Ocular Surface Microbiota in Contact Lens Users and Contact-Lens-Associated Bacterial Keratitis.

Our objectives were to investigate whether the conjunctival microbiota is altered by contact lens wear and/or bacterial keratitis and to explore the hypothesis that commensals of conjunctival microbiota contribute to bacterial keratitis. Swab samples from both eyes were collected separately from the inferior fornix of the conjunctiva of non-contact-lens users (nparticipants = 28) and contact lens users (nparticipants = 26) and from patients with contact-lens-associated bacterial keratitis (nparticipants = 9). DNA from conjunctival swab samples was analyzed with 16S rRNA gene amplicon sequencing. Pathogens from the corneal infiltrates were identified by cultivation. In total, we identified 19 phyla and 283 genera; the four most abundant genera were Pseudomonas, Enhydrobacter, Staphylococcus, and Cutibacterium. Several pathogens related to bacterial keratitis were identified in the conjunctival microbiota of the whole study population, and the same bacteria were identified by both methods in the conjunctiva and cornea for four patients with contact-lens-associated bacterial keratitis. The overall conjunctival microbiota profile was not altered by contact lens wear or bacterial keratitis; thus, it does not appear to contribute to the development of bacterial keratitis in contact lens users. However, in some individuals, conjunctival microbiota may harbor opportunistic pathogens causing contact-lens-associated bacterial keratitis.

Ocular surface microbiota in patients with aqueous tear-deficient dry eye.

PURPOSE: An altered ocular surface microbiota may contribute to the pathophysiology of dry eye disease. The aim of the study was to explore potential differences in microbiota diversity and composition in aqueous tear-deficient dry eye (with and without ocular graft-versus-host disease) compared with controls. METHODS: Swab samples from the inferior fornix of the conjunctiva were obtained from patients with aqueous tear-deficient dry eye with and without ocular graft-versus-host disease (n = 18, n = 21, respectively) and controls (n = 28). Isolated bacterial DNA from swabs were analyzed with 16S rRNA gene amplicon sequencing. RESULTS: Decreased microbiota diversity was observed in patients with aqueous tear-deficient dry eye (p ≤ 0.003) who also showed a difference in microbiota composition compared with controls (p = 0.001). Although several genera were less abundant in aqueous tear-deficient dry eye, a minimal core ocular surface microbiota comprising five genera was shared by >75% of the study participants: Enhydrobacter, Brevibacterium, Staphylococcus, Streptococcus and Cutibacterium. Pseudomonas was identified as a bacterial biomarker for controls and Bacilli for patients with aqueous tear-deficient dry eye. CONCLUSIONS: Ocular surface microbiota in patients with aqueous tear-deficient dry eye was characterized by an aberrant microbiota composition in comparison to controls, with decreased diversity and reduced relative abundances of several genera. Additionally, a few genera were present in most of the study population, indicating that a minimal core ocular surface microbiota may exist.

Relaxation of porcine retinal arterioles during acute hypoxia in vitro depends on prostaglandin and NO synthesis in the perivascular retina.

PURPOSE: Disturbances in retinal oxygenation influence retinal function, but are also accompanied by changes in the tone of retinal arterioles. However, the mechanisms underlying these tone changes have not been studied in detail. MATERIALS AND METHODS: Porcine retinal arterioles were mounted in a wire myograph, and the vasoactive effects of hypoxia and hyperoxia were studied before and after removal of the perivascular retinal tissue. Subsequently, the experiments were repeated in the presence of antagonists to prostaglandins, nitric oxide (NO), adenosine and glutamate. RESULTS: Hypoxia induced a significant concentration-dependent relaxation of U46619-contracted retinal arterioles which depended on the presence of the perivascular retinal tissue. The relaxation was significantly reduced by inhibiting the synthesis of prostaglandins and NO simultaneously. The recovery of vascular tone after hypoxia was incomplete, but increased to a normal level during the inhibition of prostaglandin synthesis. Hyperoxia induced a slight concentration-dependent contraction of retinal arterioles that was not affected by any of the antagonists used. CONCLUSIONS: Hypoxia-induced relaxation of porcine retinal arterioles in vitro depends on prostaglandins and NO and the presence of perivascular retinal tissue, whereas recovery of tone after hypoxia depends on the action of prostaglandins. Clinical intervention studies of these effects may help treating retinal diseases where disturbances in tissue oxygenation are involved in the disease pathogenesis.

GABA-induced relaxation of porcine retinal arterioles in vitro depends on inhibition from the perivascular retina and is mediated by GABAC receptors.

PURPOSE: To study the dependence of γ-aminobutyric acid (GABA)-induced relaxation of retinal arterioles on the glutamate agonist N-methyl-D-aspartate (NMDA), adenosine triphosphate (ATP), prostaglandin E2 (PGE2), and adenosine, and to characterize the type and location of GABA-receptor(s) mediating GABA-induced relaxation of retinal arterioles. METHODS: Porcine retinal arterioles were mounted in a wire myograph, and the effects of agonists and antagonists to NMDA, ATP, PGE2, and adenosine on GABA-induced relaxation were studied. Additionally, experiments were conducted to study relaxation induced by agonists to specific GABA receptors, and GABA-induced relaxation in the presence of specific GABA antagonists. Finally, immunohistochemistry was performed to identify the location of GABA(C) receptors in the porcine retina. RESULTS: GABA induced vasorelaxation during blocking of the glutamate NMDA receptor, PGE2 receptor, and ATP synthesis degradation, but not during blocking of the adenosine receptor or by agonists to any of these compounds. The vasorelaxing effect of GABA could be elicited by a specific GABA(C) agonist, but not by specific GABA(A) or GABA(B) agonists, and could be blocked by a specific GABA(C) antagonist, but not by specific GABA(A) or GABA(B) antagonists. GABA(C) receptor subunits could be identified in the ganglion cell layer, and at the border between the outer plexiform and inner nuclear layers. CONCLUSIONS: GABA-induced relaxation of porcine retinal vessels is mediated by the GABA(C) receptor in the perivascular retinal tissue, and depends on blocking of the glutamate NMDA receptor, prostaglandin E2 receptor, or ATP degradation.

ATP-induced relaxation of porcine retinal arterioles in vitro depends on prostaglandin E synthesized in the perivascular retinal tissue.

PURPOSE: It has been shown that inhibition of prostaglandin synthesis in the perivascular retinal tissue can prevent the relaxation of retinal arterioles induced by N-methyl-d-aspartic acid (NMDA) and adenosine triphosphate (ATP). The purpose of the present study was to identify the prostaglandins involved in this retina-dependent relaxation. METHODS: Porcine retinal arterioles were mounted in a myograph for isometric tone measurements. The effect of the prostaglandins (PGs) PGE(2), PGF(2α), PGD(2), and PGI(2) and of thromboxane A(2) (TXA(2)) on vascular tone was recorded before and after removal of the perivascular retina, and the specificity of the responses were confirmed by blocking with specific antagonists. Finally, the coupling between prostaglandins found to have a specific vasoactive effect, dependent on the perivascular retina, and the individual vasorelaxing effects of NMDA, ATP, and adenosine were studied. RESULTS: All prostaglandins tested showed a significant relaxation of precontracted arterioles at the highest concentrations, whereas PGF(2α) induced a significant constriction of isolated noncontracted arterioles. In the presence of perivascular retinal tissue, the dilating effect of PGE(2) increased significantly, an effect that was blocked by a prostaglandin E prostanoid (EP(1)) receptor blocker, whereas PGD(2) induced a dual response, with a significant contraction at low concentrations and a significant dilation at high concentrations. Inhibition of the cyclo-oxygenase (COX) enzyme with ibuprofen, as well as the EP(1) receptor, blocked the vasodilating effect of ATP, but not that of NMDA and adenosine, in the presence of perivascular retinal tissue. CONCLUSIONS: ATP-induced vasodilation depends on the production of PGE in the perivascular retina. However, the regulation of retinal arteriolar tone involves COX products other than PGE.

Imaging of Ca2+ responses mediated by presynaptic L-type channels on GABAergic boutons of cultured hippocampal neurons.

We have previously demonstrated that L-type Ca(2+) channels are involved in post-tetanic potentiation (PTP) of GABAergic IPSCs in cultured hippocampal neurons. Here we have used intracellular Fluo-3 to detect [Ca(2+)](i) in single GABAergic boutons in response to stimulation that evokes PTP. During control stimulation of the presynaptic GABAergic neuron at 40 Hz for 1-2 s, DeltaF/F(0) increased rapidly to a peak value and started to decline shortly after the train ended, returning to baseline within 10-20 s. The L-type channel blocker, isradipine (5 microM), had no significant effect on the amplitude or kinetics of the Ca(2+) signal. Following blockade of N- and P/Q-type Ca(2+)-channels, the amplitude was reduced by 52.9+/-3%. Isradipine caused a reduction of the remaining response (by 26.6+/-5%, P<0.01), that was fully reversible on washing. The L-type channel "agonist", BayK 8644 (8 microM), caused a significant enhancement of the peak (by 18.7%+/-7%, P<0.05). The rising phase of the Ca(2+) signal, which is related to the rate of entry of Ca(2+) into the bouton, was decreased by isradipine (by 25.5+/-6%, P<0.05) and enhanced by BayK 8644 (by 45.2%+/-16%, P<0.05). These Ca(2+) imaging experiments support the putative role of L-type channels in PTP of GABAergic synapses on cultured hippocampal neurons. We expect L-channels to be few in number, although they may couple strongly to intracellular signalling cascades that could amplify a signal that regulates synaptic vesicle turnover in the GABAergic boutons.

The vasodilating effect of acetazolamide and dorzolamide involves mechanisms other than carbonic anhydrase inhibition.

PURPOSE: Carbonic anhydrase inhibitors reduce intraocular pressure, which may protect the optic nerve from ischemia. However, carbonic anhydrase inhibitors have also been shown to dilate the blood vessels in the retina and the optic nerve head. The purpose of the present study was to investigate whether CO(2), H(+), or factors other than carbonic anhydrase inhibition are involved in this vasodilating effect. METHODS: Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a myograph for isometric force measurements. After precontraction with the prostaglandin analogue U46619, concentration-response experiments were performed with acetazolamide and dorzolamide before and after removal of the perivascular retina. The experiments were performed at normal pH and during acidosis, during normocapnia and hypercapnia, as well as in the nominal absence of CO(2) and HCO(3)(-). RESULTS: The maximum relaxation was significantly lower and the EC(50) significantly higher during normal pH compared with acidosis (P = 0.002 and P < 0.0001, respectively), but neither the maximum relaxation nor EC(50) was changed by hypercapnia (P = 0.054 and P = 0.57, respectively). The findings confirmed that carbonic anhydrase-induced vasodilation depends on the perivascular retinal tissue and that dorzolamide produces significantly more pronounced relaxation than does acetazolamide. EC(50) of carbonic anhydrase inhibitor-induced vasorelaxation and the maximum relaxation of dorzolamide were unchanged in the nominal absence of CO(2) and HCO(3)(-) (P = 0.65 and P < 0.0001, respectively). CONCLUSIONS: The vasodilating effect of carbonic anhydrase inhibitors on porcine retinal arterioles depends on the perivascular retinal tissue and acidosis, but not on hypercapnia. The effect involves mechanisms other than carbonic anhydrase inhibition.

N-methyl-D-aspartic acid causes relaxation of porcine retinal arterioles through an adenosine receptor-dependent mechanism.

PURPOSE: Disturbances in retinal perfusion due to impaired regulation of vascular tone are believed to be involved in the pathogenesis of several vision-threatening retinal diseases. Two recent studies have shown that the glutamate receptor agonist, N-methyl-D-aspartic acid (NMDA), and adenosine induce relaxation of isolated porcine retinal arterioles in vitro. However, it remains to be elucidated whether the relaxing action of the two substances are coupled. METHODS: Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a myograph for isometric tone measurements. Changes in tone were induced by increasing concentrations of NMDA in the presence of blockers of adenosine receptors and ATP hydrolysis and by increasing concentrations of adenosine in the presence of the NMDA receptor blocker DL-APV (DL-amino-5-phosphonovaleric acid). The experiments were repeated after the perivascular tissue had been removed. RESULTS: NMDA produced a relaxing effect on retinal vessels with preserved perivascular retinal tissue (P < 0.001) which disappeared after removal of the tissue. Blocking of the NMDA and adenosine receptors and hydrolysis of adenosine triphosphate (ATP) significantly reduced the vasorelaxing effect of NMDA in the presence of perivascular retinal tissue (P < 0.05 for all three comparisons). Adenosine produced a concentration-dependent relaxation that was not significantly affected by blocking the NMDA receptor with DL-APV (P = 0.088). CONCLUSIONS: The findings suggest that the vasorelaxing effect of NMDA on porcine retinal arterioles in vitro is mediated by hydrolysis of ATP to adenosine in the perivascular retinal tissue.

The relaxing effect of perivascular tissue on porcine retinal arterioles in vitro is mimicked by N-methyl-D-aspartate and is blocked by prostaglandin synthesis inhibition.

PURPOSE: Retinal hyperperfusion resulting from disturbances in the regulation of arteriolar tone is involved in the pathophysiology of a variety of retinal diseases. The mechanisms underlying this regulation of tone involve cellular components in both the vascular wall and the perivascular tissue. However, previous in vitro studies of the influence of perivascular retinal tissue on retinal tone regulation have been hampered by the release of an endogenous relaxing factor that renders the arteriole insensitive to vasoconstrictors. The purpose of the present study was to test whether N-methyl-D-aspartate (NMDA) and gamma-amino butyric acid (GABA) receptors, and a cyclooxygenase (COX) product influence this effect of perivascular retinal tissue in vitro. METHODS: Porcine retinal arterioles were mounted in a wire myograph for isometric force measurements. The contractile effect of the prostaglandin analogue U46619 was studied on vessels with preserved perivascular retinal tissue and after this tissue had been removed. The influence of the perivascular tissue was studied after addition of NMDA (a specific agonist for a subtype of the glutamate receptor), DL-amino-5-phosphonovaleric acid (DL-APV, an antagonist at the same receptor), the natural inhibitory transmitter GABA, and picrotoxin (an antagonist at ionotropic GABA receptors). These experiments were made in the absence and presence of the COX inhibitor, ibuprofen. RESULTS: U46619 caused a concentration-dependent contraction of isolated retinal arterioles. This vasoconstriction was significantly smaller in the presence of perivascular tissue. The NMDA-receptor antagonist, DL-APV, reduced this attenuating influence of the perivascular tissue on the response to U46619, and the response could be modified by NMDA and GABA, but not by picrotoxin. However, ibuprofen totally blocked the attenuating influence of the perivascular tissue on the response to U46619. CONCLUSIONS: The inhibition of vascular contractility induced by perivascular retinal tissue in vitro involves NMDA-receptors and an effect of GABA-mimetic substance on retinal tissue. The generation of these effects involves a COX product.

Variable involvement of the perivascular retinal tissue in carbonic anhydrase inhibitor induced relaxation of porcine retinal arterioles in vitro.

PURPOSE: Inhibition of carbonic anhydrase in the eye is an important treatment modality for reducing the intraocular pressure in glaucoma. However, evidence suggests that carbonic anhydrase inhibition also exerts a relaxing effect on the vessels in the optic nerve, and it has been suggested that this vasorelaxing effect is a result of an interplay between the perivascular tissue and constituents in the retinal vascular wall. However, the exact nature of this interplay is unknown. METHODS: Isolated porcine retinal arterioles and arterioles with preserved perivascular retinal tissue were mounted in a myograph. After precontraction with the prostaglandin analogue U46619, the vasorelaxing effect of the carbonic anhydrase inhibitors methyl bromopyruvate, ethyl bromopyruvate, acetazolamide, and dorzolamide were studied. RESULTS: All the examined carbonic anhydrase inhibitors induced a significant relaxation of retinal arterioles. There was no significant difference between the effect of the different carbonic anhydrase inhibitors in the presence of perivascular retinal tissue. However, in the isolated retinal arterioles the vasodilating effect of dorzolamide was significantly lower, and the vasodilating effect of acetazolamide almost disappeared. CONCLUSIONS: A further elucidation of the mechanisms of action of carbonic anhydrase-induced dilation of retinal arterioles may contribute to a better understanding of the regulation of retinal blood flow. The perivascular retinal tissue may play a significant role in diameter control of retinal arterioles.

ATP-induced relaxation of porcine retinal arterioles depends on the perivascular retinal tissue and acts via an adenosine receptor.

PURPOSE: Purinergic compounds and cyclooxygenase inhibitors are involved in the tone regulation of isolated retinal arterioles in vitro, but it is unknown whether the perivascular retinal tissue influences these effects. METHODS: Adenosine-and ATP-induced vasodilation of porcine retinal arterioles was studied in a wire myograph before and after removal of the perivascular tissue. RESULTS: Both adenosine and ATP caused relaxation of the studied arterioles. This effect depended on the perivascular tissue and could be blocked by antagonists but was unaffected by ibuprofen. CONCLUSIONS: The relaxation of porcine retinal arterioles induced by purinergic compounds is modulated by the perivascular retinal tissue.