Updating International Society for Biological and Environmental Repositories Best Practices, Fifth Edition: A New Process for Relevance in an Evolving Landscape.

The recently published fifth edition of the International Society for Biological and Environmental Repositories (ISBER) Best Practices signifies a pivotal milestone in navigating the complexities of repository management. Repositories operate within a constantly evolving landscape influenced by the changing fields of biospecimen science, technology, legal requirements, and ethical considerations. This dynamic is further amplified by unprecedented local and global challenges, such as pandemics, conflicts, and supply chain disruptions. Creating this new edition required a comprehensive approach capable of delivering a focused and coherent resource reflecting the broad horizon of its diverse users. The innovative approach used the existing phased development process and integrated the canvassing of opinions, formal evaluation, and real-time collaboration platforms. Merging these techniques enabled efficient collection and effective distillation of the latest in biobanking practices, enhancing the value of the fifth edition for repositories of specimens and associated data. The expanded document is a testament to the collective efforts of many dedicated individuals who have built upon the foundations of prior editions.

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged ≥80 years old, with a death-to-preservation time (DtoP) of ≤6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30° macula cube, 30 µm spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 ± 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Cost-Benefit and Cost-Utility Analysis of Amphotericin B Supplementation of Corneal Storage Media With Endothelial Keratoplasty-Prepared Tissue.

PURPOSE: To determine the cost-effectiveness of amphotericin B supplementation, we analyzed both current costs to treat postendothelial keratoplasty (EK) fungal infections and potential costs associated with amphotericin B supplementation. METHODS: We collected 19 US cases of post-EK fungal eye infections from the published literature and assessed the associated costs from the literature. A survey of surgeons was also conducted with questions regarding their experiences in managing these infections. RESULTS: We estimated that the costs to diagnose, manage, and treat post-EK fungal keratitis and post-EK fungal endophthalmitis are USD $21,113 and $34,850, respectively. The largest portion of the costs can be attributed to the need for additional surgical management, which is required in 79% of the cases. We estimated the total cost of amphotericin B supplementation to be $44.39 per graft with use of conventional amphotericin B and conservative assumptions regarding supplementation processes. Cost-effectiveness analysis demonstrated that amphotericin B supplementation is cost-effective at $100,000 per quality-adjusted life-year level only if amphotericin B supplementation can prevent more than 69.62% of post-EK fungal infections, assuming the incidence of post-EK fungal infection remains at the level it was between 2012 and 2017. CONCLUSIONS: We found that amphotericin B supplementation can be cost-effective under conservative assumptions if it is moderately effective in preventing post-EK fungal infections.

Donor cornea tissue in cases of drowning or water submersion: eye banks practice patterns and tissue outcomes.

Surgical use of donor corneal tissue from victims of water submersion (drowning or submersion secondary to death) remains controversial due to limited evidence about the quality of these tissues. To assess the safety of donor corneal tissue from victims of water submersion, an investigation of eye banks' practice patterns and tissue outcomes was conducted. All 79 Eye Bank Association of America accredited eye banks were contacted for a phone interview of practices regarding tissue from victims of water submersion. A retrospective review of corneal tissues from 2014 to 2016 from a large eye bank network was performed to identify all donors submerged in water. Corneal epithelial integrity, endothelial cell density (ECD), rim cultures, and adverse events were analyzed for associations with water submersion characteristics. 49 eye banks (62% response) participated in the survey. 55% of these eye banks had specific, written protocol for tissue eligibility from donors submerged in water. With or without specific protocol, eye banks reported considering water type (84%) and length of time submerged (92%) to determine eligibility. 22% of eye banks reported medical director involvement when eligibility determination was unclear. 79 tissues from 40 donors who were submerged were identified in 2014-2016 eye bank data. No donor tissues had pre-processing corneal infiltrates, positive rim cultures, or adverse events post-keratoplasty. Corneal epithelial integrity and ECD were not associated with water type or length of time submerged. In conclusion, data from a large eye bank network showed no adverse events or outcomes, indicating these tissues may be safe.

Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice.

DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis.

Interaction of tubby-like protein-1 (Tulp1) and microtubule-associated protein (MAP) 1A and MAP1B in the mouse retina.

Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.

Protein partners of dynamin-1 in the retina.

Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1−/− mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.

Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits.

PURPOSE: To compare the biological effects of riboflavin-ultraviolet A (UVA) corneal cross-linking (CXL) performed with a traditional epithelium-off method to several transepithelial methods in a rabbit model. Preliminary experiments on biomechanical rigidity were also performed. METHODS: Four treatment groups were included: (1) standard epithelium-off, (2) tetracaine transepithelial, (3) benzal-konium chloride-ethylenediaminetetraacetic acid (BKC-EDTA) transepithelial, and (4) femtosecond laser-assisted transepithelial riboflavin-UVA CXL. Six eyes from each treatment group and the untreated control group were analyzed at 24 hours and 2 months after treatment in wound healing studies. The TUNEL assay was performed to detect the extent of stromal cell death. Optical density was measured with a Scheimpflug analyzer. The corneal stiffening effect was quantitated in three eyes from each group using optical coherence elastography performed 2 months after treatments. RESULTS: Twenty-four hours after CXL, stromal cell death extended full corneal thickness with both standard epithelium-off CXL and femtosecond laser-assisted CXL, but only approximately one-third stromal depth after BKC-EDTA transepithelial CXL. Negligible stromal cell death was detected with tetracaine transepithelial CXL. Cell death results were statistically different between the BKC-EDTA transepithelial CXL and standard epithelium-off CXL groups (P < .0001). Significant corneal opacity differences were noted. Standard epithelium-off CXL had the greatest density and tetracaine transepithelial CXL had the least density compared to the control group after treatment. As measured with optical coherence elastography, a trend toward greater mean stiffening was observed with BKC-EDTA transepithelial CXL than with epithelium-off CXL, femtosecond laser-assisted CXL, or tetracaine transepithelial CXL, but the result did not reach statistical significance. All of the CXL treatment groups exhibited significantly smaller variance of stiffness compared to the control group. CONCLUSION: In the rabbit model, BKC-EDTA transepithelial CXL produced less stromal cell death and less risk of endothelial cell damage than standard epithelium-off CXL or femtosecond laser-assisted CXL. Additional study is needed to determine whether biomechanical stiffness is significantly different between the epithelium-off CXL and transepithelial CXL groups.

Histopathology and functional correlations in a patient with a mutation in RPE65, the gene for retinol isomerase.

PURPOSE: Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. METHODS: Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. RESULTS: Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. CONCLUSIONS: The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.

Immunocytochemical evidence of Tulp1-dependent outer segment protein transport pathways in photoreceptor cells.

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1-/- mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1-/- photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1-/- retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

Light-evoked responses of the retinal pigment epithelium: changes accompanying photoreceptor loss in the mouse.

Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration (Prph(Rd2/+)), which was crossed onto a Nyx(nob) background to eliminate the b-wave and most other postreceptoral ERG components. On this background, Prph(Rd2/+) mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in Prph(Rd2/+) mice than predicted by rod photoreceptor activity (Rm(P3)) or anatomical analysis. At 4 mo of age, the RPE in Prph(Rd2/+) mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease.

Tubby-like protein 1 (Tulp1) is required for normal photoreceptor synaptic development.

Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In wild type (wt) mice, Tulp1 is localized to the photoreceptor inner segment, connecting cilium and synapse. To investigate the role of Tulp1 in the synapse, we examined the pre- and postsynaptic architecture in tulp1-/- mice. We used immunohistochemistry to examine tulp1-/- mice prior to retinal degeneration and made comparisons to wt littermates and rd10 mice. In the tulp1-/- synapse, the spatial relationship between the ribbon-associated proteins, Bassoon and Piccolo, are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted, most likely a direct consequence of the malformed photoreceptor synapses. Comparable abnormalities are not seen in rd10 mice. The association of early onset and severe photoreceptor degeneration, which is preceded by synaptic abnormalities, appears to represent a phenotype not previously described. Our new evidence indicates that Tulp1 is not only critical for photoreceptor function and survival, but is essential for the proper development of the photoreceptor synapse.

Early synaptic defects in tulp1-/- mice.

PURPOSE: Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa. To investigate the role of Tulp1 in the photoreceptor synapse, the authors examined the presynaptic and postsynaptic architecture and retinal function in tulp1(-/-) mice METHODS: The authors used immunohistochemistry to examine tulp1(-/-) mice before retinal degeneration and made comparisons with wild-type (wt) littermates and retinal degeneration 10 (rd10) mice, another model of photoreceptor degeneration that has a comparable rate of degeneration. Retinal function was characterized with the use of electroretinography. RESULTS: In wt mice, Tulp1 is localized to the photoreceptor synapse. In the tulp1(-/-) synapse, the spatial relationship between the ribbon-associated proteins Bassoon and Piccolo are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted. Comparable abnormalities are not seen in rd10 mice. The leading edge of the a-wave had normal kinetics in tulp1(-/-) mice but reduced gain in rd10 mice. The b-wave intensity-response functions of tulp1(-/-) mice are shifted to higher intensities than in wt mice, but those of rd10 mice are not. CONCLUSIONS: Photoreceptor synapses and bipolar cell dendrites in tulp1(-/-) mice display abnormal structure and function. A malformation of the photoreceptor synaptic ribbon is likely the cause of the dystrophy in bipolar cell dendrites. The association of early-onset, severe photoreceptor degeneration preceded by synaptic abnormalities appears to represent a phenotype not previously described. Not only is Tulp1 critical for photoreceptor function and survival, it is essential for the proper development of the photoreceptor synapse.

Short-term constant light potentiation of large-magnitude circadian phase shifts induced by 8-OH-DPAT: effects on serotonin receptors and gene expression in the hamster suprachiasmatic nucleus.

Nonphotic phase-shifting of mammalian circadian rhythms is thought to be mediated in part by serotonin (5-HT) acting in the suprachiasmatic nucleus (SCN) circadian clock. Previously we showed that brief (1-3 days) exposure to constant light (LL) greatly potentiates nonphotic phase-shifting induced by the 5-HT agonist, (+/-)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT). Here we investigated potential mechanisms for this action of LL, including 5-HT receptor upregulation and SCN clock gene and neuropeptide gene expression. Autoradiographic analysis of ritanserin inhibition of [3H]8-OH-DPAT binding indicated that LL (approximately 2 days) did not affect 5-HT7 receptor binding in the SCN or dorsal raphe. Measurement of 5-HT1A autoreceptors in the median raphe and 5-HT1B receptors in the SCN also showed no effect of LL. In experiment 2, hamsters held under a 14-h light : 10-h dark photocycle (LD) or exposed to LL for approximately 2 days received an intraperitoneal injection of 8-OH-DPAT or vehicle at zeitgeber time (ZT) 6 or 0 and were killed after 2 h of dark exposure. 8-OH-DPAT suppressed SCN Per1 and Per2 mRNAs at both ZTs, as assessed by in situ hybridization. Per1 mRNA was also suppressed by LL alone. In addition, in situ hybridization of arginine vasopressin (AVP) mRNA and vasoactive intestinal polypeptide mRNA showed that LL significantly suppressed the former but not the latter. The LL-induced suppression of SCN Per1 mRNA and AVP mRNA may be involved in LL-induced potentiation of pacemaker resetting, especially as these data provide additional evidence that LL suppresses circadian pacemaker amplitude, thus rendering the clock more susceptible to phase-shifting stimuli.

Regulation of serotonin release in the Syrian hamster intergeniculate leaflet region.

In mammals, the thalamic intergeniculate leaflet (IGL) conveys behavioral (non-photic) phase-resetting information to the circadian clock of the suprachiasmatic nucleus. Here we report a 24 h fluctuation in in vivo serotonin release in the hamster IGL region, peaking at night. Novel wheel exposure at midday, a stimulus that can reset circadian phase, activates the release of serotonin in the IGL region. In addition, electrical stimulation of the dorsal raphe nucleus acutely increases serotonin release the IGL region, confirming a functional serotonergic projection from this nucleus to the IGL. Collectively, these findings suggest that behavioral and/or circadian changes in dorsal raphe nuclear activity could modulate serotonin-mediated activities of the IGL.

Midbrain raphe modulation of nonphotic circadian clock resetting and 5-HT release in the mammalian suprachiasmatic nucleus.

Serotonin (5-HT) is an important regulator of the mammalian circadian clock of the suprachiasmatic nucleus (SCN); however, critical questions remain concerning the control of serotonergic activity in the SCN and how this relates to the putative clock-resetting actions of 5-HT. Previously, we reported that electrical stimulation of the dorsal raphe nucleus (DRN) or median raphe nucleus (MRN) in hamsters evoked 5-HT release in the SCN. This DRN-stimulated 5-HT release was blocked by systemic injection of 5-HT antagonists, indicating a 5-HT receptor-mediated pathway from the DRN to the SCN. In the present study, targeted injections of the 5-HT1,2,7 antagonist metergoline or the selective 5-HT7 antagonist DR4004 into the DRN or MRN attenuated DRN-electrically stimulated SCN 5-HT release, supporting a multisynaptic DRN-->MRN-->SCN route. Intra-DRN and intra-MRN injections of the GABA(A) antagonist bicuculline significantly stimulated SCN 5-HT release, whereas intra-DRN or intra-MRN injections of the GABAA agonist muscimol suppressed this release. The 5-HT release induced by intra-DRN bicuculline was also blocked by co-injection of DR4004. In complementary behavioral trials, SCN 5-HT release associated with a phase-advancing sleep deprivation stimulus at midday was prevented by intra-DRN injection of metergoline. Also, phase-advance shifts induced by novel wheel access at midday were suppressed, but not blocked, by intra-DRN injection of DR4004 or muscimol. These results indicate that 5-HT7 and GABAergic receptors of the DRN and MRN regulate behaviorally induced 5-HT release in the SCN, and that DRN output modulates nonphotic phase-resetting responses.