Three-dimensional printing of the retina.

PURPOSE OF REVIEW: Biological three-dimensional printing has received a lot of media attention over recent years with advances made in printing cellular structures, including skin and heart tissue for transplantation. Although limitations exist in creating functioning organs with this method, the hope has been raised that creating a functional retina to cure blindness is within reach. The present review provides an update on the advances made toward this goal. RECENT FINDINGS: It has recently been shown that two types of retinal cells, retinal ganglion cells and glial cells, can be successfully printed using a piezoelectric inkjet printer. Importantly, the cells remained viable and did not change certain phenotypic features as a result of the printing process. In addition, recent advances in the creation of complex and viable three-dimensional cellular structures have been made. SUMMARY: Some first promising steps toward the creation of a functional retina have been taken. It now needs to be investigated whether recent findings can be extended to other cells of the retina, including those derived from human tissue, and if a complex and viable retinal structure can be created through three-dimensional printing.

Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome.

The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by ∼2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Treatment of retinal explants with a cocktail of these factors conferred retinal ganglion cell neuroprotection, with factors from the platelet-derived growth factor family being the most potent. Blockade of platelet-derived growth factor signalling with neutralizing antibody or with small molecule inhibitors of platelet-derived growth factor receptor kinase or downstream phosphatidylinositol 3 kinase eliminated retinal ganglion cell neuroprotection conferred by mesenchymal stem cell co-culture. Intravitreal injection of platelet-derived growth factor -AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data demonstrate that mesenchymal stem cells secrete a number of neuroprotective proteins and suggest that platelet-derived growth factor secretion in particular may play an important role in mesenchymal stem cell-mediated retinal ganglion cell neuroprotection. Furthermore, platelet-derived growth factor may represent an independent target for achieving retinal ganglion cell neuroprotection.

Activated retinal glia mediated axon regeneration in experimental glaucoma.

Glaucoma, a leading cause of blindness, is a neurodegenerative disease characterized by progressive loss of retinal ganglion cell axons in the optic nerve and their cell bodies in the retina. Reactive retinal glial changes have been observed in glaucoma but the role of such glial changes in the pathogenesis of the condition remains unclear. In the present study we found that retinal ganglion cells in an experimental animal model of glaucoma have an increased axon regenerative potential. Regeneration of adult rat retinal ganglion cell axons after optic nerve crush was significantly increased in vivo when combined with intraocular pressure-induced experimental glaucoma. This enhanced axon regeneration response was correlated with a significant increase in activation of glial fibrillary acidic protein+retinal glia. Using a dissociated retinal ganglion cell culture model we showed that reducing the number of activated retinal glia with a glial specific toxin, α-Aminoadipic acid, significantly reduced the growth potential of retinal ganglion cells from glaucomatous rat eyes, suggesting that activated retinal glia mediate, at least in part, the growth promoting effect. This was shown to be mediated by both membrane-bound and soluble glial-derived factors. Neurotrophin and ciliary neurotrophic/leukemia inhibitory factor blockers did not affect the regenerative potential, excluding these growth factors as principal mediators of the enhanced growth response occurring in glaucomatous retinal cultures. These observations are the first to reveal that retinal ganglion cells from glaucomatous rat eyes have an enhanced regenerative capacity. Furthermore, our results suggest that activated retinal glia mediate at least part of this response. Further work to understand and enhance the regeneration-promoting effect of activated retinal glia is required to determine if this approach could be useful as part of a therapeutic strategy to encourage optic nerve regeneration in glaucoma.

Retinal ganglion cell survival and axon regeneration in WldS transgenic rats after optic nerve crush and lens injury.

BACKGROUND: We have previously shown that the slow Wallerian degeneration mutation, whilst delaying axonal degeneration after optic nerve crush, does not protect retinal ganglion cell (RGC) bodies in adult rats. To test the effects of a combination approach protecting both axons and cell bodies we performed combined optic nerve crush and lens injury, which results in both enhanced RGC survival as well as axon regeneration past the lesion site in wildtype animals. RESULTS: As previously reported we found that the Wld(S) mutation does not protect RGC bodies after optic nerve crush alone. Surprisingly, we found that Wld(S) transgenic rats did not exhibit the enhanced RGC survival response after combined optic nerve crush and lens injury that was observed in wildtype rats. RGC axon regeneration past the optic nerve lesion site was, however, similar in Wld(S) and wildtypes. Furthermore, activation of retinal glia, previously shown to be associated with enhanced RGC survival and axon regeneration after optic nerve crush and lens injury, was unaffected in Wld(S) transgenic rats. CONCLUSIONS: RGC axon regeneration is similar between Wld(S) transgenic and wildtype rats, but Wld(S) transgenic rats do not exhibit enhanced RGC survival after combined optic nerve crush and lens injury suggesting that the neuroprotective effects of lens injury on RGC survival may be limited by the Wld(S) protein.

Can Liposomes Survive Inkjet Printing? The Effect of Jetting on Key Liposome Attributes for Drug Delivery Applications.

Purpose: Inkjet printing has the potential to enable novel personalized and tailored drug therapies based on liposome and lipid nanoparticles. However, due to the significant shear force exerted on the jetted fluids, its suitability for shear-sensitive materials such as liposomes, has not been verified. We have conducted a proof-of-concept study to examine whether the particle concentration and size distribution of placebo liposomes are affected by common inkjet/dispensing technologies. Methods: We have subjected three types of liposome-containing fluids ("inks") to two different commercial dispensing/jetting technologies, which are relevant to most drug printing approaches. The liposome jetting processes were observed in real-time using strobographic imaging techniques. The phospholipid concentrations and particle size distributions were determined before and after jetting via enzymatic colorimetric and dynamic light scattering methods, respectively. Results: Our results have shown that the jetting dynamics of the liposome inks are well predicted by the established inkjet printing regime map based on their physical properties and the jetting conditions. Importantly, although significant shear forces were confirmed during jetting, the liposome concentrations and particle size distributions in the collected samples remain largely unaffected. Conclusion: These findings, we believe, provide the essential proof-of-concept to encourage further development in this highly topical research area.

The expression of dynein light chain DYNLL1 (LC8-1) is persistently downregulated in glaucomatous rat retinal ganglion cells.

High intraocular pressure induces glaucomatous degeneration of retinal ganglion cells (RGCs). The cellular mechanisms leading to activation of the apoptosis cascade are multidimensional and only partially understood. A small dynein subunit, the light chain DYNLL1 (synonym LC8-1, PIN) has recently been shown to be an important regulator of neuron proteins known to be involved in glaucomatous RGC death including NO synthases, the pro-apoptotic protein Bim and the dynein intermediate chain. Also, DYNLL1 is a regulator of mitochondria anchorage in axons, which is impaired in glaucoma. We investigated expression of DYNLL1 and 2 and its dynein binding partner dynein intermediate chain in a rat model of chronic glaucoma. Laser capture microdissection (LCM) allowed us to collect distinct cell layers and cell bodies from the retina to gain data highly specific for retinal ganglion cells. Glaucoma was induced in 23 rats by laser treatment to the aqueous outflow tract. RNA was extracted from LCM dissected ganglion cell layers (GCL) and 100 pooled RGCs per retina. Expression levels for 1, 2 and 4 week timepoints were analysed by quantitative PCR for DYNLL1 and 2, dynein intermediate chain and GFAP. DYNLL protein abundance in RGCs was quantified in immunostained retina sections. DYNLL gene 1 but not 2 was expressed in RGCs. In the glaucoma model DYNLL1 was strongly and persistently downregulated at all timepoints. DYNLL protein was significantly less abundant at the 4 week timepoint. In contrast, the motorprotein binding partner dynein intermediate chain 1 was more stably expressed. DYNLL2 was upregulated in glia cells at 2 weeks. Expression of DYNLL1, the only form of the dynein light chain expressed in RGCs, is downregulated persistently in glaucoma, while its binding partner dynein IC-1 is unchanged. The specific lack of DYNLL1 could have an impact on the function of their regulatory binding partners and contribute in several ways to neuron dysfunction and apoptosis.

Increased synthesis of spermidine as a result of upregulation of arginase I promotes axonal regeneration in culture and in vivo.

Adult spinal axons do not spontaneously regenerate after injury. However, if the peripheral branch of dorsal root ganglion neurons is lesioned before lesioning the central branch of the same neurons in the dorsal column, these central axons will regenerate and, if cultured, are not inhibited from extending neurites by myelin-associated inhibitors of regeneration such as myelin-associated glycoprotein (MAG). This effect can be mimicked by elevating cAMP and is transcription dependent. The ability of cAMP to overcome inhibition by MAG in culture involves the upregulation of the enzyme arginase I (Arg I) and subsequent increase in synthesis of polyamines such as putrescine. Now we show that a peripheral lesion also induces an increase in Arg I expression and synthesis of polyamines. We also show that the conditioning lesion effect in overcoming inhibition by MAG is initially dependent on ongoing polyamine synthesis but, with time after lesion, becomes independent of ongoing synthesis. However, if synthesis of polyamines is blocked in vivo the early phase of good growth after a conditioning lesion is completely blocked and the later phase of growth, when ongoing polyamine synthesis is not required during culture, is attenuated. We also show that putrescine must be converted to spermidine both in culture and in vivo to overcome inhibition by MAG and that spermidine can promote optic nerve regeneration in vivo. These results suggest that spermidine could be a useful tool in promoting CNS axon regeneration after injury.

Optic nerve and vitreal inflammation are both RGC neuroprotective but only the latter is RGC axogenic.

Intravitreal inflammation, induced by either lens injury, or intravitreal injection of zymosan (IVZ), protects RGC from apoptosis and stimulates axon regeneration after optic nerve transection. Here, we investigate the differential effects of intra-optic nerve zymosan (ONZ) and IVZ injections on RGC neuroprotection and axogenesis. After both IVZ and ONZ injection, zymosan-induced inflammation promoted a similar 4-/5-fold enhancement in RGC survival, compared to optic nerve transected controls, but only IVZ promoted RGC axon regeneration. IVZ was the most effective in activating retinal astrocyte/Müller cells while regulated intramembraneous proteolysis (RIP) of p75(NTR) and inactivation of Rho (key components of the axon growth inhibitory signalling cascade) occurred in both ONZ and IVZ, but only in the latter did RGC axons regenerate. We suggest that neuroprotective factors may be transported to RGC somata by retrograde transport after ONZ and diffuse into the retina after IVZ injection, but an axogenic agent is required to initiate and maintain disinhibited RGC axon regeneration that may be an exclusive property of a Müller cell-derived factor released after IVZ.

Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells.

The optic nerve, like most mature CNS pathways, does not regenerate after injury. Through unknown mechanisms, however, macrophage activation in the eye stimulates retinal ganglion cells (RGCs) to regenerate long axons beyond the site of optic nerve injury. Here we identify the calcium (Ca(2+))-binding protein oncomodulin as a potent macrophage-derived growth factor for RGCs and other neurons. Oncomodulin binds to rat RGCs with high affinity in a cyclic AMP (cAMP)-dependent manner and stimulates more extensive outgrowth than other known trophic agents. Depletion of oncomodulin from macrophage-conditioned media (MCM) eliminates the axon-promoting activity of MCM. The effects of oncomodulin involve downstream signaling via Ca(2+)/calmodulin kinase and gene transcription. In vivo, oncomodulin released from microspheres promotes regeneration in the mature rat optic nerve. Oncomodulin also stimulates outgrowth from peripheral sensory neurons. Thus, oncomodulin is a new growth factor for neurons of the mature central and peripheral nervous systems.

Can 3D printing of oral drugs help fight the current COVID-19 pandemic (and similar crisis in the future)?

The ongoing COVID-19 crisis has highlighted the importance of a robust drug supply chain which can be quickly and flexibly ramped up to produce life-saving drug treatments. 3D printing (3DP) of oral solid dosage forms (OSDF) could be a viable part of the emergency drug production response to support vulnerable patients in rural regions and other isolated locations. In the context of the current pandemic, the suitability of different 3DP technologies will depend on the physicochemical properties, unit dose strength and BCS classification of the repurposed drug compounds currently being trialed for COVID-19. Furthermore, the deployment strategy should focus on simplifying dosage forms and formulations, scaling down the size and complexity of the printing systems and real-time quality assurance via process analytical technologies (PAT).

Risk of needle tract seeding after coaxial ultrasound-guided percutaneous biopsy for primary and metastatic tumors of the liver: report of a single institution.

PURPOSE: The objective of this study was to determine the incidence of needle track seeding after ultrasound-guided percutaneous biopsy of indeterminate liver lesions with a coaxial biopsy system without any other additional intervention or ablation therapy. METHODS: We identified 172 patients in a retrospective cohort study who underwent ultrasound-guided biopsy due to a liver mass in our institution between 2007 and 2016. The same coaxial biopsy system was used in all patients, no consecutive ablation was performed. RESULTS: None of the finally included 131 patients developed neoplastic seeding. There was one major complication (0.76%), the rest of the complications were minor (3.8%) and did not require further intervention. CONCLUSION: Needle track seeding is a rare delayed complication after percutaneous liver biopsy. Coaxial liver biopsy is a safe method to obtain multiple samples with a single punch in patients with primary or metastatic liver lesions.

Activated retinal glia promote neurite outgrowth of retinal ganglion cells via apolipoprotein E.

In the present study, we investigated the role and mechanism through which activated retinal glia stimulate retinal ganglion cell (RGC) neurite outgrowth. We have found that the level of retinal glial activation correlates directly with enhanced RGC neurite outgrowth after a preconditioning intravitreal Zymosan injection. Reduction in the number of activated glia in primary rat retinal cultures led to significantly reduced RGC neurite outgrowth. Glial-related neurite outgrowth appears to be, at least in part, mediated via apolipoprotein E (ApoE), which is expressed by activated retinal astrocytes and Müller glia. ApoE-deficient mice showed significantly reduced RGC neurite outgrowth potential after intravitreal Zymosan injection compared with age-matched wild-type animals. These observations suggest that ApoE, expressed by activated retinal glia, stimulates RGC neurite outgrowth after intravitreal Zymosan injection.

Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting.

We have investigated the differential mediators of the neurotrophic effects of intravitreal peripheral nerve grafting and lens injury on adult rat retinal ganglion cells (RGC). Lens injury and intravitreal peripheral nerve grafting both stimulated RGC neurite growth in vitro and axon regeneration past the optic nerve lesion site in vivo concomitant with activation of retinal glia and invasion of macrophages into the eye. These observations, together with the results of coculture studies using a macrophage-free intact peripheral nerve segment, a macrophage-free intact lens, a macrophage-rich peripheral nerve segment, or a macrophage-rich injured lens in retinal cultures suggest that the stimulation of RGC axon regeneration by lens injury and intravitreal peripheral nerve grafting share a common macrophage-derived component overlain by distinct lens-derived and peripheral nerve-derived neurotrophic factors, respectively. RGC axon regeneration following lens injury and intravitreal peripheral nerve grafting was similar in vivo, correlating with similar retinal glia activation whereas, in vitro, the level of RGC neurite outgrowth was significantly higher following intravitreal peripheral nerve grafting compared with lens injury, concomitant with the presence of increased numbers of activated retinal glia. This suggests that in vivo RGC axon regeneration induced by lens injury and peripheral nerve grafting may be limited, in part, by factors derived from activated retinal glia.

Regeneration of axons in the visual system.

This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Müller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75NTR, and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery.

Retinal glia promote dorsal root ganglion axon regeneration.

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.

Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing.

We have investigated whether inkjet printing technology can be extended to print cells of the adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia, and the effects on survival and growth of these cells in culture, which is an important step in the development of tissue grafts for regenerative medicine, and may aid in the cure of blindness. We observed that RGC and glia can be successfully printed using a piezoelectric printer. Whilst inkjet printing reduced the cell population due to sedimentation within the printing system, imaging of the printhead nozzle, which is the area where the cells experience the greatest shear stress and rate, confirmed that there was no evidence of destruction or even significant distortion of the cells during jet ejection and drop formation. Importantly, the viability of the cells was not affected by the printing process. When we cultured the same number of printed and non-printed RGC/glial cells, there was no significant difference in cell survival and RGC neurite outgrowth. In addition, use of a glial substrate significantly increased RGC neurite outgrowth, and this effect was retained when the cells had been printed. In conclusion, printing of RGC and glia using a piezoelectric printhead does not adversely affect viability and survival/growth of the cells in culture. Importantly, printed glial cells retain their growth-promoting properties when used as a substrate, opening new avenues for printed CNS grafts in regenerative medicine.

Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways.

Mammalian sterile 20-like kinase-3b (Mst3b, encoded by Stk24), regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using short hairpin RNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.

Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.