Background Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma. Methods Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined. Findings Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage. Conclusions The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.

PURPOSE:Optic nerve disease in chronic IOP elevation rat glaucoma models develops at different rates. This study was undertaken to investigate whether anterior chamber (AC) changes develop in two popular models in vivo and whether the changes are related to IOP. METHODS:Ten female Wistar rats and 12 male Brown-Norway rats were subjected to episcleral vein cauterization (EVC) and hypertonic saline episcleral vein sclerosis (HSEVS), respectively. Contralateral untreated eyes served as controls. IOP was recorded for a period of 5 to 6 weeks, and with the rats under anesthesia, the eyes were imaged with an ultrasound biomicroscope. Measurements of the AC depth (ACD), trabecular-iris angle (TIA), iris thickness at the thickest point near the pupillary margin (IT), angle opening distance (AOD; at 200 microm from the scleral spur), and ciliary body area (CBA) were compared between control eyes of the two strains and between experimental and control eyes within each strain. The differences were correlated with IOP history. RESULTS:Eyes subjected to EVC demonstrated greater increases in IOP than eyes subjected to HSEVS. Between rat strains, control eyes differed significantly in all the parameters studied, except for ACD. No difference was detected between experimental and control eyes in the EVC group. In contrast, experimental eyes in the HSEVS group had approximately 71% larger ACDs and approximately 32% smaller CBAs than did the contralateral control eyes (P < 0.001). ACD and CBA correlated well (R2 = 0.80 and 0.51, respectively) with IOP in the HSEVS group. Two of the experimental eyes in this group showed the presence of ultrasound-scattering material in the AC. CONCLUSIONS:Despite apparently higher IOP exposure, eyes in the EVC rat model of glaucoma do not undergo changes in the AC. In contrast, eyes subjected to HSEVS display deepening of the AC and reduction in size of the ciliary body within 5 to 6 weeks. These changes correlate to IOP exposure and may be the result of specific changes induced by the experimental intervention. These models are likely to rely on different mechanisms of pressure elevation and cannot be used interchangeably.

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9–10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1 h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold® or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95–100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p < 0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2–3 days prior to initiation of apoptotic RGC death.

BackgroundNoninvasive intraocular pressure (IOP) measurement in mice is critically important for understanding the pathophysiology of glaucoma. Rebound tonometry is one of the methods that can be used for obtaining such measurements. We evaluated the ability of the rebound tonometer (RT) to determine IOP differences among various mouse strains and whether differences in corneal thickness may affect IOP measurements in these animals. Materials and MethodsFive different commonly used mouse strains (BALB/C, CBA/CAHN, AKR/J, CBA/J, and 129P3/J) were used. IOP was measured in eyes from 12 nonsedated animals (6 male and 6 female) from each strain at 2 to 3 months of age using the RT. IOPs were measured in all animals, on 2 different days between 10 AM and 12 PM. Subsequently, a number of eyes from each strain were cannulated to provide a calibration curve specific for that strain. Tonometer readings for all strains were converted to apparent IOP values using the calibration data obtained from the calibration curve of the respective strain. For comparison purposes, IOP values were also obtained using the C57BL/6 calibration data previously reported. IOP for the 5 strains, male and female animals, and the different occasion of measurement were compared using repeat measures analysis of variance. The central corneal thickness (CCT) of another group of 8 male animals from each of the 5 strains was also measured using an optical low coherence reflectometry (OLCR) pachymeter modified for use with mice. CCT values were correlated to mean IOPs of male animals and to the slopes and intercept of individual strain calibration curves. ResultsNoninvasive IOP measurements confirm that the BALB/C strain has lower and the CBA/CAHN has higher relative IOPs than other mouse strains while the AKR/J, the CBA/J, and the 129P3/J strains have intermediate IOPs. There is a very good correlation of apparent IOP values obtained by RT with previously reported true IOPs obtained by cannulation. There was a small but statistically significant difference in IOP between male and female animals in 2 strains (129P3/J and AKR/J) with female mice having higher relative IOPs. No correlation between CCT and IOP was detected. CCT did not correlate with any of the constants describing the calibration curves in the various strains. ConclusionsNoninvasive IOP measurement in mice using the RT can be used to help elucidate IOP phenotype, after prior calibration of the tonometer. CCT has no effect on mouse IOP measurements using the RT.

Our purpose was to evaluate the accuracy, reproducibility and predictive ability of two non-invasive tonometers developed for intraocular pressure (IOP) measurements in the mouse. The prototype impact-rebound tonometer (I-R) and a prototype optical interferometry tonometer (OIT) utilizing a fiberoptic pressure sensor, were compared. Enucleated eyes from C57/BL6 mice were used for the calibration. The anterior chamber was cannulated and the IOP was adjusted in increments of 5 cm of H2O (open stopcock method). A calibration curve was generated for each individual eye along with a master calibration curve for all eyes. Two operators measured the IOP. The instruments were then used in alternating order to measure the IOP in C57/BL6 and in DBA2/J animals. The same eyes were subsequently cannulated and the error of the non-invasive tonometers was determined. Both tonometers yielded almost equivalent ex vivo calibration curves with individual R2 of 0.9878 and 0.9902 respectively. Both instruments were highly reproducible. In vivo the I-R tonometer underestimated while the OIT overestimated the IOP. This error was systematic and therefore predictable. The confidence intervals of this error were determined by comparing the IOP estimates provided by each tonometer with the measurements obtained invasively by cannulation in vivo. The 95% CI of the error were 2.36 mmHg for the I-R and 2.62 mmHg for the OIT respectively. Non-invasive tonometry in the mouse is feasible. Both non-invasive instruments provide accurate and reproducible measurements with the OIT requiring calibration curves for each individual investigator.

Background The present study was designed to investigate the effect of topical antiglaucoma drugs on Peak Pulse Blood Volume (PPBV; German abreviation: PGBV) in cynomolgus monkey eyes without (CMNG; German abreviation: RA-KTL) and with lasersurgically induced glaucoma (CMG; German abreviation: RA-LHDG).Methods CMG unilaterally received 2-3 laser treatments so as to develop the lasered-eye glaucoma model. Intraocular pressure (IOP; German abreviation: IOD) and Ocular Pulse Amplitude were measured and PPBV was determined until the glaucoma model had stabilized. Consecutively topical antiglaucoma drugs (epinephrine, paraaminoclonidine, pilocarpine, timolol) were investigated in 4-8 animals in CMNG and CMG eyes.Results SOP and PPBV were not significantly altered in CMNG. In the CMG eyes epinephrine and paraaminoclonidine did not significantly alter IOP or PPBV, whereas pilocarpine and timolol sig. (p<0.01) reduced IOP and significantly (p < 0.05) increased PPBV. Conclusion With respect to improved PPBV in the CMG eye the rank order of drug effectiveness is timolol > pilocarpine > paraaminoclonidine > epinephrine.

Background Ocular pulse amplitude (OPA) is reduced in normal tension glaucoma (NTG) patients when compared to non-glaucomatous, healthy control subjects. This might be related to a vasospastic reaction. The objective of this study was to determine if low OPA in NTG is associated with a vasospastic reaction and its response to vasodilation.Methods Nifedipine, a calcium channel blocker, vasodilator and systemic antihypertensive agent improves visual fields in NTG patients following acute and chronic dosing. The effect of 60 mg of daily orally administered nifedipine on OPA, intraocular pressure (IOP, German abbreviation: IOD), blood pressure (BP, German abbreviation: RR) and pulse rate (PR, German abbreviation:HF) were measured prior to and for 3 months after initiating nifedipine therapy in 32 NTG patients with and without a vasospastic reaction as manifested by a local cold exposure test. Before treatment, all patients had reduced OPA evaluated with the Langham Ocular Blood Flow System.Results During nifedipine treatment NTG patients with a vasospastic reaction showed a significant (p < 0.001) increase in OPA, whereas NTG patients without a vasospastic reaction showed no sig. (p > 0.05) change in OPA.Conclusion There may be two different subgroups of NTG patients, those who have a vasospastic reaction and react to nifedipine. while others lack the ability to react to nifedipine or might have a different, non-vasospastic pathology. Calcium channel blockers and other vasodilators may be useful in the treatment of vasospastic NTG patients.

• Background: Nitric oxide synthase (NOS) is present in many ocular tissues where it may have different physiological functions. This warrants a thorough characterization of NOS activity in the eye. • Methods: NOS distribution and its biochemical properties were determined in the retina, choroid, ciliary processes (CP), and trabecular meshwork (TM). • Results: Retinal NOS required NADPH (diphenyleneiodonium, a flavoprotein inhibitor, which inhibited enzyme activity with an IC50 of 0.36 μM, FAD (40 μM), FMN (40 μM), and BH4 (4 μM) as cofactors for optimal activity. Ocular NOS appeared to be regulated by free divalent cations, since its activity was inhibited by EDTA (slopes >3.0 and IC50 values of 12.8, 19.7, and 53 μM, respectively). Ocular NOS required calmodulin, since NOS activity was inhibited by trifluoperazine (calmodulin inhibitor, IC50=41 μM). NOS activity is widely distributed in the eye, (choroid >reinta >CP >TM) and is mainly cytosolic (70–95%).l-Arginine analogs inhibited NOS in the retina, choroid, and TM. In all three tissues,NG-methyl-l,-arginine displayed the highest affinity for inhibition (IC50=0.2–0.7 μM) followed by canavanine (IC50=13–33 μM), while aminoguanidine only weakly inhibited NOS (IC50=93–179 μM). • Conclusion: In all tissues, the order of potency of inhibition points to the presence of constitutive rather than inducible NOS. Moreover, it is possible that TM contains more than a single form of NOS.

The capacity of ouabain (1 mug/kg/min and 2 mug/kg/min) to induce cardiotoxicity was explored in control cats and cats after spinal cord transection. In cats receiving 1 mug/kg/min, a higher dose of ouabain was needed to produce lethal arrhythmias in spinal cats compared to intact cats. However, in cats receiving 2 mug/kg/min, there was no difference in dose to death between intact and spinal cats. At death, the serum level and ventricular contents of ouabain was higher in animals with spinal cord transection regardless of the rate of ouabain infusion. Thus it appears that determination of the myocardial tissue digitalis concentration more accurately reflects the state of digitalis sensitivity than the administered dose. Additionally, seemingly paradoxical findings with regard to dose are explicable when serum and tissue concentration are simultaneously evaluated.

The dose, serum level and tissue content of ouabain needed to produce cardiotoxicity were examined in Dial-urethane-anesthetized cats with and without spinal cord transection. At the time of ventricular fibrillation, the dose and serum level of ouabain were significantly higher in animals with spinal cord transection. The concentration of ouabain was elevated in atrium, ventricle and most other tissues of the spinal animal. Only the renal ouabain concentration exceeded that of the myocardium. Although the lower blood pressure observed in the spinal animals might reduce the efficiency of ouabain delivery to the myocardium, it could not explain the higher myocardial tissue requirements for cardiotoxicity. Heart rate, which might influence the rate of ouabain uptake by the myocardium, similarly does not adequately account for the observed changes in sensitivity. These data are consistent with an important role for the nervous system in the genesis of cardiotoxicity by ouabain.

The action of propranolol on cardiotoxicity produced by ouabain was studied in intact and isolated guinea pig hearts. The lethal event in the intact guinea pig was ventricular fibrillation; in the isolated heart it was asystole. Myocardial ouabain content at death was significantly higher in isolated preparations than in the intact heart. Propranolol was observed to alter the mode of death in the intact guinea pig from ventricular fibrillation to asystole. The myocardial ouabain content at the time of death was elevated in guinea pigs pretreated with propranolol to the level observed in isolated guinea pig heart preparations. Exposure of the isolated preparation to propranolol prolonged the time to the induction of cardiac rhythm disorders and death without changing myocardial ouabain content. These data suggest that propranolol, by eliminating neural influence transforms the cardiotoxicity of ouabain to an in vitro mechanism. In the isolated heart, propranolol prolongs the time to the induction of cardiac rhythm disorders by inhibiting the myocardial uptake of ouabain.