The effect of lumbar medial branch radiofrequency neurotomy on cobb angle progression in individuals with adult scoliosis compared to natural history: A cross-sectional study.

Background: Lumbar radiofrequency neurotomy (LRFN) effectively alleviates zygapophyseal joint-mediated pain by coagulating medial branch nerves to disrupt nociceptive signaling pathways. The concomitant denervation of multifidus fibers has led to concern that LRFN may increase segmental instability and accelerate degenerative changes in patients with certain pre-existing spinal pathologies. There is a paucity of literature evaluating whether LRFN increases the progression of spinal curvature in patients with adult scoliosis. Objective: Compare the lumbosacral Cobb angle progression rate in patients with adult scoliosis who underwent LRFN to the annual progression rate of 0.83 ± 1.1° expected by natural history. Design: Cross-sectional study. Methods: Consecutive patients diagnosed with adult scoliosis who underwent LRFN to treat zygapophyseal joint-related low back pain were identified. Patient demographics, LRFN procedure details, and radiographs confirming scoliosis were collected from electronic medical records. Pre- and post-LRFN radiographs were used to calculate the average annual rate of Cobb angle progression. Data were analyzed using a Wilcoxon signed-rank test and a linear regression model. Results: Sixty patients (mean age 69.2 ± 11.6 years; 70.0 % female) met the criteria and were included in the analyses. The mean time to radiographic follow-up was 35.0 ± 22.7 months post-LRFN. The average Cobb angle progression was 0.54 ± 3.03° per year and did not differ significantly from the known natural progression rate of 0.83 ± 1.1° per year. None of the included covariates (body mass index, LRFN laterality, and number of levels denervated) were significantly associated with the average annual Cobb angle progression rate. Conclusions: Our results suggest that LRFN has no appreciable effect on the rate of Cobb angle progression in patients with adult scoliosis.

The association of payer type on genicular radiofrequency neurotomy treatment outcomes: Results of a cross-sectional study.

Background: Genicular radiofrequency neurotomy (GRFN) is an effective treatment for a subset of individuals with chronic knee pain. Previous studies demonstrate that Medicare and Medicaid beneficiaries report worse outcomes following various interventional procedures compared with commercially insured patients. Objective: Evaluate the association of payer type on GRFN treatment outcomes. Methods: Consecutive patients who underwent GRFN at a tertiary academic center were contacted for participation. Demographic, clinical, and procedural characteristics were collected from electronic medical records. Outcome data were collected by standardized telephone survey at 6-12 months, 12-24 months and ≥24 months. Treatment success was defined as ≥50% numerical pain rating scale (NPRS) score reduction from baseline. Data were analyzed using descriptive statistics for demographic, clinical, and procedural characteristics. Logistic and Poisson regression analyses were performed to examine the association of variables of interest and pain reduction. Results: One hundred thirty-four patients treated with GRFN (mean 65.6 ± 12.7 years of age, 59.7% female) with a mean follow-up time of 23.3 ± 11.3 months were included. Payer type composition was 48.5% commercial (n = 65), 45.5% Medicare (n = 61), 3.7% Medicaid (n = 5), 1.5% government (n = 2), and 0.8% self-pay (n = 1). Overall, 47.8% of patients (n = 64) reported ≥50% NPRS score reduction after GRFN. After adjusting for age, follow-up duration, Kellgren-Lawrence osteoarthritis grade, baseline opioid use, antidepressant/antianxiety medication use, history of knee replacement, and number of RFN lesions placed, the logistic regression model showed no statically significant association between payer type and treatment outcome (OR = 2.11; 95% CI = 0.87, 5.11; p = 0.098). Discussion/conclusion: In this study, after adjusting for demographic, clinical, and procedural characteristics, we found no association between payer type and treatment success following GRFN. This observation contrasts findings from other interventional studies reporting an association between payer category and treatment success.

Relaxation of mitochondrial hyperfusion in the diabetic retina via N6-furfuryladenosine confers neuroprotection regardless of glycaemic status.

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.

Genicular nerve radiofrequency ablation for the treatment of chronic knee joint pain: a real-world cohort study with evaluation of prognostic factors.

BACKGROUND: Genicular nerve radiofrequency ablation (GNRFA) is an effective treatment for chronic knee pain. However, there has been minimal investigation of real-world, long-term outcomes and factors that predict treatment success after GNRFA. OBJECTIVES: To evaluate the effectiveness of GNRFA for chronic knee pain in a real-world population and identify predictive factors. METHODS: Consecutive patients who underwent GNRFA at a tertiary academic center were identified. Demographic, clinical, and procedural characteristics were collected from the medical record. Outcome data were numeric rating scale (NRS) pain reduction and Patient Global Impression of Change (PGIC). Data were collected by standardized telephone survey. Predictors of success were evaluated with logistic and Poisson regression analyses. RESULTS: Of the 226 total patients identified, 134 (65.6 ± 12.7; 59.7% female) were successfully contacted and analyzed, with a mean follow-up time of 23.3 ± 11.0 months. Of those, 47.8% (n = 64; 95% CI: 39.5%-56.2%) and 61.2% (n = 82; 95% CI: 52.7%-69.0%) reported ≥50% NRS score reduction and ≥2-point NRS score reduction, respectively, and 59.0% (n = 79; 95% CI: 50.5%-66.9%) reported "much improved" on the PGIC questionnaire. Factors associated with a greater likelihood of treatment success (P < .05) were higher Kellgren-Lawrence osteoarthritis grade (2-4 vs 0-1); no baseline opioid, antidepressant, or anxiolytic medication use; and >3 nerves targeted. CONCLUSION: In this real-world cohort, approximately half of the participants experienced clinically meaningful improvements in knee pain after GNRFA at an average follow-up time of nearly 2 years. Factors associated with higher likelihood of treatment success were more advanced osteoarthritis (Kellgren-Lawrence Grade 2-4); no opioid, antidepressant, or anxiolytic medication use; and >3 nerves targeted.

Aldehyde Dehydrogenase and Aldo-Keto Reductase Enzymes: Basic Concepts and Emerging Roles in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a complication of diabetes mellitus that can lead to vision loss and blindness. It is driven by various biochemical processes and molecular mechanisms, including lipid peroxidation and disrupted aldehyde metabolism, which contributes to retinal tissue damage and the progression of the disease. The elimination and processing of aldehydes in the retina rely on the crucial role played by aldehyde dehydrogenase (ALDH) and aldo-keto reductase (AKR) enzymes. This review article investigates the impact of oxidative stress, lipid-derived aldehydes, and advanced lipoxidation end products (ALEs) on the advancement of DR. It also provides an overview of the ALDH and AKR enzymes expressed in the retina, emphasizing their growing importance in DR. Understanding the relationship between aldehyde metabolism and DR could guide innovative therapeutic strategies to protect the retina and preserve vision in diabetic patients. This review, therefore, also explores various approaches, such as gene therapy and pharmacological compounds that have the potential to augment the expression and activity of ALDH and AKR enzymes, underscoring their potential as effective treatment options for DR.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Does medial branch radiofrequency neurotomy accelerate degenerative lumbar spondylolisthesis compared to natural progression? A cross-sectional cohort study.

Background: Lumbar radiofrequency neurotomy (LRFN) effectively treats lumbar zygapophyseal joint pain by coagulating medial branch nerves (MBNs) carrying nociceptive signals. MBNs also innervate deep paraspinal muscles. There is a paucity of literature on whether LRFN accelerates the progression of vertebral displacement in patients with degenerative lumbar spondylolisthesis (DLS). Objective: Compare the rate of spondylolisthesis progression in adults with DLS who underwent LRFN to the 2% annual rate of progression expected by natural history. Design: Cross-sectional cohort study. Methods: Consecutive patients with pre-existing DLS who underwent LRFN for zygapophyseal joint-mediated low back pain were identified. Patient demographics, LRFN procedure details, and radiographic images confirming Meyerding Grade (I-II) spondylolisthesis were collected from electronic medical records. The quantitative magnitude of spondylolisthesis progression and the annualized rate were calculated from pre-and post-LRFN radiographs. Data were analyzed using Wilcoxon signed-rank tests and a linear regression model. Results: 152 patients (mean age 65.9 ± 12.3 years; 59.2% female) met eligibility criteria and were included in the analyses. Average time to radiographic follow-up was 35.6 ± 24.7 months post-LRFN. The average spondylolisthesis progression rate of 1.63 ± 2.91% per year calculated for the LRFN cohort was significantly lower than the 2% annual rate of progression associated with natural history (p < 0.001). None of the included covariates, such as age, BMI, LRFN laterality, number of levels denervated, or history of prior lumbar spinal surgery, were significantly associated with the average annual rate of progression. Conclusions: Our results suggest that spondylolisthesis progression rate is no different or worse than the expected natural progression rate in patients with pre-existing DLS who have undergone LRFN.

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch­dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch­dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

Endogenous Mas-related G-protein-coupled receptor X1 activates and sensitizes TRPA1 in a human model of peripheral nerves.

Mas-related G-protein-coupled receptor X1 (MrgprX1) is a human-specific Mrgpr and its expression is restricted to primary sensory neurons. However, its role in nociception and pain signaling pathways is largely unknown. This study aims to investigate a role for MrgprX1 in nociception via interaction with the pain receptor, Transient Receptor Potential Ankyrin 1 (TRPA1), using in-vitro and in-vivo human neuronal models. MrgprX1 protein expression in human trigeminal nociceptors was investigated by the immunolabeling of the dental pulp and cultured peripheral neuronal equivalent (PNE) cells. MrgprX1 receptor signaling was monitored by Fura-2-based Ca2+ imaging using PNEs and membrane potential responses were measured using FluoVoltTM . Immunofluorescent staining revealed MrgprX1 expression in-vivo in dental afferents, which was more intense in inflamed compared to healthy dental pulps. Endogenous MrgprX1 protein expression was confirmed in the in-vitro human PNE model. MrgprX1 receptor signaling and the mechanisms through which it couples to TRPA1 were studied by Ca2+ imaging. Results showed that MrgprX1 activates TRPA1 and induces membrane depolarization in a TRPA1 dependent manner. In addition, MrgprX1 sensitizes TRPA1 to agonist stimulation via Protein Kinase C (PKC). The activation and sensitization of TRPA1 by MrgprX1 in a model of human nerves suggests an important role for this receptor in the modulation of nociception.

Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

Ion channels and myogenic activity in retinal arterioles.

Retinal pressure autoregulation is an important mechanism that protects the retina by stabilizing retinal blood flow during changes in arterial or intraocular pressure. Similar to other vascular beds, retinal pressure autoregulation is thought to be mediated largely through the myogenic response of small arteries and arterioles which constrict when transmural pressure increases or dilate when it decreases. Over recent years, we and others have investigated the signaling pathways underlying the myogenic response in retinal arterioles, with particular emphasis on the involvement of different ion channels expressed in the smooth muscle layer of these vessels. Here, we review and extend previous work on the expression and spatial distribution of the plasma membrane and sarcoplasmic reticulum ion channels present in retinal vascular smooth muscle cells (VSMCs) and discuss their contribution to pressure-induced myogenic tone in retinal arterioles. This includes new data demonstrating that several key players and modulators of the myogenic response show distinctively heterogeneous expression along the length of the retinal arteriolar network, suggesting differences in myogenic signaling between larger and smaller pre-capillary arterioles. Our immunohistochemical investigations have also highlighted the presence of actin-containing microstructures called myobridges that connect the retinal VSMCs to one another. Although further work is still needed, studies to date investigating myogenic mechanisms in the retina have contributed to a better understanding of how blood flow is regulated in this tissue. They also provide a basis to direct future research into retinal diseases where blood flow changes contribute to the pathology.

The Role of Lipoxidation in the Pathogenesis of Diabetic Retinopathy.

Lipids can undergo modification as a result of interaction with reactive oxygen species (ROS). For example, lipid peroxidation results in the production of a wide variety of highly reactive aldehyde species which can drive a range of disease-relevant responses in cells and tissues. Such lipid aldehydes react with nucleophilic groups on macromolecules including phospholipids, nucleic acids, and proteins which, in turn, leads to the formation of reversible or irreversible adducts known as advanced lipoxidation end products (ALEs). In the setting of diabetes, lipid peroxidation and ALE formation has been implicated in the pathogenesis of macro- and microvascular complications. As the most common diabetic complication, retinopathy is one of the leading causes of vision loss and blindness worldwide. Herein, we discuss diabetic retinopathy (DR) as a disease entity and review the current knowledge and experimental data supporting a role for lipid peroxidation and ALE formation in the onset and development of this condition. Potential therapeutic approaches to prevent lipid peroxidation and lipoxidation reactions in the diabetic retina are also considered, including the use of antioxidants, lipid aldehyde scavenging agents and pharmacological and gene therapy approaches for boosting endogenous aldehyde detoxification systems. It is concluded that further research in this area could lead to new strategies to halt the progression of DR before irreversible retinal damage and sight-threatening complications occur.

Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy.

Mitochondrial quality control (MQC) is crucial for regulating CNS homeostasis, and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human postmortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide therapeutic options for the management of DR-associated complications.

Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis.

Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis. Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording. Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model. Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

Intravitreal AAV2.COMP-Ang1 Attenuates Deep Capillary Plexus Expansion in the Aged Diabetic Mouse Retina.

Purpose: We determine whether intravitreal angiopoietin-1 combined with the short coiled-coil domain of cartilage oligomeric matrix protein by adeno-associated viral serotype 2 (AAV2.COMP-Ang1) delivery following the onset of vascular damage could rescue or repair damaged vascular beds and attenuate neuronal atrophy and dysfunction in the retinas of aged diabetic mice. Methods: AAV2.COMP-Ang1 was bilaterally injected into the vitreous of 6-month-old male Ins2Akita mice. Age-matched controls consisted of uninjected C57BL/6J and Ins2Akita males, and of Ins2Akita males injected with PBS or AAV2.REPORTER (AcGFP or LacZ). Retinal thickness and visual acuity were measured in vivo at baseline and at the 10.5-month endpoint. Ex vivo vascular parameters were measured from retinal flat mounts, and Western blot was used to detect protein expression. Results: All three Ins2Akita control groups showed significantly increased deep vascular density at 10.5 months compared to uninjected C57BL/6J retinas (as measured by vessel area, length, lacunarity, and number of junctions). In contrast, deep microvascular density of Ins2Akita retinas treated with AAV2.COMP-Ang1 was more similar to uninjected C57BL/6J retinas for all parameters. However, no significant improvement in retinal thinning or diabetic retinopathy-associated visual loss was found in treated diabetic retinas. Conclusions: Deep retinal microvasculature of diabetic Ins2Akita eyes shows late stage changes consistent with disorganized vascular proliferation. We show that intravitreally injected AAV2.COMP-Ang1 blocks this increase in deep microvascularity, even when administered subsequent to development of the first detectable vascular defects. However, improving vascular normalization did not attenuate neuroretinal degeneration or loss of visual acuity. Therefore, additional interventions are required to address neurodegenerative changes that are already underway.

CAMKII as a therapeutic target for growth factor-induced retinal and choroidal neovascularization.

While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti­VEGF therapy include the upregulation of other proangiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signaling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor-induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and -δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularization in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signaling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularization, while enhancing reparative angiogenesis in the ischemic retina.

Müller glial dysfunction during diabetic retinopathy in rats is reduced by the acrolein-scavenging drug, 2-hydrazino-4,6-dimethylpyrimidine.

AIMS/HYPOTHESIS: Recent studies suggest that abnormal function in Müller glial cells plays an important role in the pathogenesis of diabetic retinopathy. This is associated with the selective accumulation of the acrolein-derived advanced lipoxidation end-product, Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine), on Müller cell proteins. The aim of the current study was to identify more efficacious acrolein-scavenging drugs and determine the effects of the most potent on Müller cell FDP-lysine accumulation and neuroretinal dysfunction during diabetes. METHODS: An ELISA-based in vitro assay was optimised to compare the acrolein-scavenging abilities of a range of drugs. This identified 2-hydrazino-4,6-dimethylpyrimidine (2-HDP) as a new and potent acrolein scavenger. The ability of this agent to modify the development of diabetic retinopathy was tested in vivo. Male Sprague Dawley rats were divided into three groups: (1) non-diabetic; (2) streptozotocin-induced diabetic; and (3) diabetic treated with 2-HDP in their drinking water for the duration of diabetes. Liquid chromatography high-resolution mass spectrometry was used to detect 2-HDP reaction products in the retina. Immunohistochemistry, real-time quantitative (q)RT-PCR and electroretinography were used to assess retinal changes 3 months after diabetes induction. RESULTS: 2-HDP was the most potent of six acrolein-scavenging agents tested in vitro (p < 0.05). In vivo, administration of 2-HDP reduced Müller cell accumulation of FDP-lysine at 3 months in rats rendered diabetic with streptozotocin (p < 0.001). A 2-HDP adduct was identified in the retinas of diabetic animals treated with this compound. 2-HDP supplementation was associated with reduced Müller cell gliosis (p < 0.05), reduced expression of the oxidative stress marker haem oxygenase-1 (p < 0.001) and partial normalisation of inwardly rectifying K+ channel 4.1 (Kir4.1) expression (p < 0.001 for staining in perivascular regions and the innermost region of the ganglion cell layer). Diabetes-induced retinal expression of inflammatory markers, inflammatory signalling compounds and activation of retinal microglial cells were all reduced in 2-HDP-treated animals. Retinal neurophysiological defects in diabetic animals, as indicated by changes in the electroretinogram 7 weeks after induction of diabetes, were also reduced by 2-HDP (p < 0.05-0.01 for b-wave amplitudes at flash intensities from -10 to +10 dB; p < 0.01 for time to peak of summed oscillatory potentials at +10 dB). CONCLUSIONS/INTERPRETATION: These findings support the hypothesis that Müller cell accumulation of FDP-lysine plays an important role in the development of diabetic retinopathy. Our results also suggest that 2-HDP may have therapeutic potential for delaying or treating this sight-threatening complication.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Characterization of a Spontaneously Immortalized Murine Müller Glial Cell Line QMMuC-1.

Purpose: Müller glia are critical for the survival of retinal neurons and the integrity of retinal blood vessels. Müller glial cultures are important tools for investigating Müller glial pathophysiology. Here, we report a spontaneously immortalized Müller glial cell line originally cultured and subsequently cloned from mouse pups. The cell line, Queen's University Murine Müller glia Clone-1 (QMMuC-1), has been cultured for over 60 passages, has morphologic features like primary Müller cell (PMC) cultures and remains stable. Methods: QMMuC-1 and PMC cells were processed for immunohistochemistry, quantitative RT-PCR, Western blotting, whole cell voltage-clamping, and bioenergetic profiling. Results: Immunocytochemistry showed that QMMuC-1 express known Müller glial markers, including glutamine synthetase, glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA), Aquaporin 4, Kir4.1, interleukin 33 (IL-33), and sex determining region Y (SRY)-box2 (Sox2), but not Cone arrestin, Calbindin 1, CD68, and ionized calcium-binding adapter molecule 1 (Iba1). Compared with PMC, QMMuC-1 express higher levels of chemokine (C-C motif) ligand 2 (Ccl2), VEGFA, and glutamate aspartate transporter (GLAST), but lower levels of interleukin 6 (IL-6), brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF1), and neurotrophin 3 (NTF3). Whole-cell patch clamp recordings demonstrated characteristic inward currents in response to L-glutamate and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) by QMMuC-1 cells. The L-glutamate-induced current was significantly higher in QMMuC-1 cells compared with PMC. Bioenergetic profiling studies revealed similar levels of glycolysis and basal mitochondrial respiration between QMMuC-1 and PMC. However, mitochondrial spare capacity was significantly lower in QMMuC-1 compared with PMC. Conclusions: Our results suggest that the QMMuC-1 Müller glial cell line retains key characteristics of PMC with its unique profiles in cytokine/neurotrophic factor expression and mitochondrial respiration. QMMuC-1 has utility as an invaluable tool for understanding the role of Müller glia in physiological and pathological conditions.