Haruko Funaki

Molecular cloning of a new aquaporin from rat pancreas and liver.

A new water channel (aquaporin-8, gene symbol AQP8) was isolated from rat pancreas and liver by homology cloning. Ribonuclease protection assay showed intense expression of the gene in pancreas and liver, less intense in colon and salivary gland, and negligible in other organs. The full-length cDNA was obtained by ligation of ∼1.4-kilobase (kb) cDNA isolated from the rat liver cDNA library to ∼0.5 kb of the 5′-end fragment obtained by the rapid amplification of cDNA ends method. A major transcript of ∼1.45 kb was demonstrated in liver and colon by Northern blot analysis. Expression of the cRNA in Xenopusoocytes markedly enhanced osmotic water permeability in a mercury-sensitive manner, indicating a water channel function of this molecule. The open reading frame encoded a 263-amino acid protein with a predicted molecular size of 28 kDa. Hydropathy analysis represented six membrane-spanning domains and five connecting loops containing two sites of NPA motif as preserved in other aquaporins. Unlike other mammalian aquaporins, AQP8 has an unusual structure with a long N terminus and a short C terminus, which are found in plant aquaporin, γ-tonoplast intrinsic protein. By in situ hybridization,AQP8 mRNA expression was assumed in hepatocytes, acinal cells of pancreas and salivary gland, and absorptive colonic epithelial cells. The physiological role(s) of AQP8 remain to be elucidated.

Expression and localization of aquaporins in rat gastrointestinal tract.

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.

Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells

Aquaporin (AQP) 5 gene was recently isolated from salivary gland and identified as a member of the AQP family. The mRNA expression and localization have been examined in several organs. The present study was focused on elucidation of AQP5 expression and localization in the eye, salivary gland, and lung in rat. RNase protection assay confirmed intense expression of AQP5 mRNA in these organs but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected for total RNA isolation. AQP5 mRNA was enriched in cornea but not in other portions (retina, lens, iris/ciliary body, conjunctiva, or sclera). AQP5 was selectively localized on the surface of corneal epithelium in the eye by immunohistochemistry and immunoelectron microscopy using an affinity-purified anti-AQP5 antibody. AQP5 was also localized on apical membranes of acinar cells in the lacrimal gland and on the microvilli protruding into intracellular secretory canaliculi of the serous salivary gland. In the lung, apical membranes of type I pulmonary epithelial cells were also immunostained with the antibody. These findings suggest a role of AQP5 in water transport to prevent dehydration or to secrete watery products in these tissues.

Surgical management of retinal detachment associated with myopic macular hole: anatomic and functional status of the macula☆

PURPOSE To evaluate the postoperative status of the macula after vitreous surgery with internal limiting membrane removal for macular hole related retinal detachment in patients with severe myopia. DESIGN Interventional case series. METHODS We prospectively examined 10 eyes with retinal detachment associated with a myopic macular hole from 10 consecutive patients, and performed pars plana vitrectomy with internal limiting membrane peeling. Macular buckling was performed in one eye during the initial treatment and in three eyes during subsequent operations. The main outcome measures were the anatomic reattachment rate and the postoperative status of the macular hole. We examined the macular area pre- and postoperatively with slit-lamp biomicroscopy and with a scanning laser ophthalmoscope. Cross-sectional imaging of the macular area was conducted with optical coherence tomography. RESULTS Successful retinal reattachment was achieved in seven eyes (70%) after the initial surgery and in three eyes (30%) after additional procedures. Visual acuity remained unchanged in two eyes (20%), and improved by two or more logarithmic units of minimum angle of resolution (logMAR) measurement in eight eyes (80%). The macular hole was anatomically closed in only one eye (10%). Postoperative enlargement of the macular hole was observed in seven eyes. CONCLUSIONS In highly myopic eyes with macular hole related retinal detachment, closure of the macular hole is difficult to attain despite the complete relief of tangential traction by internal limiting membrane peeling. Results indicate the presence of a possible imbalance between the retina and the choroid-sclera complex associated with axial elongation and posterior staphyloma in highly myopic eyes.

Measurement of microcirculation in the optic nerve head by laser speckle flowgraphy and scanning laser doppler flowmetry

PURPOSE To evaluate and compare blood flow measurements by laser speckle flowgraphy and scanning laser Doppler flowmetry in the optic nerve head of normal volunteers. METHODS This prospective study included 60 eyes of 60 normal volunteers (50.0 years; range, 21 to 77 years). Measurements were taken at the temporal neuroretinal rim away from visible vessels. The square blur rate, a quantitative index of relative blood velocity, was measured by laser speckle flowgraphy. Using scanning laser Doppler flowmetry, volume, flow, and velocity were measured at the same neuroretinal rim locations. RESULTS The average square blur rate, volume, flow, and velocity were 7.11 +/- 1.65, 7.74 +/- 3.19, 151.85 +/- 70.63, and 0.53 +/- 0. 23 arbitrary units, respectively (n = 60). Square blur rate correlated significantly with flow and velocity (r =.361, P =.005; r =.359, P =.005, respectively). However, there was no significant correlation between square blur rate and volume (r =.101, P =.441). Although square blur rate decreased significantly with increasing age (r = -.375, P =.003), volume, flow, or velocity showed no significant correlation with age (r = -.249, P =.054; r = -.166, P =. 205; r = -.143, P =.275, respectively). Square blur rate also decreased significantly with mean blood pressure (r = -.315, P =. 014), but volume, flow, or velocity showed no significant correlation with mean blood pressure (r = -.159, P =.225; r = -.059, P =.654; r = -.043, P =.742, respectively). CONCLUSION We found only a weak correlation between the blood flow indexes, as measured by laser speckle flowgraphy and scanning laser Doppler flowmetry because of basic differences in the principles of measurement.

Measurement of microcirculation in optic nerve head by laser speckle flowgraphy in normal volunteers

PURPOSE To report blood flow in the optic nerve head between the right and left eyes or the superior and inferior neuroretinal rims in normal volunteers using laser speckle flowgraphy. METHODS This prospective study included 120 eyes of 60 normal volunteers (mean age, 50.0 +/- 16.9 years; range, 21 to 77 years). The square blur rate was measured by laser speckle flowgraphy (Kyushu Institute of Technology, Iizuka, Japan). The sequence of eye measurements was randomized. In each eye, measurements were taken at the neuroretinal rim away from visible vessels. Linear regression analysis, paired two-tailed t test, and two-way analysis of variance were used for statistical analysis. P values less than.05 were accepted as statistically significant. RESULTS There was a significant correlation in square blur rate between the right and left eyes (r = 0.587, P <.001). Square blur rate in the superior temporal neuroretinal rim significantly correlated with that in the inferior temporal neuroretinal rim in each of the right (r = 0.546, P <.001) and left (r = 0.465, P <.001) eyes. Square blur rate in the right eye was higher than that in the left eye (P =.049). Square blur rate in the superior neuroretinal rim was higher than that in the inferior neuroretinal rim in both the right (P =.035) and left (P =. 005) eyes. CONCLUSION There were statistically significant differences of optic nerve head blood flow in normal volunteers using laser speckle flowgraphy between the right and left eyes and between the superior and inferior temporal neuroretinal rims. These normal data can be used for understanding physiological ocular hemodynamics.

Relationship between age and the thickness of the retinal nerve fiber layer in normal subjects.

PURPOSE To determine it there are any age-dependent changes in the thickness of the retinal nerve fiber layer (RNFL) in the peripapillary area. METHODS Sixty normal volunteers (31 men, 29 women) (120 eyes) whose ages ranged from 23-75 years (mean 48.4 years) participated in this study. The thickness of the RNFL was determined using a scanning laser polarimeter along the peripapillary area with a 1.75 disc diameter and along another ring 0.8 mm away from the disc margin. RESULTS The thickness of the RNFL was not significantly correlated with age in either of the two ring areas. However, the RNFL thickness ratio of total/nasal area decreased significantly with increase in age in both rings. There was an increase in the difference of RNFL thickness between the right and left eyes of the same individual with aging, in both rings. CONCLUSIONS It was suggested that the RNFL thickness determined along both rings demonstrated almost identically the relationship between age and RNFL thickness in normal subjects.

Measurement of intraocular pressure using the NT-4000: a new non-contact tonometer equipped with pulse synchronous measurement function.

Purpose:NT-4000 (Nidek Co. Ltd., Gamagori, Japan) is a new non-contact tonometer (NCT) equipped with pulse synchronous measurement function that can measure intraocular pressure (IOP) synchronized with the ocular pulse. The purpose of this study was to evaluate the usefulness of NT-4000 in normal subjects and in patients with glaucoma and ocular hypertension. Methods:This study included 175 eyes of 175 subjects. Firstly, the IOP was measured using NT-4000 without the pulse synchronous measurement function (NTn). Secondly, the IOP at peak, middle, and trough phases of the pulse signal were measured using NT-4000 with the pulse synchronous measurement function (NTp, NTm, NTt, respectively). Additionally, the IOP was measured with Goldmann applanation tonometer (GT). The coefficient of variation (CV) of three readings in the NCT measurements was used to evaluate the intra-session reproducibility. Statistical comparisons were performed using Wilcoxon signed rank test and one-way analysis of variance with Scheffes test. Linear regression analysis was used to calculate correlation coefficients. P values less than 0.05 were accepted as statistically significant. Results:The CV of NTn, NTp, NTm, and NTt were 6.4%, 5.5%, 4.9%, and 5.2%, respectively. The CV of NTp, NTm, and NTt were significantly smaller than that of NTn (P = 0.007, P < 0.001, P < 0.001, respectively). NTp was significantly higher than NTt (P = 0.038). GT was significantly correlated with NTn, NTp, NTm, and NTt (r = 0.898, P < 0.001; r = 0.912, P < 0.001; r = 0.908, P < 0.001; r = 0.900, P < 0.001, respectively). Conclusion:NT-4000 can detect the fluctuation of IOP associated with the ocular pulse.

Optic disc topography as measured by confocal scanning laser ophthalmoscopy and visual field loss in Japanese patients with primary open-angle or normal-tension glaucoma.

Purpose:To determine whether differences in the optic disc topography and those in the relation between the optic disc topography and visual field indices exist between Japanese patients with primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). Methods:The study included consecutive Japanese patients with POAG (n = 60) or NTG (n = 60). Using the Heidelberg Retina Tomograph (HRT), we measured disc area, cup area, cup-to-disc area ratio, rim area, cup volume, rim volume, cup shape measure, and height variation contour. Each HRT parameter was measured in the total optic disc and in its four 90° quadrants (superior, temporal, inferior, and nasal). The mean deviation for the entire field and the sum of the total deviation values corresponding to each optic disc quadrant were calculated using the Humphrey full-threshold 30-2 program. Results:No significant differences were found between the POAG and NTG groups for any HRT parameter either globally or regionally. There were no significant differences between the two groups in the correlation coefficients between any HRT parameter and the corresponding visual field indices either globally or regionally. Conclusion:No significant differences were apparent between Japanese patients with POAG and NTG both in the optic disc parameters as measured by HRT and in the degree of correlation between HRT parameter and the corresponding visual field indices.

Measurement of retinal nerve fibre layer by scanning laser polarimetry and high pass resolution perimetry in normal tension glaucoma with relatively high or low intraocular pressure

AIMS To determine whether any differences may exist in the relation between the neural capacity as determined by high pass resolution perimetry and the thickness of the retinal nerve fibre layer (RNFL) in patients having normal tension glaucoma (NTG) with a relatively high intraocular pressure (IOP) between 16 and 21 mm Hg (HNTG)v those with a lower IOP below 15 mm Hg (LNTG). METHODS Scanning laser polarimetry and high pass resolution perimetry were performed in 20 eyes of 20 patients with HNTG and 21 eyes of 21 patients with LNTG. The correlation between total and regional thickness of the peripapillary RNFL and the corresponding total and regional neural capacity with linear regression analysis were evaluated. RESULTS Overall, although the total RNFL thickness was not significantly correlated with the total neural capacity, the RNFL thickness in each of the superior and inferior quadrants was significantly correlated with the corresponding regional neural capacity (r=0.44,p=0.0045; r=0.39,p=0.0126 for each). The RNFL thickness in each of the superior and inferior quadrants in the HNTG group was significantly correlated with the corresponding regional neural capacity (r=0.52,p=0.0196; r=0.49,p=0.0286 for each). No significant correlation between neural capacity and the RNFL thickness was observed either globally or regionally in the LNTG group. CONCLUSION The degree of the correlation between neural capacity as determined by high pass resolution perimetry and thickness of the RNFL as measured by scanning laser polarimetry appeared to differ in NTG patients with an IOP higher than 15 mm Hg v those with a lower IOP.