genes (Fuhrmann et al. 1999; Shao et al. 2007), their generally low sensitivity has precluded the universal use of these reporters.
To overcome these limitations, we have explored a recently discovered new luciferase for its suitability as a more sensitive reporter of gene expression in Chlamydomonas. The luciferase from the copepod marine organism Gaussia princeps represents one of the smallest and brightest bioluminescent proteins known to date (Tannous et al. 2005; Remy and Michnick 2006). In an ATP-independent reaction, it catalyzes the oxidation of the substrate coelenterazine resulting in light emission at a wavelength of 480 nm. The Gaussia luciferase gene (G-Luc) has been successfully used as a reporter of gene expression in mammalian cells (Tannous et al. 2005) and could also be split and used for the detection of protein–protein interactions by protein fragment complementation assays (Remy and Michnick 2006). It encodes a non-toxic, monomeric protein of only 185 amino acids and this small size may make it particularly suitable for expression in recalcitrant species.
We report here the development of the luciferase gene from G. princeps as a sensitive reporter gene for the in vivo monitoring of gene expression in Chlamydomonas. A codon-optimized version of the gene fused to Chlamydomonas expression signals generated more than 7-fold higher bioluminescence activity than the R-Luc gene from R. reniformis. Furthermore, G-Luc displayed drastically higher signal intensity than R-Luc in luminescence imaging (about 40-fold), thus facilitating the in vivo monitoring of responses to environmental stress stimuli in C. reinhardtii.
Materials and methods
Algal strains and culture conditions
Chlamydomonas reinhardtii strain 325 (CW15,mt+, arg7–8) was used in this study (kindly provided by Dr. Christoph F. Beck, University of Freiburg, Germany). Cultures were grown mixotrophically in Tris–acetate phosphate (TAP) medium (Harris 1989) on a rotary shaker at 23°C under continuous irradiation with white light (55 μE m−2 s−1). The TAP medium was supplemented with 100 mg l−1 of arginine when required.
Nucleic acid manipulations
The coding region of the G. princeps luciferase gene (G-Luc) was synthesized de novo according to the nuclear codon usage of C. reinhardtii (GenScript, Piscataway, NJ). The synthetic G-Luc gene (GenBank accession number EU372000) was ligated as NdeI/EcoRI fragment into the similarly digested PsaD expression cassette (Fischer and Rochaix 2001). Analogously, a codon-optimized R. reniformis luciferase gene (R-Luc; Fuhrmann et al. 2004) was cloned as NdeI/EcoRI fragment into the PsaD cassette. For inducible expression, the G-Luc coding region was excised by digestion with HincII and BamHI and inserted into an inducible expression cassette driven by the Hsp70A promoter (Shao et al. 2007; Fig. 1b).Fig. 1Codon usage optimization of the G. princes luciferase and construction of expression cassettes for the transformation of Chlamydomonas reinhardtii. a Adaptation of the G-Luc gene to the codon usage in the nuclear genome of Chlamydomonas reinhardtii. The relative frequencies of the individual codons of the native G-Luc gene (GenBank accession number AY015993) in the Chlamydomonas nuclear genome are indicated by grey bars with the most frequently used triplet in Chlamydomonas set to 100%. The synthetic G-Luc gene (diamonds) was optimized by changing all codons to the most frequently used ones in Chlamydomonas reinhardtii. bG-Luc and R-Luc expression cassettes. To comparatively assess constitutive expression levels, the G-Luc (dark grey box) and R-Luc (hatched box) coding regions were inserted into the PsaD expression cassette (PsaD promoter and 5′ UTR shown as open box; Fischer and Rochaix 2001). Both the R-Luc (Fuhrmann et al. 2004) and the G-Luc genes are optimized with regard to the codon usage in the Chlamydomonas nuclear genome. For inducible expression, the Hsp70A promoter (from position −23 to −285 with respect to the translation initiation codon of Hsp70A) was fused to a gene fragment containing the 5′ UTR and the first three exons of the Hsp70B gene (Shao et al. 2007). The positions of the three heat shock elements (HSE, black boxes) within the promoter region are indicated. Light grey bars represent exons, introns are depicted as broken lines. The third exon of Hsp70B was fused to the coding region of R-Luc and G-Luc, respectively. The 3′ UTR of the reporter gene cassettes is derived from RBCS2, a nuclear gene for the small subunit of Rubisco (Shao et al. 2007)
Nuclear transformation of Chlamydomonas
Chlamydomonas nuclear co-transformation was carried out using the glass bead method (Kindle 1990). Plasmid DNA used for transformation was purified by PEG precipitation. Prior to transformation, plasmid pCB412 containing the C. reinhardtiiARG7 gene as selectable marker was linearized by digestion with EcoRI; all Luc constructs were linearized with ScaI. Co-transformation and selection for arginine prototrophy were employed to introduce the Luc constructs into the Chlamydomonas nuclear genome. Arginine prototrophic clones were selected on TAP medium. Transformants harboring the Luc constructs were identified by luciferase assays.
Bioluminescence assays
Coelenterazine (P.J.K. GmbH; Kleinblittersdorf, Germany), the substrate for G-Luc and R-Luc, was dissolved in ethanol (1 mM stock solution). To assay luciferase activity, C. reinhardtii cultures were grown in liquid TAP medium under constant illumination (55 μE m−2 s−1) to a final cell density of 3–6 × 106 cells ml−1. After sampling, cells were spun down, resuspended in the same volume of sample buffer [1.5 mM Tris–HCl (pH 7.8), 1 mM EDTA], and frozen at −20°C for at least 20 min. After thawing, 20 μl samples were transferred to 96-well, white microtiter plates and 125 μl of the assay buffer [0.1 M K2HPO4 (pH 7.6), 0.5 M NaCl, 1 mM EDTA] was added to each well. Following incubation at room temperature for 15 min in the dark, bioluminescence was assayed using a luminometer (MicroBeta TriLux; PerkinElmer) by auto-injecting the substrate (coelenterazine 0.01 mM; 50 μl per well). The luminescence units are presented as luminescence counts per second (LCPS). The background was normalized by measuring wells containing only buffer or buffer with cells lacking the Luc gene. Inducible expression of the Luc fusion genes was normalized to the chlorophyll content of the cultures (Porra et al. 1989). The induction factor was calculated by comparison with untreated cells.
For in vivo luminescence imaging, C. reinhardtii cultures were grown in TAP medium under constant illumination (55 μE m−2 s−1) to a final density of 1–2 × 106 cells ml−1. A volume of 3 μl cell suspension were spotted onto TAP agar plates and incubated under constant illumination (55 μE m−2 s−1) for 4 days. Luminescence of the cells was visualized in the presence of the substrate (coelenterazine 0.05 mM) using an ultra sensitive Photon Counting Camera (C2400-30H; Hamamatsu). Samples imaged in the absence of the substrate served as control. The luminescence images were acquired and processed with the HPD-LIS software (Hamamatsu) using an integration time of 20 min and linear signal intensity.
RNA gel blot analyses
Total cellular RNA was extracted according to published protocols (von Gromoff et al. 1989). RNA samples (15 μg total RNA) were electrophoresed in formaldehyde-containing 1% agarose gels and blotted onto Hybond XL membranes (GE Healthcare). To produce a hybridization probe for detection of G-Luc transcripts, the coding region of the gene was excised from a plasmid clone. A Hsp70A-specific probe was prepared from a plasmid clone originally described as hsp70-2 (von Gromoff et al. 1989). The probes were purified by agarose gel electrophoresis following extraction of the DNA fragments of interest from excised gel slices using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and then radiolabeled with 32P-dCTP using the MegaPrime kit (GE Healthcare). Hybridizations were performed at 65°C in Church buffer (Church and Gilbert 1984).
Results
Design of the Gaussia luciferase as reporter gene for Chlamydomonas
Bioluminescent proteins are widely used as reporter genes to measure gene expression, determine subcellular protein localization, and study protein–protein interactions. Luciferases are nontoxic, bioluminescent reporter proteins suitable to monitor gene expression quantitatively. Unfortunately, the previously constructed Chlamydomonas-specific Renilla luciferase (Fuhrmann et al. 2004) suffers from low sensitivity, presumably due to low expression levels and/or low protein stability. As in mammalian cells, the luciferase from the marine copepod G. princeps proved to be a much more sensitive reporter than firefly and Renilla luciferases (Tannous et al. 2005); we set out to test this luciferase as a reporter gene in C. reinhardtii.
Previous work had shown that, in Chlamydomonas, adaptation of the codon usage of trangenes significantly improves expression levels (Fuhrmann et al. 1999, 2004). As the codon usage in the native luciferase from G. princeps deviated strongly from that in nuclear genes of C. reinhardtii, we adjusted all codons to the most preferred triplets in Chlamydomonas (according to the codon usage table for Chlamydomonas: http://www.kazusa.org.jp/codon; Fig. 1a). Following this codon optimization in silico, the gene was resynthesized and will be subsequently referred to as G-Luc standing for Gaussia luciferase gene (GenBank accession number EU372000). In all subsequent experiments, G-Luc was compared side-by-side to the Renilla luciferases (Fuhrmann et al. 2004), referred to as R-Luc.
Both luciferase genes were cloned into two different expression cassettes: (1) the PsaD cassette (Fischer and Rochaix 2001), whose promoter is constitutively active at least under photosynthetic conditions and (2) an inducible expression cassette driven by the Hsp70A promoter fused to the 5′ region of the Hsp70B gene (Shao et al. 2007; Fig. 1b). This heat shock gene promoter was shown previously to positively respond to a variety of inducing signals, including heat stress, light, retrograde signals from the plastid, and reactive oxygen species (von Gromoff et al. 1989; Kropat et al. 1997; Kropat and Beck 1998; Schroda et al. 2000; Shao et al. 2007). All gene constructs were introduced into arginine-auxotroph Chlamydomonas cells by glass bead-mediated co-transformation followed by selection for arginine prototrophy.
Expression and heat inducibility of the Gaussia luciferase reporter
To compare the sensitivity of the Chlamydomonas-specific G-Luc as a reporter gene with that of the previously designed R-Luc, 24 transgenic clones from each construct with the constitutive PsaD promoter were randomly chosen and the six best-expressing ones were assayed for their luciferase activities. While only three out of six best R-Luc clones had significant luciferase activity, all six G-Luc clones showed high activity (Fig. 2a). Moreover, when the activities were compared quantitatively, the G-Luc clones displayed, on average, more than 7-fold higher bioluminescence signal intensity than the R-Luc clones (Fig. 2a), indicating that the new G-Luc is more sensitive and more efficient than previously established reporter genes for Chlamydomonas. The very high luciferase activities measured indicate that G-Luc will also be suitable for reporting expression from promoters that are considerably weaker than the PsaD promoter.Fig. 2Comparison of G-Luc and R-Luc activity in transgenic Chlamydomonas reinhardtii strains. a Expression of G-Luc and R-Luc under the control of the constitutive PsaD promoter. For each construct, bioluminescence assays with six independent transformants were performed using a luminescence counter. The bars represent the mean of three independent experiments. The standard deviation is indicated. b Heat induction of the G-Luc and R-Luc reporters under the control of the Hsp70A promoter. Expression of luciferase in transformants harboring the PHsp70A-Luc constructs was induced by a temperature shift from 23 to 40°C for 1 h. After a 1 h recovery phase at room temperature, luciferase activity was assayed and the induction factors were calculated by comparison with untreated samples. Four arbitrarily chosen transformants were assayed for each luciferase construct. The bars represent the mean of three independent experiments, the standard deviation is indicated. The basal expression levels of the transformants under non-inducing conditions were 105, 5, 133 and 2 LCPS μg−1 chlorophyll for the four R-Luc clones and 4, 5, 37 and 18 LCPS μg−1 chlorophyll for the four G-Luc clones
Next we wanted to compare the two luciferase genes when expressed under the control of the inducible Hsp70A promoter. To this end, inducibility was determined by measuring luciferase activities in the uninduced and induced states for the four best-expressing co-transformants from each construct (identified among 32 randomly picked clones). Transgene expression was induced by shifting the growth temperature of the algal culture from 23 to 40°C for 1 h. While background expression under non-inducing conditions was comparably low in R-Luc and G-Luc transformants, the G-Luc transformants showed much higher bioluminescence under inducing conditions (on average more than 7-fold; Fig. 2b). This confirms the higher sensitivity of the G-Luc reporter for another expression cassette (Hsp70A promoter + RbcS terminator) and, moreover, indicates that G-Luc can be used as a highly sensitive reporter gene for measuring inducible gene expression in C. reinhardtii.
Luciferase imaging and assessment of protein stability
One of the most powerful applications of luminescent reporter proteins is their use in genetic screens for mutants in cellular signal transduction pathways. This usually requires detection of the reporter gene activity by imaging techniques to facilitate high-throughput screening of mutagenized organisms. Unfortunately, due to the lack of sufficiently sensitive reporters, this has not been possible in Chlamydomonas to date. We, therefore, were interested in testing whether the sensitivity of our new G-Luc reporter gene was sufficiently high to allow visualization of gene expression by luciferase imaging.
To this end, we assayed luciferase activity from both the constitutive and the inducible expression constructs in vivo using a photon-counting camera. Even the best-expressing clone with R-Luc controlled by the PsaD expression cassette (Fig. 2a) did not show enough luminescence to be detectable by luciferase imaging (Fig. 3a). In contrast, G-Luc activity was sufficiently strong to be readily detectable (Fig. 3a). Similar results were obtained for inducible expression from the Hsp70A promoter. While R-Luc activity was barely above the detection limit, inducibility of G-Luc expression was detected with high sensitivity (Fig. 3b).Fig. 3In vivo assay of Gaussia luciferase and Renilla luciferase activities in Chlamydomonas reinhardtii by visualizing luminescence with a photon-counting camera. Left panels show photographs of the algal colonies prior to luminescence imaging, right panels show the luminescence images. The clones with the highest luciferase expression levels in Fig. 2 were used. a For detection of luciferase activity in living algae, wild-type cells (WT) and transformants harboring the PpsaD R-Luc or PpsaD G-Luc constructs (strains R1 and G9 from Fig. 2a) were spotted in three replicas onto agar-solidified medium and grown under normal light conditions (55 μE m−2 s−1) for 4 days. The ratio of luciferase signal intensities of PpsaD G-Luc:PpsaD R-Luc was approximately 40 (40.4 ± 2.5); the signal intensity of PpsaD R-Luc was only slightly above background (PpsaD R-Luc: WT = 3.4 ± 1.5). b Luminescence of the PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken with a photon-counting camera. Luminescence intensities are color-coded with the maximum set to 1.3 × 105
Different possible explanations can account for the much better performance of G-Luc compared to R-Luc: higher expression rates, higher enzymatic activity or higher stability of the Gaussia enzyme. To distinguish between these possibilities, we performed stability assays by measuring luciferase activities in dependence on the temperature. To this end, algal cultures were subjected to 30 min of high temperature incubation followed by a 30 min recovery phase at room temperature prior to measurement of luciferase activity. If the Gaussia enzyme were indeed more stable than the Renilla enzyme, its activity should decline less sharply with temperature. This was indeed the case (Fig. 4a): while the Renilla luciferase suffered a strong temperature-dependent decline in activity, the Gaussia enzyme was much less affected, suggesting that higher enzyme stability contributes substantially to the superior performance of G-Luc.Fig. 4Monitoring the heat stress response in Chlamydomonas strains expressing luciferase constructs driven by the heat-inducible Hsp70A promoter. a Assessment of the thermostability of the Gaussia and Renilla luciferases. PpsaD-Luc transformants were subjected to 30 min of high temperature treatment as indicated followed by a 30 min recovery phase at room temperature. Luciferase activities were measured with a luminescence counter. Note the high temperature sensitivity of the Renilla enzyme with all activity being lost at temperatures above 46°C. In contrast, the Gaussia enzyme appears to be much more stable, facilitating its use as a sensitive reporter of heat stress-induced gene expression. b Visualization of the heat stress response with G-Luc. For luciferase imaging, wild-type cells (WT) and transformants harboring PHsp70A R-Luc, PHsp70A G-Luc or P∆HSEG-Luc (G-Luc fusion to a Hsp70A promoter lacking the HSE region; Shao et al. 2007) were spotted in three replicas on agar plates, grown under normal light conditions (55 μE m−2 s−1) for 4 days, photographed (left picture), and then shifted from 23 to 47°C for 15 min. After 3.5 h at room temperature (RT), the luminescence of the transformants upon substrate addition was recorded with a photon-counting camera (right picture). c Analysis of the kinetics of luciferase induction in a PHsp70A G-Luc transformant exposed to heat stress. Colonies of the PHsp70A G-Luc strain growing on a TAP agar plate were exposed to 40 or 47°C for 15 min. After 1, 3 or 5 h recovery at RT, the luminescence of the colonies was visualized using a photon-counting camera. d Comparison of the induction of G-Luc expression by heat stress with induction of the endogenous Hsp70A gene at the mRNA level. Expression from the Hsp70A promoter was induced by a 15-min incubation at 40 or 47°C, and then followed over time by incubation at room temperature (RT) for the time spans indicated. The induction kinetics of luciferase expression from the Hsp70A promoter parallels that of the endogenous Hsp70A gene
To explore the heat inducibility of G-Luc under the control of the Hsp70A promoter in somewhat greater detail, we sought to identify optimum experimental conditions for conducting genetic screens for signaling mutants. We, therefore, tested different combinations of temperatures of the heat shock and recovery times and also included a control construct, in which the heat-shock elements (HSE) were deleted from the Hsp70A promoter (Shao et al. 2007). As expected, this deletion completely abolished heat inducibility under all conditions tested (Fig. 4b and data not shown). Efficient heat induction of the G-Luc reporter was achieved in a wide temperature range, from 40 to 47°C (cp. Figs. 3b, 4b). However, heat shock at higher temperatures required longer recovery times at room temperature before luciferase activity could be visualized by imaging. Whereas following heat shock at 40°C, maximum bioluminescence was measured after 1 h recovery, a recovery phase of 3 h was required to obtain similarly high bioluminescence after a heat shock at 47°C (Fig. 4c).
Next we wanted to confirm that heat induction of luciferase activity parallels G-Luc mRNA accumulation. This was clearly the case upon both induction at 40°C and induction at 47°C (Fig. 4d). At both temperatures, mRNA levels peaked at about the same time as enzyme activities (cp. Fig. 4c, d). Moreover, the kinetics of G-Luc mRNA accumulation correlated, by and large, with heat induction of the endogenous Hsp70A gene (Fig. 4d), ultimately confirming that the luciferase reporter faithfully mirrors promoter activity.
Having established that G-Luc expression can be readily monitored by luminescence imaging, we finally wanted to provide a quantitative assessment of the superior performance of the G-Luc reporter by direct luciferase imaging of primary transformants. To this end, Petri dishes with transformed Chlamydomonas colonies were exposed to the substrate and analyzed by luminescence imaging (Fig. 5). While transformation with G-Luc produced a high number of brightly luminescing colonies, R-Luc luminescence was much lower and barely detectable (Fig. 5). These data ultimately confirm the much higher sensitivity of the G-Luc reporter and its suitability for luminescence imaging.Fig. 5In vivo assay of Gaussia luciferase and Renilla luciferase activities in primary Chlamydomonas transformants by visualizing luminescence with a photon-counting camera. Co-transformation experiments were conducted using identical amounts of linearized DNA (500 ng Luc-containing plasmid + 100 ng ARG7-containing plasmid) and identical amounts of algal cells. Upper panels show photographs of the algal colonies prior to luminescence imaging, lower panels show the luminescence images. Luminescence intensities are color-coded with the maximum set to 1.3 × 105. a Luminescence of PpsaD-Luc transformants. The ratio of total-plate luminescence of PpsaD G-Luc:PpsaD R-Luc was approximately 36. b Luminescence of PHsp70A-Luc transformants induced by heat shock. The cultures were shifted from 23 to 40°C for 1 h. After recovery at room temperature for 1 h, the luminescence image was taken. The ratio of total-plate luminescence of PHsp70A G-Luc:PHsp70A R-Luc was approximately 16. Note that a large fraction of the non-luminescing colonies is not co-transformed (i.e., harbors the selectable marker gene but not the luciferase reporter)
Discussion
In this work, we have established the luciferase from the marine copepod G. princeps as a novel and highly sensitive bioluminescent reporter in the model alga C. reinhardtii. The G-Luc reporter outperforms previously developed reporter genes for Chlamydomonas, improves the monitoring of gene expression and, most importantly, represents the first nuclear reporter gene that is sufficiently sensitive to facilitate in vivo imaging in Chlamydomonas. This expands the toolbox available for Chlamydomonas genetics and cell biology and will make possible experimental approaches that theretofore could not be taken in Chlamydomonas. First and foremost, the possibility to conduct large-scale mutant screens by bioluminescence imaging of live algal colonies will facilitate powerful genetic strategies for the isolation of novel components of all those signal transduction cascades that modify gene expression by targeting specific promoters. For example, the inducible promoter used in this study (Hsp70A) is the target of several distinct signal transduction pathways in response to heat, retrograde signals from the chloroplast, and reactive oxygen species (Shao et al. 2007). Mutagenesis of our algal strains expressing G-Luc from this promoter (Fig. 4b, c), followed by selection for mutants incapable of inducing the luciferase gene in response to a specific stress stimulus, should allow the genetic dissection of the underlying signal transduction pathways. Our preliminary results indicate that reactive oxygen species, which are much weaker inducers of the promoter than heat stress (Shao et al. 2007), induce G-Luc expression sufficiently strongly to facilitate such a screen in a microtiter plate format, although screening on agar plates will require further optimization and improvement of the assay sensitivity.
In mammalian cells, a split version of the Gaussia luciferase was successfully used for the detection of protein–protein interactions in vivo by protein fragment complementation assays (Remy and Michnick 2006). As currently no method is available to identify protein–protein interactions in Chlamydomonas cells, the development of screens for protein interaction partners seems to be a particularly promising future application of the G-Luc reporter.
It should be noted that thus far, the G-luc reporter gene has been tested only in cell wall-deficient Chlamydomonas strains, which are easily transformable. It remains to be tested whether substrate uptake or luminescence imaging are influenced by the more rigid walls present in strains with wild type-like cell wall structure.
Although the Chlamydomonas-specific G-Luc described here provides a workable reporter of gene expression that is significantly more sensitive than previously established reporter genes, our data indicate that the superior performance of the Gaussia luciferase gene is not due to its better expression in C. reinhardtii, but rather due to its very high enzyme stability. Most probably, the adaptation of other widely used reporter genes to C. reinhardtii (like the genes for the fluorescent proteins GFP and YFP) will require a general solution to the transgene expression problem in Chlamydomonas. This could be achieved by either developing novel expression tools or generating dedicated expression strains in which the suspected epigenetic transgene silencing mechanism is inactivated.

luciferase
gaussia princeps
chlamydomonas reinhardtii
reporter gene
bioluminescence
heat-inducible expression