Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush.

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-ß1, IL-1ß, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1ß, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-ß1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-ß1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Comparative Analysis of Retinal Organotypic Cultures and In Vivo Axotomized Retinas.

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.

Axonal Injuries Cast Long Shadows: Long Term Glial Activation in Injured and Contralateral Retinas after Unilateral Axotomy.

BACKGROUND: To analyze the course of microglial and macroglial activation in injured and contralateral retinas after unilateral optic nerve crush (ONC). METHODS: The left optic nerve of adult pigmented C57Bl/6 female mice was intraorbitally crushed and injured, and contralateral retinas were analyzed from 1 to 45 days post-lesion (dpl) in cross-sections and flat mounts. As controls, intact retinas were studied. Iba1+ microglial cells (MCs), activated phagocytic CD68+MCs and M2 CD206+MCs were quantified. Macroglial cell changes were analyzed by GFAP and vimentin signal intensity. RESULTS: After ONC, MC density increased significantly from 5 to 21 dpl in the inner layers of injured retinas, remaining within intact values in the contralateral ones. However, in both retinas there was a significant and long-lasting increase of CD68+MCs. Constitutive CD206+MCs were rare and mostly found in the ciliary body and around the optic-nerve head. While in the injured retinas their number increased in the retina and ciliary body, in the contralateral retinas decreased. Astrocytes and Müller cells transiently hypertrophied in the injured retinas and to a lesser extent in the contralateral ones. CONCLUSIONS: Unilateral ONC triggers a bilateral and persistent activation of MCs and an opposed response of M2 MCs between both retinas. Macroglial hypertrophy is transient.

Ly6c as a New Marker of Mouse Blood Vessels: Qualitative and Quantitative Analyses on Intact and Ischemic Retinas.

Ly6c is an antigen commonly used to differentiate between classical and non-classical monocytes/macrophages. Here we show its potential as a marker of the mouse vasculature, particularly of the retinal vascular plexuses. Ly6c was immunodetected in several tissues of C57BL/6 mice using isolectin IB4 as the control of vasculature staining. In the retina, Ly6c expression was analyzed qualitatively and quantitatively in intact, ischemic, and contralateral retinas from 0 to 30 days after the insult. Ly6c expression was observed in all organs and tissues tested, with a brighter signal and more homogeneous staining than the IB4. In the retinas, Ly6c was well expressed, allowing a detailed study of their anatomy. The three retinal plexuses were morphologically different, and from the superficial to the deep one occupied 15 ± 2, 24 ± 7, and 38 ± 1.4 percent of the retinal surface, respectively. In the injured retinas, there was extravasation of the classically activated monocyte/macrophages (Ly6chigh) and the formation of new vessels in the superficial plexus, increasing the area occupied by it to 25 ± 1%. In the contralateral retinas, the superficial plexus area decreased gradually, reaching significance at 30 days, and Ly6c expression progressively disappeared in the intermediate and deep plexuses. Although the role of Ly6c in vascular endothelial cell function is still not completely understood, we demonstrate here that Ly6c can be used as a new specific marker of the mouse vasculature and to assess, qualitatively and quantitatively, vascular changes in health and disease.

Topical bromfenac transiently delays axotomy-induced retinal ganglion cell loss.

Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on the survival of central nervous system neurons, the retinal ganglion cells (RGCs). Here we have analyzed the neuroprotective effect of topical bromfenac treatment, a nonsteroidal anti-inflammatory drug (NSAID) used in clinic to ameliorate post-operative inflammation, on axotomized rat RGCs. The left optic nerve of adult rats was subjected to optic nerve crush (ONC). Half of the rats were treated with a topical instillation of saline. On the other half, immediately after the surgery, 2 drops of bromfenac (0.09% Yellox; Bausch & Lomb) were instilled, and then every 12 h until analysis. Retinas in both groups were dissected 3, 5, 7, 9 and 14 days after ONC (n = 4-8/time point/group). Toxicity of bromfenac was assessed in intact retinas treated during 14 days (n = 6). Intact untreated retinas were used as control of the RGC population. RGCs were identified by Brn3a immunodetection and automatically quantified. Our results show that bromfenac does not cause RGC loss in intact retinas. In the injured groups, the number of RGCs at 7, 9 and 14 days after the lesion was significantly higher in treated vs. untreated retinas. To our knowledge this is the first report showing that a topical treatment with a NSAIDs delays axotomy-induced RGC loss and indicates that treatment with NSAIDs could be used as conjunctive therapy in diseases that proceed with optic nerve damage.

ß-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells.

To study the effect of taurine depletion induced by ß-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received ß-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. ß-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting ß-alanine supplements due to the limited understanding of their potential adverse effects.

Neuronal Death in the Contralateral Un-Injured Retina after Unilateral Axotomy: Role of Microglial Cells.

For years it has been known that unilateral optic nerve lesions induce a bilateral response that causes an inflammatory and microglial response in the contralateral un-injured retinas. Whether this contralateral response involves retinal ganglion cell (RGC) loss is still unknown. We have analyzed the population of RGCs and the expression of several genes in both retinas of pigmented mice after a unilateral axotomy performed close to the optic nerve head (0.5 mm), or the furthest away that the optic nerve can be accessed intraorbitally in mice (2 mm). In both retinas, RGC-specific genes were down-regulated, whereas caspase 3 was up-regulated. In the contralateral retinas, there was a significant loss of 15% of RGCs that did not progress further and that occurred earlier when the axotomy was performed at 2 mm, that is, closer to the contralateral retina. Finally, the systemic treatment with minocycline, a tetracycline antibiotic that selectively inhibits microglial cells, or with meloxicam, a non-steroidal anti-inflammatory drug, rescued RGCs in the contralateral but not in the injured retina. In conclusion, a unilateral optic nerve axotomy triggers a bilateral response that kills RGCs in the un-injured retina, a death that is controlled by anti-inflammatory and anti-microglial treatments. Thus, contralateral retinas should not be used as controls.

Microglial dynamics after axotomy-induced retinal ganglion cell death.

BACKGROUND: Microglial cells (MCs) are the sentries of the central nervous system. In health, they are known as surveying MCs because they examine the tissue to maintain the homeostasis. In disease, they activate and, among other functions, become phagocytic to clean the cellular debris. In this work, we have studied the behavior of rat retinal MCs in two models of unilateral complete intraorbital optic nerve axotomy which elicit a different time course of retinal ganglion cell (RGC) loss. METHODS: Albino Sprague-Dawley rats were divided into these groups: (a) intact (no surgery), (b) fluorogold (FG) tracing from the superior colliculi, and (c) FG tracing + crush or transection of the left optic nerve. The retinas were dissected from 2 days to 2 months after the lesions (n = 4-12 group/lesion and time point) and then were subjected to Brn3a and Iba1 double immunodetection. In each intact retina, the total number of Brn3a+RGCs and Iba+MCs was quantified. In each traced retina (b and c groups), FG-traced RGCs and phagocytic microglial cells (PMCs, FG+Iba+) were also quantified. Topographical distribution was assessed by neighbor maps. RESULTS: In intact retinas, surveying MCs are homogenously distributed in the ganglion cell layer and the inner plexiform layer. Independently of the axotomy model, RGC death occurs in two phases, one quick and one protracted, and there is a lineal and topographical correlation between the appearance of PMCs and the loss of traced RGCs. Furthermore, the clearance of FG+RGCs by PMCs occurs 3 days after the actual loss of Brn3a expression that marks RGC death. In addition, almost 50% of MCs from the inner plexiform layer migrate to the ganglion cell layer during the quick phase of RGC loss, returning to the inner plexiform layer during the slow degeneration phase. Finally, in contrast to what happens in mice, in rats, there is no microglial phagocytosis in the contralateral uninjured retina. CONCLUSIONS: Axotomy-induced RGC death occurs earlier than RGC clearance and there is an inverse correlation between RGC loss and PMC appearance, both numerically and topographically, suggesting that phagocytosis occurs as a direct response to RGC death rather than to axonal damage.

Light-induced retinal degeneration causes a transient downregulation of melanopsin in the rat retina.

In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m+RGCs) and the expression of the melanopsin protein in the retina. The m+RGCs may be more resistant than other RGCs to lesion, but the effects of an acute light exposure in this population are unknown. Albino rats were exposed to white light (3000 lux) continuously for 48 h and processed 0, 3, 7 or 30 days after light exposure (ALE). Whole-mounted retinas were immunodetected with antibodies against melanopsin, Brn3a, and rhodopsin to study the populations of m+RGC, Brn3a+RGC and rods (which are the most abundant photoreceptors in the rat retina). Three days ALE there was substantial rod loss in an arciform area of the superior retina and with time this loss expanded in the form of rings all throughout the retina. Light exposure did not affect the number of Brn3a+RGCs but diminished the numbers of m+RGCs. Immediately ALE there was a significant decrease in the mean number of immunodetected m+RGCs that was more marked in the superior retina. Later, the number of m+RGCs increased progressively and reached normal values one month ALE. Western blot analysis showed that melanopsin expression down-regulates shortly ALE and recovers thereafter, in accordance with the anatomical data. This study demonstrates that there is a transient downregulation of melanopsin expression in the RGCs during the first month ALE. Further studies would be needed to clarify the long-term effect of light exposure on the m+RGC population.

Laser-induced ocular hypertension in adult rats does not affect non-RGC neurons in the ganglion cell layer but results in protracted severe loss of cone-photoreceptors.

To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.

Minocycline Administration Does Not Have an Effect on Retinal Ganglion Cell Survival in a Murine Model of Ocular Hypertension.

This study aims to investigate two key aspects in a mouse model of ocular hypertension (OHT): first, the time course of retinal ganglion cell (RGC) death and the parallel activation of caspase-3 (a-Casp3+ cells) to narrow the therapeutic window; and second, the effect of caspase-3 and microglia inhibition by minocycline on RGC rescue in this model. RGC loss after OHT induction was significant at day 7 and progressed to 30 days. However, anatomical RGC death was preceded by significant Casp3 activation on day 3. Microglial inhibition by minocycline did not alter the course of OHT or rescue RGCs but resulted in a decrease in a-Casp3+ cells and phagocytic and total microglia. Therefore, RGC death commitment occurs earlier than their loss of Brn3a expression, microglial cells do not exacerbate RGC loss, and while this death is primarily apoptotic, apoptosis inhibition does not rescue RGCs, suggesting that alternative death pathways play a role in glaucomatous injury.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Retinal response to systemic inflammation differs between sexes and neurons.

Background: Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. Methods: A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. Results: In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1ß. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Conclusions: Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

Signalling pathways and cell death mechanisms in glaucoma: Insights into the molecular pathophysiology.

Glaucoma is a complex multifactorial eye disease manifesting in retinal ganglion cell (RGC) death and optic nerve degeneration, ultimately causing irreversible vision loss. Research in recent years has significantly enhanced our understanding of RGC degenerative mechanisms in glaucoma. It is evident that high intraocular pressure (IOP) is not the only contributing factor to glaucoma pathogenesis. The equilibrium of pro-survival and pro-death signalling pathways in the retina strongly influences the function and survival of RGCs and optic nerve axons in glaucoma. Molecular evidence from human retinal tissue analysis and a range of experimental models of glaucoma have significantly contributed to unravelling these mechanisms. Accumulating evidence reveals a wide range of molecular signalling pathways that can operate -either alone or via intricate networks - to induce neurodegeneration. The roles of several molecules, including neurotrophins, interplay of intracellular kinases and phosphates, caveolae and adapter proteins, serine proteases and their inhibitors, nuclear receptors, amyloid beta and tau, and how their dysfunction affects retinal neurons are discussed in this review. We further underscore how anatomical alterations in various animal models exhibiting RGC degeneration and susceptibility to glaucoma-related neuronal damage have helped to characterise molecular mechanisms in glaucoma. In addition, we also present different regulated cell death pathways that play a critical role in RGC degeneration in glaucoma.

Pan-retinal ganglion cell markers in mice, rats, and rhesus macaques.

Univocal identification of retinal ganglion cells (RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using retrograde tracing of retinorecipient areas. This is an invasive technique, and its use is precluded in higher mammals such as monkeys. In the past decade, several RGC markers have been described. Here, we reviewed and analyzed the specificity of nine markers used to identify all or most RGCs, i.e., pan-RGC markers, in rats, mice, and macaques. The best markers in the three species in terms of specificity, proportion of RGCs labeled, and indicators of viability were BRN3A, expressed by vision-forming RGCs, and RBPMS, expressed by vision- and non-vision-forming RGCs. NEUN, often used to identify RGCs, was expressed by non-RGCs in the ganglion cell layer, and therefore was not RGC-specific. γ-SYN, TUJ1, and NF-L labeled the RGC axons, which impaired the detection of their somas in the central retina but would be good for studying RGC morphology. In rats, TUJ1 and NF-L were also expressed by non-RGCs. BM88, ERRß, and PGP9.5 are rarely used as markers, but they identified most RGCs in the rats and macaques and ERRß in mice. However, PGP9.5 was also expressed by non-RGCs in rats and macaques and BM88 and ERRß were not suitable markers of viability.

Methods to Identify Rat and Mouse Retinal Ganglion Cells in Retinal Flat-Mounts.

The identification of distinct retinal ganglion cell (RGC) populations in flat-mounted retinas is key to investigating pathological or pharmacological effects in these cells. In this chapter, we review the main techniques for detecting the total population of RGCs and various of their subtypes in whole-mounted retinas of pigmented and albino rats and mice, four of the animal strains most studied by the scientific community in the retina field. These methods are based on the studies published by the Vidal-Sanz's laboratory.

Retinal compensatory changes after light damage in albino mice.

PURPOSE: To investigate the anatomic and functional changes triggered by light exposure in the albino mouse retina and compare them with those observed in the albino rat. METHODS: BALB/c albino mice were exposed to 3,000 lx of white light during 24 h and their retinas analyzed from 1 to 180 days after light exposure (ALE). Left pupil mydriasis was induced with topical atropine. Retinal function was analyzed by electroretinographic (ERG) recording. To assess retinal degeneration, hematoxylin and eosin staining, the TdT-mediated dUTP nick-end labeling (TUNEL) technique, and quantitative immunohistofluorescence for synaptophysin and protein kinase Cα (PKCα) were used in cross sections. Intravenous injection of horseradish peroxidase and Fluoro-Gold™ tracing were used in whole-mounted retinas to study the retinal vasculature and the retinal ganglion cell (RGC) population, respectively. RESULTS: Light exposure caused apoptotic photoreceptor death in the central retina. This death was more severe in the dorsal than in the ventral retina, sparing the periphery. Neither retinal vascular leakage nor retinal ganglion cell death was observed ALE. The electroretinographic a-wave was permanently impaired, while the b-wave decreased but recovered gradually by 180 days ALE. The scotopic threshold responses, associated with the inner retinal function, diminished at first but recovered completely by 14 days ALE. This functional recovery was concomitant with the upregulation of protein kinase Cα and synaptophysin. Similar results were obtained in both eyes, irrespective of mydriasis. CONCLUSIONS: In albino mice, light exposure induces substantial retinal damage, but the surviving photoreceptors, together with compensatory morphological/molecular changes, allow an important restoration of the retinal function.

Insights and future directions for the application of perinatal derivatives in eye diseases: A critical review of preclinical and clinical studies.

Perinatal derivatives (PnD) are gaining interest as a source for cell-based therapies. Since the eye is easily accessible to local administration, eye diseases may be excellent candidates to evaluate novel therapeutic approaches. With this work, we performed a systematic review of published preclinical and clinical studies addressing PnD in the treatment of ocular diseases. We have set two specific objectives: (i) to investigate the current level of standardization in applied technical procedures in preclinical studies and (ii) to assess clinical efficacy in clinical trials. Hereto, we selected studies that applied amniotic membrane (hAM) and mesenchymal stromal cells derived from amniotic membrane (hAMSC), placenta (hPMSC), umbilical cord (hUC-MSC) and Wharton's Jelly (hUC-WJ-MSC), excluding those where cells were not transplanted individually, following a systematic PubMed search for preclinical studies and consultation of clinical studies on https://clinicaltrials.gov and https://www.clinicaltrialsregister.eu/. Our bibliographic search retrieved 26 pre-clinical studies and 27 clinical trials. There was a considerable overlap regarding targeted ocular structures. Another common feature is the marked tendency towards (i) locally administered treatments and (ii) the PnD type. In the cornea/ocular surface, hAM was preferred and usually applied directly covering the ocular surface. For neuroretinal disorders, intra-ocular injection of umbilical or placental-derived cells was preferred. In general, basic research reported favourable outcomes. However, due to lack of standardization between different studies, until now there is no clear consensus regarding the fate of administered PnD or their mode of action. This might be accountable for the low index of clinical translation. Regarding clinical trials, only a minority provided results and a considerable proportion is in "unknown status". Nevertheless, from the limited clinical evidence available, hAM proved beneficial in the symptomatic relief of bullous keratopathy, treating dry eye disease and preventing glaucoma drainage device tube exposure. Regarding neuroretinal diseases, application of Wharton's Jelly MSC seems to become a promising future approach. In conclusion, PnD-based therapies seem to be beneficial in the treatment of several ocular diseases. However, much is yet to be done both in the pre-clinical and in the clinical setting before they can be included in the daily ophthalmic practice.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.