A Rapid Method for Postmortem Vitreous Chemistry—Deadside Analysis

Vitreous fluid is commonly collected for toxicological analysis during forensic postmortem investigations. Vitreous fluid is also often analyzed for potassium, sodium, chloride and glucose for estimation of time since death, and for the evaluation of electrolyte imbalances and hyperglycemia, respectively. Obtaining such results in the early phase of a death investigation is desirable both in regard to assisting the police and in the decision-making prior to the autopsy. We analyzed vitreous fluid with blood gas instruments to evaluate/examine the possible impact of different sampling and pre-analytical treatment. We found that samples from the right and left eye, the center of the eye as well as whole vitreous samples gave similar results. We also found imprecision to be very low and that centrifugation and dilution were not necessary when analyzing vitreous samples with blood gas instruments. Similar results were obtained when analyzing the same samples with a regular multi-analysis instrument, but we found that such instruments could require dilution of samples with high viscosity, and that such dilution might impact measurement accuracy. In conclusion, using a blood gas instrument, the analysis of postmortem vitreous fluid for electrolytes and glucose without sample pretreatment produces rapid and reliable results.

DIA Comparative Proteomic Analysis of Retro-oil Fluid and Vitreous Fluid From Retinal Detachment Patients

Objectives: There have been reports of unexplained visual loss following intra-ocular silicone oil (SiO) tamponade in retinal detachment patients, yet the underlying mechanism is unknown. The aim of this study was to investigate the mechanisms behind retinal toxicity following intra-ocular SiO tamponade in retinal detachment patients.Methods and Results: Vitreous fluid samples were acquired from 27 patients (27 eyes). Twelve eyes for data-independent acquisition (DIA) were divided into four groups: pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RD group), SiO removal after successful retinal reattachment (SO group), cataract surgery after successful retinal reattachment with sterilized air tamponade (FA group), and PPV for epiretinal membrane (ERM group). The remaining 15 eyes were used for enzyme-linked immunosorbent assay analysis. DIA was combined with two-dimensional liquid chromatography–tandem mass spectrometry to find expression changes in the proteome of vitreous. Mean number mass spectra, statistically differentially expressed proteins, gene ontology (GO), pathway representations, and protein interactions were analyzed. GO analysis showed that the protein categories of synapse organization, cell adhesion, and regulation of cell migration in the SO group were differentially expressed compared to the control or FA groups (p < 0.05). Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, lysosome and cell adhesion were found to be significantly enriched in the SO group compared to the FA and control groups (p < 0.05). Cadherin 2, transferrin, and lysosome function may partially contribute to silicone oil-related vision loss.Conclusion: Vision loss-inducing novel molecular signatures and pathways that may be associated with SiO toxicity were identified. Transferrin may be a potential visual outcome biomarker for SiO tamponade.

Postoperative proliferative vitreoretinopathy development is linked to vitreal CXCL5 concentrations

The specific changes linked to de novo development of postoperative PVR have remained elusive and were the object of the underlying study. Vitreous fluid (VF) was obtained at the beginning of vitrectomy from 65 eyes that underwent vitrectomy for primary rhegmatogenous retinal detachment (RRD) without preoperative PVR. Eyes developing postoperative PVR within 6 months after re-attachment surgery were compared to those which did not regarding the preoperative concentrations of 43 cytokines and chemokines in the VF, using multiplex beads analysis. For all comparisons Holm’s correction was applied in order to control for multiple comparisons. Twelve out of 65 eyes (18.5%) developed PVR postoperatively. While 12 of the chemokines and cytokines presented concentration differences on a statistical level of p < 0.05 (CXCL5, CCL11, CCL24, CCL26, GM-CSF, IFN-γ, CCL8, CCL7, MIF, MIG/CXCL9, CCL19, and CCL25), CXCL5 was the only cytokine with sufficiently robust difference in its VF concentrations to achieve significance in eyes developing postoperative PVR compared to eyes without PVR. CXCL5 may represent a potent biomarker for the de novo development of postoperative PVR. In line with its pathophysiological role in the development of PVR, it might serve as a basis for the development of urgently needed preventive options.

Intraocular Viral Communities Associated With Post-fever Retinitis

The virome of ocular fluids is naive. The results of this study highlight the virome in the vitreous fluid of the eye of individuals without any ocular infection and compare it with the virome of the vitreous fluid of individuals with retinitis. A total of 1,016,037 viral reads were generated from 25 vitreous fluid samples comprising control and post-fever retinitis (PFR) samples. The top 10 viral families in the vitreous fluids comprised of Myoviridae, Siphoviridae, Phycodnaviridae, Herpesviridae, Poxviridae, Iridoviridae, Podoviridae, Retroviridae, Baculoviridae, and Flaviviridae. Principal coordinate analysis and heat map analysis clearly discriminated the virome of the vitreous fluid of the controls from that of the PFR virome. The abundance of 10 viral genera increased significantly in the vitreous fluid virome of the post-fever retinitis group compared with the control group. Genus Lymphocryptovirus, comprising the human pathogen Epstein-Barr virus (EBV) that is also implicated in ocular infections was significantly abundant in eight out of the nine vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. Human viruses, such as Hepacivirus, Circovirus, and Kobuvirus, were also significantly increased in abundance in the vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis and the network analysis depicted an increase in the immune response by the host in the post-fever retinitis group compared with the control group. All together, the results of the study indicate changes in the virome in the vitreous fluid of patients with the post-fever retinitis group compared to the control group.

Late-onset Endophthalmitis Caused by Streptococcus Cristatus after Trabeculectomy

Purpose: To report a case of late-onset bleb-associated endophthalmitis caused by streptococcus cristatus (S. cristatus) after trabeculectomy.Case summary: A 59-year-old woman presented with left ocular pain and visual disturbance. She had undergone trabeculectomy of the left eye 2 years ago. At the initial visit, she was only able to count fingers from the left eye. Whitish thin bleb, conjunctival injection, and inflammation were observed in the left anterior chamber, and the fundus could not be visualized. Despite administering topical and systemic antibiotics, the chamber reaction worsened, and hypopyon and inflammatory membrane were noticed the next day. Vitrectomy with phacoemulsification and intravitreal antibiotic injection was performed, and the intravitreal antibiotic injections were repeated over the next two days. Bacterial culture of vitreous fluid showed a growth of S. cristatus 5 days after the surgery, and inflammation improved after 7 days. One month later, she was still only able to count fingers from the left eye, but there were no signs of endophthalmitis.Conclusions: Postoperative endophthalmitis after glaucoma filtering surgeries caused by S. cristatus has not been reported previously from Korea. To the best of our knowledge, this is the first Korean case of late-onset bleb-associated endophthalmitis caused by S. cristatus. If gram-positive cocci are grown in samples from the patients with endophthalmitis after trabeculectomy, resident oral microflora such as S. cristatus should be considered as the causative pathogen.

Intravitreal Tissue Plasminogen Activator Injection for Treatment-Resistant Diabetic Macular Edema of the Vitrectomized Eye

Diabetic macular edema (DME) is the main cause of visual loss in patients with diabetic retinopathy. DME has been treated using intravitreal anti-vascular endothelial growth factor (VEGF) drugs, steroids, laser photocoagulation, vitreoretinal surgery, and their combinations. These modalities are generally effective in preserving vision, but they sometimes produce only limited responses in patients with persistent or refractory DME. The levels of various inflammatory factors, including cytokines, chemokines, and extracellular matrices, as well as VEGF in the vitreous fluid, are increased in patients with DME. Excessive fibrinogen/fibrin levels in the vitreous fluid or fibrin deposition in the retina also contribute to DME pathogenesis. Tissue plasminogen activator (t-PA) promotes the degradation of fibrinogen or fibrin. Intravitreal t-PA injection is a commonly used treatment for subretinal hemorrhage secondary to age-related macular degeneration. Intravitreal t-PA injections have previously been used to restore vision by inducing posterior vitreous detachment in patients with DME. Herein, we describe the visual outcomes of intravitreal t-PA injection in a 78-year-old woman with treatment-resistant DME in her vitrectomized eye after several previous treatments. Before the injection, her best-corrected visual acuity (BCVA) was 0.7 logMAR and central foveal retinal thickness (CRT) was 735 μm. At 1 month after the injection, her BCVA was 0.8 logMAR and CRT was 558 μm, and 3 months later, her BCVA was 0.8 logMAR and CRT was 207 μm. Her BCVA was sustained, and CRT showed gradual improvements. These findings suggested the effectiveness of intravitreal t-PA injections for DME in the vitrectomized eye.

Correlations Between Different Angiogenic and Inflammatory Factors in Vitreous Fluid of Eyes With Proliferative Diabetic Retinopathy

Purpose: To investigate the expression of various angiogenesis and inflammation mediators in the vitreous fluid of eyes with proliferative diabetic retinopathy (PDR).Methods: A total of 38 eyes with PDR and 37 control eyes were included. Vitreous fluid was collected during vitrectomy. Vitreous levels of colony stimulating factor-1 receptor (CSF-1R), syndecan-1, placental growth factor (PIGF), and angiopoietin-like protein 4 (ANGPTL-4) were measured by multiplex immunoassay. Vitreous levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) were measured by cytometric beads array. Levels of these mediators were compared between the PDR and control eyes. Correlations between levels of different mediators and between these mediators and kidney function metrics in the PDR group were also analyzed.Results: Vitreous levels of syndecan-1, PIGF, ANGPTL-4, VEGF, and IL-8 were significantly higher in the PDR group compared to the control group (all p &lt; 0.05). Levels of VEGF were significantly correlated with levels of syndecan-1, PIGF, and ANGPTL-4 (r = 0.370 to 0.497, all p &lt; 0.05). Significant positive correlations were detected between levels of any two of the following mediators including syndecan-1, PIGF, ANGPTL-4, and IL-8 (r = 0.370 to 0.906, all p &lt; 0.05). Apart from VEGF, levels of these mediators were positively correlated with serum creatinine and blood urea nitrogen (r = 0.328 to 0.638, all p &lt; 0.05), and negatively correlated with fasting blood glucose and estimated glomerular filtration rate (r = −0.325 to −0.603, all p &lt; 0.05).Conclusions: Correlations between different angiogenesis and inflammation mediators were observed in eyes with PDR, suggesting cross-talks of different angiogenesis and inflammation pathways in the pathogenesis of PDR. The levels of angiogenesis and inflammation in PDR are correlated with kidney damage, indicating possible common pathways in diabetic retinopathy and nephropathy.

The molecular hallmarks of primary and secondary vitreoretinal lymphoma

Vitreoretinal lymphoma (VRL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) considered a variant of primary central nervous system lymphoma (PCNSL). Diagnosis of VRL requires examination of vitreous fluid, but cytologic differentiation from uveitis remains difficult. Due to its rarity and difficulty in obtaining diagnostic material, little is known about the genetic profile of VRL. The aim of our study was to investigate the mutational profile of a large series of primary and secondary VRL. Targeted next generation sequencing using a custom panel containing the most frequent mutations in PCNSL was performed on 34 vitrectomy samples of 31 patients with VRL and negative controls with uveitis. In a subset of cases, genome-wide copy number alterations (CNA) were assessed using the Oncoscan platform. Mutations in MYD88 (74%), PIM1 (71%), CD79B (55%), IGLL5 (52%), TBL1XR1 (48%), ETV6 (45%) and 9p21/CDKN2A deletions (85%) were the most common alterations, with similar frequencies in primary (15), synchronous (3) or secondary (13) VRL. This mutational spectrum is similar to MYD88mut/CD79Bmut (MCD or cluster 5) DLBCL with activation of Toll-like and B-cell receptor pathways and CDKN2A loss, confirming their close relationship. Oncoscan analysis demonstrated a high number of CNAs (mean 18.6/case). Negative controls lacked mutations or CNAs. Using cell free DNA of vitreous fluid supernatant, mutations present in cellular DNA were reliably detected in all examined cases. Mutational analysis is a highly sensitive and specific tool for the diagnosis of VRL and can also be applied successfully to cell free DNA derived from the vitreous.

Human U90926 orthologous long non-coding RNA as a novel biomarker for visual prognosis in herpes simplex virus type-1 induced acute retinal necrosis

Acute retinal necrosis (ARN) is a form of infectious uveitis caused by alpha herpesviruses, including herpes simplex virus type 1 (HSV-1). We previously found that the long non-coding RNA (lncRNA) U90926 is upregulated in murine retinal photoreceptor cells following HSV-1 infection, leading to host cell death. However, to date, an orthologous transcript has not been identified in humans. We investigated U90926 orthologous transcript in humans and examined its utility as a prognostic marker for visual acuity in patients with ARN. We identified two human orthologous transcripts (1955 and 592 bases) of lncRNA U90926. The amount of the longer human U90926 transcript was approximately 30- and 40-fold higher in the vitreous fluid of patients with ARN than in those with sarcoidosis and intraocular lymphoma, respectively. Furthermore, the expression of the longer human U90926 transcript in the vitreous fluid was highly correlated with the final best-corrected logarithm of the minimum angle of resolution visual acuity in patients with ARN (r = 0.7671, p = 0.0079). This suggests higher expression of the longer human U90926 transcript in the vitreous fluid results in worse visual prognosis; therefore, expression of the longer human U90926 transcript is a potential negative prognostic marker for visual acuity in patients with ARN.