Lipid mediators in glaucoma: Unraveling their diverse roles and untapped therapeutic potential.

Glaucoma is a complex neurodegenerative disease characterized by optic nerve damage and visual field loss, and remains a leading cause of irreversible blindness. Elevated intraocular pressure (IOP) is a critical risk factor that requires effective management. Emerging research underscores dual roles of bioactive lipid mediators in both IOP regulation, and the modulation of neurodegeneration and neuroinflammation in glaucoma. Bioactive lipids, encompassing eicosanoids, specialized pro-resolving mediators (SPMs), sphingolipids, and endocannabinoids, have emerged as crucial players in these processes, orchestrating inflammation and diverse effects on aqueous humor dynamics and tissue remodeling. Perturbations in these lipid mediators contribute to retinal ganglion cell loss, vascular dysfunction, oxidative stress, and neuroinflammation. Glaucoma management primarily targets IOP reduction via pharmacological agents and surgical interventions, with prostaglandin analogues at the forefront. Intriguingly, additional lipid mediators offer promise in attenuating inflammation and providing neuroprotection. Here we explore these pathways to shed light on their intricate roles, and to unveil novel therapeutic avenues for glaucoma management.

Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes.

Intravitreal (ITV) drug delivery is a new cornerstone for retinal therapeutics. Yet, predicting the disposition of formulations in the human eye remains a major translational hurdle. A prominent, but poorly understood, issue in pre-clinical ITV toxicity studies is unintended particle movements to the anterior chamber (AC). These particles can accumulate in the AC to dangerously raise intraocular pressure. Yet, anatomical differences, and the inability to obtain equivalent human data, make investigating this issue extremely challenging. We have developed an organotypic perfusion strategy to re-establish intraocular fluid flow, while maintaining homeostatic pressure and pH. Here, we used this approach with suitably sized microbeads to profile anterior and posterior ITV particle movements in live versus perfused porcine eyes, and in human donor eyes. Small-molecule suspensions were then tested with the system after exhibiting differing behaviours in vivo. Aggregate particle size is supported as an important determinant of particle movements in the human eye, and we note these data are consistent with a poroelastic model of bidirectional vitreous transport. Together, this approach uses ocular fluid dynamics to permit, to our knowledge, the first direct comparisons between particle behaviours from human ITV injections and animal models, with potential to speed pre-clinical development of retinal therapeutics.

Aqueous humor endothelin-1 and total retinal blood flow in patients with non-proliferative diabetic retinopathy.

PurposeThe purpose of this study was to determine the association between aqueous ET-1 levels and total retinal blood flow (TRBF) in patients with non-insulin-dependent type 2 diabetes mellitus (T2DM) and early non-proliferative diabetic retinopathy (NPDR).Patients and methodsA total of 15 age-matched controls and 15 T2DM patients with NPDR were recruited into the study. Aqueous humor (~80-120 µl) was collected before cataract surgery to measure the levels of ET-1 using suspension multiplex array technology. Four weeks post surgery, six images were acquired to assess TRBF using the prototype RTVue Doppler FD-OCT (Optovue, Inc., Fremont, CA, USA) with a double circular scan protocol. At the same visit, forearm blood was collected to determine plasma glycosylated hemoglobin (A1c) levels.ResultsAqueous ET-1 was significantly elevated in the NPDR group compared with the control group (3.5±1.8 vs 2.2±0.8, P=0.02). TRBF was found to be significantly reduced in the NPDR group compared with the control group (34.5±9.1 vs 44.1±4.6 µl/min, P=0.002). TRBF and aqueous ET-1 were not correlated within the NPDR group (r=-0.24, P=0.22). In a multivariate analysis, high A1c was associated with reduced TRBF and aqueous ET-1 levels across control and NPDR groups (P<0.01).ConclusionAqueous ET-1 levels were increased while TRBF was reduced in patients with NPDR compared with the control group. Although not directly associated, the vasoconstrictory effects of ET-1 are consistent with a reduced TRBF observed in early DR. ET-1 dysregulation may contribute to a reduction in retinal blood flow during early DR.

Alkaline and alkaline/oxidation pre-treatments of spruce wood (Picea abies)--impact on the quality of kraft pulp.

A comparable series of specimens from spruce wood were pre-treated with sodium hydroxide, sodium hydroxide and hydrogen peroxide, or per-acetic acid sequences. The pre-treatments reduced the yield of pulps and their Kappa number noticeably, diminished the degree of polymerization moderately, and increased their brightness. One-step peroxide bleaching of pulps from the pre-treated spruce wood resulted in their higher brightness compared to bleached pulp from sound wood. From the viewpoint of improved properties of pulp, the most efficient were the sodium hydroxide/per-acetic acid and per-acetic acid/sodium hydroxide sequences. The pre-treatments did not influence mechanical strength of the obtained pulps significantly.

The effects of toxicological agents on the optics and mitochondria of the lens and the mitochondria of the corneal epithelium.

This review describes how the morphology and distribution of the mitochondria of the epithelium and the superficial fibre layers of the lens were studied using confocal scanning laser microscopy. This research was correlated with an effort to use the optical properties of the intact lens in culture as a proxy for the cornea in measuring ocular toxicity. In turn, this work led to the confocal study of the in vitro and then the in vivo cornea and their possible use in using confocal microscopy to evaluate the effect of various treatments on the integrity of the surface of the eye. Finally, confocal examination of the mitochondria of the lens has provided an avenue to the study of mitochondrial dynamics.

Dose-response of the cultured bovine lens to butyl, methyl and propyl parabens.

Pre-screening of cosmetic ingredients is vital for consumer safety. Previous in vivo techniques, such as the Draize test, have proved to be unreliable in predicting ocular irritancy and therefore there is a need for alternate testing methodologies. One such test is the scanning laser in vitro assay system which quantifies irritancy based on the focusing ability of the cultured bovine lens. In combination with confocal microscopy, a more thorough documentation of ocular irritancy can be achieved. This study investigates the response of cultured bovine lenses over time to butyl, methyl and propyl parabens, which are common antimicrobial agents found in cosmetic and ophthalmic products. The focusing ability of the lens was measured with an automated laser scanner over a period of 96 h. At 120 h post-treatment, the lenses were analysed by using a confocal laser scanning microscope to determine the characteristics of nuclei, and the morphology and distribution of mitochondria within the lenses. Irritancy to the three parabens was investigated at both an optical and cellular level. Each of the parabens was tested at 0.002% and 0.2%, where the 0.2% butyl paraben was found to be the most irritating.

Optical quality changes of the ocular lens during induced parr-to-smolt metamorphosis in Rainbow Trout (Oncorhynchus mykiss). Ocular lens optical quality during induced salmonid metamorphosis.

The effect of an induced salmonid parr-to-smolt metamorphosis ('smoltification') on the optical quality of the ocular lens was studied. In two separate experiments, rainbow trout (Oncorhynchus mykiss) parr were fed thyroxine in their diet to induce the metamorphosis. Lenses were excised at regular samplings during the treatment period and optically scanned using a custom scanning laser monitor. Radioimmunoassay was used to measure serum titers of thyroxine and 3,5,3'-triiodo-L: -thyronine. It was found that lens optical quality was consistently negatively correlated with 3,5,3'-triiodo-L: -thyronine levels, but not with thyroxine levels. To test if thyroid hormones are directly responsible for the change in optical quality, rainbow trout lenses were cultured for 72 h in a medium containing 3,5,3'-triiodo-L: -thyronine, but no effect was observed. The significance of these findings in the contexts of the fishes' visual capabilities and smolting physiology is discussed.

Long-term lens organ culture system with a method for monitoring lens optical quality.

The Scan Tox System is a method for monitoring lens optical quality (focus or lack of focus) in culture conditions, which mimic conditions inside the eye. The ocular lens is an ideal organ for long-term culture experiments because it has no direct blood supply and no connection to the nervous system. The Scan Tox System makes it possible to keep lenses for long-term studies of up to a few weeks. The use of cultured lenses, mainly bovine, replaces the need for testing the effects of potentially damaging agents on live animals. This optical monitoring apparatus uses a computer-operated scanning laser beam, a video-camera system and a video frame analyzer to record the focal length and transmittance of the cultured lens. The scanner is designed to measure the focal length at points across the diameter of the lens. The lens container permits the lens to be exposed to a vertical laser beam from below. The laser source projects its light onto a plain mirror, which is mounted at 45 degrees C on a carriage assembly. The mirror reflects the laser beam directly up through the test lens. The mirror carriage is connected to a positioning motor, which moves the laser beam across the lens. The camera sees the cross section of the beams and, by examining the image at each position of the mirror, Scan Tox software is able to measure the quality of the lens by calculating the back vertex distance for each beam position. The cultured lenses continue to maintain their original refractive function. When foreign substances are introduced to a cultured lens, the Scan Tox System measures the resulting optical response. This provides a very sensitive means to follow early damage to the eye lens. Because the lens is maintained in an intact state in solutions that are similar to those inside the eye, the lens retains its normal recuperative powers. So in addition to measuring early damage, this system allows measurement of recovery from damage.

Optical response of the cultured bovine lens; testing opaque or partially transparent semi-solid/solid common consumer hygiene products.

This study determines the relative ocular lens irritancy of 16 common partially transparent or non-transparent consumer hygiene products. The irritancy was found by measuring the changes in the sharpness of focus [referred to as the back vertex distance (BVD) variability] of the cultured bovine lens using a scanning laser In Vitro Assay System. This method consists of a laser beam that scans across the lens, and a computer, which then analyses the average focal length (mm), the BVD variability (mm), and the intensity of the beam transmitted. Lenses were exposed to the 16 hygiene products and the lens' focusing ability was monitored over 192 h. The products are semi-solids or solids (e.g. gels, lotions, shampoos). They are categorized into six groups: shampoos, body washes, lotions, toothpastes, deodorant, and anti-perspirant. Damage (measured by > 1 mm BVD variability) occurred slower for the shampoos, especially in the case of baby shampoo. The results indicate that shampoos exhibit the lowest level of ocular lens toxicity (irritability) while the deodorant is the most damaging.

Optical function and mitochondrial metabolic properties in damage and recovery of bovine lens after in vitro carbonyl cyanide m-chlorophenylhydrazone treatment.

In order to elucidate the correlation between lens optical function and metabolic function, in vitro bovine lens optical quality and mitochondrial integrity was measured following treatment with carbonyl cyanide m-chlorophenylhydrazone (the mitochondrial depolarizing agent, CCCP). The results indicate that in vitro exposure to CCCP resulted in concentration and time-dependent loss of sharp focus. The concentrations tested included 65.0, 32.5, 16.25 and 8.125 microm CCCP. Lenses treated with two lower concentrations show recovery from damage at the 24-h scan point. In lenses treated with 65 microM CCCP, mitochondria in lens epithelial and superficial cortical fibre cells appeared short and swollen. The results of this study indicate that lens optical function and mitochondrial integrity are closely correlated.

Suppression of post-vitrectomy lens changes in the rabbit by novel benzopyranyl esters and amides.

This study reports for the first time a therapeutic modality for the suppression of posterior subcapsular cataract (PSC) formation in an animal model (rabbit) of vitrectomy. This therapeutic modality may also have the potential to attenuate/prevent the high incidence of loss of vision due to cataract formation in patients that undergo vitrectomy. Unilateral, partial vitrectomy was performed on 2.5 month old Dutch Belted rabbits with vitreous replaced by either commercially available BSS((R)) or BSS PLUS((R)) (n=16). Alternatively, vitreous was replaced with a proprietary, modified BSS PLUS((R)) irrigating solution containing 1.25 microM AL-8417 (n=12), 5.0 microM AL-12615 (n=5) or 5.0 microM AL-17052 (n=9). Age matched, non-operated rabbits were used as controls (n=16). Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) quality analysis 6 months following surgery. Results demonstrate that vitreous replacement with an irrigating solution that contains the ester-linked benzopyran, AL-8417, the amide-linked benzopyran pro-drug, AL-17052, or its active metabolite, AL-12615, prevented abnormal post-vitrectomy lens growth, or fiber formation. Focal length variability (FLV) assessments (sharpness of focus) confirmed the beneficial drug effects detected morphologically, with FLV being essentially equal to that of age-matched, non-surgical controls. In contrast, lenses of animals with vitreous replaced solely with BSS((R)) or BSS PLUS((R)) exhibited significantly higher FLV than both age-matched controls and animals that underwent vitrectomy with drug-containing irrigating solutions. The ability of AL-8417, AL-17052 and its active metabolite, AL-12615, to suppress vitrectomy-induced posterior lens fiber changes appears to reside in their unique pharmacological profile, acting as antioxidant, anti-inflammatory and cytostatic agents.

Impairment of eye lens cell physiology and optics by broadband ultraviolet A-ultraviolet B radiation.

The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate-acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin-streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively.

Action spectrum and recovery for in vitro UV-induced cataract using whole lenses.

PURPOSE: To establish the in vitro action spectrum for acute UV cataractogenesis using whole cultured lenses. The recovery pattern of the induced cataract was also investigated. METHODS: Aseptically dissected porcine lenses were cultured in glass chambers. At 1 week, lenses were exposed to a predetermined UV energy (J/cm(2)) at specific wavebands ranging from 270 to 370 nm at 5- and 10-nm intervals. The UV energy was generated by a PRA integrated arc lamp system using a water-cooled 1000 W, high-pressure xenon lamp. The lamp output was limited using a deionized water filter, a monochromator, and secondary optics. An electronic shutter was used to control the exposure time. The median effective dose, ED(50) (i.e., UV energy threshold) for each waveband was statistically determined using probit analysis. Irradiated spots (3.06 mm(2)) on the lenses were monitored every 6 to 12 hours up to 48 hours postirradiation for any UV-induced opacity with a dissecting microscope and photomicrography. The ED(50)s were plotted against wavelengths to obtain the action spectrum. RESULTS: The threshold values for 270, 300, and 365 nm were 0.057, 0.069, and 137.19 J/cm(2), respectively. Permanent UV-induced cataract was obtained at twice the threshold values for UVB and UVA. CONCLUSIONS: An action spectrum for in vitro UV-induced cataract using whole cultured lens is established. These data are comparable to published in vitro (with isolated lens epithelial cells) and in vivo action spectra. The recovery pattern appears to be similar to the in vivo situation.

Refractive state and accommodation in the eyes of free-swimming versus restrained juvenile lemon sharks (Negaprion brevirostris).

Optical measurements of the refractive state of the eyes of various shark species typically have depicted sharks as hyperopic (far-sighted) with little evidence of accommodation (i.e. the ability to change focus for visualizing objects at different distances from the eye). In this study, we used infrared video retinoscopy to measure the refractive state in juvenile lemon sharks (Negaprion brevirostris). This technique allows dynamic measurement of refractive state in free-swimming animals as they pass by an aquarium window. We found that unrestrained lemon sharks are focused emmetropically relative to a 1-m distant photorefractor for the lateral visual field. However, when restrained either right side up or upside down (the latter inducing tonic immobility), the sharks become increasingly hyperopic, an artifact also reported in some other vertebrates. In addition, unrestrained lemon sharks display small amplitude accommodative excursions. Thus, refractive state measurements on restrained sharks in general may not reflect the natural, resting state of the shark eye, but rather, an induced hyperopia and lack of accommodative function. Such an artifact may be present in other vertebrate species, underscoring the need to obtain measurements of refractive state in unrestrained animals.

High-fructose feeding of streptozotocin-diabetic rats is associated with increased cataract formation and increased oxidative stress in the kidney.

We examined the effects of high-fructose (FR) feeding on the development of diabetic complications in the lens and the kidney of streptozotocin (STZ)-diabetic rats. Male Wistar Furth rats were treated with one of two doses of STZ (HIGH STZ, 55 mg/kg body weight; MOD STZ, 35 mg/kg body weight) or vehicle alone (SHAM) and were then assigned to a control (CNTL) or 400 g FR/kg diet for 12 weeks. At the end of the study, body weight, plasma glucose and insulin concentrations differed among STZ groups (HIGH v. MOD v. SHAM, P < 0.001) but did not differ due to diet. Plasma FR concentrations were significantly higher in FR-fed v. CNTL-fed groups (P < 0.0001) and in HIGH-STZ groups v. MOD-STZ and SHAM groups (P < 0.0004 and P < 0.0001 respectively). Focal length variability of the lens, a quantitative measure of cataract formation, was increased in the HIGH STZ, FR group compared with the HIGH STZ, CNTL group (P < 0.01). The concentration of H2O2 in kidney microsomes was significantly higher in HIGH STZ, FR rats v. HIGH STZ, CNTL rats (P < 0.01). Micro-albuminuria was not observed in any of the groups examined, and there was no evidence of extensive histological damage in the kidney from any rats. Under conditions of severe hyperglycaemia, high FR intake promotes the development of cataracts in the lens of the eye, and results in increased concentrations of substances indicative of oxidative stress in the kidney. Although FR has been suggested as a carbohydrate source for diabetics, a high FR diet coupled with hyperglycaemia produces effects that may promote some of the complications associated with diabetes.

The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy.

This study was conducted first, to characterize structural changes in rabbit lenses after vitrectomy; and second, to assess whether such changes correlate with a quantifiable compromise in optical function. Unilateral, partial vitrectomies were performed on 2.5 month old Dutch Belted rabbits (n = 64). Age matched non-operated rabbits (n = 32) were used as controls. Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) analysis at 1.5, 3, 6 and 12 months post-surgery (n = 16 lenses from operated animals and n = 8 lenses from non-operated controls at each time point). Results demonstrate that in rabbits lens growth, or fiber formation, is compromised after vitrectomy. From 1.5 to 12 months after surgery, lenses had progressively more crooked posterior line sutures with sub-branches of increasing size and number in successive growth shells. Quantification of lens optical quality specifically along and/or between these atypical suture branches and sub-branches revealed a significant increase in focal length variability (sharpness of focus) after vitrectomy. A peripheral zone of fibers with abnormal posterior ends was produced surrounding the pre-surgical lens mass. This additional zone of aberrant fibers was associated with a quantifiable degradation in lens optics. Studies on the prevention of post-vitrectomy lens changes in this rabbit model may yield useful information applicable to the human condition.

Effects of experimentally induced ametropia on the morphology and optical quality of the avian crystalline lens.

PURPOSE: To examine the effects of refractive error on avian lens morphology and optical quality. METHODS: Hatchling white leghorn chicks were unilaterally goggled for 7 days with either a form-deprivation goggle (n = 12), a -10 D defocus goggle (n = 12), or a +10 D defocus goggle (n = 12) to induce myopia and hyperopia. Optical quality of lenses (focal length and focal length variability) from treated and contralateral control eyes was assessed using a scanning laser apparatus. Lens morphology was examined by light and electron microscopy. RESULTS: Although the induction of refractive errors did not significantly alter lens size, shape, paraxial focal length, or average focal length, average focal length variability increased. Lenses from eyes goggled with form-deprivation and +10 D defocus goggles demonstrated a twofold increase in average focal length variability, when compared with their contralateral controls. The morphology of the lens is not altered by these experimental manipulations. CONCLUSIONS: This study provides evidence that the refractive development of the chick lens is not independent of the refractive development of the ocular globe and that chick lenticular development is influenced by both genetics and visual experience.

Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B.

Recent evidence supports the idea that matrix metalloproteinases (MMPs) act as morphogenetic regulators in embryonic and adult events of tissue remodeling. MMP activity is controlled primarily at the level of gene expression. In a recent study we characterized the transcriptional promoter of the MMP gene, gelatinase B (gelB), in transgenic mice, demonstrating the requirement for DNA sequences between -522 and +19 for appropriate activity. In this study we investigated factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea. Pax-6 is a homeobox and paired domain transcription factor that acts at the top of the hierarchy of genes controlling eye development. Pax-6 is also expressed in the adult eye. We show here that the tissue expression pattern of Pax-6 overlaps extensively with gelB promoter activity in the developing and adult eye. In addition Pax-6 is observed to be upregulated in repairing corneal epithelium, as is gelB promoter activity. In cell culture transfection experiments, we identified two promoter regions which mediate positive response to Pax-6. By electrophoretic mobility shift assay, we further pinpoint two Pax-6 binding sites within these response regions and demonstrate direct interaction of the Pax-6 paired domain with one of these sites. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner.

Age-related changes in human ciliary muscle.

PURPOSE: To examine the role of the ciliary muscle in accommodation and presbyopia. METHODS: Sixteen pairs of eyes from donors aged 1 to 107 years were treated with atropine or pilocarpine and then processed for light microscopy. Seven pairs were analyzed for treatment effects, including morphometric measurements of muscle dimensions, muscle fiber group area, and the percentage of connective tissue. RESULTS: The ciliary muscle shortened in length in response to drug treatment at all ages. The ciliary muscle of older subjects contained greater amounts of connective tissue and was shorter, wider and the internal apical edge moved forward. CONCLUSIONS: This study confirms previous reports that the human ciliary muscle retains its ability to contract throughout the lifespan. However, the ciliary muscle also displayed age-related changes, which may be partly accounted for by the forces exerted from the aging lens and zonules.