Painless Indirect Argon Laser in High Risk Proliferative Diabetic Retinopathy.

Background: Proliferative retinopathy is an important cause of vision loss in diabetic patients. Incomplete panretinal photocoagulation (PRP) can lead to recurrent proliferation of new vessels. Patients and Methods: We retrospectively analysed the outcome of patients with high risk proliferative diabetic retinopathy (PDR) previously treated with slit lamp PRP who underwent indirect fill in argon laser treatment with scleral indentation under anesthesia for persistent neovascular proliferation. Results: Seventeen eyes of ten patients were included. The mean age at diabetes onset was 17.3 years SD 16.2 (range 2-44). All patients reported long standing poor glycemic control (mean HbA1c: 8.5 % SD 1.3 range 5.9-10.2). The area of retinal ischemia decreased significantly from 15 ± 7.5 disk areas (DA) before fill-in laser to 3.2 ± 4.2 DA after fill-in laser (p = 0.001). The new vessels also regressed significantly after laser treatment 8.6 ± 6.1 DA before treatment versus 6.5 ± 6.4 DA after laser treatment, (p = 0.044). Quiescent PDR was reached in 10 eyes (58.8 %) at the last visit. Conclusions: Fill-in indirect argon laser under general anesthesia should be considered to achieve further new vessels regression in high risk PDR patients. Scleral indentation and absence of pain may allow for more extensive laser application.

Use of the dried blood spot sampling process coupled with fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry: application to fluoxetine, norfluoxetine, reboxetine, and paroxetine analysis.

The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 microL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30 min at 60 degrees C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5 min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500 ng mL(-1) for fluoxetine and norfluoxetine and 20 to 500 ng mL(-1) for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20 pg mL(-1) for all the analytes. The stability of DBS was also evaluated at -20 degrees C, 4 degrees C, 25 degrees C, and 40 degrees C for up to 30 days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractive-derivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studies.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

Acid-sensing ion channel 1a drives AMPA receptor plasticity following ischemia and acidosis in hippocampal CA1 neurons

The CA1 region of the hippocampus is particularly vulnerable to ischemic damage. While NMDA receptors play a major role in excitotoxicity, it is thought to be exacerbated in this region by two forms of post-ischemic AMPA receptor (AMPAR) plasticity - namely, anoxic long-term potentiation (a-LTP), and a delayed increase in the prevalence of Ca2+ -permeable GluA2-lacking AMPARs (CP-AMPARs). The acid-sensing ion channel 1a (ASIC1a) which is expressed in CA1 pyramidal neurons, is also known to contribute to post-ischemic neuronal death and to physiologically induced LTP. This raises the question - does ASIC1a activation drive the post-ischemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD), and dissociated cultures of hippocampal pyramidal neurons (HPN) exposed to low pH (acidosis). We find that both a-LTP and the delayed increase in the prevalence of CP-AMPARs are dependent on ASIC1a activation during ischemia. Indeed, acidosis alone is sufficient to induce the increase in CP-AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP-AMPARs. By demonstrating that ASIC1a activation contributes to post-ischemic AMPAR plasticity, our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells, and provide insight into the role of ASIC1a and CP-AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP-AMPAR plasticity, and that inhibiting ASIC1a affords neuroprotection.

Extracellular Subunit Interactions Control Transitions between Functional States of Acid-sensing Ion Channel 1a.

Acid-sensing ion channels (ASICs) are neuronal, voltage-independent Na(+) channels that are transiently activated by extracellular acidification. They are involved in pain sensation, the expression of fear, and in neurodegeneration after ischemic stroke. Our study investigates the role of extracellular subunit interactions in ASIC1a function. We identified two regions involved in critical intersubunit interactions. First, formation of an engineered disulfide bond between the palm and thumb domains leads to partial channel closure. Second, linking Glu-235 of a finger loop to either one of two different residues of the knuckle of a neighboring subunit opens the channel at physiological pH or disrupts its activity. This suggests that one finger-knuckle disulfide bond (E235C/K393C) sets the channel in an open state, whereas the other (E235C/Y389C) switches the channel to a non-conducting state. Voltage-clamp fluorometry experiments indicate that both the finger loop and the knuckle move away from the β-ball residue Trp-233 during acidification and subsequent desensitization. Together, these observations reveal that ASIC1a opening is accompanied by a distance increase between adjacent thumb and palm domains as well as a movement of Glu-235 relative to the knuckle helix. Our study identifies subunit interactions in the extracellular loop and shows that dynamic changes of these interactions are critical for normal ASIC function.