JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 30, Number 7, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0130
Diameters and Wall-to-Lumen Ratio of Retinal Arterioles in Patients with Retinal Vein Occlusion Before and After Treatment with Dexamethasone Intravitreal Implants Francesco Semeraro,1 Andrea Russo,1 Damiano Rizzoni,2 Paola Danzi,1 Francesco Morescalchi,1 and Ciro Costagliola 3
Abstract
Background: To evaluate the diameters and wall-to-lumen ratio (WLR) of retinal arterioles in patients with retinal vein occlusion (RVO) before and after a 0.7 mg dexamethasone (DEX) intravitreal implant and compare it with a matched control group of normal eyes. Methods: This was a single-site, multi-investigator, prospective, open-label, observational study in 15 patients with vision loss due to branch or central RVO treated with a single injection of DEX implant. An age-matched control group of 16 normal eyes was recruited. External and internal arteriolar diameters, WLR, and wall thickness were assessed in vivo using scanning-laser Doppler flowmetry. Visual acuity (VA) and central macular thickness (CMT) were evaluated. Results: Mean internal diameter showed a significant reduction in post-treatment RVO patients compared with pre-treatment RVO patients (56.0 – 18.0 mm vs. 67.9 – 16.9 mm, respectively; P = 0.02). Mean WLR in pretreatment RVO patients was 0.47 – 0.19, showing an increase to 0.63 – 0.23 3 months after treatment (P = 0.037). No significant difference was found in arteriolar external diameter between normotensive, pretreatment, and post-treatment subjects. Conclusion: Treatment with a DEX implant in RVO patients led to significant improvements in both VA and CMT. These changes were accompanied by reductions in arteriolar lumen diameter, which could contribute to decreased capillary leakage and macular swelling.
Introduction
R
etinal vein occlusion (RVO) is a common cause of vision loss in older people and the second most common retinal vascular disease after diabetic retinopathy.1 Macular edema is the major culprit for vision loss in both central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). Although full pathogenesis has not yet been completely understood, an excessive vascular endothelial growth factor (VEGF) production along with release of inflammatory factors would play a key role.2 Multiple strategies have been investigated for the treatment of the macular edema, including laser photocoagulation,3 anti-VEGF injections,4,5 and corticosteroid injections.6 However, repeated treatments are often required to control macular edema and increase the chance of visual improvement. To avoid this drawback, a dexamethasone (DEX) 1 2 3
delivery system has recently been made available (Ozurdex; Allergan, Inc., Irvine, CA) to provide the sustained delivery of preservative-free DEX in the vitreous cavity and retina.7–10 High steroid levels have been reported to induce small-resistance artery remodeling with an increase of the wall-to-lumen ratio (WLR),11,12 but the retinal arteriolar modifications after DEX intravitreal implant have not yet been evaluated. The noninvasive measurement of WLR in retinal arterioles by using scanning laser Doppler flowmetry has recently been introduced and represents a noninvasive and easily repeatable procedure for evaluating the retinal arterioles.13,14 Here, we evaluated the internal and external diameters as well as the WLR of retinal arterioles in 15 patients with RVO before and after an injection of the DEX implant compared with an age-matched control group of normal eyes in non-hypertensive subjects.
Eye Clinic, Department of Neurological and Vision Sciences, University of Brescia, Brescia, Italy. Medical Clinic, Department of Medical and Surgical Sciences, University of Brescia, Brescia, Italy. Eye Clinic, Department of Health Sciences, University of Molise, Campobasso, Italy.
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Methods Study design This was a single-site, multi-investigator, prospective, open-label, observational study of eyes with vision loss due to macular edema associated with CRVO or BRVO. Patients with RVO attending the retina clinics of the ‘‘Spedali Civili di Brescia’’ were enrolled over a 6-month period, from June to November 2011, and were treated within 6 months of diagnosis through an intravitreal injection of DEX (Ozurdex). An age-matched control group of 16 normal eyes in healthy and normotensive subjects was subsequently recruited over a 1-month period, from December 2011 to January 2012, among patients attending routine eye examinations, to compare retinal arterioles diameters and WLR. None of the control patients was under any therapy. Informed consent was obtained from each participant before enrollment in the study. The local ethics committee (Clinica Oculistica, University of Brescia) approved the protocol, and it was registered with clinicaltrials.gov (identifier: NCT01789437). The study was conducted in accordance with Good Clinical Practice guidelines and in compliance with the Declaration of Helsinki.
Inclusion and exclusion criteria Subjects with RVO were eligible if the following criteria were met: ability to provide written informed consent and comply with study assessments for the full duration of the study; age > 20 years; and decreased visual acuity (VA) as a result of clinically detectable macular edema associated with either CRVO or BRVO. The duration of macular edema was required to be between 4 and 24 weeks for both CRVO and BRVO; retinal thickness in the central subfield (as measured using optical coherence tomography) had to be > 350 mm in the study eye. The exclusion criteria were as follows: diabetes; previous intravitreal anti-VEGF therapy or intravitreal steroid therapy; previous photodynamic therapy or focal laser; active retinal or optic disc neovascularization; active or history of choroidal neovascularization; presence of rubeosis iridis; any active infection; glaucoma, current ocular hypertension, or a history of steroid-induced intraocular pressure (IOP) increase in either eye; or concurrent eye disease in the study eye that could compromise VA (e.g., choroidal neovascularization, diabetic retinopathy, and epiretinal membrane).
Study treatment Both BRVO and CRVO patients were treated with a 0.7 mg DEX implant. On day 0, a topical anesthetic (lidocaine 4%) was administered, and the eye was prepared according to the standard clinical practice for intravitreal injections. The implant was inserted into the vitreous cavity
FIG. 1. Reflection (A) and perfusion (B) images of a representative hypertensive RVO patient compared with a representative normotensive patient (C: reflection image; D: perfusion image). RVO, retinal vein occlusion.
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through the pars plana between 3.5 mm and 4.0 mm posterior to the limbus, by using the appropriate applicator.
Clinical evaluation RVO patients were evaluated at baseline and on days 30, 60, 90, 150, and 180 after the treatment. The following assessments were performed at each visit: adverse ocular events (e.g., uncontrolled inflammation, endophthalmitis, retinal tears and/or retinal detachment, eye pain, eye itch, eye redness, cataract, and iritis); best-corrected VA (EarlyTreatment Diabetic Retinopathy Study, ETDRS); IOP (measured with Goldmann applanation tonometer); ophthalmic examination at complete dilation; and central macular thickness (CMT) measured using OCT (Spectralis OCT; Heidelberg Engineering, GmbH, Heidelberg, Germany). Study OCT scans were performed using real-time eye tracking software (AutoRescan, Spectralis OCT; Heidelberg Engineering GmbH) in order to ensure that the same tissue slices were analyzed at each follow-up visit. Control group patients underwent a comprehensive ophthalmological evaluation, including best-corrected VA, IOP, ophthalmic examination at complete dilation, OCT-measured CMT, and scanning laser Doppler flowmetry evaluation of arteriolar diameters.
Evaluation of retinal arteriolar morphology The WLR of the retinal arterioles was assessed using scanning laser Doppler flowmetry at 670 nm (Heidelberg Retina Flowmeter; Heidelberg Engineering), an established method used to investigate retinal perfusion.13,15,16 The method is described in detail elsewhere.14 Briefly, a retinal arteriole of 80–140 mm in size in the superficial retinal layer in a retinal sample of 2.56 · 0.64 · 0.30 mm was scanned within 2 s, at a resolution of 256 points · 64 lines · 128 lines (Fig. 1). Measurements were performed in the juxtapapillary area of the eye, 2–3 mm temporal superior to the optic nerve; each procedure measured 1 arteriole, and the mean of 3 measurements was considered for further analysis.15 For cases with BRVO, the site of measurement was located 2–3 mm distal to the crossing site. Only the arterioles that could be unambiguously discriminated and clearly identified on the temporal superior side of the optic nerve were selected. Images of arterioles without sharp contrast to the retina or with crossing and overlapping venules, curved arterioles, or arterioles with more than 1 bifurcation on the image and images with more than 4 eye movements were excluded. The examination was performed with the patient in the sitting position after 20 min of rest, at room temperature and daylight conditions between 08.00 and 14.00 h, but before lunch. Analyses of diameters were performed offline with an automatic full-field perfusion imaging analysis
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Table 1. Demographic and Baseline Characteristics of the Retinal Vein Occlusion and Normotensive Patients
No. of patients % Female % Male Mean age Mean visual acuity (LogMAR) Mean CMT (mm) Mean IOP (mm Hg) Smokers Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dL) Serum triglycerides (mg/dL) Superior temporal crossing site (%) Inferior temporal crossing site (%)
BRVO
CRVO
Normotensive
10 60 40 67.2 – 10.8 0.38 – 0.19 622.1 – 165.5 12.9 – 3.4 3/10 142 – 17.2 89.3 – 11.5 203 – 33.2 118 – 70.5 60 40
5 60 40 64.2 – 17.9 0.72 – 0.26 845.2 – 216.3 16.4 – 2.9 1/5 138 – 16.4 88.2 – 10.2 202 – 31.7 124 – 72.4 — —
16 56.3 43.7 66.4 – 14 0.01 – 0.03 211.2 – 8.4 14.7 – 2.2 2/16 124 – 13.2 71.6 – 9.2 208 – 33.9 98.4 – 37.1 — —
Values are expressed as means – SD. BRVO, branch retinal vein occlusion; CMT, central macular thickness; CRVO, central retinal vein occlusion; IOP, intraocular pressure.
program (Nirox Optoelectronics, Brescia, Italy).17 Outer arteriole diameter was measured in reflection images, and lumen diameter was measured in perfusion images.15,18,19 WLR was calculated using the formula: (arteriole diameter lumen diameter)/lumen diameter.15,18,19 The reproducibility of the measurement of WLR is quite good. We calculated intraobserver (10 patients, 2 readings of the same observer 1 day apart) and interobserver (10 patients, 2 simultaneous observers) coefficients of variation. The values we obtained were 13% and 11%, respectively. Our data are quite similar to those reported in a recently published reliability analysis of the measurement of WLR with scanning laser Doppler flowmetry, showing an intraobserver coefficient of variation of *8%–9% and an interobserver coefficient of variation of *9%–10%.20
Outcome measures Outcomes were analyzed at 5 time points: 30, 60, 90, 150, and 180 days. The outcome measures were as follows: adverse ocular events; mean change in VA; and mean change in CMT. The measurement of the diameters of the retinal arterioles and the calculation of WLR were performed at baseline and at 3 months after treatment in RVO patients.
Statistical analysis All of the results were normally distributed and are expressed as means – SD in the text and tables. A repeatedmeasures ANOVA was performed, using Greenhouse–Geisser and Bonferroni corrections, to detect differences between the outcome measures. A 1-way ANOVA with a Tukey post hoc test was done to detect differences in arteriolar parameters between groups of pre-treatment, post-treatment, and control patients. All analyses were performed using SPSS version 17 software, and significance was defined as a P-value of < 0.05.
All RVO patients were found to be hypertensive and treated with antihypertensive drugs (diuretics in 8% of patients, angiotensin-converting enzyme inhibitors in 44%, calcium channel blockers in 20%, angiotensin receptor blockers in 30%, b-blockers in 12%, and doxazosin in 5%). An age-matched control group of 16 normal eyes in normotensive subjects was subsequently recruited. The demographic and baseline characteristics of the study population are listed in Table 1.
Artery remodeling Arteriolar diameters in normotensive, BRVO, and CRVO patients are presented in Tables 2 and 3. Both BRVO and CRVO patients showed a significantly increased pre-treatment mean wall thickness (13.8 – 4.2, P = 0.04 and 18.5 – 5.3, P = 0.001, respectively) and WLR (0.45 – 0.15, P = 0.04 and 0.52 – 0.26, P = 0.02, respectively) in comparison to normotensive subjects. Conversely, no significant differences in internal or external arteriolar diameter were observed when BRVO and CRVO patients were compared with normotensive patients. Three months after an injection of the DEX implant, external arteriolar diameter decreased in both BRVO and CRVO patients, but not to a significant extent (P = 0.42 and P = 0.31, respectively). There was a slight decrease, though not statistically significant, in internal arteriolar diameter in both BRVO and CRVO patients (P = 0.06 and P = 0.12). However, this reduction became statistically significant when RVO patients were analyzed together (P = 0.02). Wall thickness did not change significantly in either BRVO (P = 0.33) or CRVO (P = 0.46) patients. WLR increased significantly in BRVO patients (P = 0.002) but did not change significantly in CRVO patients (P = 0.88). The increase in WLR was significant when RVO patients were analyzed together (P = 0.037).
Results A total of 15 patients that met all inclusion and exclusion criteria were enrolled and completed the study (66.2 – 13 years); 5 of these patients were diagnosed with CRVO (33.3%), while the other 10 were diagnosed with BRVO (66.7%).
Visual acuity One month after DEX injection, mean VA significantly increased to 0.32 – 0.22 LogMAR (P = 0.017), although efficacy peaked after 2 months (0.23 – 0.17 LogMAR; P = 0.0001).
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Table 2. Arteriolar Diameters in Normotensive, Branch Retinal Vein Occlusion, and Central Retinal Vein Occlusion Patients
Normotensive BRVO pretreatment BRVO post-treatment CRVO pretreatment CRVO post-treatment
External diameter (mm)
Internal diameter (mm)
Wall thickness (mm)
WLR
No. of patients
93.6 – 19 90 – 15.7 82.1 – 7.6 116.1 – 14.7 101.3 – 22.5
74.4 – 15.5 62.4 – 11.4 47.7 – 7.5 79.1 – 21.7 72.9 – 22.2
9.6 – 3.8 13.8 – 4.2 17.2 – 2.5 18.5 – 5.3 14.2 – 5.3
0.26 – 0.11 0.45 – 0.15 0.74 – 0.16 0.52 – 0.26 0.42 – 0.2
16 10 10 5 5
Values are expressed as means – SD. WLR, wall-to-lumen ratio.
Three months after the injection, VA started declining to 0.26 – 0.21 LogMAR (P = 0.001), reaching 0.36 – 0.22 LogMAR (P = 0.049) 5 months later. After 6 months, the mean VA was 0.38 – 0.20 LogMAR, and the difference from baseline was no longer statistically significant (P = 0.262). The data are shown in Fig. 2.
Central macular thickness The mean decrease in CMT was statistically significant after 30 days (460.1 – 211.9 mm; P = 0.016) and peaked at 2 months after treatment (427.6 – 166.6 mm; P = 0.003). Three months after the injections, CMT returned to 541.0 – 201.4 mm (P = 0.036). The decrease reached non-significant values, 628.2 – 150.8 mm (P = 1) and 608.6 – 196.3 mm (P = 1) after 5 and 6 months, respectively. The CMT data are shown in Fig. 2.
Safety analysis The only adverse events that we encountered were anterior chamber cells (n = 5 patients) and mild ocular hypertension. Mean IOP peaked 2 months after treatment, but the change from baseline was never significant throughout the entire duration of the study. Three patients required IOPlowering medication (timolol-dorzolamide fixed combination) due to an IOP increase more than 25 mm Hg. The mean IOP fluctuations are shown in Fig. 3. We did not notice either an increase in the incidence or progression of cataracts in any of the 11 phakic eyes (clinically assessed during slit-lamp examination using the LOCS III grading system21) or retinal neovascularization during the study.
Discussion The results of in vivo scanning laser Doppler flowmetry showed arteriolar eutrophic inward remodeling, secondary to
hypertension, in RVO patients, as compared with an agematched control group of normal eyes in non-hypertensive subjects. A further reduction in internal arteriolar diameter was observed at 3 months after an injection of the DEX implant. Essential and secondary hypertension are known to alter the microcirculation, including low-resistance arteries and arterioles.17 These changes mainly involve arterial wall thickening and reduction of the internal diameter of lowresistance arteries with a consequent increase in media-tolumen ratio without any significant change in the total amount of wall tissue. This process is known as inward eutrophic remodeling.22 This process can also result in vascular wall hypertrophy, which is the growth of smooth muscle cells13,23 with increased arterial rigidity and possible compression of the retinal vein. Ozurdex in an implant made of a solid biodegradable polymer, which enables dual-phase pharmacokinetics, initially releasing a burst of DEX to rapidly achieve a therapeutic concentration followed by sustained release at lower concentrations.24 The concentration of DEX in the vitreous peaks at 213 – 49 ng/mL between days 60 and 90.24 A high sustained concentration of DEX, which is 5 times more potent than triamcinolone acetonide, is likely to contribute to further remodeling of the retinal arterioles, as high steroid levels have been reported to induce small-resistance artery remodeling25 with an associated increase of the WLR.11,12 This process is probably mediated by the stimulation of vascular smooth muscle cell growth.12 It was recently demonstrated that glucocorticoids might activate rapid mineralocorticoid receptor signaling in vascular smooth muscle cells, which involves MAPK/ERK-dependent pathways,26 suggesting that glucocorticoids may contribute to vascular remodeling via mineralocorticoid receptor signaling regardless of circulating aldosterone levels.27 The results from this study show a reduction in arteriolar internal diameter in both BRVO and CRVO patients after
Table 3. Arteriolar Diameters in Normal Eyes and Retinal Vein Occlusion Patients, Before and After Intravitreal Dexamethasone Injection
Normotensive Pretreatment Post-treatment P-value
External diameter (mm)
Internal diameter (mm)
Wall thickness (mm)
WLR
93.6 – 19 98.7 – 19.6 88.5 – 16.4 0.07
74.4 – 15.5 67.9 – 16.9 56.0 – 18.0a 0.02
9.6 – 3.8 15.4 – 5a 16.2 – 3.7a 0.661
0.26 – 0.11 0.47 – 0.19a 0.63 – 0.23a 0.037
Values are expressed as means – SD. P-value documents the difference between pre- and post-treatment groups. a Statistically significant differences compared with normotensive subjects.
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FIG. 2. Mean VA and CMT modifications over time. VA, visual acuity; CMT, central macular thickness. the injection of a DEX implant, although the WLR was significantly increased only in BRVO patients. This difference might be partially due to the small number of patients in the CRVO sample as well as to the greater pre- and posttreatment external diameters found in CRVO patients. Macular edema in RVO patients is caused by increased capillary pressure from venous outflow obstruction and by the blood–retinal barrier loss associated with retinal ischemia due to vascular stasis and defective capillary perfusion.28 The key mechanism of altered blood–retinal barrier function is changed permeability of retinal endothelial cells caused by events such as increased capillary pressure, elevated levels of growth factors, cytokines, inflammation, loss of pericytes, and alteration of Muller cells. As a result of this process, paracellular and transcellular transport across the retinal vascular wall increases because of disruption of endothelial tight junctions and changes in endothelial caveolar transcellular transport, respectively. Corticosteroids such as triamcinolone and DEX administered by an intravitreal injection have been widely used off-label in patients with macular edema secondary to RVO
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and diabetes, and they have a noticeably beneficial anatomical effect on retinal swelling and vascular leakage as detected by OCT and fluorescein angiography.9,10 Results from this study confirm the efficacy of DEX implant, with noteworthy improvements in both VA and CMT in BRVO and CRVO patients peaking at 2 months after the injection, in line with previously published data.8–10 CMT improvements after intravitreal DEX correlated directly with reduced macular leakage, as recently reported by Sadda et al.,29 supporting the idea that macular swelling results from excess fluid leakage from nearby capillaries. As it is in other vascular beds, the rules of Starling determine water homeostasis and edema formation in the retina. Net water transport over the endothelium is determined by the sum of hydrostatic pressure and osmotic pressure of the luminal and extraluminal compartments.28 With regard to hydrostatic pressure, the results from this study show a small but significant reduction in internal arteriolar diameter after treatment with intravitreal DEX. The remodeling induced by Ozurdex may be considered an undesirable effect of steroids. However, this lumen reduction, according to Poiseuille’s relationship, results in an increase in arteriolar flow resistance. We, therefore, speculate that the resultant reduction in capillary hydrostatic pressure could modify Starling’s equilibrium, thereby contributing, at least partially and temporarily, to the reduction in vascular leakage observed in the retina after corticosteroid administration. With regard to the osmotic pressure, steroid effects on the blood–retinal barrier have not been fully elucidated. Despite the well-known direct barrier-enhancing effect of steroids on cell culture models of the blood–brain barrier or blood– retinal barrier,30–33 steroids may also act indirectly by inhibition of inflammatory cells or cytokines,34 via a direct effect on tight junction molecules or other functions of endothelial cells, and by preventing the osmotic swelling of Muller cells caused by an alteration of aquaporin 4.35,36 Ocular steroid-related side-effects most commonly include cataract formation and elevated IOP.37 Our safety findings resemble those reported in the Geneva Study, which found that only 20% (3 out of 15) of patients required IOPlowering therapy during the first 6 months,9,10 whereas no cataract progression was recorded.
Study limitations
FIG. 3. Mean IOP progress over time. IOP, intraocular pressure.
Since our control group consisted of healthy subjects, we could not observe the natural history of WLR in RVO without DEX implantation. Therefore, we cannot assert with certainty that the observed changes in WLR are purely due to the DEX implant. Second, the sample size is quite small. However, a post hoc power analysis calculation showed that the sample size of 15 patients provided a power of *0.98, for demonstrating an effect size of 1 mm between pre- and post-treatment arteriolar diameters, at a significance level of 0.05. Third, this study included patients with BRVO and patients with CRVO. Nevertheless, this study was aimed at evaluating the remodeling of retinal arterioles after the DEX implant, and there is no compelling evidence that remodeling differs in either BRVO or CRVO. Lastly, a longer follow up with repeated assessments at different time points is needed to observe the long-term
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arteriolar modifications after intravitreal DEX implants. The current cohort will continue to be followed up for 1 year to evaluate both the arteriolar remodeling and the safety. In conclusion, we found that the administration of DEX intravitreal implant in RVO patients led to a remodeling in the diameter of the retinal arteriolar lumen. However, this further arteriolar remodeling does not seem to have a detrimental effect on the retinal circulation. It rather contributes to reduced capillary leakage and macular swelling, by modifying Starling’s equilibrium. Further studies are needed to confirm these vascular changes over a longer term in larger samples.
Author Disclosure Statement
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10.
11. 12.
No competing financial interests exist.
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Longitudinal Study of Cataract Study Group. Arch. Ophthalmol. 111:831–836, 1993. Mulvany, M.J., Baumbach, G.L., Aalkjaer, C., Heagerty, A.M., Korsgaard, N., Schiffrin, E.L., and Heistad, D.D. Vascular remodeling. Hypertension. 28:505–506, 1996. Heagerty, A.M., Aalkjaer, C., Bund, S.J., Korsgaard, N., and Mulvany, M.J. Small artery structure in hypertension: dual processes of remodeling and growth. Hypertension. 21:391–397, 1993. London, N.J., Chiang, A., and Haller, J.A. The dexamethasone drug delivery system: indications and evidence. Adv. Ther. 28:351–366, 2011. Iglarz, M., Touyz, R.M., Viel, E.C., Amiri, F., and Schiffrin, E.L. Involvement of oxidative stress in the profibrotic action of aldosterone. Interaction wtih the renin-angiotension system. Am. J. Hypertens. 17:597–603, 2004. Molnar, G.A., Lindschau, C., Dubrovska, G., Mertens, P.R., Kirsch, T., Quinkler, M., Gollasch, M., Wresche, S., Luft, F.C., Muller, D.N., and Fiebeler, A. Glucocorticoid-related signaling effects in vascular smooth muscle cells. Hypertension. 51:1372–1378, 2008. van den Meiracker, A.H., and Batenburg, W.W. Corticosteroid-dependent, aldosterone-independent mineralocorticoid-receptor activation in the heart. J. Hypertens. 26:1307–1309, 2008. Klaassen, I., Van Noorden, C.J., and Schlingemann, R.O. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog. Retin Eye Res. 34:19–48, 2013. Sadda, S., Danis, R.P., Pappuru, R.R., Keane, P.A., Jiao, J., Li, X.Y., and Whitcup, S.M. Vascular changes in eyes treated with dexamethasone intravitreal implant for macular edema after retinal vein occlusion. Ophthalmology. 120:1423–1431, 2013. Felinski, E.A., Cox, A.E., Phillips, B.E., and Antonetti, D.A. Glucocorticoids induce transactivation of tight junction genes occludin and claudin-5 in retinal endothelial cells via a novel cis-element. Exp. Eye Res. 86:867–878, 2008. Kashiwamura, Y., Sano, Y., Abe, M., Shimizu, F., Haruki, H., Maeda, T., Kawai, M., and Kanda, T. Hydrocortisone enhances the function of the bloodenerve barrier through the up-regulation of claudin-5. Neurochem. Res. 36:849– 855, 2011.
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32. Nagasawa, K., Chiba, H., Fujita, H., Kojima, T., Saito, T., Endo, T., and Sawada, N. Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells. J. Cell Physiol. 208:123–132, 2006. 33. Wisniewska-Kruk, J., Hoeben, K.A., Vogels, I.M., Gaillard, P.J., Van Noorden, C.J., Schlingemann, R.O., and Klaassen, I. A novel co-culture model of the bloode retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes. Exp. Eye Res. 96:181–190, 2012. 34. McAllister, I.L., Vijayasekaran, S., Chen, S.D., and Yu, D.Y. Effect of triamcinolone acetonide on vascular endothelial growth factor and occludin levels in branch retinal vein occlusion. Am. J. Ophthalmol. 147:838–846, 2009. 35. Pannicke, T., Iandiev, I., Wurm, A., Uckermann, O., Vom, H.F., Reichenbach, A., Wiedemann, P., Hammes, H.P., and Bringmann, A. Diabetes alters osmotic swelling characteristics and membrane conductance of glial cells in rat retina. Diabetes. 55:633–639, 2006. 36. Cui, B., Sun, J.H., Xiang, F.F., Liu, L., and Li, W.J. Aquaporin 4 knockdown exacerbates streptozotocin-induced diabetic retinopathy through aggravating inflammatory response. Exp. Eye Res. 98:37–43, 2012. 37. Becerra, E.M., Morescalchi, F., Gandolfo, F., Danzi, P., Nascimbeni, G., Arcidiacono, B., and Semeraro, F. Clinical evidence of intravitreal triamcinolone acetonide in the management of age-related macular degeneration. Curr. Drug Targets. 12:149–172, 2011.
Received: July 4, 2013 Accepted: April 4, 2014 Address correspondence to: Dr. Andrea Russo Eye Clinic Department of Neurological and Vision Sciences University of Brescia Piazzale Spedali Civili, 1 Brescia 25125 Italy E-mail: [email protected]
Diameters and Wall-to-Lumen Ratio of Retinal Arterioles in Patients with Retinal Vein Occlusion Before and After Treatment with Dexamethasone Intravitreal Implants Francesco Semeraro,1 Andrea Russo,1 Damiano Rizzoni,2 Paola Danzi,1 Francesco Morescalchi,1 and Ciro Costagliola 3
Abstract
Background: To evaluate the diameters and wall-to-lumen ratio (WLR) of retinal arterioles in patients with retinal vein occlusion (RVO) before and after a 0.7 mg dexamethasone (DEX) intravitreal implant and compare it with a matched control group of normal eyes. Methods: This was a single-site, multi-investigator, prospective, open-label, observational study in 15 patients with vision loss due to branch or central RVO treated with a single injection of DEX implant. An age-matched control group of 16 normal eyes was recruited. External and internal arteriolar diameters, WLR, and wall thickness were assessed in vivo using scanning-laser Doppler flowmetry. Visual acuity (VA) and central macular thickness (CMT) were evaluated. Results: Mean internal diameter showed a significant reduction in post-treatment RVO patients compared with pre-treatment RVO patients (56.0 – 18.0 mm vs. 67.9 – 16.9 mm, respectively; P = 0.02). Mean WLR in pretreatment RVO patients was 0.47 – 0.19, showing an increase to 0.63 – 0.23 3 months after treatment (P = 0.037). No significant difference was found in arteriolar external diameter between normotensive, pretreatment, and post-treatment subjects. Conclusion: Treatment with a DEX implant in RVO patients led to significant improvements in both VA and CMT. These changes were accompanied by reductions in arteriolar lumen diameter, which could contribute to decreased capillary leakage and macular swelling.
Introduction
R
etinal vein occlusion (RVO) is a common cause of vision loss in older people and the second most common retinal vascular disease after diabetic retinopathy.1 Macular edema is the major culprit for vision loss in both central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). Although full pathogenesis has not yet been completely understood, an excessive vascular endothelial growth factor (VEGF) production along with release of inflammatory factors would play a key role.2 Multiple strategies have been investigated for the treatment of the macular edema, including laser photocoagulation,3 anti-VEGF injections,4,5 and corticosteroid injections.6 However, repeated treatments are often required to control macular edema and increase the chance of visual improvement. To avoid this drawback, a dexamethasone (DEX) 1 2 3
delivery system has recently been made available (Ozurdex; Allergan, Inc., Irvine, CA) to provide the sustained delivery of preservative-free DEX in the vitreous cavity and retina.7–10 High steroid levels have been reported to induce small-resistance artery remodeling with an increase of the wall-to-lumen ratio (WLR),11,12 but the retinal arteriolar modifications after DEX intravitreal implant have not yet been evaluated. The noninvasive measurement of WLR in retinal arterioles by using scanning laser Doppler flowmetry has recently been introduced and represents a noninvasive and easily repeatable procedure for evaluating the retinal arterioles.13,14 Here, we evaluated the internal and external diameters as well as the WLR of retinal arterioles in 15 patients with RVO before and after an injection of the DEX implant compared with an age-matched control group of normal eyes in non-hypertensive subjects.
Eye Clinic, Department of Neurological and Vision Sciences, University of Brescia, Brescia, Italy. Medical Clinic, Department of Medical and Surgical Sciences, University of Brescia, Brescia, Italy. Eye Clinic, Department of Health Sciences, University of Molise, Campobasso, Italy.
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Methods Study design This was a single-site, multi-investigator, prospective, open-label, observational study of eyes with vision loss due to macular edema associated with CRVO or BRVO. Patients with RVO attending the retina clinics of the ‘‘Spedali Civili di Brescia’’ were enrolled over a 6-month period, from June to November 2011, and were treated within 6 months of diagnosis through an intravitreal injection of DEX (Ozurdex). An age-matched control group of 16 normal eyes in healthy and normotensive subjects was subsequently recruited over a 1-month period, from December 2011 to January 2012, among patients attending routine eye examinations, to compare retinal arterioles diameters and WLR. None of the control patients was under any therapy. Informed consent was obtained from each participant before enrollment in the study. The local ethics committee (Clinica Oculistica, University of Brescia) approved the protocol, and it was registered with clinicaltrials.gov (identifier: NCT01789437). The study was conducted in accordance with Good Clinical Practice guidelines and in compliance with the Declaration of Helsinki.
Inclusion and exclusion criteria Subjects with RVO were eligible if the following criteria were met: ability to provide written informed consent and comply with study assessments for the full duration of the study; age > 20 years; and decreased visual acuity (VA) as a result of clinically detectable macular edema associated with either CRVO or BRVO. The duration of macular edema was required to be between 4 and 24 weeks for both CRVO and BRVO; retinal thickness in the central subfield (as measured using optical coherence tomography) had to be > 350 mm in the study eye. The exclusion criteria were as follows: diabetes; previous intravitreal anti-VEGF therapy or intravitreal steroid therapy; previous photodynamic therapy or focal laser; active retinal or optic disc neovascularization; active or history of choroidal neovascularization; presence of rubeosis iridis; any active infection; glaucoma, current ocular hypertension, or a history of steroid-induced intraocular pressure (IOP) increase in either eye; or concurrent eye disease in the study eye that could compromise VA (e.g., choroidal neovascularization, diabetic retinopathy, and epiretinal membrane).
Study treatment Both BRVO and CRVO patients were treated with a 0.7 mg DEX implant. On day 0, a topical anesthetic (lidocaine 4%) was administered, and the eye was prepared according to the standard clinical practice for intravitreal injections. The implant was inserted into the vitreous cavity
FIG. 1. Reflection (A) and perfusion (B) images of a representative hypertensive RVO patient compared with a representative normotensive patient (C: reflection image; D: perfusion image). RVO, retinal vein occlusion.
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through the pars plana between 3.5 mm and 4.0 mm posterior to the limbus, by using the appropriate applicator.
Clinical evaluation RVO patients were evaluated at baseline and on days 30, 60, 90, 150, and 180 after the treatment. The following assessments were performed at each visit: adverse ocular events (e.g., uncontrolled inflammation, endophthalmitis, retinal tears and/or retinal detachment, eye pain, eye itch, eye redness, cataract, and iritis); best-corrected VA (EarlyTreatment Diabetic Retinopathy Study, ETDRS); IOP (measured with Goldmann applanation tonometer); ophthalmic examination at complete dilation; and central macular thickness (CMT) measured using OCT (Spectralis OCT; Heidelberg Engineering, GmbH, Heidelberg, Germany). Study OCT scans were performed using real-time eye tracking software (AutoRescan, Spectralis OCT; Heidelberg Engineering GmbH) in order to ensure that the same tissue slices were analyzed at each follow-up visit. Control group patients underwent a comprehensive ophthalmological evaluation, including best-corrected VA, IOP, ophthalmic examination at complete dilation, OCT-measured CMT, and scanning laser Doppler flowmetry evaluation of arteriolar diameters.
Evaluation of retinal arteriolar morphology The WLR of the retinal arterioles was assessed using scanning laser Doppler flowmetry at 670 nm (Heidelberg Retina Flowmeter; Heidelberg Engineering), an established method used to investigate retinal perfusion.13,15,16 The method is described in detail elsewhere.14 Briefly, a retinal arteriole of 80–140 mm in size in the superficial retinal layer in a retinal sample of 2.56 · 0.64 · 0.30 mm was scanned within 2 s, at a resolution of 256 points · 64 lines · 128 lines (Fig. 1). Measurements were performed in the juxtapapillary area of the eye, 2–3 mm temporal superior to the optic nerve; each procedure measured 1 arteriole, and the mean of 3 measurements was considered for further analysis.15 For cases with BRVO, the site of measurement was located 2–3 mm distal to the crossing site. Only the arterioles that could be unambiguously discriminated and clearly identified on the temporal superior side of the optic nerve were selected. Images of arterioles without sharp contrast to the retina or with crossing and overlapping venules, curved arterioles, or arterioles with more than 1 bifurcation on the image and images with more than 4 eye movements were excluded. The examination was performed with the patient in the sitting position after 20 min of rest, at room temperature and daylight conditions between 08.00 and 14.00 h, but before lunch. Analyses of diameters were performed offline with an automatic full-field perfusion imaging analysis
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Table 1. Demographic and Baseline Characteristics of the Retinal Vein Occlusion and Normotensive Patients
No. of patients % Female % Male Mean age Mean visual acuity (LogMAR) Mean CMT (mm) Mean IOP (mm Hg) Smokers Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dL) Serum triglycerides (mg/dL) Superior temporal crossing site (%) Inferior temporal crossing site (%)
BRVO
CRVO
Normotensive
10 60 40 67.2 – 10.8 0.38 – 0.19 622.1 – 165.5 12.9 – 3.4 3/10 142 – 17.2 89.3 – 11.5 203 – 33.2 118 – 70.5 60 40
5 60 40 64.2 – 17.9 0.72 – 0.26 845.2 – 216.3 16.4 – 2.9 1/5 138 – 16.4 88.2 – 10.2 202 – 31.7 124 – 72.4 — —
16 56.3 43.7 66.4 – 14 0.01 – 0.03 211.2 – 8.4 14.7 – 2.2 2/16 124 – 13.2 71.6 – 9.2 208 – 33.9 98.4 – 37.1 — —
Values are expressed as means – SD. BRVO, branch retinal vein occlusion; CMT, central macular thickness; CRVO, central retinal vein occlusion; IOP, intraocular pressure.
program (Nirox Optoelectronics, Brescia, Italy).17 Outer arteriole diameter was measured in reflection images, and lumen diameter was measured in perfusion images.15,18,19 WLR was calculated using the formula: (arteriole diameter lumen diameter)/lumen diameter.15,18,19 The reproducibility of the measurement of WLR is quite good. We calculated intraobserver (10 patients, 2 readings of the same observer 1 day apart) and interobserver (10 patients, 2 simultaneous observers) coefficients of variation. The values we obtained were 13% and 11%, respectively. Our data are quite similar to those reported in a recently published reliability analysis of the measurement of WLR with scanning laser Doppler flowmetry, showing an intraobserver coefficient of variation of *8%–9% and an interobserver coefficient of variation of *9%–10%.20
Outcome measures Outcomes were analyzed at 5 time points: 30, 60, 90, 150, and 180 days. The outcome measures were as follows: adverse ocular events; mean change in VA; and mean change in CMT. The measurement of the diameters of the retinal arterioles and the calculation of WLR were performed at baseline and at 3 months after treatment in RVO patients.
Statistical analysis All of the results were normally distributed and are expressed as means – SD in the text and tables. A repeatedmeasures ANOVA was performed, using Greenhouse–Geisser and Bonferroni corrections, to detect differences between the outcome measures. A 1-way ANOVA with a Tukey post hoc test was done to detect differences in arteriolar parameters between groups of pre-treatment, post-treatment, and control patients. All analyses were performed using SPSS version 17 software, and significance was defined as a P-value of < 0.05.
All RVO patients were found to be hypertensive and treated with antihypertensive drugs (diuretics in 8% of patients, angiotensin-converting enzyme inhibitors in 44%, calcium channel blockers in 20%, angiotensin receptor blockers in 30%, b-blockers in 12%, and doxazosin in 5%). An age-matched control group of 16 normal eyes in normotensive subjects was subsequently recruited. The demographic and baseline characteristics of the study population are listed in Table 1.
Artery remodeling Arteriolar diameters in normotensive, BRVO, and CRVO patients are presented in Tables 2 and 3. Both BRVO and CRVO patients showed a significantly increased pre-treatment mean wall thickness (13.8 – 4.2, P = 0.04 and 18.5 – 5.3, P = 0.001, respectively) and WLR (0.45 – 0.15, P = 0.04 and 0.52 – 0.26, P = 0.02, respectively) in comparison to normotensive subjects. Conversely, no significant differences in internal or external arteriolar diameter were observed when BRVO and CRVO patients were compared with normotensive patients. Three months after an injection of the DEX implant, external arteriolar diameter decreased in both BRVO and CRVO patients, but not to a significant extent (P = 0.42 and P = 0.31, respectively). There was a slight decrease, though not statistically significant, in internal arteriolar diameter in both BRVO and CRVO patients (P = 0.06 and P = 0.12). However, this reduction became statistically significant when RVO patients were analyzed together (P = 0.02). Wall thickness did not change significantly in either BRVO (P = 0.33) or CRVO (P = 0.46) patients. WLR increased significantly in BRVO patients (P = 0.002) but did not change significantly in CRVO patients (P = 0.88). The increase in WLR was significant when RVO patients were analyzed together (P = 0.037).
Results A total of 15 patients that met all inclusion and exclusion criteria were enrolled and completed the study (66.2 – 13 years); 5 of these patients were diagnosed with CRVO (33.3%), while the other 10 were diagnosed with BRVO (66.7%).
Visual acuity One month after DEX injection, mean VA significantly increased to 0.32 – 0.22 LogMAR (P = 0.017), although efficacy peaked after 2 months (0.23 – 0.17 LogMAR; P = 0.0001).
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Table 2. Arteriolar Diameters in Normotensive, Branch Retinal Vein Occlusion, and Central Retinal Vein Occlusion Patients
Normotensive BRVO pretreatment BRVO post-treatment CRVO pretreatment CRVO post-treatment
External diameter (mm)
Internal diameter (mm)
Wall thickness (mm)
WLR
No. of patients
93.6 – 19 90 – 15.7 82.1 – 7.6 116.1 – 14.7 101.3 – 22.5
74.4 – 15.5 62.4 – 11.4 47.7 – 7.5 79.1 – 21.7 72.9 – 22.2
9.6 – 3.8 13.8 – 4.2 17.2 – 2.5 18.5 – 5.3 14.2 – 5.3
0.26 – 0.11 0.45 – 0.15 0.74 – 0.16 0.52 – 0.26 0.42 – 0.2
16 10 10 5 5
Values are expressed as means – SD. WLR, wall-to-lumen ratio.
Three months after the injection, VA started declining to 0.26 – 0.21 LogMAR (P = 0.001), reaching 0.36 – 0.22 LogMAR (P = 0.049) 5 months later. After 6 months, the mean VA was 0.38 – 0.20 LogMAR, and the difference from baseline was no longer statistically significant (P = 0.262). The data are shown in Fig. 2.
Central macular thickness The mean decrease in CMT was statistically significant after 30 days (460.1 – 211.9 mm; P = 0.016) and peaked at 2 months after treatment (427.6 – 166.6 mm; P = 0.003). Three months after the injections, CMT returned to 541.0 – 201.4 mm (P = 0.036). The decrease reached non-significant values, 628.2 – 150.8 mm (P = 1) and 608.6 – 196.3 mm (P = 1) after 5 and 6 months, respectively. The CMT data are shown in Fig. 2.
Safety analysis The only adverse events that we encountered were anterior chamber cells (n = 5 patients) and mild ocular hypertension. Mean IOP peaked 2 months after treatment, but the change from baseline was never significant throughout the entire duration of the study. Three patients required IOPlowering medication (timolol-dorzolamide fixed combination) due to an IOP increase more than 25 mm Hg. The mean IOP fluctuations are shown in Fig. 3. We did not notice either an increase in the incidence or progression of cataracts in any of the 11 phakic eyes (clinically assessed during slit-lamp examination using the LOCS III grading system21) or retinal neovascularization during the study.
Discussion The results of in vivo scanning laser Doppler flowmetry showed arteriolar eutrophic inward remodeling, secondary to
hypertension, in RVO patients, as compared with an agematched control group of normal eyes in non-hypertensive subjects. A further reduction in internal arteriolar diameter was observed at 3 months after an injection of the DEX implant. Essential and secondary hypertension are known to alter the microcirculation, including low-resistance arteries and arterioles.17 These changes mainly involve arterial wall thickening and reduction of the internal diameter of lowresistance arteries with a consequent increase in media-tolumen ratio without any significant change in the total amount of wall tissue. This process is known as inward eutrophic remodeling.22 This process can also result in vascular wall hypertrophy, which is the growth of smooth muscle cells13,23 with increased arterial rigidity and possible compression of the retinal vein. Ozurdex in an implant made of a solid biodegradable polymer, which enables dual-phase pharmacokinetics, initially releasing a burst of DEX to rapidly achieve a therapeutic concentration followed by sustained release at lower concentrations.24 The concentration of DEX in the vitreous peaks at 213 – 49 ng/mL between days 60 and 90.24 A high sustained concentration of DEX, which is 5 times more potent than triamcinolone acetonide, is likely to contribute to further remodeling of the retinal arterioles, as high steroid levels have been reported to induce small-resistance artery remodeling25 with an associated increase of the WLR.11,12 This process is probably mediated by the stimulation of vascular smooth muscle cell growth.12 It was recently demonstrated that glucocorticoids might activate rapid mineralocorticoid receptor signaling in vascular smooth muscle cells, which involves MAPK/ERK-dependent pathways,26 suggesting that glucocorticoids may contribute to vascular remodeling via mineralocorticoid receptor signaling regardless of circulating aldosterone levels.27 The results from this study show a reduction in arteriolar internal diameter in both BRVO and CRVO patients after
Table 3. Arteriolar Diameters in Normal Eyes and Retinal Vein Occlusion Patients, Before and After Intravitreal Dexamethasone Injection
Normotensive Pretreatment Post-treatment P-value
External diameter (mm)
Internal diameter (mm)
Wall thickness (mm)
WLR
93.6 – 19 98.7 – 19.6 88.5 – 16.4 0.07
74.4 – 15.5 67.9 – 16.9 56.0 – 18.0a 0.02
9.6 – 3.8 15.4 – 5a 16.2 – 3.7a 0.661
0.26 – 0.11 0.47 – 0.19a 0.63 – 0.23a 0.037
Values are expressed as means – SD. P-value documents the difference between pre- and post-treatment groups. a Statistically significant differences compared with normotensive subjects.
EFFECTS OF DEXAMETHASONE INTRAVITREAL IMPLANT ON RETINAL ARTERIOLES
FIG. 2. Mean VA and CMT modifications over time. VA, visual acuity; CMT, central macular thickness. the injection of a DEX implant, although the WLR was significantly increased only in BRVO patients. This difference might be partially due to the small number of patients in the CRVO sample as well as to the greater pre- and posttreatment external diameters found in CRVO patients. Macular edema in RVO patients is caused by increased capillary pressure from venous outflow obstruction and by the blood–retinal barrier loss associated with retinal ischemia due to vascular stasis and defective capillary perfusion.28 The key mechanism of altered blood–retinal barrier function is changed permeability of retinal endothelial cells caused by events such as increased capillary pressure, elevated levels of growth factors, cytokines, inflammation, loss of pericytes, and alteration of Muller cells. As a result of this process, paracellular and transcellular transport across the retinal vascular wall increases because of disruption of endothelial tight junctions and changes in endothelial caveolar transcellular transport, respectively. Corticosteroids such as triamcinolone and DEX administered by an intravitreal injection have been widely used off-label in patients with macular edema secondary to RVO
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and diabetes, and they have a noticeably beneficial anatomical effect on retinal swelling and vascular leakage as detected by OCT and fluorescein angiography.9,10 Results from this study confirm the efficacy of DEX implant, with noteworthy improvements in both VA and CMT in BRVO and CRVO patients peaking at 2 months after the injection, in line with previously published data.8–10 CMT improvements after intravitreal DEX correlated directly with reduced macular leakage, as recently reported by Sadda et al.,29 supporting the idea that macular swelling results from excess fluid leakage from nearby capillaries. As it is in other vascular beds, the rules of Starling determine water homeostasis and edema formation in the retina. Net water transport over the endothelium is determined by the sum of hydrostatic pressure and osmotic pressure of the luminal and extraluminal compartments.28 With regard to hydrostatic pressure, the results from this study show a small but significant reduction in internal arteriolar diameter after treatment with intravitreal DEX. The remodeling induced by Ozurdex may be considered an undesirable effect of steroids. However, this lumen reduction, according to Poiseuille’s relationship, results in an increase in arteriolar flow resistance. We, therefore, speculate that the resultant reduction in capillary hydrostatic pressure could modify Starling’s equilibrium, thereby contributing, at least partially and temporarily, to the reduction in vascular leakage observed in the retina after corticosteroid administration. With regard to the osmotic pressure, steroid effects on the blood–retinal barrier have not been fully elucidated. Despite the well-known direct barrier-enhancing effect of steroids on cell culture models of the blood–brain barrier or blood– retinal barrier,30–33 steroids may also act indirectly by inhibition of inflammatory cells or cytokines,34 via a direct effect on tight junction molecules or other functions of endothelial cells, and by preventing the osmotic swelling of Muller cells caused by an alteration of aquaporin 4.35,36 Ocular steroid-related side-effects most commonly include cataract formation and elevated IOP.37 Our safety findings resemble those reported in the Geneva Study, which found that only 20% (3 out of 15) of patients required IOPlowering therapy during the first 6 months,9,10 whereas no cataract progression was recorded.
Study limitations
FIG. 3. Mean IOP progress over time. IOP, intraocular pressure.
Since our control group consisted of healthy subjects, we could not observe the natural history of WLR in RVO without DEX implantation. Therefore, we cannot assert with certainty that the observed changes in WLR are purely due to the DEX implant. Second, the sample size is quite small. However, a post hoc power analysis calculation showed that the sample size of 15 patients provided a power of *0.98, for demonstrating an effect size of 1 mm between pre- and post-treatment arteriolar diameters, at a significance level of 0.05. Third, this study included patients with BRVO and patients with CRVO. Nevertheless, this study was aimed at evaluating the remodeling of retinal arterioles after the DEX implant, and there is no compelling evidence that remodeling differs in either BRVO or CRVO. Lastly, a longer follow up with repeated assessments at different time points is needed to observe the long-term
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arteriolar modifications after intravitreal DEX implants. The current cohort will continue to be followed up for 1 year to evaluate both the arteriolar remodeling and the safety. In conclusion, we found that the administration of DEX intravitreal implant in RVO patients led to a remodeling in the diameter of the retinal arteriolar lumen. However, this further arteriolar remodeling does not seem to have a detrimental effect on the retinal circulation. It rather contributes to reduced capillary leakage and macular swelling, by modifying Starling’s equilibrium. Further studies are needed to confirm these vascular changes over a longer term in larger samples.
Author Disclosure Statement
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10.
11. 12.
No competing financial interests exist.
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Received: July 4, 2013 Accepted: April 4, 2014 Address correspondence to: Dr. Andrea Russo Eye Clinic Department of Neurological and Vision Sciences University of Brescia Piazzale Spedali Civili, 1 Brescia 25125 Italy E-mail: [email protected]
