Morphology and morphometry of the semitendinosus distal tendon in adults and foetuses.

BACKGROUND: The distal tendon of the semitendinosus is often used as a graft in orthopedic reconstructive surgery. Knowledge of the exact morphology of this tendon, and also the ability to predict its morphometric data are certainly helpful when planning the procedure of surgery. Comparison of the semitendinosus distal tendon anatomy in adults and foetuses may be scientifically relevant. There are no scientific reports on this tendon anatomy in foetuses. MATERIALS AND METHODS: Seventy semitendinosus muscles from cadavers were obtained using standard dissection techniques (50 muscles were obtained from adults and 20 from foetuses). Moreover, ultrasound examinations of 20 muscles were performed in living individuals. RESULTS: Two main parts of the distal tendon were distinguished - the external part not covered with muscle fibres and the internal part, which is partially or entirely hidden within the muscle belly (venter). The average length of the distal tendon was 32.34 cm, while the average lengths of the external and internal parts were 9.65 cm and 12.59 cm, respectively. The external part was solid and cylindrical. The internal part was flat and rolled like a trough, thus making the tendon a poor transplant material. Similarly, the distal tendon in foetuses consisted of two parts, including the external and internal part. CONCLUSIONS: The proportions between the lengths of different muscle parts were very similar in adults and foetuses.

Anatomy and morphometry of the distal gracilis muscle tendon in adults and foetuses.

Ten human gracilis muscles obtained from adults and ten gracilis muscles collected from human foetuses between the 15th and 21st week of gestation were examined. The results of this preparatory study show that the gracilis muscle in adults is narrow and long - 482 mm on average. The distal tendon of gracilis muscle is long, 294 mm on average. It can be divided into two sections - external part, outside the muscle belly, and internal, intramuscular, part. The latter one is partially covered by muscle fibres and some of it is completely hidden inside the muscle belly, which is on average 76 mm long. Presence of an intramuscular part of the distal tendon was also demonstrated in the foetal material. Moreover, very strong correlations between particular muscle lengths were noted in foetuses. (Folia Morphol 2018; 77, 1: 138-143).

Tension pneumocephalus following suboccipital sitting craniotomy in the pediatric population.

BACKGROUND: Sitting craniotomy often results in entrapment of air in fluid-filled intracranial cavities. Gas under pressure exerts a deleterious effect on adjacent nervous tissue, resulting in clinical deterioration. AIM OF STUDY: To assess the incidence of tension pneumocephalus (TP) and to define risk factors associated therewith. MATERIAL AND METHOD: Analysis included 100 consecutive patients (57 boys, 43 girls, mean age 9.7 y) undergoing suboccipital sitting craniotomy since 2012 to 2014. RESULTS: In our material (n=100) TP was seen in 7 cases, asymptomatic pneumocephalus (AP) in 77 and no pneumocephalus (NP) in 16. Tumor types encountered were typical for pediatric population. In the TP group (n=7) the ratio of low-grade to high-grade tumors was 5:2, in the AP group (n=77) 2:1 and in the NP group (n=16) 1:1. Preoperative hydrocephalus was present in 21 cases (21%, mean incidence), thereof 3 in the TP group (3/7; 42.8%), 12 in AP group (12/77; 15.5%) and 6 in the NP group (6/16; 37.5%). All TP patients received an emergency external drainage, thereof 4 required a permanent ventriculo-peritoneal shunt (57.1%), while AP and NP patients combined (n=93) required a permanent shunt in 4 cases only (4.3%). TP-associated morbidity (n=2) consisted in a significant deterioration of neurological condition. CONCLUSIONS: TP is a relatively rare but potentially serious complication of suboccipital sitting craniotomy. Risk factors for TP are low-grade tumor and pre-existing long-standing hydrocephalus. TP requires emergency decompression by temporary external drainage. TP patients significantly more often require a permanent CSF shunt.

Anatomy of the long peroneal muscle of the leg.

BACKGROUND: The aim of the work was to perform a morphometric analysis of the long peroneal muscle (LPM) of the leg and explore the relationship between muscle belly and tendon. MATERIALS AND METHODS: Ten lower limbs (8 right and 2 left) were fixed in formaldehyde and dissected using standard technique. The LPM was exposed from the proximal attachment to the top of a lateral malleolus. RESULTS: The tendon was subsequently freed and various measurements were taken. The tendon of the LPM enters deep into the muscle belly. Muscle fibres surround the tendon and descend as far down as 4 cm above the lateral malleolus. Muscle fibres insert mainly along posterior border of the tendon and on its medial surface, leaving lateral surface only partly covered. CONCLUSIONS: The LPM contains a long intramuscular segment of the tendon and area of the musculotendinous junction varies along the LPM. It makes the idea of uniform pennation pattern of the LPM unlikely.

Decellularized Amniotic Membrane Scaffold as a Pericardial Substitute: An In Vivo Study.

BACKGROUND: In the development of new biomaterials for pericardium substitute, acellular amniotic membrane (AAM) presents potential for new applications in regenerative medicine. We studied an AAM as a pericardial substitute to achieve a suitable, cost effective, abundant matrix for the purpose of using it as graft for tissue repair. METHODS: Twenty Wistar rats were randomly divided into 2 groups (n = 10/group) and had their pericardiums excised. In the experimental group, the excised pericardium segment was substituted by a 7-mm-diameter patch of decellularized AAM sutured to the lesion area. After 4 weeks, the heart's outer layer of both groups was evaluated. The structure and component characteristics of the scaffold were determined with the use of hematoxylin and eosi, Alizarin Red S, and immumohistochemical staining and scanning electron microscopy. RESULTS: Histopathologic examination of the AAM patches revealed that the integrity of the AAM was preserved, and no calcification was observed on the surface of the myocardium. We also observed thicker pericardium repair tissue in the AAM group compared with the control group. AAM patches, by virtue of their low immunogenicity, evoked minimal host-versus-graft reaction. CONCLUSIONS: We conclude that AAM appears to be an ideal substitute for pericardium lesions, because it is integrated into the biologic tissue owing to its low immunogenicity and its ability to diminish the occurrence of adhesions and scarring, increasing the pericardium thickness.

Anatomy of sartorius muscle.

A sartorius muscle is the longest muscle of the human system. It runs over 2 joints- hip and knee joints. In the study 10 sartorius muscles were examined. They were dissected free of lower human limbs. Dimensions of limbs which these muscles come from and dimensions of the muscles and their component parts were examined. The attention was paid mainly to parts of tendon located inside the muscle belly. The results show that they are either of a comparable length (distal tendon) or several times longer (proximal tendon) than visible parts located outside of the muscle. Moreover, a complex structure of the distal tendon which includes 2 tracts of different places of insertion was stated. Inferior tract inserted in the same place as muscle tendons: semitendinosus and gracilis. The superior tract inserted transversely against the former one. The tendon of the sartorius muscle forms additionally an aponeurosis whose fibres enter into the deep fascia of crus. The muscle belly is characterised with various width on different levels of its length. In half of casess word-like distal segment of belly is formed.

Multiple metastases of carcinoma basocellulare into spinal column.

Basal cell carcinoma presents a relatively low potential and local malignancy and very slow growth giving only occasionally metastatic spreading. The frequency of occurrence of metastatic dissemination is estimated in the literature depending on examined population from 0.028% to 0.55%. Metastases are most often found in lymph nodes, lungs bones and internal organs: liver, spleen, kidneys, adrenal glands, pleura and the peritoneum. Authors present a case of a 69-years old female with an extensive basal cell carcinoma of the head convexity, infiltrating the subcutaneous tissue, periostium, bone and dura mater, giving distant metastases to other bone and soft tissue structures of a thoracic spine, which was confirmed by biopsy and histopathological findings of neoplasm tissue in spine. The primary lesion was successfully treated surgically. Despite administered radiotherapy of metastases in spine, progress of the disease during 1-year period was observed. The patient was alive with metastatic tumours present at last follow-up. Basing on the review of the literature and our case report we can distinguish following factors which may increase the risk of occurrence of basal cell carcinoma metastases: the great extent of the primary lesion, deep penetration to stromal tissue, blood and lymph vessel invasion, long history of tumour occurrence and the presence of metatypia in histopathological findings. The above-mentioned case fulfils the criteria of carcinoma basocellulare metastases proposed by Latters and Kessel and may be included to the general registration list of this cancer in the world.

Experimental study of the effects of different stapling devices in healing of the mechanically sutured bronchial stump.

OBJECTIVE: The invention of the mechanical suture of the bronchial stump resulted in the significant decrease of the incidence of bronchial fistulas. Bronchial fistula constitutes the most dangerous complication of the pulmonary resection. In connection with some negative opinions in world literature regarding the safety of applying some types of mechanical suture, the multi-factor analysis of efficacy of bronchial stump closure following the total pneumonectomy by two different types of stapling devices was performed. METHODS: The experimental study was performed on 22 sheep. Each sheep underwent left pneumonectomy. In group I the bronchus was closed by the hinged-jaw stapling device (TA-Premium, Auto-Suture). In group II the bronchus was closed by the stapling device of parallel pattern (RLV 30 Ethicon). The macroscopic parameters (i.e. linear structure of staples, degree of staples closure, the symmetry of staples closure in the medial and lateral part of bronchial stump) as well as microscopic parameters (i.e. degree of inflammatory reaction, degree disorder in collagen fibers system, degree of disorders in cartilaginous system, degree of vascular proliferation and nervous regeneration) were evaluated. RESULTS: In three cases of group I the serious abnormalities in staples closure in the medial part of the bronchial stump were revealed. Abnormalities were found also in microscopic evaluation of the specimens. In the whole group the inflammatory reaction predominated in the medial part of bronchial stump near the hinge of the cartridge (P value <0.05). The disorder in the collagen fibers system as well as in the stratified structure of muscular fibers and cartilaginous system was proved. On the other hand, in group II all staples were properly closed in adequate linear structure, without any symmetry in both medial and lateral end of the bronchial stump. The microscopic findings were only the subtle inflammatory process and a slight disarrangement in muscular, collagen and cartilaginous systems. CONCLUSION: The listed abnormalities of mechanical, hinged-jaw suture of bronchial stump seem to be due to the inaccurate placement of staples, their incomplete closure, and excessive damage to the sutured tissues. We conclude that the application of the hinged-jaw mechanical suture of the bronchial stump might result in higher incidence of bronchial fistula after pneumonectomy.

Perioperative management of a post heart transplantation patient with renal failure qualified to lung resection.

Since the first heart transplantation (1967, Christian Bernard), hundreds of similar procedures have been performed all over the world. Considerable advance made in immunosuppressive treatment improved survival rate and long-term efficiency of treatment improved survival rate and long-term efficiency of treatment. Many of these patients suffer from ailments requiring operations which are not connected with the transplanted organ. The present study describes a case of a post heart transplantation patient qualified to lung resection, in whom renal insufficiency occurred in the course of immunosuppressive therapy.

Protection from oxidative insult in glutathione depleted lens epithelial cells.

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2 and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2 when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15-20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0. 05 mm H2O2 (for 50,000 cells) or 2 doses of 0.5 mm H2O2 (for 800,000 cells). After exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7. When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mm H2O2 the H2O2 was cytotoxic, whereas cells treated with H2O2 alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2 from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2 exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H2O2 cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2 was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H2O2 via glutathione peroxidase.

Heme oxygenase synthesis is induced in cultured lens epithelium by hyperbaric oxygen or puromycin.

We showed previously that treatment of cultured rabbit lens epithelial cells (LECs) with hyperbaric oxygen (HBO) produced DNA strand-breaks, caused reversible inhibition of protein synthesis and induced the synthesis of a 32 kD protein. In the present work, we employed immunostaining procedures to identify the 32 kD protein as heme oxygenase-1 (HO-1). Increased synthesis of the enzyme was observed as early as 12 hr after HBO-treatment, reached a maximum at 18 hr and was not detectable at 36 hr. Exposure of the cells to hemin also increased the synthesis of HO-1. An HBO-induced inhibition of protein synthesis and the subsequent induction of HO-1 was also observed in the capsule-epithelium of cultured rabbit lenses. For both LECs and the cultured lens, only HO-1 and not heme oxygenase-2 was HBO-inducible. Use of the antioxidant dimethylthiourea with HBO-treated lenses or LECs did not alter the observed effects on protein synthesis or the induction of HO-1. In contrast to results obtained with 50 atm O2, a pressure of 25 atm O2 inhibited protein synthesis only slightly and failed to induce synthesis of the 32 kD protein (although, as shown previously, identical exposure of LECs to 25 atm O2 significantly damaged DNA). Inhibition of protein synthesis in LECs and cultured lenses with the use of puromycin also induced synthesis of HO-1. Both hemin (10 micron), a source of iron, and 50 atm O2 produced a three-fold increase in the concentration of ferritin, a natural iron chelator, in LECs two days after exposure; no effects on ferritin levels were observed after 1 or 3 days. The finding that the increase in ferritin concentration occurred in the cells significantly after hemin- or HBO-induced synthesis of heme oxygenase indicates that chelatable iron rather than the heme molecule itself may have been the primary agent responsible for inducing ferritin synthesis. The data suggest that HBO-induced synthesis of HO-1 in the lens epithelium may be the result of an inhibition of protein synthesis, possibly leading to an accumulation of heme, rather than a direct protective response against oxidative stress.

Regional differences in the distribution of catalase in the epithelium of the ocular lens.

Oxidative stress is thought to play a major role in cataract formation. The present experiments are aimed at gaining a better understanding of the systems that protect the lens from damage by reactive oxygen species. The aqueous humor normally contains hydrogen peroxide (H2O2), a compound capable of generating reactive oxygen species. The systems protecting the ocular lens from oxidative damage are primarily confined to the epithelium, a single layer of cells on the anterior side of the organ directly beneath the lens capsule. When cultured rabbit lenses were challenged with a single dose of 0.2 mM H2O2, cells in the peripheral region of the epithelium survived; those in the central region died. Here we investigate the histochemical and immunoperoxidase distributions of catalase, an enzyme which detoxifies H2O2, in cells from the peripheral and central regions of the epithelium on flat mount preparations of the epithelium. In a flat mount, the entire population of lens epithelial cells can be viewed on one preparation. The reaction product for catalase activity and its immunoperoxidase localization were more intense in peripheral epithelial cells than in cells throughout the central epithelium. Treatment of cultured lens epithelial cells or rabbit lenses with 3-aminotriazole or potassium cyanide, inhibitors of catalase, reduced or abolished the histochemical reaction product. Ultrastructural cytochemistry confirmed the presence of catalase in microperoxisomes of the epithelial cells from whole lenses. The decreased level of catalase throughout the central epithelium may account for the increased susceptibility of these cells to H2O2-induced cell death.

Effects of cyclosporine A on ozone-induced pulmonary lesion formation: pharmacologic elimination of the T-lymphocyte regulatory response.

To assess the involvement of T-lymphocytes in ozone-induced lung damage, CD-1 mice were exposed to air or 0.7 ppm ozone (1.37 mg O3/m3 air) in the presence and absence of the immunosuppressive drug cyclosporine A (CSA). Mice were thus divided into four treatment groups for both the 4 and 14 day exposure times: 1) AIR + VEH, 2) AIR + CSA, 3) O3 + VEH, and 4) O3 + CSA. Thy-1.2 positive cells (T-lymphocytes) per pulmonary lesion were determined and quantitative histomorphometric analysis of lesion volume was performed. By Day 14, the number of T-lymphocytes per lesion in O3 + VEH (vehicle) animals had increased to approximately 3.5 times that seen at Day 4. At 4 and 14 days of O3 + CSA treatment, the number of T cells per lesion was half that seen in O3 + VEH mice. At Day 4, lesion size and appearance were comparable in O3 + VEH and O3 + CSA animals, while at Day 14, O3 + CSA treatment resulted in larger and more cellular lesions that contained a greater proportion of polymorphonuclear cells, and the lesions extended further into the lung periphery. Inflammatory cells were observed in areas of epithelial cell proliferation and in alveolar spaces distal to the small airway terminus. After 14 days, lesion volume was approximately twice as great following O3 + CSA administration than with O3 treatment alone. These results are consistent with effects seen in another model of immunosuppression, the nude mouse, and they implicate a regulatory role for T-lymphocytes in the response to ozone.

Hydrogen peroxide affects specific epithelial subpopulations in cultured rabbit lenses.

PURPOSE: To investigate the effect of hydrogen peroxide on the epithelial cells of cultured rabbit lenses. METHODS: Lenses were cultured in minimum essential medium containing a single dose of 0.03, 0.1, or 0.2 mM H2O2. Three hours later the medium was replaced with peroxide-free minimum essential medium. Lenses were also treated with 0.5 mM 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU) to lower the activity of glutathione reductase and then exposed to 0.03 mM H2O2 maintained nearly constant by glucose oxidase. After H2O2 treatment, lenses were fixed and whole mounts of the epithelium were prepared or lenses were processed for electron microscopy. RESULTS: Cells exposed to a single dose of 0.03 mM H2O2 appeared normal; 0.1 mM H2O2 was not cytotoxic. Exposure to 0.2 mM H2O2 elicited swelling in cells in the pre-equatorial region (30 minutes) followed by the formation of islands of cells in the pre-equatorial region at 1 hour. Central epithelial cells appeared normal at 1 hour, were swollen at 3 hours and dead at 24 hours. By 48 hours, dead cells were found in the pre-equatorial and central regions. Cells in the peripheral region of the epithelium did not exhibit cytotoxicity. If lenses were pretreated with BCNU and then challenged with a maintained level of 0.03 mM H2O2, cytotoxicity was induced in the central and pre-equatorial regions. Cells in the peripheral region survived BCNU-H2O2 treatment. CONCLUSIONS: Cells in the peripheral region of cultured lenses were more resistant to H2O2 cytotoxicity than cells in the central and pre-equatorial regions. The antioxidant defense or repair systems for H2O2-induced damage do not appear to be uniformly distributed in subpopulations of the lens epithelium.

Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.

PURPOSE: To determine if lens epithelial lines can be established from cryopreserved whole rabbit lenses and from cryopreserved capsule-epithelial preparations (CEPs). METHODS: Lenses or freshly isolated CEPs were cryopreserved and subsequently thawed. Thawed whole lenses were cultured for 48 hours in growth medium and fixed, and whole mounts were examined for mitosis. In addition, CEPs were peeled from cryopreserved lenses and placed in tissue culture. Viability of cryopreserved cells was assessed measuring attachment efficiency and growth. RESULTS: Whole mounts from cryopreserved lenses that were thawed and placed in organ culture in a serum-containing medium exhibited numerous mitotic figures. Freshly isolated CEPs that were cryopreserved and CEPs from cryopreserved lenses generated cell lines. Attachment efficiency was 90% within 3 hours of plating. When 50,000 cells from cryopreserved CEPs were cultured in growth medium, 10(6) cells were noted after 7 days of culture. The cells completed 27 population doublings and showed no sign of senescence. CONCLUSIONS: Rabbit lens epithelial cell lines can be initiated from cryopreserved lenses or CEPs.

Lens epithelium: a primary target of UVB irradiation.

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

The superoxide dismutase mimic TEMPOL protects cultured rabbit lens epithelial cells from hydrogen peroxide insult.

The superoxide dismutase mimic, 4-hydroxy TEMPO (TEMPOL), was used to investigate the mechanism by which H2O2 damages cultured rabbit lens epithelial cells and to identify some of the targets of H2O2 insult. Most studies aimed at determining the mechanism by which H2O2 exerts its cytotoxic effect have used iron chelators to prevent the generation of the damaging hydroxyl radical. Since TEMPOL does not chelate transition metals, we were afforded an additional means of investigating the mechanism by which H2O2 exerts its cytotoxicity. Cells at low or high density were cultured in MEM containing 5 mM TEMPOL and exposed to a single sub-lethal dose of 0.05 or 0.5 mM H2O2, respectively. Analysis of EPR spectra indicated that TEMPOL was stable in MEM, did not destroy H2O2 and penetrated the intracellular fluid. TEMPOL prevented or curtailed the H2O2-induced inhibition of cell growth, blebbing of the cell membrane, the decrease in NAD+, the activation of poly ADP-ribose polymerase, an enzyme involved in DNA repair, and limited the induction of single strand breaks in DNA normally brought about by H2O2. TEMPOL did not prevent the H2O2-induced decrease in reduced glutathione, lactate production, and the activity of glyceraldehyde 3-phosphate dehydrogenase, or the H2O2-induced increases in oxidized glutathione and hexose monophosphate shunt activity. Addition of TEMPOL 1-15 min after exposure of cells to H2O2 offered partial protection from the inhibition of cell division. TEMPOL at 5 mM did not inhibit cell growth. These results, coupled with our other findings suggest that some of the H2O2-induced damage in cultured rabbit LECs is mediated by intracellular redox-active metals involved in the Haber-Weiss cycle. Cellular changes not protected by TEMPOL, including attack of H2O2 on the thiol groups of GSH (mediated through glutathione peroxidase) and G3PDH, are likely brought about by H2O2 itself and not by reactions of oxygen free-radicals generated from H2O2.

Hyperbaric oxygen inhibits the growth of cultured rabbit lens epithelial cells without affecting glutathione level.

Studies on human patients and experimental animals indicate that hyperbaric O2 can opacify the lens nucleus and damage the lens epithelium in vivo. Here we investigate the effects of hyperbaric O2 on cultured rabbit lens epithelial cells (LECs). When the cells were exposed to 50 atm O2 (99% O2 + 1% CO2) for 3 hr there were no immediate effects on morphology, viability and transport processes (uptake of 86Rb and 14C-alpha AIB). In addition, the O2 treatment did not lower the high level of reduced glutathione or increase the low level of oxidized glutathione. However, 50 atm O2 did produce a near doubling in the glycolytic rate which maintained ATP at levels only slightly lower than normal. Although the 3-hr O2 treatment was not lethal, it completely inhibited cell division for 2 days. After 2 days, growth was initiated and, at day 7 the rate of growth was faster than the controls (control cells were treated with ambient air or 50 atm N2 for 3 hr). Cells treated with 8 atm O2 for 3 hr exhibited a slowed rate of growth, relative to controls, while exposure to 2 atm O2, did not inhibit mitosis. Changes in morphology (multilayering and elongation) of cells exposed to 50 atm O2, but not the controls, were evident 7 days after the 3-hr exposure. The incorporation of [35S]methionine into individual polypeptides and [3H]thymidine into DNA was significantly inhibited immediately following a 3-hr treatment with 50 atm O2, but both parameters recovered within 2 days. DNA strand breaks were observed in LECs following hyperbaric O2 treatment as low as 4 atm O2 for 3 hr and increased with higher pressures of O2, but not N2. Treatment with 50 atm O2 nearly doubled the activity of the DNA repair enzyme, poly-ADP-ribose polymerase, and decreased the level of its substrate NAD+; the latter effect was reduced by 3-aminobenzamide, an inhibitor of the enzyme. Thus, although LECs tolerated brief exposures to high pressures of O2 without cell death, DNA damage occurred at relatively low pressures of O2. All of the effects of hyperbaric O2 on LECs occurred without any alteration of the normal levels of reduced and oxidized glutathione. It appears that GSH is important in maintaining cell viability during exposure to an elevated level of O2, but that it is incapable of preventing O2-induced effects on growth and DNA.

Tempol and deferoxamine protect cultured rabbit lens epithelial cells from H2O2 insult: insight into the mechanism of H2O2-induced injury.

In order to investigate the mechanism by which H2O2 damages the epithelium, 8 x 10(5) rabbit lens epithelial cells were treated with TEMPOL or deferoxamine and exposed to a single sublethal dose of 0.5 mM H2O2. TEMPOL is a SOD mimic, has a characteristic EPR spectrum and is metal independent. EPR spectra indicated that TEMPOL was not destroyed by H2O2, catalyzed the destruction of the superoxide anion, and penetrated the cells. Cells treated with H2O2 showed membrane blebbing, growth inhibition, an increase in GSSG, a dose-dependent decrease in GSH, ATP, NAD+, and in the activity of G3PDH, and in lactate production. H2O2 stimulated the hexose mono-phosphate shunt and induced single strand breaks in DNA. Treatment with TEMPOL or deferoxamine prevented or curtailed H2O2-induced inhibition of growth, the decrease in NAD+, the induction of single strand breaks in DNA, and membrane blebbing, but not the other biochemical parameters investigated. Both TEMPOL and deferoxamine prevent Fe+2-mediated generation of the damaging hydroxyl radical. TEMPOL reacts with superoxide and thus prevents it from recycling Fe+3 to Fe+2. It also oxidizes DNA-Fe+2 to DNA-Fe+3. Deferoxamine chelates intracellular Fe+3 and prevents its reduction to Fe+2. These compounds which limit the availability of Fe+2 by different means indicate that transition metals (including those bound to DNA) mediate certain of the damaging effects of H2O2.