Joana Galvao

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

Diabetes differentially affects the content of exocytotic proteins in hippocampal and retinal nerve terminals

Diabetes has been associated with cognitive and memory impairments, and with alterations in color and contrast perception, suggesting that hippocampus and retina are particularly affected by this disease. A few studies have shown that diabetes differentially affects neurotransmitter release in different brain regions and in retina, and induces structural and molecular changes in nerve terminals in both hippocampus and retina. We now detailed the impact over time of diabetes (2, 4 and 8 weeks of diabetes) on a large array of exocytotic proteins in hippocampus and retina.The exocytotic proteins density was evaluated by immunoblotting in purified synaptosomes and in total extracts of hippocampus and retina from streptozotocin-induced diabetic and age-matched control animals. Diabetes affected differentially the content of synaptic proteins (VAMP-2, SNAP-25, syntaxin-1, synapsin-1 and synaptophysin) in hippocampal and retinal nerve terminals. Changes were more pronounced and persistent in hippocampal nerve terminals. In general, the alterations in retina occurred earlier, but were transitory, with the exception of synapsin-1, since its content decreased at all time points studied. The content of synaptotagmin-1 and rabphilin 3a in nerve terminals of both tissues was not affected. In total extracts, no changes were detected in the retina, whereas in hippocampus SNAP-25 and syntaxin-1 content was decreased, particularly when more drastic changes were also detected in nerve terminals. These results show that diabetes affects the content of several exocytotic proteins in hippocampus and retina, mainly at the presynaptic level, but hippocampus appears to be more severely affected. These changes might influence neurotransmission in both tissues and may underlie, at least partially, previously detected physiological changes in diabetic humans and animal models. Since diabetes differentially affects exocytotic proteins, according to tissue and insult duration, functional studies will be required to assess the physiological impairment induced by diabetes on the exocytosis in central synapses.

In vivo imaging of retinal ganglion cell apoptosis.

Apoptosis, or programmed cell death, plays a vital role in normal development and ageing. However, dysregulation of this process is responsible for many disease states including; cancer, autoimmune and neurodegeneration. For this reason, in vivo visualisation of apoptosis may prove a useful tool for both laboratory research and clinical diagnostics. Glaucoma comprises a distinctive group of chronic optic neuropathies, characterised by the progressive loss of retinal ganglion cells (RGCs). Early diagnosis of glaucoma remains a clear and unmet need. Recently, there have been significant advances in the detection of apoptosis in vivo using fluorescent probes to visualise single RGCs undergoing apoptosis, specifically DARC (Detection of Apoptotic Retinal Cells) [1] and capQ technology [2(••)].

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.

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.

Effects of 3,4-Methylenedioxymethamphetamine Administration on Retinal Physiology in the Rat

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is known to produce euphoric states, but may also cause adverse consequences in humans, such as hyperthermia and neurocognitive deficits. Although MDMA consumption has been associated with visual problems, the effects of this recreational drug in retinal physiology have not been addressed hitherto. In this work, we evaluated the effect of a single MDMA administration in the rat electroretinogram (ERG). Wistar rats were administered MDMA (15 mg/kg) or saline and ERGs were recorded before (Baseline ERG), and 3 h, 24 h, and 7 days after treatment. A high temperature (HT) saline-treated control group was also included. Overall, significantly augmented and shorter latency ERG responses were found in MDMA and HT groups 3 h after treatment when compared to Baseline. Twenty-four hours after treatment some of the alterations found at 3 h, mainly characterized by shorter latency, tended to return to Baseline values. However, MDMA-treated animals still presented increased scotopic a-wave and b-wave amplitudes compared to Baseline ERGs, which were independent of temperature elevation though the latter might underlie the acute ERG alterations observed 3 h after MDMA administration. Seven days after MDMA administration recovery from these effects had occurred. The effects seem to stem from specific changes observed at the a-wave level, which indicates that MDMA affects subacutely (at 24 h) retinal physiology at the outer retinal (photoreceptor/bipolar) layers. In conclusion, we have found direct evidence that MDMA causes subacute enhancement of the outer retinal responses (most prominent in the a-wave), though ERG alterations resume within one week. These changes in photoreceptor/bipolar cell physiology may have implications for the understanding of the subacute visual manifestations induced by MDMA in humans.