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Journal of Neuroscience Methods Aug 2024Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is...
BACKGROUND
Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met.
NEW METHOD
This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo.
RESULTS
We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture.
COMPARISON WITH EXISTING METHODS
Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation.
CONCLUSIONS
This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.
Topics: Animals; Organoids; Retina; Exosomes; Perfusion; Mice; Mice, Inbred C57BL; Tissue Culture Techniques; Cell Survival
PubMed: 38823594
DOI: 10.1016/j.jneumeth.2024.110181 -
Acta Neuropathologica Communications May 2024Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also...
Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.
Topics: Animals; Mice, Inbred C57BL; Mice; Dimethyl Sulfoxide; Biomarkers; Mice, Transgenic; Tomography, Optical Coherence; Retinal Rod Photoreceptor Cells; Contrast Sensitivity; Disease Models, Animal; Retinal Pigment Epithelium; Vision, Ocular
PubMed: 38822433
DOI: 10.1186/s40478-024-01799-8 -
Scientific Reports May 2024In the animal kingdom, threat information is perceived mainly through vision. The subcortical visual pathway plays a critical role in the rapid processing of visual...
In the animal kingdom, threat information is perceived mainly through vision. The subcortical visual pathway plays a critical role in the rapid processing of visual information-induced fear, and triggers a response. Looming-evoked behavior in rodents, mimicking response to aerial predators, allowed identify the neural circuitry underlying instinctive defensive behaviors; however, the influence of disk/background contrast on the looming-induced behavioral response has not been examined, either in rats or mice. We studied the influence of the dark disk/gray background contrast in the type of rat and mouse defensive behavior in the looming arena, and we showed that rat and mouse response as a function of disk/background contrast adjusted to a sigmoid-like relationship. Both sex and age biased the contrast-dependent response, which was dampened in rats submitted to retinal unilateral or bilateral ischemia. Moreover, using genetically manipulated mice, we showed that the three type of photoresponsive retinal cells (i.e., cones, rods, and intrinsically photoresponsive retinal ganglion cells (ipRGCs)), participate in the contrast-dependent response, following this hierarchy: cones > > rods > > > ipRGCs. The cone and rod involvement was confirmed using a mouse model of unilateral non-exudative age-related macular degeneration, which only damages canonical photoreceptors and significantly decreased the contrast sensitivity in the looming arena.
Topics: Animals; Rats; Mice; Male; Retinal Ganglion Cells; Photic Stimulation; Female; Contrast Sensitivity; Behavior, Animal; Retinal Cone Photoreceptor Cells; Mice, Inbred C57BL; Visual Perception; Fear; Retina; Visual Pathways
PubMed: 38822033
DOI: 10.1038/s41598-024-63458-1 -
Tissue & Cell Jun 2024In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals...
In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis.
Topics: Animals; Retina; Microscopy, Electron, Scanning; Sepia; Embryo, Nonmammalian; Neurogenesis; Photoreceptor Cells
PubMed: 38820948
DOI: 10.1016/j.tice.2024.102417 -
Neural Regeneration Research Feb 2025Retinal aging has been recognized as a significant risk factor for various retinal disorders, including diabetic retinopathy, age-related macular degeneration, and...
Retinal aging has been recognized as a significant risk factor for various retinal disorders, including diabetic retinopathy, age-related macular degeneration, and glaucoma, following a growing understanding of the molecular underpinnings of their development. This comprehensive review explores the mechanisms of retinal aging and investigates potential neuroprotective approaches, focusing on the activation of transcription factor EB. Recent meta-analyses have demonstrated promising outcomes of transcription factor EB-targeted strategies, such as exercise, calorie restriction, rapamycin, and metformin, in patients and animal models of these common retinal diseases. The review critically assesses the role of transcription factor EB in retinal biology during aging, its neuroprotective effects, and its therapeutic potential for retinal disorders. The impact of transcription factor EB on retinal aging is cell-specific, influencing metabolic reprogramming and energy homeostasis in retinal neurons through the regulation of mitochondrial quality control and nutrient-sensing pathways. In vascular endothelial cells, transcription factor EB controls important processes, including endothelial cell proliferation, endothelial tube formation, and nitric oxide levels, thereby influencing the inner blood-retinal barrier, angiogenesis, and retinal microvasculature. Additionally, transcription factor EB affects vascular smooth muscle cells, inhibiting vascular calcification and atherogenesis. In retinal pigment epithelial cells, transcription factor EB modulates functions such as autophagy, lysosomal dynamics, and clearance of the aging pigment lipofuscin, thereby promoting photoreceptor survival and regulating vascular endothelial growth factor A expression involved in neovascularization. These cell-specific functions of transcription factor EB significantly impact retinal aging mechanisms encompassing proteostasis, neuronal synapse plasticity, energy metabolism, microvasculature, and inflammation, ultimately offering protection against retinal aging and diseases. The review emphasizes transcription factor EB as a potential therapeutic target for retinal diseases. Therefore, it is imperative to obtain well-controlled direct experimental evidence to confirm the efficacy of transcription factor EB modulation in retinal diseases while minimizing its risk of adverse effects.
PubMed: 38819040
DOI: 10.4103/NRR.NRR-D-23-02033 -
Stem Cell Research & Therapy May 2024X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of...
BACKGROUND
X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS.
METHODS
hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy.
RESULTS
ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.
CONCLUSIONS
The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.
Topics: Retinoschisis; Humans; Induced Pluripotent Stem Cells; Eye Proteins; Genetic Therapy; Organoids; Mutation; Retina; Male; Cell Differentiation
PubMed: 38816767
DOI: 10.1186/s13287-024-03767-4 -
International Journal of Biological... Jun 2024We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout...
We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.
Topics: Animals; Apoptosis; Diabetes Mellitus, Experimental; Ependymoglial Cells; Carrier Proteins; Mice; Signal Transduction; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Mice, Knockout; Photoreceptor Cells; Gene Knockdown Techniques; Brain-Derived Neurotrophic Factor; Thioredoxins; Male; Diabetic Retinopathy; Photoreceptor Cells, Vertebrate; Disease Models, Animal
PubMed: 38815945
DOI: 10.1016/j.ijbiomac.2024.132731 -
Experimental Eye Research Jul 2024Photobiomodulation (PBM) therapy uses light of different wavelengths to treat various retinal degeneration diseases, but the potential damage to the retina caused by...
Photobiomodulation (PBM) therapy uses light of different wavelengths to treat various retinal degeneration diseases, but the potential damage to the retina caused by long-term light irradiation is still unclear. This study were designed to detect the difference between long- and short-wavelength light (650-nm red light and 450-nm blue light, 2.55 mW/cm, reference intensity in PBM)-induced injury. In addition, a comparative study was conducted to investigate the differences in retinal light damage induced by different irradiation protocols (short periods of repeated irradiation and a long period of constant irradiation). Furthermore, the protective role of PARP-1 inhibition on the molecular mechanism of blue light-induced injury was confirmed by a gene knockdown technique or a specific inhibitor through in vitro and in vivo experiments. The results showed that the susceptibility to retinal damage caused by irradiation with long- and short-wavelength light is different. Shorter wavelength lights, such as blue light, induce more severe retinal damage, while the retina exhibits better resistance to longer wavelength lights, such as red light. In addition, repeated irradiation for short periods induces less retinal damage than constant exposure over a long period. PARP-1 plays a critical role in the molecular mechanism of blue light-induced damage in photoreceptors and retina, and inhibiting PARP-1 can significantly protect the retina against blue light damage. This study lays an experimental foundation for assessing the safety of phototherapy products and for developing target drugs to protect the retina from light damage.
Topics: Animals; Poly (ADP-Ribose) Polymerase-1; Mice; Light; Retina; Retinal Degeneration; Mice, Inbred C57BL; Radiation Injuries, Experimental; Disease Models, Animal; Blotting, Western; Male; Low-Level Light Therapy; Blue Light
PubMed: 38815794
DOI: 10.1016/j.exer.2024.109946 -
Frontiers in Molecular Neuroscience 2024The complex nature of the retina demands well-organized signaling to uphold signal accuracy and avoid interference, a critical aspect in handling a variety of visual... (Review)
Review
The complex nature of the retina demands well-organized signaling to uphold signal accuracy and avoid interference, a critical aspect in handling a variety of visual stimuli. A-kinase anchoring proteins (AKAPs), known for binding protein kinase A (PKA), contribute to the specificity and efficiency of retinal signaling. They play multifaceted roles in various retinal cell types, influencing photoreceptor sensitivity, neurotransmitter release in bipolar cells, and the integration of visual information in ganglion cells. AKAPs like AKAP79/150 and AKAP95 exhibit distinct subcellular localizations, impacting synaptic transmission and receptor sensitivity in photoreceptors and bipolar cells. Furthermore, AKAPs are involved in neuroprotective mechanisms and axonal degeneration, particularly in retinal ganglion cells. In particular, AKAP6 coordinates stress-specific signaling and promotes neuroprotection following optic nerve injury. As our review underscores the therapeutic potential of targeting AKAP signaling complexes for retinal neuroprotection and enhancement, it acknowledges challenges in developing selective drugs that target complex protein-protein interactions. Overall, this exploration of AKAPs provides valuable insights into the intricacies of retinal signaling, offering a foundation for understanding and potentially addressing retinal disorders.
PubMed: 38813437
DOI: 10.3389/fnmol.2024.1412407 -
Frontiers in Molecular Neuroscience 2024Dm9 neurons in have been proposed as functional homologs of horizontal cells in the outer retina of vertebrates. Here we combine genetic dissection of neuronal circuit...
Dm9 neurons in have been proposed as functional homologs of horizontal cells in the outer retina of vertebrates. Here we combine genetic dissection of neuronal circuit function, two-photon calcium imaging in Dm9 and inner photoreceptors, and immunohistochemical analysis to reveal novel insights into the functional role of Dm9 in early visual processing. Our experiments show that Dm9 receive input from all four types of inner photoreceptor R7p, R7y, R8p, and R8y. Histamine released from all types R7/R8 directly inhibits Dm9 via the histamine receptor Ort, and outweighs simultaneous histamine-independent excitation of Dm9 by UV-sensitive R7. Dm9 in turn provides inhibitory feedback to all R7/R8, which is sufficient for color-opponent processing in R7 but not R8. Color opponent processing in R8 requires additional synaptic inhibition by R7 of the same ommatidium via axo-axonal synapses and the second histamine receptor HisCl1. Notably, optogenetic inhibition of Dm9 prohibits color opponent processing in all types of R7/R8 and decreases intracellular calcium in photoreceptor terminals. The latter likely results from reduced release of excitatory glutamate from Dm9 and shifts overall photoreceptor sensitivity toward higher light intensities. In summary, our results underscore a key role of Dm9 in color opponent processing in and suggest a second role of Dm9 in regulating light adaptation in inner photoreceptors. These novel findings on Dm9 are indeed reminiscent of the versatile functions of horizontal cells in the vertebrate retina.
PubMed: 38813436
DOI: 10.3389/fnmol.2024.1347540