[Risk factors for complications of endoscopic full-thickness resection of upper gastrointestinal submucosal tumors].

Objective: To analyze the risk factors for complications of endoscopic full-thickness resection (EFTR) of upper gastrointestinal submucosal tumors (SMTs). Methods: This was a retrospective observational study. The indications for EFTR included: (1) SMTs originating from the muscularis propria layer and growing out of the cavity or infiltrating the deep part of the muscularis propria layer; (2) SMTs diameter <5 cm; and (3) tumor identified as closely adherent to the serous layer during endoscopic submucosal dissection or endoscopic mucosal resection. This study included patients with SMTs originating from the muscularis propria layer in upper digestive tract, diagnosed preoperatively by endoscopic ultrasonography or computed tomography, who were successfully treated with EFTR. Those with incomplete clinical data were excluded. The clinical data of 154 patients with upper gastrointestinal SMTs who underwent EFTR at the Department of Gastroenterology, First Affiliated Hospital of Soochow University from January 2016 to January 2022 were retrospectively analyzed. Post-EFTR complications (such as delayed perforation, delayed bleeding, and postoperative infection, including electrocoagulation syndrome) were monitored and the risk factors for them were analyzed. Results: Among the 154 study patients, 33 (21.4%) developed complications, including delayed bleeding in three (1.9%), delayed perforation in two (1.3%), and postoperative infection in 28 (18.2%). One patient with bleeding was classified as having a major complication (hospitalized for more than 10 days because of complication). According to univariate analysis, complication was associated with tumor diameter >15 mm, operation time >90 minutes, defect closure method(purse string suture), and diameter of resected specimen ≥20 mm (all P<0.05). Multivariate logistic regression analysis showed that operation time >90 minutes (OR=6.252, 95%CI: 2.530-15.446, P<0.001) and tumor diameter >15 mm (OR=4.843, 95%CI: 1.985-11.817, P=0.001) were independent risk factors for complications after EFTR in patients with upper gastrointestinal SMTs. The independent risk factors for postoperative infection in these patients were operation time>90 minutes (OR=4.993, 95%CI:1.964-12.694, P=0.001) and purse string suture (OR=7.142, 95%CI: 1.953-26.123, P=0.003). Conclusion: Patients with upper gastrointestinal SMTs undergoing EFTR with tumor diameter >15 mm or operation time >90 minutes have a significantly increased risk of postoperative complications. Postoperative monitoring is important for these patients with SMTs.

miR-25 Mediates Retinal Degeneration Via Inhibiting ITGAV and PEDF in Rat.

BACKGROUND: Age-related macular degeneration (AMD) is the main cause of irreversible blindness in the elderly. Oxidative stress in retinal pigment epithelium (RPE) is deemed to play a pivotal role in the pathogenesis of AMD. miR-25 functions as an essential modulator in response to oxidative-stress in several cell types, but its function in RPE cells is poorly understood. OBJECTIVE: To explore the roles of miR-25 in RPE cells and in the development of AMD. METHODS: A rat model of retinal degeneration was induced by sodium iodate (SI). Subretinal injection of antagomiR-25 was performed for the intervention while the scramble as control. Visual responses were recorded with Electroretinogram (ERG). TUNEL assay was performed to detect apoptosis. Phagosome quantification in vivo was performed to evaluate RPE cell function. Oxygen-glucose deprivation treatment was performed to mimic in vitro oxidative stress. Gene expression at mRNA level and protein level were performed by quantitative polymerase chain reaction (qPCR) and Western Blot, respectively. The pigment epithelium derived factor (PEDF) level in the cultured medium was measured by Enzyme-linked immunosorbent assay (ELISA). The interaction between miR-25 and integrin αV (IGTAV) / PEDF 3'UTR was examined by dual luciferase assay. Chromatin immunoprecipitation (ChIP) assay was performed to examine its transcriptional regulation of miR-25. RESULTS: Oxidative stress up-regulated miR-25 in RPE cells in very early stage, accompanied by decreased phagocytosis and reduced growth factor secretion in those cells. Such changes preceded RPE cell apoptosis and visual impairment in the SItreated rats. Furthermore, antagomiR-25 intervention effectively rescued RPE cells from degeneration in such model. The increased miR-25 was confirmed to mediate RPE degeneration through direct targeting IGTAV and PEDF. On the other hand, upstream, miR-25 was found to be up-regulated by STAT3 signaling under oxidative stress in both in vivo and in vitro models. CONCLUSION: Our findings demonstrate that, in SI-treated rats, oxidative stress activates STAT3 signaling which up-regulates miR-25 expression, in a very early stage. The increased miR-25 then inhibits ITGAV and PEDF expressions, resulting in RPE phagocytosis dysfunction and then RPE apoptosis and visual impairment as observed in patients with AMD. These findings lead us to a better understanding of AMD pathogenesis, and suggest that miR-25 could be a potential therapeutic target for oxidative stress related RPE diseases, like AMD.

Human Umbilical Cord Mesenchymal Stem Cells: Subpopulations and Their Difference in Cell Biology and Effects on Retinal Degeneration in RCS Rats.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential candidates for treating retinal degeneration (RD). OBJECTIVE: To further study the biology and therapeutic effects of the hUC-MSCs on retinal degeneration. METHODS: Two hUC-MSC subpopulations, termed hUC-MSC1 and hUC-MSC2, were isolated by single-cell cloning method and their therapeutic functions were compared in RCS rat, a RD model. RESULTS: Although both subsets satisfied the basic requirements for hUC-MSCs, they were significantly different in morphology, proliferation rate, differentiation capacity, phenotype and gene expression. Furthermore, only the smaller, fibroblast-like, faster growing subset hUC-MSC1 displayed stronger colony forming potential as well as adipogenic and osteogenic differentiation capacities. When the two subsets were respectively transplanted into the subretinal spaces of RCS rats, both subsets survived, but only hUC-MSC1 expressed RPE cell markers Bestrophin and RPE65. More importantly, hUC-MSC1 showed stronger rescue effect on the retinal function as indicated by the higher b-wave amplitude on ERG examination, thicker retinal nuclear layer, and decreased apoptotic photoreceptors. When both subsets were treated with interleukin-6, mimicking the inflammatory environment when the cells were transplanted into the eyes with degenerated retina, hUC-MSC1 expressed much higher levels of trophic factors in comparison with hUC-MSC2. CONCLUSION: The data here, in addition to prove the heterogeneity of hUC-MSCs, confirmed that the stronger therapeutic effects of hUC-MSC1 were attributed to its stronger anti-apoptotic effect, paracrine of trophic factors and potential RPE cell differentiation capacity. Thus, the subset hUC-MSC1, not the other subset or the ungrouped hUC-MSCs should be used for effective treatment of RD.

Erythropoietin Protects Retina Against Ceramide 2-Induced Damage in Rat.

BACKGROUND: Ceramide plays critical roles in cell proliferation, senescence and apoptosis, and is implicated in neurodegenerative diseases, etc. To clarify if ceramide plays some roles in retinal diseases, we established in vivo and in vitro retinal injury models with ceramide 2 (C2) treatment. In addition, Erythropoietin (EPO), which showed protective effects on retinal cells and blood-retinal barrier (BRB), was also tested for its protection and possible mechanism(s) in these models. METHODS: Male Sprague-Dawley rats were divided into four groups, i.e., normal control, vehicle control, C2 treatment, and C2+EPO treatment. After intravitreal injection, the rats were examined for eye fundus, electroretinogram, histological study, and immunostaining, etc. In vitro, retinal neuronal cell line (R28) and the primary human retinal microvascular endothelial cells (HRMECs) were treated with C2, cell viability assay, transendothelial electrical resistance (TEER) and BRB-related molecules were studied to test the protective effect of EPO. RESULTS: Intravitreal C2-treatment caused significant vision loss in rats, as reflected by reduced b-wave amplitude, increased TUNEL positive cells and GFAP immunostaining in retina. Another major retinal injury observed was BRB breakdown following C2- treatment. Such C2-induced injuries were further confirmed by in vitro study. When HRMECs were treated with C2, the TEER was significantly reduced. The mechanisms for C2 to induce such injuries might be through evidently increased expressions of the related molecules like plasmalemma vesicle-associated protein (PLVAP or PV-1), ecto- 5'-nucleotidase (CD73) and intercellular adhesion molecule-1 (ICAM-1), as observed in C2-treated R28 cells. All these injuries induced by C2 were significantly prevented by EPO both in vivo and in vitro, and its protective mechanisms here might be, in addition to neuroprotective, closely related to its maintenance of BRB integrity, through reducing the expressions of PV-1, CD73 and ICAM-1. CONCLUSION: C2 could induce severe retinal injury, and such injuries could be effectively prevented by EPO treatment.

Subpopulations of Bone Marrow Mesenchymal Stem Cells Exhibit Differential Effects in Delaying Retinal Degeneration.

Bone marrow mesenchymal stem cells (BMSCs) have a therapeutic role in retinal degeneration (RD). However, heterogeneity of BMSCs may be associated with differential therapeutic effects in RD. In order to confirm this hypothesis, two subsets of rat BMSCs, termed rBMSC1 and rBMSC2, were obtained, characterized and functionally evaluated in the treatment of RD of Royal College of Surgeons (RCS) rats. Both subpopulations expressed mesenchymal stem cells (MSC) markers CD29 and CD90, but were negative for hemacyte antigen CD11b and CD45 expression. In comparison with rBMSC2, rBMSC1 showed higher rate of proliferation, stronger colony formation, and increased adipogenic potential, whereas rBMSC2 exhibited higher osteogenic potential. Microarray analysis showed differential gene expression patterns between rBMSC1 and rBMSC2, including functions related to proliferation, differentiation, immunoregulation, stem cell maintenance and division, survival and antiapoptosis. After subretinal transplantation in RCS rats, rBMSC1 showed stronger rescue effect than rBMSC2, including increased b-wave amplitude, restored retinal nuclear layer thickness, and decreased number of apoptotic photoreceptors, whereas the rescue function of rBMSC2 was essentially not better than the control. Histological analysis also demonstrated that rBMSC1 possessed a higher survival rate than rBMSC2 in subretinal space. In addition, treatment of basic fibroblast growth factor, an accompanying event in subretinal injection, triggered more robust increase in secretion of growth factors by rBMSC1 as compared to rBMSC2. Taken together, these results have suggested that the different therapeutic functions of BMSC subpopulations are attributed to their distinct survival capabilities and paracrine functions. The underlying mechanisms responsible for the different functions of BMSC subpopulation may lead to a new strategy for the treatment of RD.

Human Adipose-Derived Stem Cells Delay Retinal Degeneration in Royal College of Surgeons Rats Through Anti-Apoptotic and VEGF-Mediated Neuroprotective Effects.

UNLABELLED: Stem cell therapy is a promising therapeutic approach for retinal degeneration (RD). Our study investigated the effects of human adipose derived stem cell (hADSCs) on Royal College of Surgeons (RCS) rats. METHODS: Green fluorescent protein (GFP)-labeled hADSCs were transplanted subretinally into RCS rats at postnatal (PN) 21 days to explore potential therapeutic effects, while adeno-associated viral vector (AAV2)-vascular endothelial growth factor (VEGF) and siVEGF-hADSCs were used to aid the mechanistic dissections. Visual function was evaluated by Electroretinogram (ERG) recording. Potential transdifferentiations were examined by Immunofluorescence (IF) and gene expressions were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Apoptotic retinal cells were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay and the cytokines secreted by hADSCs were measured by Enzyme-linked Immunosorbent Assay (ELISA). RESULTS: The visual function of RCS rats began to decrease one week after their eyes opened at PN week 3 and almost lost in PN 5 weeks, accompanied by the loss of retinal outer nuclear layer (ONL). Subretinal transplantation of hADSCs significantly improved the visual function 2 weeks after the transplantation and such therapeutic effect persisted up to 8 weeks after the treatment (PN 11 weeks), with 3-4 rows of photoreceptors remained in the ONL and reduced apoptosis. Consistent with these phenotypic changes, the gene expression of rod photoreceptor markers Rhodopsin (Rho), Crx and Opsin (Opn1) in RCS rats showed obvious decreasing trends over time after PN 3 weeks, but were elevated with hADSC treatment. hADSC transplantation also repressed the expressions of Bax, Bak and Caspase 3, but not the expression of anti-apoptotic genes, including Bcl-2 and Bcl-XL. Finally, substantial VEGF, hepatocyte growth factor (HGF) and pigment epithelium-derived factor (PEDF) secretions from hADSCs were detected, while endogenous Vegf expression level decreased over time in RCS rats. The treatment of AAV2-VEGF showed comparable therapeutic effects as hADSCs but siRNA knockdown of VEGF in hADSCs essentially abolished the therapeutic effects. CONCLUSIONS: Subretinal transplantation of hADSCs in RCS rats effectively delayed the retinal degeneration, enhanced the retinal cell survival and improved the visual function. Mechanistically this was mainly due to hADSC dependent anti-apoptotic and neuroprotective effects through its secretion of growth and neurotrophic factors including VEGF. Clinical application of hADSCs merits further investigation.

Altered expression of metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in cervical disc herniation patients.

The aim of the current study was to examine matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in patients with cervical disc herniation (CDH). A total of 127 specimens from CDH patients undergoing posterior spinal surgery were obtained for the case group, which was divided into three subgroups: lateral protrusion (N = 102), median protrusion (N = 18), and paramedian protrusion (N = 7). Another 55 specimens from subjects who had cervical spine trauma and underwent spinal canal decompression were obtained for the control group. Routine hematoxylin and eosin staining was performed for pathological diagnosis. Immunohistochemical (IHC) analysis was used to determine MMP-2 and TIMP-2 expression. Under light microscopy, MMP-2-positive cells presented brown-yellow or dark brown staining in the cell membrane or cytoplasm. MMP-2 expression in the case group was significantly higher than that in controls (P < 0.05). Furthermore, MMP-2 expression in the lateral and median protrusion groups was significantly higher compared to that in the paramedian protrusion group (both P < 0.05), while there was no apparent difference in MMP-2 expression between the lateral and median protrusion groups (P > 0.05). IHC results showed that TIMP-2 expression in cases was significantly lower than that in controls (P < 0.05). Spearman correlation analysis indicated that MMP- 2 was negatively correlated with TIMP-2 expression (r = -0.418, P < 0.001). In conclusion, MMP-2 expression increased, whereas TIMP- 2 expression decreased in CDH patients, suggesting that MMP-2 and TIMP-2 expression may contribute to CDH development.

The ratio of transforming growth factor-ß1/bone morphogenetic protein-7 in the progression of the epithelial-mesenchymal transition contributes to rat liver fibrosis.

This study was designed to show whether rat liver epithelial cells could undergo epithelial-mesenchymal transition (EMT), thereby directly contributing to liver fibrosis. The role of the ratio of transforming growth factor-ß1 (TGF-ß1)/bone morphogenetic protein-7 (BMP-7) was evaluated in the progression of EMT or mesenchymal-epithelial transition. Primary rat liver epithelial cells were stimulated with different ratios of TGF-ß1/BMP-7 and examined for evidence of transition to a mesenchymal or epithelial phenotype. Liver sections were labeled to detect antigens associated with liver epithelial cells [E-cadherin (E-cad)], EMT [fibroblast-specific protein-1 (FSP-1), vimentin], myofibroblasts [α-smooth muscle actin (α-SMA)], and intracellular signal-transduction mediated by forming liver fibrosis undergo EMT, resulting in the formation of invasive fibroblasts; this process may be driven or impeded by a response to local TGF-ß1 or BMP-7. BMP-7 downregulated α-SMA and phosphorylated Smad2/3. Stimulation of cultured cells with TGF-ß1 induced the expression of pSmad2/3, FSP-1, and α-SMA. Stimulation of cultured cells with BMP-7 induced the expression of E-cad. We demonstrated that the cells upregulated E-cad release compared with untreated cells, but TGF-ß1 was different. We found that the equilibrium of the ratio of TGF-ß1/BMP-7 was 1/10. In summary, the mechanism for this process was not determined. Demonstration of the contribution of what the ratio of TGF-ß1/BMP-7 induced to EMT to the chronic liver diseases would provide a new basis for understanding pathogenesis and potential treatment.

Subretinal transplantation of rat MSCs and erythropoietin gene modified rat MSCs for protecting and rescuing degenerative retina in rats.

For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cell based gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i.e., rat mesenchymal stem cells (rMSCs) alone, erythropoietin (EPO) gene modified rMSCs (EPO-rMSCs) or doxycycline (DOX) inducible EPO expression rMSCs (Tet-on EPO-rMSCs), were transplanted into the subretinal spaces of SI-treated rats. The rMSCs were prepared for transplantation after 3 to 5 passages or modified with EPO gene. During the 8 weeks after the transplantation, the rats treated with rMSCs alone or with two types of EPO-rMSCs were all monitored with fundus examination, fundus fluorescein angiography (FFA) and electroretinogram. The transplantation efficiency of donor cells was examined for their survival, integration and differentiation. Following the transplantation, labeled donor cells were observed in subretinal space and adopted RPE morphology. EPO concentration in vitreous and retina of SI-treated rats which were transplanted with EPO-rMSCs or Tet-on EPO-rMSCs was markedly increased, in parallel with the improvement of retinal morphology and function. These findings suggest that rMSCs transplantation could be a new therapy for degenerative retinal diseases since it can protect and rescue RPE and retinal neurons, while EPO gene modification to rMSCs could be an even better option.

Neuroprotective effect of grafting GDNF gene-modified neural stem cells on cerebral ischemia in rats.

Previous studies indicated the beneficial effects of glial cell line-derived neurotrophic factor (GDNF) and transplanted neural stem cells (NSCs) on stroke. Here, we explored whether transplantation of neural stem cells (NSCs) modified by GDNF gene provides a better therapeutic effect than native NSCs after stroke. Primary rat NSCs were transfected with GDNF plasmid (GDNF/NSCs, labeled by green fluorescent protein from AdEasy-1, GFP). Adult rats were subjected to two-hour middle cerebral artery occlusion and reperfusion, followed by infusion of NSCs (labeled with5-bromo-2'-deoxyuridine before infusion, BrdU), GDNF/NSCs and saline at 3 days after reperfusion (NSCs group, GDNF/NSCs group, control group), respectively. All rats were sacrificed at 1, 2, 3, 5, and 7 weeks after reperfusion. Modified Neurological Severity Scores (mNSS) test and H and E staining were respectively performed to evaluate neurological function and lesion volume. Immunohistochemistry was used to identify implanted cells and observe the expressions of Synaptophysin (Syp) and postsynaptic density-95 (PSD-95) and caspase-3. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) was employed to observe apoptotic cells. Western blotting was used to detect brain-derived neurotrophic factor (BDNF) and NT-3 protein expression. Significant recovery of mNSS was found in GDNF/NSCs rats at 2 and 3 weeks after reperfusion compared with NSCs rats. Lesion volume in the NSCs and GDNF/NSCs groups was reduced significantly compared with control group. The number of NSCs in the GDNF/NSCs group was significantly increased in comparison with NSCs group. Moreover, Syp-immunoreactive product at 2 and 3 weeks after reperfusion and PSD-95 immunoreactive product in the GDNF/NSCs group were significantly increased compared with NSCs group. In contrast, caspase-3 positive cells and TUNEL-positive cells in the GDNF/NSCs group were significantly decreased compared with NSCs group. Significant increase of BDNF protein in the GDNF/NSCs and NSCs groups was observed compared to the control group at different time points of reperfusion, and GDNF/NSCs grafting significantly increased BDNF protein expression compared to NSCs grafting. In addition, significant increase of NT-3 protein in GDNF/NSCs and NSCs groups was detected only at 1 week of reperfusion compared to control group. The results demonstrate that grafting NSCs modified by GDNF gene provides better neuroprotection for stroke than NSCs grafting alone.

BubR1 deficiency results in enhanced activation of MEK and ERKs upon microtubule stresses.

UNLABELLED: Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo-insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. OBJECTIVE: Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. METHOD AND RESULTS: Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper-activation of ERKs and MEK in BubR1(+/-) murine embryonic fibroblasts (MEF) compared to that of wild-type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. CONCLUSION: Our studies suggest that there exists a cross-talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross-talk.

Inducible expression of Na+/myo-inositol cotransporter mRNA in anterior epithelium of bovine lens: affiliation with hypertonicity and cell proliferation.

Bovine lenses were pretreated with physiological (314 +/- 6 mosm) or hypertonic medium (> 450 mosm) for 20 hours prior to introduction of the lenses into modified Ussing chambers (providing a leakproof seal at the lens equator). The anterior (epithelial-containing) and posterior (cell-free) aspects of the mounted intact lens could thus be independently bathed with myo-[3H]inositol. Myo-inositol uptake as a function of concentration across the anterior aspect was indicative of an active carrier-mediated component and at high concentration, significant myo-inositol influx due to passive diffusion. Myo-inositol uptake was markedly stimulated across the anterior aspect and inhibitable by ouabain but not stimulated across the posterior aspect, when lenses were exposed to hyperosmotic medium by the addition of raffinose or sodium chloride. Myo-inositol rapidly desaturates across both aspects of the lens consistent with its efflux being largely a function of passive diffusional leakout from the extracellular space. In order to corroborate the apparent hypertonic-induced upregulation of gene expression inferred by kinetic analysts, the level of Na+/myo-inositol cotransporter mRNA was determined by reverse transcription and quantitative PCR using harvested anterior epithelial cells from intact lenses exposed to physiologic or hypertonic medium for 20 hours. RNA samples were microextracted from both the central and equatorial regions of the lenticular epithelium of the anterior lens capsule. The abundance of Na+/myo-inositol cotransporter mRNA was similar from epithelium of the central zone irrespective of medium tonicity but was markedly elevated from the equatorial zone of osmotically-stressed lenses. While both the quiescent epithelial cells of the central region and actively dividing epithelial cells of the equatorial zone of the intact lens express Na+/myo-inositol contransporter mRNA, only the equatorial epithelium responds to hypertonic insult with enhanced uptake of myo-inositol due to increased transcription of the Na+/myo-inositol cotransporter gene, suggesting a possible role for the state of cell proliferation.

Inhibition of naphthalene cataract in rats by aldose reductase inhibitors.

Naphthalene-induced cataract in rat lenses can be completely prevented by AL01576, an aldose reductase inhibitor (ARI). In an attempt to understand the mechanism of this inhibition, several ARIs were examined to compare their efficacies in preventing naphthalene cataract, using both in vitro and in vivo models. Two classes of ARIs were tested: One group including AL01576, AL04114 (a AL01576 analog) and Sorbinil contained the spirohydantoin group, while Tolrestat contained a carboxylic acid group. Furthermore, to clarify if aldose reductase played a role in naphthalene-induced cataractogenesis in addition to its role in sugar cataract formation, a new dual cataract model was established for ARI evaluations. This was achieved by feeding rats simultaneously with high galactose and naphthalene or incubating rat lenses in culture media containing high galactose and naphthalene dihydrodiol. Under these conditions, both cortical cataract and perinuclear cataract developed in the same lens. It was found that at the same dosage of 10 mg/kg/day, both AL01576 and AL04114 completely prevented all morphological and biochemical changes in the lenses of naphthalene-fed rats. Sorbinil was less efficacious, while Tolrestat was inactive. AL01576 showed a dose-response effect in preventing naphthalene cataract and at 10 mg/kg/day, it was also effective as an intervention agent after cataractogenesis had begun. With the dual cataract model, Tolrestat prevented the high galactose-induced cortical cataract but showed no protection against the naphthalene-induced perinuclear cataract. AL01576, on the other hand, prevented both cataract formations. Results for dulcitol and glutathione levels were in good agreement with the morphological findings. AL04114, and ARI as potent as AL01576 but without its property for cytochrome P-450 inhibition, displayed similar efficacy in preventing naphthalene cataract. Based on these results, it was concluded that the prevention of the naphthalene cataract probably results from inhibition of the conversion of naphthalene dihydrodiol to 1,2-dihydroxynaphthalene and that the effect of the ARIs cannot be explained by their inhibition of the dihydrodiol dehydrogenase activity of aldose reductase.

Further studies on the dynamic changes of glutathione and protein-thiol mixed disulfides in H2O2 induced cataract in rat lenses: distributions and effect of aging.

To further investigate the role of protein-thiol mixed disulfides in cataractogenesis, an in vitro H2O2 cataract model was used with rat lenses to study the effect of aging, and the dynamic changes in the cortex, nucleus and the lens protein fractions. A group of lenses was exposed to H2O2-containing media (0.6 mM) for 1 to 3 days so that cortical cataract was induced gradually. Another group of lenses was first subjected to H2O2 exposure for one day and then recovered in the oxidant-free media for one or two days. These lenses were examined for the distribution of free glutathione and protein-thiol mixed disulfides (protein-glutathione and protein-cysteine) in the cortical and nuclear regions as well as in the water soluble and water insoluble fractions. Similar to the results reported earlier, the glutathione depletion in the whole lens occurred immediately and extensively during the 3-day H2O2 exposure. This loss was evenly distributed in the cortical and nuclear fractions. The level of protein-glutathione increased rapidly and continued throughout the 3 days. Most of the accumulation was found in the cortex and in both lens protein fractions. The protein-cysteine modification responded more slowly and less to oxidative stress. The delayed formation occurred mainly in the nucleus and in both lens protein fractions. In the recovery group, glutathione depletion was less drastic in the cortical and nuclear regions, but the elevated protein-glutathione in both regions and both protein fractions spontaneously decreased to its respective basal level within 1 day. Protein-cysteine on the other hand remained quite high, and in some cases it continued to rise in the absence of oxidation. Aging showed little effect on the response of rat lenses to oxidative stress. Similar patterns in glutathione and protein-thiol mixed disulfides occurred in both age groups (1, 23 months) and in both chronic oxidative stress and recovery conditions.

The possible mechanism of naphthalene cataract in rat and its prevention by an aldose reductase inhibitor (ALO1576).

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALO1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20-30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate activity [3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na+/K(+)-ATPase. ALO1576 (10 mg kg-1 day-1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.

Establishment of a naphthalene cataract model in vitro.

In the past, almost all studies on naphthalene cataract were based on in vivo experiments. Such studies are laborious and time-consuming and are complicated by systemic toxicity arising from the metabolites of naphthalene. In order to study the direct effects of naphthalene metabolites on the lens, we established an in vitro 'naphthalene cataract' model system by exposing rat lens to naphthalene dihydrodiol (2.5 x 10(5) M) containing medium for 48 hr. Under these conditions, we analysed several biochemical parameters including the glutathione level, protein mixed disulfides, protein patterns on SDS-gels, active transport, NA+/K(+)-ATPase activities and the measurement of naphthalene metabolites in the cultured lenses. The results showed that both the morphological and biochemical changes were very similar to those observed in lenses of rats fed naphthalene (1 g kg-1 day-1). Furthermore, ALO1576 completely blocked the in vitro changes as it did in vivo. Therefore, this model system can be used as a new tool to investigate the mechanism of naphthalene cataract formation. Other naphthalene metabolites such as 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene and 1,2-naphthoquinone were also studied in vitro and the results showed that the effects of these naphthalene metabolites were very different from those observed in naphthalene cataracts in vivo.

Effects of naphthalene metabolites on cultured cells from eye lens.

Naphthalene is toxic to the eye and results in opacification of the lens. To investigate the metabolic events that may be occurring in the lens epithelial cells, a cell line of lens from a transgenic mouse was incubated with various metabolites of naphthalene. Naphthoquinone at 50 microM was toxic to most cells with a depletion of glutathione levels noted within 6 h of incubation. At 10 microM, naphthoquinone caused an increase in specific activity of the enzyme DT-diaphorase. This enzyme is thought to be a defense against quinones since semiquinone formation is thought to be lessened. Naphthalene-1,2-dihydrodiol at 50 microM also caused an increase in the specific activity of the DT-diaphorase, while at 10 microM no apparent change occurred in the cells. Although there was evidence of metabolic alterations in the cells with the metabolites of naphthalene, the protein profile by two-dimensional gel electrophoresis did not change and there was no indication of an increase in carbonyl formation in the soluble proteins of the cells. These experiments indicate that the metabolites of naphthalene can cause alteration in the metabolism of the lens cells but may not cause apparent changes in the major proteins within the lens epithelium.

[The blood galactokinase activity in Chinese with reference to congenital cataract].

The galactokinase (GK) activity of whole blood in Chinese volunteers and patients with congenital cataracts was studied by radioassay after Beutler. The GK activity was found to be highest 78.42 +/- 22.01 mU/g.Hb, in the newborn, and is decreased with age up to 4 years to remain constant thereafter at a mean normal value of 27.79 +/- 4.98 mU/g.Hb, which was consistent with Beutler's results. The frequency of GK heterozygotes in normal Chinese was found to be 1/234(0.43%). Among 55 children with congenital cataracts developed during the first year after birth, 4 (7.30%) were heterozygotes. Furthermore, the frequency of heterozygotes was found to be 3/16 (18.80%) in infants with nuclear cataracts, which differed very significantly from that of the normal Chinese. The probable association of congenital cataracts with decreased GK activity was discussed.