Human Vitreous Humor Research Articles

A new approach to assess post-mortem interval: A machine learning-assisted label-free ATR-FTIR analysis of human vitreous humor

A crucial issue in forensics is determining the post-mortem interval (PMI), the time between death and the finding of a body. Despite various methods already employed for its estimation, only approximate values are currently achievable. Vitreous humor (VH) is an avascular tissue between the lens and the retina, mainly composed by a collagen fibers network, hyaluronic acid, and hyalocytes. Recently, it has received interest in forensic medicine, being easy to collect and susceptible to low microbiological contamination and putrefaction. Based on this evidence and thanks to the ability of Attenuated Total Reflectance – Fourier Transform InfraRed (ATR–FTIR) spectroscopy to perform fast analyses on a minimal sample amount, in this study, a new analytical approach to reliably estimate PMI is proposed combining ATR-FTIR analysis of VH human samples with multivariate statistical procedures, such as Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA), for discriminant classification. Regression procedures, including Partial Least Squares Regression (PLSR), were performed: extremely positive results were obtained, and the most discriminant spectral features were highlighted (peaks at 1665, 1630, 1585, 1400, 1220, 1200, 1120, 854, 835, and 740 cm−1) and associated to PMI classes (average accuracy over 80 %). Specific and reliable markers able to correlate the macromolecular composition of VH with the PMI were identified, revealing a post-mortem protein degradation and amino acids deamination (decrease of proteins and increase of free amino acids and NH3), an increase of lactate, which diffuses from the retina to the VH, and changes in the hyaluronic acid component.

Revealing new insights: Two-center evidence of microplastics in human vitreous humor and their implications for ocular health

Microplastics (MPs), an emerging environmental contaminant, have raised growing health apprehension due to their detection in various human biospecimens. Despite extensive research into their prevalence in the environment and the human body, the ramifications of their existence within the enclosed confines of the human eye remain largely unexplored. Herein, we assembled a cohort of 49 patients with four ocular diseases (macular hole, macular epiretinal membrane, retinopathy and rhegmatogenous retinal detachment) from two medical centers. After processing the samples with an optimized method, we utilized Laser Direct Infrared (LD-IR) spectroscopy and Pyrolysis Gas Chromatography/Mass Spectrometry (Py-GC/MS) to analyze 49 vitreous samples, evaluating the characteristics of MPs within the internal environment of the human eye. Our results showed that LD-IR scanned a total of 8543 particles in the composite sample from 49 individual vitreous humor samples, identifying 1745 as plastic particles, predominantly below 50 μm. Concurrently, Py-GC/MS analysis of the 49 individual samples corroborated these findings, with nylon 66 exhibiting the highest content, followed by polyvinyl chloride, and detection of polystyrene. Notably, correlations were observed between MP levels and key ocular health parameters, particularly intraocular pressure and the presence of aqueous humor opacities. Intriguingly, individuals afflicted with retinopathy demonstrated heightened ocular health risks associated with MPs. In summary, this research provides significant insights into infiltration of MP pollutants within the human eye, shedding light on their potential implications for ocular health and advocating for further exploration of this emerging health risk.

Low expression of RBP4 in the vitreous humour of patients with proliferative diabetic retinopathy who underwent Conbercept intravitreal injection.

Intravitreal injection of anti-VEGF antibodies has been widely used in the treatment of proliferative diabetic retinopathy (PDR). However, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. To explore proteins closely related to fibrosis, we conducted proteomic analysis of human vitreous humour collected from PDR patients who have or have not intravitreal Conbercept (IVC) injection. Sixteen vitreous humour samples from PDR patients with preoperative IVC and 20 samples from those without preoperative IVC were examined. An immunodepletion kit was used to remove high-abundance vitreous proteins. Conbercept-induced changes were determined using a tandem mass tag-based quantitative proteomic strategy. Enzyme-linked immunosorbent assays were performed to confirm the concentrations of selected proteins and validate the proteomic results. Based on a false discovery rate between 0.05% and -0.05% and a fold-change > 1.5, 97 proteins were altered (49 higher levels and 48 lower levels) in response to IVC. Differentially expressed proteins were found in the extracellular and intracellular regions and were found to be involved in VEGF binding and VEGF-activated receptor activity. Protein-protein interactions indicated associations with fibrosis, neovascularisation and inflammatory signalling pathways. We found the low levels of RBP4 in the vitreous humour of PDR patients with IVC injection, as revealed by ELISA and proteomic profiling. Moreover, RBP4 significantly restored the mitochondrial function of HRMECs induced by AGEs and down regulated the level of glycolysis. Our study is the first to report that RBP4 decreases in the vitreous humour of PDR patients who underwent Conbercept treatment, thereby verifying the role of RBP4 in glucose metabolism. Results provide evidence for the potential mechanism underlying Conbercept-related fibrosis.

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS.

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

Use of cadaveric vitreous humor as an innovative substrate for diatoms research and forensic diagnosis of drowning.

The search for diatoms is the test that most of all may contribute to the forensic diagnosis of drowning. Diatoms can be extracted through different methods, which are all complex and long. In this preliminary study, we assessed human vitreous humor as an innovative substrate on which to research diatoms. Sampling and analyses were performed on 3 groups of 10 corpses each. The first one was composed of drowned victims. The second one of victims of traffic accidents with subsequent projection in water; based on the severe traumatic injuries reported at autopsy, we considered such victims as ideally assimilable to bodies immersed in water postmortem. The third group was composed of subjects who died for natural causes and without any relationship with water. The vitreous humor was centrifugated and cytocentrifuged and spotted on two different histological slides: one was left blank and the other one was assessed with hematoxylin and eosin staining. Microscopic observation successfully revealed diatoms in all cases of the first group; in the second group diatoms were not detected in 6 cases; in the third group, no diatoms at all were observed. Diatoms were always qualitatively and quantitatively consistent with those found in the corresponding viscera and waters, which had been investigated through classical acid digestion. Our analyses have demonstrated that the vitreous humor behaves in a completely similar way to the other viscera in cases of drowning. Although further investigations are necessary, vitreous humor has proved to be an innovative, suitable, and reliable substrate for the forensic research of diatoms.

Development of an ultra-sensitive human IL-33 biomarker assay for age-related macular degeneration and asthma drug development

BackgroundOver the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding.ResultsWe tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals.ConclusionsUnlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.

Mfsd2a overexpression alleviates vascular dysfunction in diabetic retinopathy

Diabetic retinopathy (DR) is one of the common complications in diabetic patients. Nowadays, VEGF pathway is subject to extensive research. However, about 27% of the patients have a poor visual outcome, with 50% still having edema after two years’ treatment of diabetic macular edema (DME) with ranibizumab. Docosahexaenoic acid (DHA), the primary ω-3 long-chain polyunsaturated fatty acid (LC-PUFA), reduces abnormal neovascularization and alleviates neovascular eye diseases. A study reported that fish oil reduced the incidence of retinopathy of prematurity (ROP) by about 27.5% in preterm infants. Although ω-3 LC-PUFAs protects against pathological retinal neovascularization, the treatment effectiveness is low. It is interesting to investigate why DHA therapy fails in some patients. In human vitreous humor samples, we found that the ratio of DHA and DHA-derived metabolites to total fatty acids was higher in vitreous humor from DR patients than that from macular hole patients; however, the ratio of DHA metabolites to DHA and DHA-derived metabolites was lower in the diabetic vitreous humor. The expression of Mfsd2a, the LPC-DHA transporter, was reduced in the oxygen-induced retinopathy (OIR) model and streptozotocin (STZ) model. In vitro, Mfsd2a overexpression inhibited endothelial cell proliferation, migration and vesicular transcytosis. Moreover, Mfsd2a overexpression in combination with the DHA diet obviously reduced abnormal retinal neovascularization and vascular leakage, which is more effective than Mfsd2a overexpression alone. These results suggest that DHA therapy failure in some DR patients is linked to low expression of Mfsd2a, and the combination of Mfsd2a overexpression and DHA therapy may be an effective treatment.

Spectroscopy as a useful tool for the identification of changes with time in post-mortem vitreous humor for forensic toxicology purposes

Vitreous humor (VH) is an alternative biological matrix with a great advantage of longer availability for analysis due to the lack of many enzymes. The use of VH in forensic toxicology may have an added benefit, however, this application requires rapid, simple, non-destructive, and relatively portable analytical analysis methods. These requirements may be met by Fourier transform infrared spectroscopy technique (FT-IR) equipped with attenuated total reflection accessory (ATR). FT-IR spectra of vitreous humor samples, deposited on glass slides, were collected and subsequent chemometric data analysis by means of Hierarchical Cluster Analysis and Principal Component Analysis was conducted. Differences between animal and human VH samples and human VH samples stored for diverse periods of time were detected. A kinetic study of changes in the VH composition up to 2 weeks showed the distinction of FT-IR spectra collected on the 1st and 14th day of storage. In addition, data obtained for the most recent human vitreous humor samples—collected 3 and 2 years before the study, presented successful discrimination of all time points studied. The method introduced was unable to detect mephedrone addition to VH in the concentration of 10 µg/cm3.Graphic abstract

A novel regulatory network of linc00174/miR-150-5p/VEGFA modulates pathological angiogenesis in diabetic retinopathy.

Diabetic retinopathy (DR) has been regarded as a sight-threatening vascular complication of diabetes mellitus. Accumulating evidence has identified the involvement of long non-coding RNAs (lncRNAs) in DR pathogenesis. We aim to investigate the role and underlying mechanism of linc00174 in the DR process. Samples of human vitreous humour from proliferative DR and non-diabetic individuals were collected to examine the levels of linc00174. Human retinal microvascular endothelial cells (HRMECs) exposed with high glucose (HG) were employed to simulate the pathological statues of DR. Short hairpin RNA specifically targeting linc00174 was applied. CCK-8, transwell, and matrigel tube formation were performed to evaluate cell proliferation, migration, and angiogenesis. Bioinformatics analysis and luciferase reporter assay were conducted to verify the linc00174/miR-150-5p/vascular endothelial growth factor A (VEGFA) regulatory network. Western blotting was employed to determine the expression of VEGFA. Linc00174 was significantly elevated in patients with DR, as well as HG-stimulated HRMECs, of which knockdown repressed HG-induced proliferation, migration, and angiogenesis. miR-150-5p was identified as a downstream effector to be involved in linc00174-mediated protective effects. miR-150-5p directly bound to the 3' untranslated region of VEGFA. The linc00174/miR-150-5p/VEGFA axis was confirmed in retinal vascular dysfunction. The linc00174 deteriorates diabetic retinal microangiopathy via regulating miR-150-5p/VEGFA pathway, indicating a novel therapeutic target for DR treatment.

Cytokine Levels in Human Vitreous in Proliferative Diabetic Retinopathy.

In this study, we compare the vitreous cytokine profile in patients with proliferative diabetic retinopathy (PDR) to that of patients without PDR. The identification of novel cytokines involved in the pathogenesis of PDR provides candidate therapeutic targets that may stand alone or work synergistically with current therapies in the management of diabetic retinopathy. Undiluted vitreous humor specimens were collected from 74 patients undergoing vitrectomy for various vitreoretinal disorders. Quantitative immunoassay was performed for a panel of 36 neuroinflammatory cytokines in each specimen and assessed to identify differences between PDR (n = 35) and non-PDR (n = 39) patients. Levels of interleukin-8 (IL-8), IL-15, IL-16, vascular endothelial growth factor (VEGF), VEGF-D, c-reactive protein (CRP), serum amyloid-A (SAA), and intracellular adhesion molecule-1 (ICAM1) were significantly increased in the vitreous of PDR patients compared to non-PDR patients (p < 0.05). We report novel increases in IL-15 and IL-16, in addition to the expected VEGF, in the human vitreous humor of patients with PDR. Additionally, we confirm the elevation of ICAM-1, VCAM-1, SAA, IL-8 and CRP in the vitreous of patients with PDR, which has previously been described.

Vitreous metabolomics profiling of proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) with retinal neovascularisation (NV) is a leading cause of vision loss. This study identified a set of metabolites that were altered in the vitreous humour of PDR patients compared with non-diabetic control participants. We corroborated changes in vitreous metabolites identified in prior studies and identified novel dysregulated metabolites that may lead to treatment strategies for PDR. We analysed metabolites in vitreous samples from 43 PDR patients and 21 non-diabetic epiretinal membrane control patients from Japan (age 27-80years) via ultra-high-performance liquid chromatography-mass spectrometry. We then investigated the association of a novel metabolite (creatine) with retinal NV in mouse oxygen-induced retinopathy (OIR). Creatine or vehicle was administered from postnatal day (P)12 to P16 (during induced NV) via oral gavage. P17 retinas were quantified for NV and vaso-obliteration. We identified 158 metabolites in vitreous samples that were altered in PDR patients vs control participants. We corroborated increases in pyruvate, lactate, proline and allantoin in PDR, which were identified in prior studies. We also found changes in metabolites not previously identified, including creatine. In human vitreous humour, creatine levels were decreased in PDR patients compared with epiretinal membrane control participants (false-discovery rate <0.001). We validated that lower creatine levels were associated with vascular proliferation in mouse retina in the OIR model (p = 0.027) using retinal metabolomics. Oral creatine supplementation reduced NV compared with vehicle (P12 to P16) in OIR (p = 0.0024). These results suggest that metabolites from vitreous humour may reflect changes in metabolism that can be used to find pathways influencing retinopathy. Creatine supplementation could be useful to suppress NV in PDR. Graphical abstract.

Eicosanoid Profiles in the Vitreous Humor of Patients with Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy is a potentially blinding sequela of uncontrolled diabetes that involves a complex interaction of pro-angiogenic and inflammatory pathways. In this study, we compared the levels of pro-angiogenic arachidonic acid-derived mediators in human vitreous humor obtained from eyes with high-risk proliferative diabetic retinopathy versus controls. The results indicated that lipoxygenase and cytochrome P450-derived eicosanoids were elevated (5-HETE, 12-HETE, 20-HETE, and 20-COOH-AA), and there appeared to be no differences in levels measured in eyes with tractional retinal detachments versus those without. These results provide further insight into the pathogenesis of this disease and for the development of future potential therapeutic agents that target arachidonic acid metabolites to treat diabetic retinopathy.

Determination of Ethyl Glucuronide and Ethyl Sulfate in Human Whole Blood and Vitreous Humor by LC-MS-MS and Applications to the Interpretation of Postmortem Ethanol Findings.

Identifying the source of ethanol in a decedent remained a complicated problem for forensic toxicologists because of postmortem ethanol formation. As ethanol's non-oxidative metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) have the potential to distinguish between antemortem ethanol consumption and postmortem ethanol formation, due to their high sensitivity and selectivity. In the current study, a simple and quick liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of EtG and EtS in human whole blood and vitreous humor (VH). A total of 20μL of the sample was precipitated by methanol, and the analytes were detected by LC-MS/MS in a run of 6min. This method achieved high sensitivity (limits of detection: 2ng/mL for both EtG and EtS), with linearity in the range of 5-10,000ng/mL in both whole blood and VH. Deviations in accuracy, inter- and intra-day precision were all lower than 15% at three quality control levels. Subsequently, this method was applied to 62 real forensic cases. Only blood samples were available in 52 cases. Paired blood and VH samples were present in 10 cases. The concentrations of EtG and EtS in blood were in the range of 0-22,264.8ng/mL and 0-2,126.0ng/mL, respectively. In one case with both blood and VH, the blood ethanol concentration was 1.22mg/mL, with EtG and EtS both below limits of quantification (5ng/mL) in VH, and no EtG and EtS found in whole blood. The results suggested that EtG and EtS were useful markers for the interpretation of ethanol resource in postmortem blood and VH.

Post-mortem changes in metabolomic profiles of human serum, aqueous humor and vitreous humor.

Application of metabolomic methods to forensic studies may expand the limits of the post-mortem interval (PMI) estimation, and improve the accuracy of the estimation. To this end, it is important to determine which tissue is the most suitable for analysis, and which compounds are the most promising candidates for PMI estimation. This work is aimed at the comparison of human serum, aqueous humor (AH), and vitreous humor (VH) as perspective tissues for metabolomic-based PMI estimation, at the determination of most promising PMI biomarkers, and at the development of method of PMI estimation based on the measurement of concentrations of PMI biomarkers. Quantitative metabolomic profiling of samples of the human serum, AH, and VH taken at different PMIs has been performed with the use of NMR spectroscopy. It is found that the metabolomic changes in anatomically isolated ocular fluids are slower and smoother than that in blood. A good positive time correlation (Pearson coefficient r > 0.5) was observed for several metabolites, including hypoxanthine, choline, creatine, betaine, glutamate, and glycine. A model for PMI estimation based on concentrations of several metabolites in AH and VH is proposed. The obtained results demonstrate that the metabolomic analysis of AH and VH is more suitable for the PMI estimation than that of serum. The compounds with good positive time correlation can be considered as potential PMI biomarkers.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Lactate determination in human vitreous humour by capillary electrophoresis and time of death investigation.

Forensic inquests, particularly, in assessing time since death currently recognize the importance of the analysis of vitreous humour (VH) biomarkers. Present research, studies, and validates the determination of lactate (La) in VH by CZE with indirect UV detection. The BGE (pH 8.9) consisted of Tris buffer (37mM) containing 4-methoxybenzoic acid (4mM) and alkyl-trimethyl-ammonium bromide (1.2mM). Each VH specimen was diluted with a butyric acid solution (internal standard 0.057mM) and La and butyrate were separated within 3-5min (30kV). The La LOQ and LOD were 4 and 2mM, respectively. The calibration curve linearity ranged from 4 to 80mM; intra- and interruns precisions were less than 10% for standard as well as for VH specimen, respectively. To investigate postmortem interval (PMI) and VH lactate level correlation, human VH specimens were collected during autopsy (n = 40) and stored at -20°C until assay. La levels ranged from 16 to 42mM; PMI values ranged from 10 to 141 h. La (mM) and PMI (h) correlation was statistically significant (r2 = 0.527; p < 0.05). In conclusion, the present CZE analysis is efficacious to determine VH La as a biomarker for PMI investigation.

Association of Cognitive Function with Amyloid-β and Tau Proteins in the Vitreous Humor.

The eye may serve as source for diagnostic testing for early detection of Alzheimer's disease (AD). Examination of amyloid-β (Aβ) and tau protein content in human vitreous and its correlation to neuro-cognition may improve ocular-based AD detection methods. To evaluate levels of Aβ and tau protein in human vitreous humor and investigate the clinical predictive role of these proteins as early diagnostic markers of AD. A prospective, single-center, multi-surgeon cohort study. Vitreous humor samples from 80 eyes were measured quantitatively for Aβ40-42, pTau, and tTau. Linear regression was used to test associations between AD biomarker levels, Mini-Mental State Exam (MMSE), and serum apolipoprotein E (APOE) allele status, with adjustment for age, sex, and education level of patients. Lower MMSE scores were significantly associated with lower levels of vitreous Aβ40 (p = 0.015), Aβ42 (p = 0.0066), and tTau (p = 0.0085), and these biomarkers were not associated with any pre-existing eye conditions. Presence of the ɛ4 allele and the ɛ2 allele approached significance with reduced Aβ40 level (p = 0.053) and increased p-Tau level (p = 0.056), respectively. Patients with poor cognitive function have significantly lower vitreous humor levels of AD-related biomarkers Aβ40, Aβ42, and tTau. These biomarkers do not correlate with underlying eye conditions, suggesting their specificity in association with cognitive change. This is the first study to our knowledge to correlate cognition with AD-related proteins in the vitreous humor. Results suggest ocular proteins may have a role for early dementia detection in individuals at risk for AD.

Rheological Properties and Age-Related Changes of the Human Vitreous Humor.

The vitreous humor is a fragile, transparent hydrogel situated between the lens and the retina, occupying 80% of the eye's volume. Due to its viscoelastic behavior, the vitreous serves as a mechanical damper for the eye, absorbing impacts, and protecting the lens and retina. The vitreous liquefies with age, which compromises its function as a shock absorber and causes complications including retinal detachment, macular holes, and vitreous hemorrhage. Studies on the viscoelastic properties of the vitreous have been limited. Rheological testing of the vitreous has commonly been done on non-primate mammalian species. Human vitreous rheological properties have been previously reported; however, various measurement techniques were used, resulting in data that differed by orders of magnitude. Shear rheometry is commonly used to characterize soft tissues and hydrogels such as the vitreous humor. However, no human vitreous rheological data have been reported using this technique, preventing direct comparison to other published work. Additionally, no age-related changes in the mechanical properties of the human vitreous humor have been reported. Human vitreous samples (n = 39, aged 62 ± 15 years) were tested using a shear rheometer. Small amplitude oscillatory shear and creep experiments were performed. The linear viscoelastic region of the human vitreous was found to be below 1% strain. The solid phase of the old human vitreous was found to be stiffer than the young human vitreous and the porcine vitreous. The stiffness of the human vitreous gel also appeared to be positively correlated with age. Vitreous dehydration due to a decrease in hyaluronic acid concentration with age was proposed to cause the stiffening of the solid phase of the vitreous gel. Vitreous liquefaction, therefore, might be characterized as a simultaneous increase in liquid volume and localized stiffening of the vitreous gel. The phase separation of the vitreous humor with age has been hypothesized as the cause of many vitreous-related complications. This study provides viscoelastic properties and age-related changes of the human vitreous humor, which will aid in the design of biomimetic vitreous substitutes, enhancement in analyzing intravitreal transport of therapeutics, and understanding the pathological conditions of the vitreous humor.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Diffusion through the ex vivo vitreal body – Bovine, porcine, and ovine models are poor surrogates for the human vitreous

The human vitreous humour is a complex gel structure whose composition and physical properties can vary considerably from person to person and also change with age. To date, the viscoelastic properties of the human vitreous gel has not been thoroughly investigated and despite many years of intensive research, an ideal vitreous substitute remains a challenge. Understanding the physical structure and properties of the vitreous is of fundamental and therapeutic interest, providing a clear insight into diffusion and transport of administered ophthalmic drug molecules into the vitreous. A number of mammalian surrogates, mainly bovine, porcine and ovine vitreous humours have been used in the literature as a means of studying ophthalmic drug transport and diffusion. In this study, the mechanical, physical and rheological properties of ovine, porcine, and bovine surrogates were investigated and compared to human vitreous. In addition, a bespoke Franz cell construct was used to compare the diffusion of a model drug (fluorescein) through vitreous samples. Despite the similarity in rheological properties between bovine, porcine and human vitreous samples, diffusion of fluorescein through the different vitreous samples revealed great differences in values of steady-state flux and diffusion coefficient. In addition, a first-generation vitreous mimic, composed of 4.5 mg/mL hyaluronic acid with complex viscosity of 0.3 ± 0.01 Pa has been evaluated and was demonstrated to be a better mimic of the human vitreous than the mammalian samples investigated.