Functioning of the central nervous system is critically dependent on precise blood delivery achieved through the formation of blood flow patterns or differences in flux between vessels. Nevertheless, how these blood flow patterns are generated is a fundamental question that is incompletely understood. Here, we show, in live imaging experiments on mice, that specialized pericytes connected via tunneling nanotubes regulate flux patterns by modulating the response of different capillaries in opposing ways, homogenizing blood flow, which is vital to supply functionally-opposite neuronal areas of the retina. We also identify that this capacity for stimulus-induced flow homogenization is lost after the ablation of interpericyte tunneling nanotubes via laser or during ischemia-reperfusion injury, a pathological condition that leads to widespread disruption of interpericyte tunneling nanotubes. These findings contribute to understanding neurovascular coupling and blood delivery in the retina.

Preclinical studies of optic nerve injury models have led to significant insight into the mechanism underlying retinal ganglion cell neurodegeneration. During the process of ganglion cell injury, morphological changes can occur prior to gross structural changes and cell death. Similarly, following injury, functional changes can occur in the absence of substantive structural changes. These more subtle effects can often be detected using functional tools such as the electroretinogram. Moreover, the electroretinogram is a sensitive and complementary means to quantify treatment efficacy. Here, we describe in vivo electroretinography for assessing ganglion cell injury in rodent models.

To characterize how optic nerve tissue sheath (ONSp) responds to changes in intracerebral fluid infusion rate and consider if low rate intracerebral ventricular (ICV) infusion changes diurnal intracranial pressure (ICP) in rats. Anesthetized adult male rats underwent cannula placement into the lateral ventricles, for infusion and ICP monitoring. ICV infusion rate controlled ICP. A second syringe pump delivered sodium fluorescein for quantification of cerebrospinal fluid (CSF) flow to the optic nerve with fluorescence imaging (n = 14). Cannula were placed in lateral ventricle and under the optic nerve sheath (n = 6) for simultaneous pressure measurement at different ICV infusion rates, and at normal (n = 6, 95.6 ± 5.9mmHg) or high blood pressure (n = 4, 158.0 ± 9.5mmHg, intravenous Angiotensin II). Four rats were implanted with sub-dural sensors for awake ICP monitoring. Animals had ICV infusion of sterile saline at a low rate (2.5 ml/hr) for the first 4 weeks followed by a high rate (10 ml/hr) for week 5. Higher ICV flow rates produce predictable increases in ICP, which result in an increased ONSp. CSF pressure was 73 ± 3

Glaucoma, characterized by the loss of retinal ganglion cells (RGCs), is a leading cause of blindness for which there are no neuroprotective therapies. To explore observations of elevated homocysteine in glaucoma, we elevate vitreous homocysteine, which increases RGC death by 6% following ocular hypertension. Genetic association with higher homocysteine does not affect glaucoma-associated outcomes from the UK Biobank and serum homocysteine levels have no effect on glaucomatous visual field progression. This supports a hypothesis in which elevated homocysteine is a pathogenic, rather than causative, feature of glaucoma. Further exploration of homocysteine metabolism in glaucoma animal models demonstrates early and sustained dysregulation of genes involved in one-carbon metabolism and the interaction of essential cofactors and precursors (B6, B9, B12, and choline) in whole retina and optic nerve head and RGCs. Supplementing these provides neuroprotection in an acute model and prevents neurodegeneration and protects visual function in a chronic model of glaucoma.

The high metabolic demand of retinal neurons requires a precisely regulated vascular system that can deliver rapid changes in blood flow in response to neural need. In the retina, this is achieved via the action of a coordinated group of cells that form the neurovascular unit. While cells such as pericytes, Müller cells, and astrocytes have long been linked to neurovascular coupling, more recently the resident microglial population have also been implicated. In the healthy retina, microglia make extensive contact with blood vessels, as well as neuronal synapses, and are important in vascular patterning during development. Work in the brain and retina has recently indicated that microglia can directly regulate the local vasculature. In the retina, the fractalkine-Cx3cr1 signalling axis has been shown to induce local capillary constriction within the superficial vascular plexus via a mechanism involving components of the renin-angiotensin system. Furthermore, aberrant microglial induced vasoconstriction may be at the centre of early vascular reactivity changes observed in those with diabetes. This review summarizes the recent emerging evidence that microglia play multiple roles in retinal homeostasis especially in regulating the vasculature. We highlight what is known about the role of microglia under normal circumstances, and then build on this to discuss how microglia contribute to early vascular compromise during diabetes. Further understanding of the mechanisms of microglial-vascular regulation may allow alternate treatment strategies to be devised to reduce vascular pathology in diseases such as diabetic retinopathy.

SIGNIFICANCE:Previous evidence showed that transient receptor potential vanilloid 4 (TRPV4) inhibition was protective of retinal ganglion cell (RGC) loss after chronic intraocular pressure (IOP) elevation in young animals. However, the role of TRPV4 in mechanosensing IOP changes in the aging eye is not well understood. PURPOSE:This study compared the recovery of retinal function and structure after acute IOP elevation in 3- and 12-month-old mouse eyes with and without TRPV4 inhibition. METHODS:We examined retinal TRPV4 expression in 2-month-old rodent eyes using immunohistochemistry and transcript analysis of isolated macroglia and RGCs. To modulate TRPV4, mice were treated daily with either vehicle or a TRPV4 antagonist (HC-067047 10 mg/kg) delivered intraperitoneally for 7 days before and 7 days after IOP elevation (50 mmHg for 30 minutes). Retinal function and structure were assessed using dark-adapted full-field electroretinography and optical coherence tomography, respectively. RESULTS:We showed that Müller cells strongly expressed TRPV4. Seven days after IOP elevation, RGC functional recovery was significantly poorer in older mice treated with TRPV4 antagonist compared with age-matched vehicle controls (-54 ± 7% vs. -24 ± 10%, p=0.046) and their younger TRPV4 antagonist-treated counterparts (-5 ± 5%, p<0.001). CONCLUSIONS:This study showed that there was an age-related deficit in RGC functional recovery from IOP elevation with TRPV4 inhibition.

Introduction:To consider how blood pressure and intracranial pressure modify the way that retinal function responds to intraocular pressure elevation in rats. Methods:Six groups of adult Long-Evans rats (n = 7-11 eyes/group, total animals 25) were anesthetized and underwent acute pressure modification. Blood pressure (BP) was measured via a femoral artery cannula and elevated by angiotensin II infusion into the femoral vein. Intracranial pressure (ICP) was set to 0 mmHg, 5 mmHg, or 25 mmHg in three separate groups of rats via a cannula in the lateral ventricle. At each ICP (-5 mmHg, 5 mmHg, or 25 mmHg) and BP setting (normal or high), intraocular pressure (IOP) was increased from 10 mmHg to 90 mmHg in 10 mmHg steps. At each IOP level, ganglion retinal function was assessed using the electroretinogram. Results:Compared with normal blood pressure groups, animals with high blood pressure had significantly smaller baseline ganglion cell-mediated scotopic threshold responses (STR). Animals with high ICP had larger scotopic threshold response (STR) amplitudes than the normal and low ICP groups. Both high BP and high ICP rendered retinal function less susceptible to IOP elevation; however, the effect was greater for high BP. Conclusion:Retinal function is critically dependent on ocular perfusion pressure; excessive low or high perfusion attenuates function. The ocular perfusion pressure (BP-IOP) relationship largely accounts for the effect of IOP and BP modulation on retinal function but could not account for differences in ganglion cell function between ICP levels.

Age-related neuronal adaptations are known to help maintain function. This study aims to examine gross age-related in vivo retinal functional adaptations (using electroretinography) in young and middle aged C57BL/6J and Thy1-YFPh mice and to relate this to in vivo retinal structure (using optical coherence tomography). Electroretinography responses were generally larger in Thy1-YFPh mice than in C57BL/6J mice, with similar in vivo retinal layer thicknesses except for longer inner/outer photoreceptor segment in Thy1-YFPh mice. Relative to 3-month-old mice, 12-month-old mice showed reduced photoreceptor (C57BL/6J 84.0±2.5 %; Thy1-YFPh 80.2±5.2 %) and bipolar cell (C57BL/6J 75.6±2.3 %; Thy1-YFPh 68.1±5.5 %) function. There was relative preservation of ganglion cell function (C57BL/6J 79.7±3.7 %; Thy1-YFPh 91.7±5.0 %) with age, which was associated with increased b-wave (bipolar cell) sensitivities to light. Ganglion cell function was correlated with both b-wave amplitude and sensitivity. This study shows that there are normal age-related adaptations to preserve functional output. Different mouse strains may have varied age-related adaptation capacity and should be taken into consideration when examining age-related susceptibility to injury.

Myopia (short-sightedness) is the most common ocular disorder. It generally develops after over-exposure to aberrant visual environments, disrupting emmetropization mechanisms that should match eye growth with optical power. A pre-screening of strongly associated myopia-risk genes identified through human genome-wide association studies implicates efemp1 in myopia development, but how this gene impacts ocular growth remains unclear. Here, we modify efemp1 expression specifically in the retina of zebrafish. We found that under normal lighting, efemp1 mutants developed axial myopia, enlarged eyes, reduced spatial vision and altered retinal function. However, under myopia-inducing dark-rearing, compared to control fish, mutants remained emmetropic and showed changes in retinal function. Efemp1 modification changed the expression of efemp1, egr1, tgfb1a, vegfab and rbp3 genes in the eye, and changed the inner retinal distributions of myopia-associated EFEMP1, TIMP2 and MMP2 proteins. Efemp1 modification also impacted dark-rearing- induced responses of vegfab and wnt2b genes and above-mentioned myopia-associated proteins. Together, we provided robust evidence that light-dependent ocular growth is regulated by efemp1.

Pathogenic variants in HCN1 causing cation leak result in a severe developmental and epileptic encephalopathy (DEE). Current treatment options for patients with HCN1 -DEE are limited and are insufficient to fully address both the seizures and clinical comorbidities of this disorder. Org 34167 is a brain penetrant broad-spectrum HCN channel inhibitor that has completed phase I clinical trials. We used a range of assays at molecular, cellular, network and behavioural levels to explore the potential of Org 34167 as a precision medicine for HCN1 -DEE. Org 34167 restored the voltage sensitivity of the DEE HCN1M305L mutated channel, significantly reducing cation leak. It also restored Ih-mediated ‘sag’, hyperpolarised the resting membrane potential and reduced firing of layer V neurons from the Hcn1M294L mouse model of HCN1 -DEE, which was engineered based on the HCN1M305L pathogenic variant. Additionally, Org 34167 reduced neuronal epileptiform activity and restored retinal light sensitivity in these mice, suggesting it may improve both seizures and other clinical comorbidities. However, Org 34167-mediated tremors were noted at therapeutic doses. Org 34167 was also effective at reducing cation leak caused by five additional HCN1 pathogenic variants, suggesting broader utility. Overall, these data demonstrate that a small molecule HCN inhibitor can restore channel and consequent physiological functions, positioning it as a promising precision therapeutic approach for HCN1 -DEE. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations aCSF : Artificial cerebrospinal fluid AP : Action potential ASM : Anti-seizure medication AUC : Area under the curve DEE : Developmental and epileptic encephalopathy ECoG : Electrocorticography ERG : Electroretinography HCN : Hyperpolarisation-activated, Cyclic Nucleotide-gated channel HEK : Human embryonic kidney Ih : Hyperpolarisation-activated current i.p. : Intraperitoneal IV : Current-voltage SEM : Standard error of the mean V0.5 : Half-maximal activation voltage WT : Wild-type

Pregnancy introduces a multitude of changes in the body, including hormonal fluctuations and metabolic changes, which can lead to atypical ocular signs and symptoms. Ocular manifestations range from fluctuations in vision, to microstructural changes in the retina and choroid, to dry eye disease. This narrative review highlights the range of pregnancy-related effects on the eye and vision that are likely to present in the context of routine eyecare. Specifically, physiological ocular changes and pathological ocular changes that manifest for the first time, or are exacerbated, in uncomplicated pregnancy are discussed. The literature has evolved from simply noting differences in the eye between pregnant and non-pregnant groups, to refining knowledge of the proposed underlying pathophysiology with the advent of newer technologies in eyecare. A particular focus of this review is navigating when pregnancy changes in the eye occur or peak during the gestational period, and whether the changes are short-lived or might extend past pregnancy. While many pregnancy-associated changes are temporary and resolve post-partum, it is also recognised that some changes persist after pregnancy, with a notable absence of literature on ocular changes with loss or termination of pregnancy. Currently or previously pregnant women (or those planning to become pregnant), and other health professionals, should be educated about the importance of seeking eyecare before, during and after pregnancy.

Myopia is predicted to impact approximately 5 billion people by 2050, necessitating mechanistic understanding of its development. Myopia results from dysregulated genetic mechanisms of emmetropization, caused by over-exposure to aberrant visual environments; however, these genetic mechanisms remain unclear. Recent human genome-wide association studies have identified a range of novel myopia-risk genes. To facilitate large-scale in vivo mechanistic examination of gene-environment interactions, this study aims to establish a myopia model platform that allows efficient environmental and genetic manipulations. We established an environmental zebrafish myopia model by dark-rearing. Ocular biometrics including relative ocular refraction were quantified using optical coherence tomography images. Spatial vision was assessed using optomotor response (OMR). Retinal function was analyzed via electroretinography (ERG). Myopia-associated molecular contents or distributions were examined using RT-qPCR or immunohistochemistry. Our model produces robust phenotypic changes, showing myopia after 2 weeks of dark-rearing, which were recoverable within 2 weeks after returning animals to normal lighting. 2-week dark-reared zebrafish have reduced spatial-frequency tuning function. ERG showed reduced photoreceptor and bipolar cell function (a- and b-waves) after only 2 days of dark-rearing, which worsened after 2 weeks of dark-rearing. We also found dark-rearing-induced changes to expression of myopia-risk genes, including egr1, vegfaa, vegfab, rbp3, gjd2a and gjd2b, inner retinal distribution of EFEMP1, TIMP2 and MMP2, as well as transiently reduced PSD95 density in the inner plexiform layer. Coupled with the gene editing tools available for zebrafish, our environmental myopia model provides an excellent platform for large-scale investigation of gene-environment interactions in myopia development.

Retinal hyperspectral imaging (HSI) is a non-invasive in vivo approach that has shown promise in Alzheimer’s disease. Parkinson’s disease is another neurodegenerative disease where brain pathobiology such as alpha-synuclein and iron overaccumulation have been implicated in the retina. However, it remains unknown whether HSI is altered in in vivo models of Parkinson’s disease, whether it differs from healthy aging, and the mechanisms which drive these changes. To address this, we conducted HSI in two mouse models of Parkinson’s disease across different ages; an alpha-synuclein overaccumulation model (hA53T transgenic line M83, A53T) and an iron deposition model (Tau knock out, TauKO). In comparison to wild-type littermates the A53T and TauKO mice both demonstrated increased reflectivity at short wavelengths ~ 450 to 600 nm. In contrast, healthy aging in three background strains exhibited the opposite effect, a decreased reflectance in the short wavelength spectrum. We also demonstrate that the Parkinson’s hyperspectral signature is similar to that from an Alzheimer’s disease model, 5xFAD mice. Multivariate analyses of HSI were significant when plotted against age. Moreover, when alpha-synuclein, iron or retinal nerve fibre layer thickness were added as a cofactor this improved the R2 values of the correlations in certain groups. This study demonstrates an in vivo hyperspectral signature in Parkinson’s disease that is consistent in two mouse models and is distinct from healthy aging. There is also a suggestion that factors including retinal deposition of alpha-synuclein and iron may play a role in driving the Parkinson’s disease hyperspectral profile and retinal nerve fibre layer thickness in advanced aging. These findings suggest that HSI may be a promising translation tool in Parkinson’s disease.

Purpose: To investigate neurovascular function in eyes with age-related macular degeneration (AMD). Methods: Subjects with bilateral large drusen (intermediate AMD) and healthy controls >= 50 years old were recruited. The vasculature within the central 6 x 6-mm retinal area was captured using optical coherence tomography angiography (OCTA) and segmented to return superficial plexus, deep plexus, choriocapillaris, and choroid. OCTA scans were acquired without flicker light stimulation (conventional OCTA) and during flicker light stimulation to increase retinal activity and metabolic demand (functional OCTA). Vascular area density (VAD) and the vascular reactivity index (VRI; change in VAD induced by flicker stimulation) were determined and compared between control and AMD eyes. Results: Thirty-five subjects (19 AMD cases and 16 healthy controls) participated in the study. In healthy eyes, flicker stimulation induced an increase in VAD (positive VRI, vasodilation) in the superficial plexus (P < 0.001) and deep plexus (P < 0.001). There was a trend for increased VAD in the choriocapillaris (P = 0.077), but there was no change in the choroid (P = 0.654). In AMD eyes, there was no change in VAD in response to flicker stimulation in any of the vascular layers examined (P >= 0.294). Linear mixed models confirmed that AMD was associated with a reduced VRI in the superficial plexus (P < 0.001) and deep plexus (P < 0.001). Conclusions: Eyes with large drusen show a reduction in retinal vascular reactivity compared to healthy eyes, which suggests that there is impairment of retinal neurovascular function in intermediate AMD. Translational Relevance: Functional OCTA could be used to study neurovascular function in retinal diseases.