Regulation of nitric oxide production by δ-opioid receptors during glaucomatous injury.

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve

Measurement of protein nitrosylation in retina by Western blotting using nitrotyrosine antibodies in response to glaucomatous injury.

<p>Animals were treated with SNC-121 (1 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. Retina extracts were collected at the 7^th day, post injury, and analyzed using anti-nitrotyrosine antibodies and appropriate secondary antibodies (HRP-conjugated; dilution 1∶3000). The signal was captured using enhanced chemiluminescent reagent and the Biorad Versadoc imaging system. Data shown are representative of four independent experiments. Data are expressed as mean ± SEM. *<i>P</i><0.05; n = 4.</p

Measurements of nitrate-nitrite in normal and glaucomatous eyes in the presence of AG.

<p>Animals were treated with AG (25 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. The absorbance was measured at 550 nm and absorbance was plotted against the nitrate standards. The amount of nitrate-nitrite is expressed as millimolar nitrates-nitrites per milligram protein. Data are expressed as mean ± SEM, (*<i>P</i><0.05; n = 4–6).</p

Changes in iNOS expression in glaucomatous retina at 7 and 42 days, post injury.

<p>(A) Brown Norway rats were treated with SNC-121 (1 mg/kg, i.p.) 30 minutes after glaucomatous injury and continued for 7 days, once daily. Retina extracts were collected at the 7^th day, post injury, and analyzed using anti-iNOS antibodies and appropriate secondary antibodies (HRP-conjugated; dilution 1∶3000). The signal was captured using enhanced chemiluminescent reagent and the Biorad Versadoc imaging system. Data are expressed as mean ± SEM. *<i>P</i><0.05; n = 4. (B) The eyes of Brown Norway rats were removed 42-days post glaucomatous injury. Cryosections of retina were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Data shown in this Figure are a representation of at least four independent experiments.</p

Pattern ERG recordings in normal and glaucomatous eyes.

<p>Changes observed in Pattern ERGs of untreated, AG or SNC-121-treated glaucomatous rat eyes are shown as a percentage of the contralateral control eye. In these experiments, Brown Norway rats were treated with AG (25 mg/kg, i.p.) or SNC-121 (1 mg/kg, i.p.), individually or together simultaneously, 30 minutes after hypertonic saline injections. Animals were treated with naltrindole 30 minutes after glaucomatous injury followed by SNC-121 treatment. The drug treatment was continued once daily for 7 days in all groups. Data are mean ± SE; *<i>P</i><0.05; n = 7–8.</p

Changes in iNOS expression in glaucomatous optic nerve at 42 days, post injury.

<p>The optic nerve (non-myelinated; 2 mm post globe) of Brown Norway rats was removed 42-days post glaucomatous injury. Contralateral optic nerve was used as the normal control. Cryosections were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Data shown in this Figure are a representation of at least four independent experiments. A total of 10 animals were used in this experiment. Comparable staining for iNOS was seen in at least 4 animals in each treatment group.</p

Retina flat mounts of normal and glaucomatous eyes.

<p>Fluorescence micrographs of flatmounted retinas depicting Fluorogold-labeled retina ganglion cells (RGCs) in normal and glaucomatous eyes in the absence or presence of drugs (<b>A</b>). Briefly, 3 µL of a 5% solution of Fluorogold was injected into the superior colliculus of anesthetized animals. Seven days post injection, animals were euthanized and retinas were prepared as flatmounts, vitreous-side facing up. Fluorescent RGCs were visualized under Zeiss microscopy. Bar = 20 µm. Total RGC tallies in glaucomatous eyes in the absence or presence of drugs. RGCs were counted in 8 microscopic fields of identical size (150 µm² area) for each retina, using Image J software (<b>B</b>). *<i>P</i><0.05; n = 6 for each group.</p

Changes in iNOS expression in glaucomatous optic nerve at 7 days, post injury.

<p>The optic nerve (non-myelinated; 2 mm post globe) of Brown Norway rats was removed 7-days post glaucomatous injury. Contralateral optic nerve was used as the normal control. Cryosections were immunostained by anti-iNOS antibodies as indicated horizontally. Ocular treatments are indicated vertically. Green color indicates staining for iNOS and blue nuclei for DAPI. There was no positive staining when primary antibodies were omitted (data not shown). Data shown here are representations of at least four independent experiments. A total of 10 animals were used in this experiment. Comparable staining for iNOS was seen in at least 4 animals in each treatment group.</p

Missed appointments in primary care: questionnaire and focus group study of health professionals.

BACKGROUND: The issue of missed appointments in primary care is important for patients and staff. Little is known about how missed appointments, and the people who miss them, are managed in primary care, or about effective strategies for managing missed appointments. AIMS: To understand the perceptions of primary care staff as to why patients miss appointments, to determine how these perceptions influence their management, and to explore the merit of different management strategies. Design of study: A postal questionnaire survey and focus group interviews. SETTING: General practices in Yorkshire. RESULTS: Missed appointments were regarded as an important problem. Patient factors rather than practice factors were perceived as most important in causing missed appointments. Intervention strategies appeared to be driven by perceptions of why patients miss appointments. Negative attitudes, embodied in terms such as "offenders" to refer to those who missed appointments were prevalent, and favoured intervention strategies included punishing the patient in some way. Receptionists believed that general practitioners should address the issue of the missed appointment with the patient. General practitioners felt guarded about addressing missed appointments with their patients in case it affected the doctor-patient relationship. CONCLUSION: People who miss appointments were viewed negatively by primary care staff, and most of the reasons for missed appointments were focused on patients. These beliefs underpinned intervention strategies aimed mainly at punishment. Since there is no evidence base concerning interventions that are effective in reducing missed appointments, these negative attitudes may not be beneficial to staff or their patients

Regulation of Nitric Oxide Production by δ-Opioid Receptors during Glaucomatous Injury

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve