Efficacy of the 5-HT1A agonist tandospirone citrate in improving symptoms of patients with functional dyspepsia: a randomized controlled trial.

OBJECTIVES: Functional dyspepsia (FD) is a common condition in the general population; however, its treatment remains a challenge. The aim of this study was to examine the efficacy of tandospirone citrate, a new partial agonist of the 5-hydroxytryptamine 1A (5-HT1A) receptor, in improving the symptoms of patients with FD. METHODS: In this double-blind, placebo-controlled, multicenter study, FD patients were randomized to treatment with 10 mg t.i.d. tandospirone citrate or to placebo for 4 weeks. The primary end point was change in abdominal symptom scores. The difference in the proportion of responders (a total abdominal symptom score of 0 or 1) was also assessed. The quality-of-life questionnaire, the SF-8, and a psychological test questionnaire, the State-Trait Anxiety Inventory (STAI), were completed at baseline and at weekly intervals. RESULTS: Data were available for 144 patients: 73 for tandospirone and 71 for placebo. Improvements in total abdominal scores were significantly larger with tandospirone than placebo at weeks 1, 2, and 4. Significantly greater improvements in the tandospirone group were observed in upper abdominal pain (P=0.02) and discomfort (P=0.002) at week 4. The proportion of responders was significantly greater in the active treatment arm at weeks 3 (P=0.017) and 4 (P=0.0016). Significant improvements in STAI (P<0.0001) were reported in both arms, as well as in the majority of questions in the SF-8 (P=0.04). No serious adverse events were reported, with similar rates in both study arms. CONCLUSIONS: Despite a considerable placebo effect, the benefits of tandospirone were shown in terms of improvement in abdominal symptom scores.

Beta-catenin mutation in carcinoma of the uterine endometrium.

Beta-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator downstream of the Wnt signaling pathway. Activation of the pathway by stabilization of beta-catenin has been shown to be important in the development of colorectal carcinoma, which is mainly caused by inactivating mutations of the adenomatous polyposis coli tumor suppressor gene or by activating mutations in exon 3 of the beta-catenin gene. Here, we analyzed mutations in exon 3 of the beta-catenin gene in endometrial carcinoma cases in which loss of heterozygosity at the adenomatous polyposis coli tumor suppressor gene locus has been rarely reported. We found that 10 of 76 cases had beta-catenin gene mutations. All mutations identified were single-base missense mutations on serine/threonine residues (codons 33, 37, 41, and 45), altering the glycogen synthase kinase-3beta phosphorylation consensus motif, which participates in the degradation of beta-catenin. To determine whether these beta-catenin mutations actually led to stabilization of this protein, expression of beta-catenin was analyzed immunohistochemically, and 9 of 10 cases with the beta-catenin mutation and 20 of 66 cases without it showed accumulation of beta-catenin in the cytoplasm and/or nucleus. In total, 38% of cases showed accumulation of beta-catenin. These data indicate that stabilization of beta-catenin due to mutations in exon 3 of the beta-catenin gene and other mechanisms may have an important role in development of endometrial carcinomas.

Clinical evaluation of microcystic macular edema in patients with glaucoma.

PurposeTo investigate the prevalence of microcystic macular edema (MME) in patients with glaucoma and the relationship between glaucomatous visual field defects and MME.Patients and methodsWe analyzed 636 eyes of 341 glaucoma patients who underwent spectral domain optical coherence tomography (SD-OCT). MME was defined as vacuoles observed in the inner nuclear layer (INL) on SD-OCT. Quantitative assessment of MME area was performed using en-face imaging obtained swept-source OCT (SS-OCT) and Adobe Photoshop CS6 Extended software. These values were compared with the visual field results with the Humphrey field analyzer.ResultsMME was observed in 1.6% of eyes. The visual field mean deviation (MD), pattern standard deviation (PSD) and visual acuity was significantly worse (P= 0.023, P=0.037, and P=0.018, respectively) in eyes with MME. The average MME area was 2.38±1.43%. There was no significant correlation between visual field deficits and MME area.ConclusionsThe MME detection rate based on general inspection was 1.6%. MME in glaucomatous eyes were associated with worse MD, PSD, and visual acuity. Further research is needed to increase the number of cases to allow for more detailed analysis.

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Topically administered timolol and dorzolamide reduce intraocular pressure and protect retinal ganglion cells in a rat experimental glaucoma model.

AIMS: This study sought to elucidate the effects of timolol and dorzolamide on intraocular pressure (IOP) and retinal ganglion cell (RGC) death in an experimental model of glaucoma in rat. METHODS: Mild elevation of IOP was induced in rats by intracameral injection of India ink and subsequent laser trabecular photocoagulation. IOP was measured before the surgical procedures and weekly thereafter. Timolol (0.5%), timolol XE (0.5%), dorzolamide (1%), and artificial tears (vehicle) were topically applied daily. Retinal sections were prepared for histology to determine RGC number. RESULTS: Timolol, timolol XE, and dorzolamide induced a significant reduction in IOP (p<0.05) and counteracted the reduction in RGC number that occurred in vehicle treated glaucomatous eyes (p<0.05). The coefficient of correlation between RGC number and IOP was significant in the dorzolamide treated group (r = -0.908, p<0.005), but not in other groups (p>0.05). CONCLUSIONS: Both timolol formulation and dorzolamide reduced IOP and protected RGCs in a rat model of experimental glaucoma. It cannot be ruled out that timolol might protect RGCs by additional mechanisms other than simply lowering of IOP.

Isolation, overexpression and disruption of a Saccharomyces cerevisiae YNK gene encoding nucleoside diphosphate kinase.

Nucleoside diphosphate kinase (NDPK) is the major enzyme responsible for the synthesis of all nucleoside triphosphates except ATP. A gene (YNK) encoding NDPK was isolated from the yeast Saccharomyces cerevisiae. The coding region consists of 459 bp encoding 153 amino acid (aa) residues. The M(r) of NDPK, calculated from the deduced aa sequence, is 17,166. Yeast NDPK was 59% and 58% identical to the rat NDPK alpha and beta, respectively. Overexpression of YNK in yeast showed high NDPK activity. The overproduced NDPK cross-reacted with anti-NDPK antibody raised against rat NDPK by Western blot analysis. Despite the fact that NDPK has features of a housekeeping enzyme, disruption of the YNK locus in a haploid strain was neither lethal nor significantly affected phenotypic behaviors such as growth rate, spore formation, mating ability and morphology. Yeast with a defective YNK still possessed NDPK activity to approximately 10% of the wild-type level. Possible sources of the remaining enzyme activity are discussed.

The Saccharomyces cerevisiae SSD1 gene is involved in the tolerance to high concentration of Ca2+ with the participation of HST1/NRC1/BFR1.

The SSD1 gene of Saccharomyces (S.) cerevisiae is a polymorphic gene involved in many aspects of the yeast cell growth (Sutton et al., 1991). We found that ssd1 null mutant shows increased sensitivities of growth to trifluoperazine (TFP) and high concentration of Ca2+. A high-copy suppressor gene, HST1, for the TFP and Ca2+ sensitivities of ssd1 null mutant was cloned and sequenced. The HST1 gene encodes a polypeptide of 915 amino acids, and is identical to the NRC1/BRF1 gene in databases. The HST1 disrupted cells were viable, but they grew slowly in the presence of high levels of Ca2+, with notable morphological change. In addition, disruption of the gene in a ssd1 null mutant further increased the sensitivities of the cells to TFP and Ca2+. The results indicated the possibility that the SSD1 gene is involved in the tolerance mechanism to high concentration of Ca2+, and the HST1 gene participates with SSD1 by its functional redundancy in Ca2+ tolerance.

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Pharmacokinetics and metabolism of 123I-BMIPP fatty acid analog in healthy and CD36-deficient subjects.

UNLABELLED: Some have suggested that CD36, which is a multifunctional receptor with a molecular weight of 88 kDa, functions as a long-chain fatty acid (LCFA) transporter. We recently reported on a complete myocardial accumulation defect of the radiolabeled LCFA analog (123)I-15-(p-iodophenyl)-(R,S)-methylpentadecanoic acid (BMIPP) in patients with CD36 deficiency. In this study, we investigated the pharmacokinetics of BMIPP in patients with a myocardial accumulation defect of BMIPP accompanied by CD36 deficiency. METHODS: Five patients (3 men, 2 women) with CD36 deficiency and 3 healthy men were investigated. Serial myocardial images were obtained every 70 s for 20 min (dynamic acquisition) and at 30, 60, 120, 180, and 240 min (static acquisition) after an intravenous bolus injection of 148 MBq BMIPP. Whole-body imaging was performed 60 min after injection. Plasma levels of BMIPP and its final metabolite, piodophenylacetic acid, at 2, 5, 10, 30, 60, 120, and 240 min after administration were determined. RESULTS: In the CD36-deficient patients, myocardial images could not be obtained for up to 240 min after administration, and cardiac pool images showing only the cardiac chambers were obtained. The heart-to-mediastinum ratio was significantly lower in the CD36-deficient patients than in the healthy volunteers (1.71 +/- 0.11 versus 2.95 +/- 0.22, P < 0.05). Hepatic uptake of BMIPP was nearly double in CD36-deficient patients. The elimination of BMIPP from the circulation was retarded in the CD36-deficient patients. CONCLUSION: We suggest that CD36 deficiency leads to decreased myocardial accumulation of BMIPP and retardation of BMIPP elimination from the circulation. The accumulation defect is probably caused by a defect in LCFA uptake into the myocardium through CD36.

Sulfated proteoglycans in the human lamina cribrosa.

The sulfated proteoglycans in the normal human lamina cribrosa were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, three types of cuprolinic blue-positive proteoglycan filaments with different sizes were associated with collagen fibers. These filaments, which were partially sensitive to chondroitinase AC and chondroitinase B, were completely removed by chondroitinase ABC and were identified as chondroitin/dermatan sulfate proteoglycans. In addition, small punctate and filamentous structures that stained with cuprolinic blue were associated with the basal laminae of astrocytes and blood vessels. Enzyme chondroitinase ABC had no effect, but heparinase digested all of these basement membrane-associated structures, indicating that they represented heparan sulfate proteoglycan molecules. Keratanase did not affect any of the cuprolinic blue-positive materials. This investigation illustrates the ultrastructural distribution and morphology of proteoglycans in the human lamina cribrosa and provides baseline information for future studies regarding the roles of proteoglycan molecules in diseases such as glaucoma.

Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells.

PURPOSE: To investigate the response of trabecular meshwork cells to phagocytic events. METHODS: Cultured bovine trabecular meshwork cells were established and exposed to latex microspheres for 40 to 44 hours. After phagocytosis, the cohesiveness of cells to their underlying matrix was measured by the susceptibility to trypsin, as indicated by the time needed to be liberated from culture plates. The amounts of two cell attachment proteins, fibronectin and laminin, in both the phagocytically challenged and the control cultures were measured at various postphagocytosis time points with an enzyme-linked immunosorbent assay. The fibronectin and laminin network was visualized with immunostaining. The mRNA levels were analyzed by Northern blot. Zymography using gelatin-containing gels was also performed to examine the gelatinase activities. RESULTS: Compared with controls, cells in phagocytically challenged cultures were more sensitive to trypsin. At the 4- and 8-hour postphagocytosis time points, the trypsinization time needed to suspend cells from tissue culture plates was significantly shorter for phagocytically challenged cells. Also, at these two time points, reduced amounts of fibronectin and laminin, as well as disruption of the fibronectin-laminin network, were observed in the phagocytically challenged trabecular meshwork cultures. The mRNA level for fibronectin was reduced, and a slightly increased gelatinase activity was noted. The fibronectin and laminin levels returned to normal by 24 hours. CONCLUSIONS: Results suggest that after phagocytosis, trabecular meshwork cells exhibit a short-term loss in cell-matrix cohesiveness. Such a loss may be related to diminished levels of cell attachment proteins.

Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin).

PURPOSE: To determine if the cornea synthesizes alpha 1-proteinase inhibitor (alpha 1-antitrypsin). METHODS: Human corneas were placed in organ culture for 24 hours in the presence of 35S-methionine to radiolabel corneal proteins. Monoclonal antibodies were used to precipitate labeled alpha 1-proteinase inhibitor. The immunologically isolated inhibitor was electrophoresed on polyacrylamide gels and visualized by autoradiography or by staining for protein. Human corneas were also fixed with formalin and imbedded in paraffin. Sections were probed with 3H-labeled complementary DNA probes to the coding region of alpha 1-proteinase inhibitor. RESULTS: Metabolically labeled alpha 1-proteinase inhibitor was recovered from organ-cultured corneas and the cornea-conditioned medium. Specific messenger RNA was observed in the cornea by in situ hybridization most prominently in corneal epithelial cells. CONCLUSIONS: alpha 1-Proteinase inhibitor is synthesized and released by human corneal epithelial cells. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor without total dependence on a supply of the inhibitor from the vascular system.

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Cathepsin G, acid phosphatase, and alpha 1-proteinase inhibitor messenger RNA levels in keratoconus corneas.

PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. The authors have shown, in corneas obtained from patients with keratoconus, that lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha 1-proteinase inhibitor (alpha 1-PI) are reduced. This study was undertaken to examine further the gene expression of cathepsin G, acid phosphatase, and alpha 1-PI in keratoconus corneas. METHODS: Corneal buttons were collected from patients with keratoconus, normal subjects, and patients with other corneal diseases. In situ hybridization was performed on paraffin sections using a tritium-labeled probe for cathepsin G or alpha 1-PI. Competitive polymerase chain reaction (PCR) was used to determine the messenger RNA (mRNA) levels for lysosomal acid phosphatase and alpha 1-PI in epithelial and stromal cells of keratoconus corneas. RESULTS: Silver grains, indicative of positive in situ hybridization products, were observed in all three cell types of normal corneas for both DNA probes. Compared with normal and other diseased controls, the labeling was enhanced for cathepsin G but was diminished for alpha 1-PI in the epithelium of keratoconus corneas. Competitive PCR showed that the mRNA level for acid phosphatase was higher and that the mRNA level for alpha 1-PI was lower in keratoconus corneas. CONCLUSIONS: These results indicate that the mRNA level for degradative enzymes in increased and that for alpha 1-PI it is reduced in keratoconus corneas. This study provides the first evidence that the altered expression of multiple enzymes and inhibitors in keratoconus occurs at the gene level. Furthermore, it implicates a possible role of coordinated transcriptional regulation of gene expressions in keratoconus.

Lysosomal enzyme activities in conjunctival tissues of patients with keratoconus.

OBJECTIVE: To examine the lysosomal enzyme activities in the conjunctival tissues of patients with keratoconus. METHODS: Tissues collected from 11 patients with keratoconus, eight patients with senile cataract, three patients with Fuchs' corneal dystrophy, and 11 normal control subjects were processed for histochemical staining for two lysosomal hydrolases, acid esterase and acid phosphatase. RESULTS: The epithelium of all conjunctival specimens stained positively for the two enzymes. The staining in the conjunctival tissues of patients with keratoconus was more prominent than that seen in specimens from either normal control subjects or patients with other diseases. CONCLUSION: Our results suggest that the conjunctival epithelium may be altered in keratoconus. Elevation of lysosomal enzyme levels has been demonstrated in the epithelium of corneas with keratoconus, implicating a role of this layer in the disease. The conjunctival abnormality seems to corroborate the corneal epithelial theory. It also adds one dimension to the pathogenesis of keratoconus.

Three-dimensional scanning electron microscopic study of keratoconus corneas.

OBJECTIVE: To examine the 3-dimensional collagen fibril organization in the Bowman layer of keratoconus corneas. METHODS: Eight keratoconus corneas, 8 corneas with other diseases, and 5 normal human corneas were studied. A cell maceration method in combination with scanning electron microscopy was used to examine the collagen network in the Bowman layer. RESULTS: In normal corneas, the surface of the Bowman layer was smooth and collagen fibrils were regularly arranged. By contrast, sharply edged defects in the Bowman layer were found in keratoconus corneas. Lattice-like configurations of the ruptured Bowman layer and collagenous scar tissue were observed, to varying degrees, in all keratoconus corneas examined. None of the other diseased corneas exhibited the ruptures. CONCLUSIONS: Scanning electron microscopy demonstrated alterations in the Bowman layer specific to keratoconus. Fragmentation of the Bowman layer may be an early change leading to keratoconus conditions.

Effect of nicardipine, a Ca2+ channel blocker, on pyruvate dehydrogenase activity and energy metabolites during cerebral ischemia and reperfusion in gerbil brain.

The purpose of this study was to determine if nicardipine, a calcium ion channel blocker, affects pyruvate dehydrogenase (PDH) activity and improves energy metabolism during cerebral ischemia and reperfusion. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 60 min followed by reperfusion up to 120 min in gerbils. Nicardipine (1 mg/kg) or saline (vehicle-treated) was given to gerbils 30 min prior to the occlusion of the common carotid arteries. PDH activity and metabolites (ATP, PCr, and lactate) were measured in cortex prior to ischemia, immediately following ischemia, and after each reperfusion period. After 60 min ischemia, PDH activity increased in both groups, and was significantly higher in the nicardipine-treated group. After 20 min reperfusion, PDH activity in the nicardipine-treated group recovered to control levels, whereas, the PDH activity in the vehicle-treated group remained elevated, and was higher than the nicardipine-treated animals. At 60 and 120 min reperfusion, the activities in the vehicle-treated group were significantly below control levels, there were no differences, however, between the two groups. ATP and PCr concentrations were markedly depleted immediately after ischemia in both groups. ATP levels at 20 min reperfusion and PCr levels at 60 min reperfusion were significantly higher in the nicardipine-treated group. Lactate concentrations in both groups increased 7-8 fold, similarly, immediately after ischemia. During reperfusion, the lactate remained elevated in both groups, though the levels in the nicardipine-treated group were lower than those in the vehicle-treated group, but not significantly. Nicardipine treatment normalized PDH activity quickly and improved energy metabolism after reperfusion.

Effect of hyperglycemia on pyruvate dehydrogenase activity and energy metabolites during ischemia and reperfusion in gerbil brain.

The effects of hyperglycemia on brain pyruvate dehydrogenase (PDH) and metabolites (ATP, PCr, and lactate) were investigated at 20 min ischemia, 0, 20, and 60 min, and 4 h reperfusion. During reperfusion, PDH activities were suppressed corresponding to the poor recovery of ATP and PCr concentrations and the increase in lactate concentration in the hyperglycemic group, suggesting that preischemic hyperglycemia may impair metabolism by suppressing PDH activity.

The effect of duration of cerebral ischemia on brain pyruvate dehydrogenase activity, energy metabolites, and blood flow during reperfusion in gerbil brain.

The objective of this study was to determine whether the duration of an ischemic insult effects the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) in relation to the recovery of metabolites and regional cerebral blood flow (rCBF) immediately after ischemia and during reperfusion in gerbil cortex. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 20 or 60 min, followed by reperfusion up to 120 min. Immediately after ischemia PDH activity increased threefold regardless of ischemic duration. In the 60-min ischemic group, PDH remained activated, the recovery of high energy phosphates and the clearance of lactate were poor, and the rCBF was 48% of controls after 20-min reperfusion, decreasing gradually to 26% at 120-min reperfusion. In the 20-min ischemic group, PDH activity normalized quickly, the restoration of energy phosphates was good, there was a quick reduction in lactate within the first 60 min of reperfusion, and the rCBF was 65% of control at 20-min reperfusion, and remained over 48% of control throughout reperfusion. Recovery of metabolism after reperfusion did not parallel the changes in rCBF in either group, most noticeably in the 60-min ischemic group. The slow normalization of PDH activity reflected the poor recovery of metabolites in the 60-min ischemic group, indicating that PDH activity is important in the resynthesis of energy metabolites during reperfusion. In conclusion, prolonging the ischemic insult effected PDH activity during reperfusion, impaired recovery of energy metabolites, and worsened the recovery of rCBF.

Three-dimensional appearance of Bowman's layer after radial keratotomy.

PURPOSE: To examine the 3-dimensional collagen fibrillar architecture of Bowman's layer after radial keratotomy (RK). SETTING: Department of Ophthalmology, Niigata University School of Medicine, Niigata, Japan. METHODS: This study used monkey eyes in which 0.3 mm deep radial incisions were made on the cornea 2 weeks and 1, 6, and 12 months before the animals were killed. Corneal buttons were immersed in a fixative and the cells macerated with sodium hydroxide 10%. Scanning electron microscopy (SEM) was performed according to standard procedures. A part of the specimens was embedded in epoxy resin for light microscopic (LM) observation for comparison. RESULTS: The 3-dimensional collagen fibrillar architecture of Bowman's layer was revealed by SEM. The rupture of Bowman's layer could be seen 12 months after surgery and there was no continuity of collagen fibrils in the ruptured area. In LM observations, the width of the stromal incisions gradually became narrower near 12 months after surgery. CONCLUSION: Our cell-maceration/SEM method showed that the rupture of Bowman's layer remained up to 12 months after RK. This suggests that discontinuity of Bowman's layer may be responsible for globe rupture after RK.