Eduardo Normando

Unexpected low-dose toxicity of the universal solvent DMSO

Dimethyl sulfoxide (DMSO) is an important aprotic solvent that can solubilize a wide variety of otherwise poorly soluble polar and nonpolar molecules. This, coupled with its apparent low toxicity at concentrations <10%, has led to its ubiquitous use and widespread application. Here, we demonstrate that DMSO induces retinal apoptosis in vivo at low concentrations (5 μl intravitreally dosed DMSO in rat from a stock concentration of 1, 2, 4, and 8% v/v). Toxicity was confirmed in vitro in a retinal neuronal cell line, at DMSO concentrations >1% (v/v), using annexin V, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), and AlamarBlue cell viability assays. DMSO concentrations > 10% (v/v) have recently been reported to cause cellular toxicity through plasma membrane pore formation. Here, we show the mechanism by which low concentrations (2–4% DMSO) induce caspase‐3 independent neuronal death that involves apoptosis‐inducing factor (AIF) translocation from mitochondria to the nucleus and poly‐(ADP‐ribose)‐polymerase (PARP) activation. These results highlight safety concerns of using low concentrations of DMSO as a solvent for in vivo administration and in biological assays. We recommend that methods other than DMSO are employed for solubilizing drugs but, where no alternative exists, researchers compute absolute DMSO final concentrations and include an untreated control group in addition to DMSO vehicle control to check for solvent toxicity.—Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. Unexpected low‐dose toxicity of the universal solvent DMSO. FASEB J. 28, 1317–1330 (2014). www.fasebj.org

Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo.

Nerve cell death is the key event in all neurodegenerative disorders, with apoptosis and necrosis being central to both acute and chronic degenerative processes. However, until now, it has not been possible to study these dynamically and in real time. In this study, we use spectrally distinct, well-recognised fluorescent cell death markers to enable the temporal resolution and quantification of the early and late phases of apoptosis and necrosis of single nerve cells in different disease models. The tracking of single-cell death profiles in the same living eye over hours, days, weeks and months is a significant advancement on currently available techniques. We identified a numerical preponderance of late-phase versus early-phase apoptotic cells in chronic models, reinforcing the commonalities between cellular mechanisms in different disease models. We showed that MK801 effectively inhibited both apoptosis and necrosis, but our findings support the use of our technique to investigate more specific anti-apoptotic and anti-necrotic strategies with well-defined targets, with potentially greater clinical application. The optical properties of the eye provide compelling opportunities for the quantitative monitoring of disease mechanisms and dynamics in experimental neurodegeneration. Our findings also help to directly observe retinal nerve cell death in patients as an adjunct to refining diagnosis, tracking disease status and assessing therapeutic intervention.

Tracking Longitudinal Retinal Changes in Experimental Ocular Hypertension Using the cSLO and Spectral Domain-OCT

PURPOSE Involvement of the outer retina is controversial in glaucoma. The aim of this study was to test, first, whether the outer retina is affected in experimental ocular hypertension (OHT) and, second, whether whole retinal thickness can be used as a surrogate marker of glaucomatous change. METHODS OHT was surgically induced in 20 Dark Agouti rats. Animals were imaged using a modified Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany) at baseline and at 3 and 8 weeks after OHT induction. Measurements were recorded for whole and individual retinal layer thickness in four regions-temporal, superior, nasal, and inferior-around the optic nerve head. RESULTS Whole retinal thickness in normal eyes was 172.19 ± 5.17 μm, with no significant regional differences. OHT caused a significant reduction in whole retinal thickness and the outer nuclear layer (ONL) at 3 and 8 weeks (P < 0.05), along with the expected thinning of the retinal nerve fiber layer (RNFL). Whole retinal thickness correlated well with RNFL (P = 0.035) and ONL (P ≤ 0.001) changes. Sensitivity of RNFL and ONL to IOP exposure appeared greater at 3 than at 8 weeks. In addition, regional profiles were significantly altered in the ONL and RNFL after OHT induction. CONCLUSIONS Adaptation of the Spectralis OCT enables tracking of structural damage in experimental rat OHT. Here the authors show evidence of glaucomatous damage in the outer retinal layers of this model with significant regional changes and highlight whole retinal thickness in the rat as a useful surrogate marker of inner and outer retinal changes. The authors believe that the OCT data can provide useful information with regard to clinical management.

Topical Delivery of Avastin to the Posterior Segment of the Eye In Vivo Using Annexin A5-associated Liposomes

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.

Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging

Aim To evaluate near-infrared (NIR) autofluorescence (AF) in patients with geographic atrophy (GA) secondary to age-related macular degeneration and to investigate the origin of the signal by in vivo and histological analysis in rats and in a human donor eye. Methods Confocal scanning laser ophthalmoscopy in vivo imaging, including blue (excitation: 488 nm, emission 500–700 nm) and NIR (excitation: 790 nm, emission >810 nm) AF was performed in 21 eyes of 18 GA patients. Pigmented and albino rats underwent with the same device both in vivo and post-mortem imaging. For the latter, cryostat prepared retinal cross-sections were imaged using an additional customised magnification lens. Finally, cross-sections of a 49-year old human donor eye were recorded. Results Atrophic areas in GA were characterised by low NIR AF intensities. In the junctional zone of atrophy, focal areas of increased intensity were seen which appeared to seldom correlate to blue AF findings. Confocal live scanning in pigmented rats identified the maximum of the NIR AF signal in the outer retina, with histological confirmation of the signal origin localised to the retinal pigment epithelium and sclera in both animals and human donor eye. No NIR AF was found in the retina of young non-pigmented rats. Discussion This study further underscores the assumption that melanin is the main source of NIR AF in the healthy retina. Increased NIR AF intensities in the junctional zone in GA may represent accumulation of melanolipofuscin, which may reflect disease activity and thus may allow for early identification of patients at high-risk of GA enlargement.

Real-time imaging of single neuronal cell apoptosis in patients with glaucoma

See Herms and Schön (doi:10.1093/brain/awx100) for a scientific commentary on this article. Glaucoma is often diagnosed late when vision loss has already occurred. Cordeiro et al. report a new fluorescent marker for retinal imaging that can safely visualise real-time in vivo neuronal apoptosis in patients. Increased labelling is observed in patients with progressive neurodegenerative disease compared to healthy controls.

Advances in retinal ganglion cell imaging

Glaucoma is one of the leading causes of blindness worldwide and will affect 79.6 million people worldwide by 2020. It is caused by the progressive loss of retinal ganglion cells (RGCs), predominantly via apoptosis, within the retinal nerve fibre layer and the corresponding loss of axons of the optic nerve head. One of its most devastating features is its late diagnosis and the resulting irreversible visual loss that is often predictable. Current diagnostic tools require significant RGC or functional visual field loss before the threshold for detection of glaucoma may be reached. To propel the efficacy of therapeutics in glaucoma, an earlier diagnostic tool is required. Recent advances in retinal imaging, including optical coherence tomography, confocal scanning laser ophthalmoscopy, and adaptive optics, have propelled both glaucoma research and clinical diagnostics and therapeutics. However, an ideal imaging technique to diagnose and monitor glaucoma would image RGCs non-invasively with high specificity and sensitivity in vivo. It may confirm the presence of healthy RGCs, such as in transgenic models or retrograde labelling, or detect subtle changes in the number of unhealthy or apoptotic RGCs, such as detection of apoptosing retinal cells (DARC). Although many of these advances have not yet been introduced to the clinical arena, their successes in animal studies are enthralling. This review will illustrate the challenges of imaging RGCs, the main retinal imaging modalities, the in vivo techniques to augment these as specific RGC-imaging tools and their potential for translation to the glaucoma clinic.

Direct optic nerve sheath (DONS) application of Schwann cells prolongs retinal ganglion cell survival in vivo.

Cell-based therapies are increasingly recognized as a potential strategy to treat retinal neurodegenerative disease. Their administration, however, is normally indirect and complex, often with an inability to assess in real time their effects on cell death and their migration/integration into the host retina. In the present study, using a partial optic nerve transection (pONT) rat model, we describe a new method of Schwann cell (SC) delivery (direct application to injured optic nerve sheath, SC/DONS), which was compared with intravitreal SC delivery (SC/IVT). Both SC/DONS and SC/IVT were able to be assessed in vivo using imaging to visualize retinal ganglion cell (RGC) apoptosis and SC retinal integration. RGC death in the pONT model was best fitted to the one-phase exponential decay model. Although both SC/DONS and SC/IVT altered the temporal course of RGC degeneration in pONT, SC/DONS resulted in delayed but long-lasting effects on RGC protection, compared with SC/IVT treatment. In addition, their effects on primary and secondary degeneration, and axonal regeneration, were also investigated, by histology, whole retinal counting, and modelling of RGC loss. SC/DONS was found to significantly reduce RGC apoptosis in vivo and significantly increase RGC survival by targeting secondary rather than primary degeneration. Both SC/DONS and SC/IVT were found to promote RGC axonal regrowth after optic nerve injury, with evidence of GAP-43 expression in RGC somas and axons. SC/DONS may have the potential in the treatment of optic neuropathies, such as glaucoma. We show that SC transplantation can be monitored in real time and that the protective effects of SCs are associated with targeting secondary degeneration, with implications for translating cell-based therapies to the clinic.

Focus on: Amyloid β

Amyloid b (Ab) [P05067 Swiss-Prot], one of the proteins shown to be associated with the onset of Alzheimer’s disease (AD), commonly exits in three forms comprising of either 38, 40 or 42 amino acids (Walsh and Selkoe, 2007). Ab is produced via the amyloidogenic pathway through posttranslational cleavage of the membrane bound Ab precursor protein (APP) by two secretases. b-Secretase cleaves APP producing the soluble sAPPb and the membrane bound C99, which is subsequently cleaved by g-secretase yielding Ab (Fig.1). The sites of g-secretase cleavagewithin C99 determine the species of Ab produced. Whilst all three types of Ab are generated in healthy individuals, increased production of the protein, particularly Ab42 which is more prone to self-aggregation, has been linked to the neuropathology of AD. Unlike the amyloidgenic pathway, the non-amyloidgenic processing of APP involves cleavage within the protein’s Ab domain by a-secretase, therefore preventing the formation of the potentially pathogenic protein. The production of Ab and the formation of amyloid plaques in the brain are influenced by several genetic risk factors.

Non-amyloidogenic effects of α2 adrenergic agonists: implications for brimonidine-mediated neuroprotection.

The amyloid beta (Aβ) pathway is strongly implicated in neurodegenerative conditions such as Alzheimers disease and more recently, glaucoma. Here, we identify the α2 adrenergic receptor agonists (α2ARA) used to lower intraocular pressure can prevent retinal ganglion cell (RGC) death via the non-amyloidogenic Aβ-pathway. Neuroprotective effects were confirmed in vivo and in vitro in different glaucoma-related models using α2ARAs brimonidine (BMD), clonidine (Clo) and dexmedetomidine. α2ARA treatment significantly reduced RGC apoptosis in experimental-glaucoma models by 97.7% and 92.8% (BMD, P<0.01) and 98% and 92.3% (Clo, P<0.01)) at 3 and 8 weeks, respectively. A reduction was seen in an experimental Aβ-induced neurotoxicity model (67% BMD and 88.6% Clo, both P<0.01, respectively), and in vitro, where α2ARAs significantly (P<0.05) prevented cell death, under both hypoxic (CoCl2) and stress (UV) conditions. In experimental-glaucoma, BMD induced ninefold and 25-fold and 36-fold and fourfold reductions in Aβ and amyloid precursor protein (APP) levels at 3 and 8 weeks, respectively, in the RGC layer, with similar results with Clo, and in vitro with all three α2ARAs. BMD significantly increased soluble APPα (sAPPα) levels at 3 and 8 weeks (2.1 and 1.6-fold) in vivo and in vitro with the CoCl2 and UV-light insults. Furthermore, treatment of UV-insulted cells with an sAPPα antibody significantly reduced cell viability compared with BMD-treated control (52%), co-treatment (33%) and untreated control (27%). Finally, we show that α2ARAs modulate levels of laminin and MMP-9 in RGCs, potentially linked to changes in Aβ through APP processing. Together, these results provide new evidence that α2ARAs are neuroprotective through their effects on the Aβ pathway and sAPPα, which to our knowledge, is the first description. Studies have identified the need for α-secretase activators and sAPPα-mimetics in neurodegeneration; α2ARAs, already clinically available, present a promising therapy, with applications not only to reducing RGC death in glaucoma but also other neurodegenerative processes involving Aβ.