Nanosecond laser therapy reverses pathologic and molecular changes in age-related macular degeneration without retinal damage.

Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression.

Immunolocalization of the P2X4 receptor on neurons and glia in the mammalian retina.

Extracellular adenosine 5'-triphosphate (eATP) acts as a neurotransmitter within the retina and brain, activating a range of ionotropic P2X and metabotropic P2Y receptors. In this study, the specific localization of the P2X4 receptor (P2X4-R) subunit was evaluated in the retina using fluorescence immunohistochemistry and pre-embedding immuno-electron microscopy. Punctate P2X4-R labeling was largely localized to the inner and outer plexiform layers of mouse, rat and cat retinae. In the mouse outer retina, double-labeling of P2X4-R with the horizontal cell marker, calbindin, revealed P2X4-R immunoreactivity (P2X4-R-IR) on horizontal cell somata and processes. In the inner retina, P2X4-R expression was found closely associated with rod and cone bipolar cell terminals, and the punctate labeling was observed on calretinin-positive amacrine cells. Using immuno-electron microscopy, P2X4-Rs were observed on processes post-synaptic to photoreceptor and bipolar cell terminals, likely representing horizontal, amacrine and ganglion cells, respectively. Furthermore, P2X4-R expression was also observed on Müller cells, astrocytes and microglia. These data suggest a role for P2X4-Rs in the lateral inhibitory pathways of the retina, modulating neuronal function of photoreceptors and bipolar cells. The expression on macro- and microglial cells implicates a role for P2X4-Rs in glial signaling, tissue homeostasis and immunosurveillance within the mammalian retina.

Characterization of retinal function and glial cell response in a mouse model of oxygen-induced retinopathy.

Retinal neovascularization, such as that occurring in proliferative diabetic retinopathy and retinopathy of prematurity, can have serious effects on visual function. By using a mouse model of neovascularization, oxygen-induced retinopathy (OIR), the interplay among angiogenesis, neuronal function, and the macro- and micro-glial response was explored. OIR was induced by exposure of mice to 75% oxygen from postnatal day 7 (P7) to P11 and then room air until P18. Controls were reared in room air. Blood vessel development was assessed by using fluorescence histochemistry. Aberrant intravitreal neovascularization was present across all eccentricities of retina in mice with OIR, whereas the number of vessels present in the deep plexus was reduced in the central regions. Neuronal function of both the rod and cone pathways, assessed by using the electroretinogram, was found to be significantly reduced in OIR. This may in part be explained by an alteration in photoreceptor outer segment morphology and also a loss of neurons and their synapses in the inner nuclear and plexiform layers of the central retina. In addition, there was an increase in the number of gliotic Müller cells and microglia in mice with OIR and the increase in the number of these cells correlated with the absence of the deep plexus. This indicates that the activity of both macro- and microglia is altered in regions where the deep plexus blood supply is deficient. Treatments or genetic manipulations directed toward amelioration of proliferative retinopathy need to address not only the vascular changes but also the alterations in neuronal and macro- and microglial function.

Neuronal and glial cell expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the rat retina.

The bio-active peptide, angiotensin II (Ang II), has been suggested to exert a neuromodulatory effect on inner retinal neurons. In this study, we examined the distribution of angiotensin receptors (ATRs) in the developing and mature rat retina and optic nerve using immunofluorescence immunocytochemistry. Double-labeling experiments were performed with established markers to identify different retinal cell populations. In adult retinae, ATRs were observed on neurons involved in "ON" pathways of neurotransmission. Angiotensin II type 1 receptors (AT(1)Rs) were expressed by a sub-population of "ON" cone bipolar cells that also labeled for G alpha(0) and islet-1. Extra-neuronal expression of AT(1)Rs was evident on retinal astrocytes, Müller cells and blood vessels. Immunoreactivity for the angiotensin II type 2 receptor (AT(2)R) was observed on conventional and displaced GABAergic amacrine cells. Co-localization studies showed that AT(2)R-expressing amacrine cells constituted at least two separate sub-populations. Cell counts revealed that all wide-field amacrine cells expressing protein kinase C-alpha were also AT(2)R-positive; a further subset of amacrine cells expressing AT(2)Rs and stratifying in sublamina "b" of the inner plexiform layer (IPL) was identified. Developmental expression of AT(1)Rs was dynamic, involving multiple inner neuronal classes. At postnatal day 8 (P8), AT(1)R immunoreactivity was observed on putative ganglion cells. The characteristic bipolar cell labeling observed in adults was not evident until P13. In contrast, AT(2)Rs were detected as early as P2 and localized specifically to amacrine cells from this age onward. These data provide further evidence for the potential role of angiotensin II in the modulation of retinal neurons and glia. The differential pattern of expression of these receptors across these cell types is similar to that observed in the brain and suggests that a similar functional role for Ang II may also exist within the retina.

Diisopropylfluorophosphate alters retinal neurotransmitter levels and reduces experimentally-induced myopia.

The excessive enlargement of the eye seen in myopia is known to be regulated, in part, by retinal neurotransmitters. One excitatory retinal neurotransmitter, acetylcholine, has been implicated in the signal cascade through the effectiveness of the muscarinic antagonist, atropine, in preventing myopia development. The present study therefore examined whether an indirect cholinomimetic, diisopropylfluorophosphate (DFP), could increase the level of experimental myopia, induced by the deprivation of pattern vision. Injections of either phosphate-buffered DFP or phosphate-buffered saline were made every 48 h into the vitreous chamber of one eye of chicks. The injected eye was then deprived of pattern vision by a translucent occluder. The fellow eye remained untreated and acted as a genetic control. At the end of the treatment period (8 days) axial ocular dimensions and cycloplegic refractive error were measured. To investigate the effects of DFP on retinal neurotransmitter levels, measurements of acetylcholine and dopamine contents were made on retinal tissue following either a single or multiple DFP injections, using reverse phase high performance liquid chromatography (HPLC). Rather than potentiating myopia and vitreous chamber elongation, a significant reduction in myopia (58%) was observed in DFP-injected deprived eyes, compared to saline controls. However, open eyes injected with DFP showed no difference in refraction or vitreous chamber depth compared to contralateral control eyes or saline controls. HPLC analysis revealed increased steady-state content of acetylcholine (+34 +/- 6 ng/mg protein, mean +/- SEM, P<0.01, equivalent to a 54% increase) and dopamine (+377 +/- 83 pg/mg protein, P<0.01, a 36% increase) in DFP-treated eyes compared to contralateral control eyes following a single DFP injection. No changes in either acetylcholine or dopamine content were found in saline-treated control animals. Injection of dopamine antagonists, in combination with DFP, indicated that the DFP-induced reduction in myopia is mediated, at least in part, through a D2 receptor mechanism. Findings argue against a direct cholinergic 'stop-go' pathway controlling ocular growth. Instead the reduction of induced myopia could be related to the action of DFP (directly or indirectly) on dopamine levels in the retina.

Angiotensin-induced enhancement of excitatory junction potentials evoked by periarteriolar nerve stimulation and vasoconstriction in rat mesenteric arteries are both mediated by the angiotensin AT1 receptor.

The aims of the present study were to determine the angiotensin II (AngII) receptor subtype(s) involved in vasoconstriction and enhancement of sympathetic neurotransmission in rat isolated mesenteric arteries. Vasoconstriction was assessed in mesenteric artery ring preparations suspended under 0.5 g of tension in a myograph. In control arteries, with an intact endothelium, AngII (1 nmol/l-3 micromol/l) caused a concentration-dependent contraction. The pEC(50) for AngII was 7.6 +/- 0.2 and the maximum response of 0.24 +/- 0.07 g was reached with 100 nmol/l. In the presence of indomethacin (3.0 micromol/l) and N(omega)-nitro-L-arginine (NOLA) (100 micromol/l) to remove the influence of endothelium-derived prostaglandins and nitric oxide, the maximum response evoked by AngII was increased to 0.48 +/- 0.1 g and the pEC(50) was 7.6 +/- 0.3. The AT1 receptor antagonist losartan (30 nmol/l) competitively blocked the AngII-induced contractions with an estimated pA(2) of 8.2 in both the control arteries and in arteries treated with indomethacin and NOLA. The AT2 receptor antagonist PD 123319 (1 micromol/l) did not affect AngII-induced contractions under either condition. Conventional intracellular microelectrode recording techniques were used to investigate the effects of AngII on excitatory junction potentials (EJP) evoked by stimulation of periarteriolar sympathetic nerves. Stimulation with trains of 10 pulses delivered at 0.9 Hz evoked EJP which were blocked by tetrodotoxin (0.1 micromol/l), guanethidine (30 micromol/l) and the P(2X) receptor desensitizing agent alpha,beta-methylene ATP (30 micromol/l) suggesting the EJP were mediated by ATP, or a related purine, released from sympathetic nerves. AngII (0.3- 100 nmol/l) did not affect the resting membrane potential or the amplitude of the first EJP, but did enhance the amplitude of the plateau EJP later in the train. A maximum 49.2 +/- 3.9% enhancement of the plateau EJP amplitude was elicited by 10 nmol/l AngII and the pEC(50) was 9.1 +/- 0.1. The facilitatory effect of AngII on EJP amplitude was not altered in the presence of indomethacin and NOLA. Losartan (30 nmol/l) competitively blocked the AngII-induced enhancement of plateau EJP amplitude, with an estimated pA(2) of 8.6. PD 123319 did not alter the enhancement of plateau EJP amplitude by AngII. The results from the present study show that both the vasoconstriction and enhancement of plateau EJP amplitude by AngII in rat mesenteric arteries are blocked by the AT1 receptor antagonist losartan and are unaffected by the AT2 receptor antagonist PD 123319.

Using Medicaid data to estimate state- and county-level prevalence of asthma among low-income children.

OBJECTIVES: Asthma is one of the most common illnesses among children, yet there is little reliable information on the number of children at the state and county level who are living with asthma. This study examines the prevalence of asthma among low-income children in North Carolina using Medicaid paid claims and enrollment data. METHODS: Claims paid by Medicaid during state fiscal year 1997-1998 with a diagnosis of asthma or for a prescription drug used to treat asthma are examined to estimate prevalence among children ages 0-14 years. Percentages of enrolled children with asthma are presented by age, race, and rural/urban residence, and the costs of asthma treatment are calculated. RESULTS: More than 12% of North Carolina children ages 0-14 years on Medicaid had an indication of asthma. Prevalence rates were found to be highest among younger children, some minority groups, and residents of rural areas. More than $23 million was paid by Medicaid during the fiscal year for asthma-related services for children ages 0-14 years. CONCLUSIONS: State Medicaid databases are a useful means of studying the prevalence of asthma and other health conditions in low-income populations. Strengths and weaknesses of the proposed methodology are discussed. Existing administrative data systems can provide quick updates of prevalence rates at the state and county level, enhancing the ability to study trends in illness over time.

abnormal chromatin (abc), a maternal-effect locus in Drosophila melanogaster.

Mutations in the maternal-effect gene abnormal chromatin (abc) in Drosophila melanogaster result in a variety of defects involving nuclear replication/division. Three recessive alleles of this gene, which maps near 51F on chromosome 2, all result in female sterility. They cause slower embryonic development that is usually abnormal from the earliest nuclear divisions and arrested by the sixth one. Nuclei tend to be large and erratically distributed, some intensely staining. Mitotic asynchrony is common. Few embryos reach the gastrula stage and none hatch. With the weakest allele, fsPL, bridges between nuclei are common; abnormal chromatin clumps that resemble yolk nuclei occur before the other nuclei reach the surface; and spindle anomalies and DNA wads with numerous centrosomes are seen. Females with the stronger alleles, fsA5 and fs27, lay fewer eggs and a smaller proportion of embryos reach blastoderm; developmental arrest occurs earlier, usually with several large nuclei distributed along the length of the embryo. Chorion defects occur in all three mutants. Mitotic asynchrony, nuclear bridging, endoreduplication and nuclear behavior aberrant from the first division suggest that the abc gene product operates in DNA replication/nuclear division. Larval (homozygous F1) neuroblast chromosome structure and mitotic indices are normal, indicating that any mitotic function is strictly maternal, i.e. abc is not a general mitotic gene. Thus abc is one of a few known genes with a maternal effect that appears to function in the embryonic cell cycle.

Genetics of 51D-52A, a region containing several maternal-effect genes and two maternal-specific transcripts in Drosophila.

Two genomic clones exhibiting a maternal-specific pattern of expression map to cytological region 52A. To elucidate the function of these clones we have undertaken a mutagenesis of the cytological region 51D-52A. This paper presents the results of this screen and the preliminary analysis of female-sterile and lethal mutations isolated. A total of twelve complementation groups have been identified, four of which are defined exclusively by female-sterile alleles. Only one visible mutation was isolated, a recessive temperature-sensitive allele of Thickened-arista (Tarts). Several of the seven lethal loci display an embryonic lethal phase. Three of the four female-sterile loci affect chorion structure with one resulting in underamplification of the chorion genes, and two (possibly three) of the four female-steriles affect nuclear division/DNA replication. Thus it appears that this is a "developmentally important" region, possibly representing a clustering of genes involved in either DNA replication or nuclear division.