Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results.

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (ß-HB). Prior work, based on detecting ß-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export ß-HB across the apical membrane. Here, we compare the accuracy of measuring ß-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of ß-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete ß-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming ß-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce ß-HB. Altogether, we substantiate ß-HB secretion in RPE but find that the secretion is equal apically and basally, RPE ß-HB can derive from ketogenic amino acids or fatty acids, and accurate ß-HB assessment requires mass spectrometric analysis.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Rebound Inverts the Staphylococcus aureus Bacteremia Prevention Effect of Antibiotic Based Decontamination Interventions in ICU Cohorts with Prolonged Length of Stay.

Could rebound explain the paradoxical lack of prevention effect against Staphylococcus aureus blood stream infections (BSIs) with antibiotic-based decontamination intervention (BDI) methods among studies of ICU patients within the literature? Two meta-regression models were applied, each versus the group mean length of stay (LOS). Firstly, the prevention effects against S. aureus BSI [and S. aureus VAP] among 136 studies of antibiotic-BDI versus other interventions were analyzed. Secondly, the S. aureus BSI [and S. aureus VAP] incidence in 268 control and intervention cohorts from studies of antibiotic-BDI versus that among 165 observational cohorts as a benchmark was modelled. In model one, the meta-regression line versus group mean LOS crossed the null, with the antibiotic-BDI prevention effect against S. aureus BSI at mean LOS day 7 (OR 0.45; 0.30 to 0.68) inverted at mean LOS day 20 (OR 1.7; 1.1 to 2.6). In model two, the meta-regression line versus group mean LOS crossed the benchmark line, and the predicted S. aureus BSI incidence for antibiotic-BDI groups was 0.47; 0.09-0.84 percentage points below versus 3.0; 0.12-5.9 above the benchmark in studies with 7 versus 20 days mean LOS, respectively. Rebound within the intervention groups attenuated and inverted the prevention effect of antibiotic-BDI against S. aureus VAP and BSI, respectively. This explains the paradoxical findings.

CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.

The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)-ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12-ATG5-ATG16L1 machinery establishes a high level of regulatory control.

Kinetic investigation reveals an HIV-1 Nef-dependent increase in AP-2 recruitment and productivity at endocytic sites.

The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.

Competition between Dissolved Organic Matter and Freshwater Plankton Control Methylmercury Isotope Fractionation during Uptake and Photochemical Demethylation.

Isotope fractionation related to photochemical reactions and planktonic uptake at the base of the food web is a major uncertainty in the biological application of mercury (Hg) stable isotopes. In freshwater systems, it is unclear how competitive interactions among methylmercury (MeHg), dissolved organic matter (DOM), and phytoplankton govern the magnitude of mass-dependent and mass-independent fractionation. This study investigated how DOM alters rates of planktonic MeHg uptake and photodegradation and corresponding Hg isotope fractionation in the presence of freshwater phytoplankton species, Raphidocelis subcapitata. Outdoor sunlight exposure experiments utilizing R. subcapitata were performed in the presence of different DOM samples using environmentally relevant ratios of MeHg-DOM thiol groups. The extent of Δ199Hg in phytoplankton incubations (2.99‰ St. Louis River HPOA, 1.88‰ Lake Erie HPOA) was lower compared to paired abiotic control experiments (4.29 and 2.86‰, respectively) after ∼30 h of irradiation, resulting from cell shading or other limiting factors reducing the extent of photodemethylation. Although the Δ199Hg/Δ201Hg ratio was uniform across experiments (∼1.4), Δ199Hg/δ202Hg slopes varied dramatically (from -0.96 to 15.4) across incubations with R. subcapitata and DOM. In addition, no evidence of Hg isotope fractionation was observed within R. subcapitata cells. This study provides a refined examination of Hg isotope fractionation markers for key processes occurring in the lower food web prior to bioaccumulation, critical for accurately accounting for the photochemical processing of Hg isotopes across a wide spectrum of freshwater systems.

Medium Depth Influences O2 Availability and Metabolism in Human RPE Cultures.

Purpose: Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. Methods: We altered the availability of O2 to human primary and induced pluripotent stem cell-derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, 13C6-glucose and 13C5-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation. Results: Medium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. Conclusions: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood." As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.

Indirect (herd) effects of topical antibiotic prophylaxis and oral care versus non-antimicrobial methods increase mortality among ICU patients: realigning Cochrane review data to emulate a three-tier cluster randomised trial.

OBJECTIVE: This study aimed to estimate the direct effects to recipients and indirect (herd) effects to non-recipients of each of topical antibiotic prophylaxis (TAP) and oral care methods on patient mortality within randomised concurrent controlled trials (RCCT) using Cochrane review data. DESIGN: Control and intervention groups from 209 RCCTs of TAP (tier 3), oral care (tier 2) each versus non-antimicrobial (tier 1) ventilator-associated pneumonia (VAP) prevention interventions arranged to emulate a three-tiered cluster randomised trial (CRT). Eligible RCCTs were those including ICU patients with >50% of patients receiving >24 hours of mechanical ventilation (MV) with mortality data available as abstracted in 13 Cochrane reviews. EXPOSURES: Direct and indirect exposures to either TAP or oral care within RCCTs versus non-antimicrobial VAP prevention interventions. MAIN OUTCOMES AND MEASURES: The ICU mortality within control and intervention groups, respectively, within RCCTs of either TAP or oral care versus that within non-antimicrobial VAP prevention RCCTs serving as benchmark. RESULTS: The ICU mortality was 23.9%, 23.0% and 20.3% for intervention groups and 28.7%, 25.5% and 19.5% for control groups of RCCTs of TAP (tier 1), oral care (tier 2) and non-antimicrobial (tier 3) methods of VAP prevention, respectively. In a random effects meta-regression including late mortality data and adjusting for group mean age, year of study publication and MV proportion, the direct effect of TAP and oral care versus non-antimicrobial methods were 1.04 (95% CI 0.78 to 1.30) and 1.1 (95% CI 0.77 to 1.43) whereas the indirect effects were 1.39 (95% CI 1.03 to 1.74) and 1.26 (95% CI 0.89 to 1.62), respectively. CONCLUSIONS: Indirect (herd) effects from TAP and oral care methods on mortality are stronger than the direct effects as made apparent by the three-tiered CRT. These indirect effects, being harmful to concurrent control groups by increasing mortality, perversely inflate the appearance of benefit within RCCTs.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Acetyl-CoA carboxylase Inhibition increases RPE cell fatty acid oxidation and limits apolipoprotein efflux.

Purpose: In age-related macular degeneration (AMD) and Sorsby's fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating ß-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate ß-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. Methods: We probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13 C6-glucose and 13 C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13 C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). Results: ACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsby's fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. Conclusions: Firsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining GUV-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Proline provides a nitrogen source in the retinal pigment epithelium to synthesize and export amino acids for the neural retina.

It is known that metabolic defects in the retinal pigment epithelium (RPE) can cause degeneration of its neighboring photoreceptors in the retina, leading to retinal degenerative diseases such as age-related macular degeneration. However, how RPE metabolism supports the health of the neural retina remains unclear. The retina requires exogenous nitrogen sources for protein synthesis, neurotransmission, and energy metabolism. Using 15N tracing coupled with mass spectrometry, we found human RPE can utilize the nitrogen in proline to produce and export 13 amino acids, including glutamate, aspartate, glutamine, alanine, and serine. Similarly, we found this proline nitrogen utilization in the mouse RPE/choroid but not in the neural retina of explant cultures. Coculture of human RPE with the retina showed that the retina can take up the amino acids, especially glutamate, aspartate, and glutamine, generated from proline nitrogen in the RPE. Intravenous delivery of 15N proline in vivo demonstrated 15N-derived amino acids appear earlier in the RPE before the retina. We also found proline dehydrogenase, the key enzyme in proline catabolism is highly enriched in the RPE but not the retina. The deletion of proline dehydrogenase blocks proline nitrogen utilization in RPE and the import of proline nitrogen-derived amino acids in the retina. Our findings highlight the importance of RPE metabolism in supporting nitrogen sources for the retina, providing insight into understanding the mechanisms of the retinal metabolic ecosystem and RPE-initiated retinal degenerative diseases.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Using live cell and STORM, imaging, nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture, and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Trends in ICU mortality and underlying risk over three decades among mechanically ventilated patients. A group level analysis of cohorts from infection prevention studies.

BACKGROUND: Has either the underlying risk or the mortality incidence among ICU patients receiving mechanical ventilation (MV) in the literature changed in recent decades? Interpreting ICU mortality trends requires an adjusted analysis accounting for changes in underlying patient risk. METHODS: Control and intervention groups from 147 randomized concurrent control trials (RCCT) of various VAP prevention interventions, as listed primarily within 13 Cochrane reviews and 63 observational studies listed primarily within four systematic reviews. Eligible studies were those including ICU patients with > 50% of patients receiving > 24 h of MV with mortality data available. ICU mortality (censored day 21 or before) or late (after day 21) mortality together with group-mean age, and group-mean APACHE II scores were extracted from all groups. These incidences were summarized in five meta-regression models versus publication year being variously adjusted for age, APACHE II scores, type of study intervention and other group level parameters. RESULTS: Among 210 studies published between 1985 and 2021, 169 being found in systematic reviews, the increase per decade in mean mortality incidence, group-mean APACHE II scores, and group-mean age, were < 1 percentage point (p = 0.43), 1.83 (95% CI; 0.51-3.15) points, and 3.9 (95% CI; 1.1-6.7) years, respectively. Only in the model with risk adjustment for both group-mean age and group-mean APACHE II score was a significant decline in mortality apparent. In all models, the mortality incidence among concurrent control groups of decontamination studies was paradoxically five percentage points higher than benchmark and showed greater dispersion. CONCLUSION: Mortality incidence has changed little over 35 years among ICU infection prevention studies whilst the patient age and underlying disease severity, measured as APACHE II, have both increased. The paradoxically high mortality among concurrent control groups within studies of decontamination methods of infection prevention remains unaccounted for.

Aerobic Glycolysis in Photoreceptors Supports Energy Demand in the Absence of Mitochondrial Coupling.

Metabolism is adapted to meet energetic needs. Based on the amount of ATP required to maintain plasma membrane potential, photoreceptor energy demands must be high. The available evidence suggests that photoreceptors primarily generate metabolic energy through aerobic glycolysis, though this evidence is based primarily on protein expression and not measurement of metabolic flux. Aerobic glycolysis can be validated by measuring flux of glucose to lactate. Aerobic glycolysis is also inefficient and thus an unexpected adaptation for photoreceptors to make. We measured metabolic rates to determine the energy-generating pathways that support photoreceptor metabolism. We found that photoreceptors indeed perform aerobic glycolysis and this is associated with mitochondrial uncoupling.

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.