BACKGROUND/AIMS:The neuroprotective effect of nicotinamide (NAM) supplementation has been demonstrated in both animal models and clinical trials. We conducted a trial to assess whether oral NAM improves retinal function in normal-tension glaucoma (NTG) participants receiving intraocular pressure (IOP)-lowering therapy. METHODS:Fifty-three NTG participants (untreated IOP ≤18 mm Hg) were enrolled in a double-masked, placebo-controlled crossover randomised clinical trial. Participants were randomly assigned to receive either oral placebo or NAM, followed by crossover without a washout period. Each treatment was administered for 12 weeks (NAM/placebo 1 g/day for 6 weeks, then 2 g/day for the subsequent 6 weeks). Retinal function was evaluated using full-field electroretinography and visual field testing. RESULTS:After 12 weeks, the amplitude changes of the photopic negative response in peak-to-trough (PhNRPT) and the B-wave were significantly greater in the NAM group (3.121±3.968 and 2.112±3.220 μV, respectively) compared with the placebo group (0.996±4.190 and 0.305±3.279 μV, respectively; p=0.045 and p=0.032). PhNRPT improved beyond twice the 95% coefficient of variation in 29.0% of the NAM group and 19.3% of the placebo group. No significant intergroup differences were observed in changes in mean deviation, pattern SD or visual field index after 12 weeks. CONCLUSION:Oral NAM supplementation in NTG participants induced functional improvement, as measured by PhNRPT and B-wave amplitude. Given that NTG is characterised by lower IOP, which may show a weaker correlation with electrophysiological activity and a slower progression rate compared with high-tension glaucoma, further long-term studies are needed to clarify the effects of NAM in this population. TRIAL REGISTRATION NUMBER:NCT06078605.

The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying retinal ganglion cell loss. Early reports of the selective vulnerability of larger retinal ganglion cells (RGCs) in human studies did not translate simply to the loss of a particular class of RGC but more likely reflected shrinkage and degeneration across all RGC classes. Subsequent studies of nonhuman primate (NHP) models of glaucoma indicated some selectivity with great damage to the magnocellular vs parvocellular pathways. More recently, rodent models of experimental glaucoma have highlighted a selective vulnerability of OFF-centered RGCs-particularly those with transient responses. Selectivity for OFF pathway damage is also seen as a trend in a rat model of glaucoma. These data support the concept that some RGCs are more vulnerable to the effects of glaucoma damage. This chapter covers some of the methods to elucidate RGC damage and the relevance of model selection to mimic human glaucoma rather than just RGC death.

Background: Retinal ganglion cell (RGC) loss is crucial in eye diseases like glaucoma. Axon damage and dendritic degeneration precede cell death, detectable within optical coherence tomography (OCT) resolution, indicating their correlation with neuronal degeneration. The purpose of this study is to evaluate the optical changes of early retinal degeneration. Methods: The detection of optical changes in the axotomised retinal explants was completed in six C57BL/6J mice. OCT images were acquired up to 120 min from enucleation. A grey-level co-occurrence-based texture analysis was performed on the inner plexiform layer (IPL) to monitor changes in the optical speckles using a principal component analysis (PCA) and a support vector machine (SVM). In parallel tests, retinal transparency was confirmed by a comparison of the modulation transfer functions (MTFs) at 0 and 120 min. Results: Quantitative confirmation by analysis of the MTFs confirmed the non-degradation of optical transparency during the imaging period: MTF (fx) = 0.267 ± 0.02. Textural features in the IPL could discriminate between the optical signals of RGC degeneration. The mean accuracy of the SVM classification was 86.3%; discrimination was not enhanced by the combination of the SVM and PCA (81.9%). Conclusions: Optical changes in the IPL can be detected using OCT following RGC axotomy. High-resolution OCT can provide an index of retinal neuronal integrity and its degeneration.

OBJECTIVE:To validate and update the Ocular Hypertension Treatment Study-European Glaucoma Prevention Study (OHTS-EGPS) model predicting risk of conversion from ocular hypertension (OHT) to glaucoma using electronic medical records (EMR). DESIGN:Evaluation and update of a risk prediction algorithm using EMRs and linked visual field (VF) tests. PARTICIPANTS:Newly diagnosed OHT patients attending hospital glaucoma services in England. Inclusion criteria are as follows: intraocular pressure (IOP) 22 to 32 mmHg (either eye); normal baseline VF test, defined as Glaucoma Hemifield Test (GHT) "within normal range" in a reliable VF test; at least 2 VF tests in total; no significant ocular comorbidities. METHODS:Risk factors are as follows: age, ethnicity, sex, IOP, vertical cup-to-disc ratio, central corneal thickness, VF pattern standard deviation, family history of glaucoma, systemic hypertension, diabetes mellitus, and glaucoma treatment. Glaucoma conversion was defined as 2 consecutive and reliable VF tests with GHT "outside normal limits" and/or need for glaucoma surgery. For validation, the OHTS-EGPS model was applied to predict a patient's risk of developing glaucoma in 5 years. In the updating stage, the OHTS model was refitted by re-estimating the baseline hazard and regression coefficients. The updated model was cross-validated and several variants were explored. MAIN OUTCOME MEASURES:Measures of discriminative ability (c-index) and calibration (calibration slope) were calculated and pooled across hospitals using random effects meta-analysis. RESULTS:From a total of 138 461 patients from 10 hospital glaucoma services in England, 9030 patients with OHT fitted the inclusion criteria. A total of 1530 (16.9%) patients converted to glaucoma during this follow-up period. The OHTS-EGPS model provided a pooled c-index of 0.61 (95% confidence interval: 0.60-0.63), ranging from 0.55 to 0.67 between hospitals. The pooled calibration slope was 0.45 (0.38-0.51), ranging from 0.25 to 0.64 among hospitals. The overall refitted model performed better than the OHTS-EGPS model, with a pooled c-index of 0.67 (0.65-0.69), ranging from 0.65 to 0.75 between hospitals. CONCLUSIONS:We performed an external validation of the OHTS-EGPS model in a large English population. Refitting the model achieved modest improvements in performance. Given the poor performance of the OHTS-EGPS model in our population, one should use caution in its application to populations that differ from those in the OHTS and EGPS. FINANCIAL DISCLOSURE(S):Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Background/Aims To assess the cost-effectiveness of making treatment decisions for patients with ocular hypertension (OHT) based on a risk prediction (RP) tool in the United Kingdom.Methods A discrete event simulation model was constructed to compare the cost-effectiveness of an alternative care pathway in which the treatment decision was guided by a validated RP tool in secondary care against decision-making based on the standard care (SC). Individual patient sampling was used. Patients diagnosed with OHT and with an intraocular pressure of 24 mm Hg or over entered the model with a set of predefined individual characteristics related to their risk of conversion to glaucoma. These characteristics were retrieved from electronic medical records (n=5740). Different stages of glaucoma were modelled following conversion to glaucoma.Results Almost all (99%) patients were treated using the RP strategy, and less than half (47%) of the patients were treated using the SC strategy. The RP strategy produced higher cost but also higher quality-adjusted life years (QALYs) than the SC strategy. The RP strategy was cost-effective compared with the SC strategy in the base-case analysis, with an incremental cost-effectiveness ratio value of £11 522. The RP strategy had a 96% probability of being cost-effective under a £20 000 per QALY threshold.Conclusions The use of an RP tool for the management of patients with OHT is likely to be cost-effective. However, the generalisability of the result might be limited due to the high-risk nature of this cohort and the specific RP threshold used in the study.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

Background/aimsTo elicit the preferences and calculate the willingness to pay (WTP) of patients with ocular hypertension (OHT) for eye monitoring services in the UK.MethodsPatients with OHT aged at least 18 years recruited from four NHS ophthalmology departments were included in the study. Patients’ preferences and WTP for an OHT monitoring service in the National Health Service were elicited using a discrete choice experiment (DCE) within a postal survey based on six attributes: (1) how OHT monitoring is organised, (2) monitoring frequency, (3) travel time from home, (4) use of a risk calculator for conversion to glaucoma, (5) risk of developing glaucoma in the next 10 years and (6) cost of monitoring. We used a sequential mixed-methods approach to design the survey.Results360 patients diagnosed with OHT were recruited with a mean age of 69 years. In the DCE, reducing the risk of conversion to glaucoma was the most important factor influencing respondents’ choice of monitoring service. Respondents preferred hospital-based monitoring services to community optometrist monitoring, and annual monitoring compared with more frequent (every 6 months) and less frequent (every 18 or 24 months) monitoring. These results can be monetised using WTP. Results of heterogeneity analysis suggest that patients with prior experience in community optometrist monitoring preferred this to hospital-based monitoring.ConclusionsAlthough hospital-based monitoring is generally preferred, patients with prior experience in community services have a different opinion, suggesting that patients who are unfamiliar with community optometry services may need additional support to accept monitoring in this setting.

A compromised capacity to maintain NAD pools is recognized as a key underlying pathophysiological feature of neurodegenerative diseases. NAD acts as a substrate in major cell functions including mitochondrial homeostasis, cell signalling, axonal transport, axon/Wallerian degeneration, and neuronal energy supply. Dendritic degeneration is an early marker of neuronal stress and precedes cell loss. However, little is known about dendritic structural preservation in pathologic environments and remodelling in mature neurons. Retinal ganglion cell dendritic atrophy is an early pathological feature in animal models of the disease and has been demonstrated in port-mortem human glaucoma samples. Here we report that a nicotinamide (a precursor to NAD through the NAD salvage pathway) enriched diet provides robust retinal ganglion cell dendritic protection and preserves dendritic structure in a rat model of experimental glaucoma. Metabolomic analysis of optic nerve samples from the same animals demonstrates that nicotinamide provides robust metabolic neuroprotection in glaucoma. Advances in our understanding of retinal ganglion cell metabolic profiles shed light on the energetic shift that triggers early neuronal changes in neurodegenerative diseases. As nicotinamide can improve visual function short term in existing glaucoma patients, we hypothesize that a portion of this visual recovery may be due to dendritic preservation in stressed, but not yet fully degenerated, retinal ganglion cells.

The progressive and irreversible degeneration of retinal ganglion cells (RGCs) and their axons is the major characteristic of glaucoma, a leading cause of irreversible blindness worldwide. Nicotinamide adenine dinucleotide (NAD) is a cofactor and metabolite of redox reaction critical for neuronal survival. Supplementation with nicotinamide (NAM), a precursor of NAD, can confer neuroprotective effects against glaucomatous damage caused by an age-related decline of NAD or mitochondrial dysfunction, reflecting the high metabolic activity of RGCs. However, oral supplementation of drug is relatively less efficient in terms of transmissibility to RGCs compared to direct delivery methods such as intraocular injection or delivery using subconjunctival depots. Neither method is ideal, given the risks of infection and subconjunctival scarring without novel techniques. By contrast, extracellular vesicles (EVs) have advantages as a drug delivery system with low immunogeneity and tissue interactions. We have evaluated the EV delivery of NAM as an RGC protective agent using a quantitative assessment of dendritic integrity using DiOlistics, which is confirmed to be a more sensitive measure of neuronal health in our mouse glaucoma model than the evaluation of somatic loss via the immunostaining method. NAM or NAM-loaded EVs showed a significant neuroprotective effect in the mouse retinal explant model. Furthermore, NAM-loaded EVs can penetrate the sclera once deployed in the subconjunctival space. These results confirm the feasibility of using subconjunctival injection of EVs to deliver NAM to intraocular targets.

Disease-modifying treatments for Alzheimer’s disease and related dementias (ADRD) will be most effective early in the disease process. Clinical use of these therapies will require practical, widely accessible biomarkers. Plasma-based protein biomarkers hold promise for identifying core AD pathology, but this complex disease likely requires other markers to identify people at highest risk of progression to dementia. Studies support eye exams as a synergistic strategy since retinal imaging would be cost effective, but significant hurdles remain. The timing and subtypes of retinal biomarkers have not yet been determined and the identification of ADRD-specific biomarkers independent of common ocular diseases is challenging. Here, we introduce a new program, Model-EyeD, to evaluate the eye as an accessible biomarker in the mouse model and human subjects. For familial AD and mixed dementia, we have backcrossed the APP SAA knock-in allele (humanized Abeta with Swedish, Artic, Austrian, mutations) from C57BL/6J (B6) to genetically diverse NZO/HILtJ (prone to obesity and diabetes), and WSB/EiJ (susceptible to cerebrovascular deficits) strains. To study late-onset AD (LOAD), we are using strains created by the IU/JAX/PITT MODEL-AD Center, including the common 677C>T variant in methylene tetrahydrofolate reductase gene ( Mthfr 677C>T ), a cardiovascular and cerebrovascular risk factor. A battery of ocular assays (pattern electroretinograms, fluorescence angiography, optical coherence tomography) are performed on four to 24 months old male and female mice. Results are being compared to MRI, PET/CT, cognition and blood biomarkers. Unlike previous transgenic amyloid models, B6. APP SAA and WSB. APP SAA mice show no retinal amyloid deposition or retinal vascular deficits at 12 months. NZO (in the absence of amyloid deposition) show retinopathy lesions (cotton wool spots, vascular leakage), suggesting NZO. APP SAA are ideal to study interactions between metabolic syndrome retinopathy and mixed dementia. Young B6. Mthfr 677C>T mice show cerebral and retinal vascular deficits. Aging studies are ongoing. Results will be confirmed in human subjects with clinical and biomarker-confirmed AD and mixed AD/small vessel vascular disease. Our extensive collection of genetically diverse mouse models, coupled with our alignment to data from human subjects, show promise in developing eye-based protocols to identify early stages of different clinical phenotypes of ADRD in humans.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer's disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with β-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

This study is about the quantification and validation of BDNF levels in mouse serum and plasma using a sensitive immunoassay. While BDNF levels are readily detectable in human serum, the functional implications of these measurements are unclear as BDNF released from human blood platelets is the main contributor to the serum levels of BDNF. As mouse platelets do not contain BDNF, this confounding factor is absent in the mouse. Accordingly, BDNF levels in mouse serum and plasma were found to be indistinguishable at 9.92 ± 1.97 pg/mL for serum and 10.58 ± 2.43 pg/mL for plasma ( p = 0.473). These levels are approximately a thousand times lower than those measured in human serum and pre-adsorption with anti-BDNF, but not with anti-NGF or anti-NT3 monoclonal antibodies, markedly reduced the BDNF signal. These results open the possibility to explore the relevance of BDNF levels as a biomarker in accessible body fluids using existing mouse models mimicking human pathological conditions.

classifi cation in retinal degenerative diseases where the dendritic architecture is altered remains problematic

Unlike humans, mice lack brain-derived neurotrophic factor in their platelets. Want et al. used a transgenic mouse model, engineered to express brain-derived neurotrophic factor in blood platelets and demonstrate significant neuroprotection for lesioned retinal ganglion cells compared with wild-type mice. In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using 'humanized' mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 +/- 11.36 ng/mL for homozygous and 17.02 +/- 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 +/- 258, 1776 +/- 435 and 1763 +/- 256 versus 1406 +/- 315 in the wild-type control group (P <= 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing similar to 15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 +/- 690 and 1921 +/- 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing similar to 50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

Gene gun DiOlistic labelling enables the detailed visualization of retinal ganglion cells (RGCs) dendritic structure. Since the level of labelling is independent of cellular health, it is useful for the characterization of neuronal structure in degenerating neurons where expressed reporters may be inadequate. The method uses compressed helium gas to fire tungsten or gold microparticles coated in carbocyanine dyes (DiD, DiI, DiO) into flat mounted retinas. Here we describe the methods to optimize labelling and ensure a high yield of adequately labelled cells, with a focus on retinal ganglion cells.