Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats.

Pulmonary emphysema is a common disease in which destruction of the lung's gas-exchange structures (alveoli) leads to inadequate oxygenation, disability and frequently death; lung transplantation provides its only remediation. Because treatment of normal rats with all-trans-retinoic acid increases the number of alveoli, we tested whether a similar effect would occur in rats with emphysema. Elastase was instilled into rat lungs, producing changes characteristic of human and experimental emphysema: increased lung volume reflecting a loss of lung elastic recoil, larger but fewer alveoli and diminished volume-corrected alveolar surface area due to destruction of alveolar walls. Treatment with all-trans-retinoic acid reversed these changes providing nonsurgical remediation of emphysema and suggesting the possibility of a similar effect in humans.

The understanding of congruent and incongruent referential gaze in 17-month-old infants: an eye-tracking study comparing human and robot.

Several studies have shown that the human gaze, but not the robot gaze, has significant effects on infant social cognition and facilitate social engagement. The present study investigates early understanding of the referential nature of gaze by comparing-through the eye-tracking technique-infants' response to human and robot's gaze. Data were acquired on thirty-two 17-month-old infants, watching four video clips, where either a human or a humanoid robot performed an action on a target. The agent's gaze was either turned to the target (congruent) or opposite to it (incongruent). The results generally showed that, independent of the agent, the infants attended longer at the face area compared to the hand and target. Additionally, the effect of referential gaze on infants' attention to the target was greater when infants watched the human compared to the robot's action. These results suggest the presence, in infants, of two distinct levels of gaze-following mechanisms: one recognizing the other as a potential interactive partner, the second recognizing partner's agency. In this study, infants recognized the robot as a potential interactive partner, whereas ascribed agency more readily to the human, thus suggesting that the process of generalizability of gazing behaviour to non-humans is not immediate.

Alveolar cells: incorporation of carbohydrate into protein and evidence for intracellular protein transport.

Alveolar cells incubated with radioactive glucosamine, galactose, and mannose incorporate radioactivity into protein, that is, into material insoluble in cold and hot trichloroacetic acid and not extracted by lipid solvents. This incorporation is incompletely inhibited by puromycin hydrochloride. The kinetics of the subcellular distribution of radioactivity are consistent with a precursor-product relationship between microsomal protein and the protein of particles sedimenting at 15,000 g. It is thus suggested that alveolar cells incorporate these substrates intact into protein at the microsomal level with subsequent transfer of this newly formed material to particles sedimenting at 15,000 g.

In vivo protein secretion by lung. Evidence for active secretion and interspecies differences.

The present study is an attempt to determine (a) if the lung actively secretes protein into the surface-active fraction of lung lavage returns; (b) if there are interspecies differences in this secretory activity; and (c) if the amount of nonradioactive protein in the lavage surface-active fraction shows interspecies variation. I found that pilocarpine stimulates the release of radioactive protein into the lavage surface-active fraction of rabbits and that this pilocarpine effect is completely blocked by atropine. Inhibition of lung oxygen consumption by iodoacetate is associaged with a dose-dependent inhibition of the pilocarpine-induced secretion. Microtubules may be involved in this secretory process because colchicine inhibits the pilocarpine effect. Of the radioactive protein in the total surface-active fraction (tissue plus lavage returns), a greater percent appears in the lavage surface-active fraction at 2 and 4 h, after a pulsed injection [U-14C] leucine, in the mouse than in the rat, which in turn has a greater amount than the rabbit. There is also a difference in the amount of nonradioactive protein per square meter of alveolar surface area in the lavage surface-active fraction of different species: mouse greater than rabbit greater than cat greater than dog. The amount of nonradioactive protein per square meter of alveolar surface area in the lavage surface-active fraction is directly proportional to the species respiratory rate; the log of the nonradioactive protein in the lavage surface-active fraction is inversely proportional to the log of the species alveolar diameter. I conclude that the lung actively secretes protein into the lavage surface-active fraction, that this secretion is under neurohumoral regulation, and that respiratory rate and alveolar size may influence this secretory activity and the amount of protein in this surface-active fraction.

Hyperoxia: influence on lung mechanics and protein synthesis.

We studied the time-course of the influence of in vivo hyperoxia on lung mechanics and on protein synthesis. After 24 h of exposure to greater than 98% O2 at 1 atm there were no alterations in descending pressure-volume curves (air or saline) of lungs excised from O2-exposed rats compared to control rats. After 48 h of hyperoxia there was a decrease in lung compliance. To study protein synthesis, as indicated by L-[U-(24)C] leucine incorporation into protein, lung slices were incubated with L-[U-(14)C]leucine and surface-active material then obtained by ultracentrifugation of lung homogenates. We measured radioactivity in total protein and in protein in the surface-active fraction. There were no alterations in incorporation after 12 h of hypertoxia. After 24 h of hyperoxia there were significant decreases (P<0.05) in L-[U-(14)C]leucine incorporation into total protein and into protein of the surface-active fraction. After 48 h of hyperoxia incorporation into protein of the surface-active fraction was decreased to a greater extent than incorporation into total protein, 63+/-4% and 75+/-5%, respectively, (P<0.025). These studies show that hyperoxia produces a major decrease in protein synthesis, including synthesis of protein in a surface-active fraction, before the onset of any detectable changes in the static compliance of excised lungs.

Adaption to hyperoxia. Influence on protein synthesis by lung and on granular pneumocyte ultrastructure.

We studied the influence of prolonged exposure to hyperoxia (O(2) > 98%) on protein synthesis and on the ultrastructure of the granular pneumocyte. To study protein synthesis, as indicated by l-[U-(14)C]-leucine incorporation into protein, lung slices were incubated with radioactive leucine and a surface-active fraction was obtained by ultracentrifugation of lung homogenates. We found that, following an initial depression in protein synthesis after 48 h of hyperoxia, protein synthesis in rats exposed to oxygen for 96 h rose to greater than control levels. This increase in protein synthesis was noted in whole lung protein and in protein present in the surface-active fraction. Stereologic ultrastructural analysis of granular pneumocytes revealed that the lamellar bodies occupy the same percentage of cytoplasmic volume in oxygen-exposed and control rats after 96 h; a previous study had shown lamellar bodies of oxygen-exposed rats to occupy less volume than those of control rats after 48 h of exposure at which time protein synthesis was also depressed. After 96 h of exposure there is a greater amount of rough endoplasmic reticulum in the granular pneumocytes of oxygen-exposed rats. These studies show that after 96 h of hyperoxia the lung has recovered its ability to synthesize protein including protein in the surface-active fraction and that these biochemical changes are associated with consistent ultrastructural alterations in the granular pneumocyte.

Tolerance of rats to hyperoxia. Lung antioxidant enzyme gene expression.

Tolerance to hyperoxia usually requires an increase of lung antioxidant enzyme (AOE) activity. We used rats with different degrees of tolerance to > 95% O2 to evaluate the importance of individual AOEs for tolerance; we also explored the regulation of AOE gene expression. During exposure of adult rats to > 95% O2, lung manganese superoxide dismutase (MnSOD) activity fell approximately 50% despite a threefold increase of MnSOD mRNA concentration; addition of a reducing agent to lung extracts from O2-exposed rats partially restored MnSOD activity. Endotoxin induced tolerance to O2 (a) without elevating Cu,Zn superoxide dismutase activity, (b) with increases of catalase and glutathione peroxidase (GP) activity of the same magnitude as occurred in O2-saline rats, but (c) with MnSOD activity 1.5-1.9-fold higher than in air-saline rats and 1.4-3.6-fold higher than in O2-saline rats. Endotoxin elevated the concentration of MnSOD and GP mRNAs without increasing their stability. O2 elevated MnSOD mRNA concentration, and increased its stability. O2 plus endotoxin increased the concentration and stability of MnSOD, catalase, and GP mRNAs. These data suggest that in adult rats tolerance to hyperoxia requires increased MnSOD activity; the data show gene expression and regulation vary among the AOEs, and that increased stability of the AOEs' mRNAs plays an important role in AOE gene expression and in tolerance to hyperoxia.

Potection from oxygen toxicity with endotoxin. Role of the endogenous antioxidant enzymes of the lung.

Endotoxin treatment of adult rats before hyperoxic exposure significantly increases their survival rate in >95% O(2) (J. Clin. Invest.61: 269, 1978). In this study, we wished to determine: (a) whether endotoxin would protect against O(2) toxicity if it were administered after the animals were already in >95% O(2) for 12-48 h; and (b) the relationship between the endogenous antioxidant enzymes of the lung and the protective effect of endotoxin treatment. Our results showed that adult rats given a single 500 mug/kg dose of endotoxin up to 36 h after the onset of O(2) exposure had significantly increased survival rates and decreased lung fluid accumulation compared to untreated animals in O(2) (P < 0.05). (Survival, 16/49 [untreated rats]; 18/20 [endotoxin at 12 h after the start of O(2) exposure]; 25/26 [endotoxin-24 h]; 15/20 [endotoxin-36 h].)Endotoxin-treated animals in O(2) showed increases in pulmonary superoxide dismutase, catalase, and glutathione peroxidase activities before the usual time of onset of measurable pulmonary edema in untreated animals in O(2). When diethyldithiocarbamate was used to block the superoxide dismutase enzyme rise in the endotoxin-treated rats in O(2), the protective action of endotoxin against pulmonary O(2) toxicity was nullified. In endotoxin-treated, O(2)-exposed mice, there were no lung antioxidant enzyme increases, and no protective effect from O(2) toxicity was achieved. We conclude that, in the rat, a single dose of endotoxin given even 36 h after the onset of hyperoxic exposure results in marked protection against O(2)-induced lung damage; and the increased lung antioxidant enzyme activity in the endotoxin-treated rats appears to be an essential component of this protective action.

Hyperoxia: a stereologic ultrastructural examination of its influence on cytoplasmic components of the pulmonary granular pneumocyte.

We used the technique of lineal analysis to study the influence of 48 h of hyperoxia on cytoplasmic organelles of pulmonary granular pneumocytes with particular reference to their lamellar bodies. We undertook this study because lamellar bodies are considered to be storage granules for pulmonary surfactant and because we had found that hyperoxia decreased [(14)C]leucine incorporation into protein of a surface-active lung fraction. We found that for lamellar bodies the percent cytoplasmic volume was 12.8+/-1.5 (mean+/-SEM) and 8.4+/-2.2, the organelle area (mum(2)) per organelle was 0.98+/-0.13 and 0.62+/-0.10 and the organelle volume (mum(2)) was 0.35+/-0.04 and 0.18+/-0.01, for air- and oxygen-exposed rats, respectively, (P=<0.05). The surface density of the lamellar body membrane was 7.05+/-0.47 and 9.36+/-0.96 (P=<0.05) for air- and oxygen-exposed rats. There were no differences in lamellar body number per cytoplasmic area or per pneumocyte between air- and oxygen-exposed rats. There were no statistical differences in these parameters between mitochondria of air- or oxygen-exposed rats. The surface density of the rough endoplasmic reticulum was the same in both groups. This study indicates that granular pneumocytes of rats exposed to hyperoxia have the same number of lamellar bodies as control rats but the lamellar bodies are smaller. This findings in consistent with the hypothesis that the hyperoxia-induced decrease in protein synthesis by lung represents at least in part a decreased synthesis of the secretory lipoprotein-pulmonary surfactant.

Studies on the regulation of secretion in Clara cells with evidence for chemical nonautonomic mediation of the secretory response to increased ventilation in rat lungs.

Using electron microscopy and morphometric methods to assess secretion, we previously found that two times tidal volume ventilation of isolated perfused rat lung stimulates secretion by bronchiolar Clara cells; this effect is not prevented by beta-adrenergic blockade (J. Clin. Invest. 1981. 67: 345-351.). In this study we used the isolated perfused rat lung and the anesthetized mechanically ventilated rat, to further study the mechanism by which large tidal volumes stimulate secretion by Clara cells. With the perfused lung we found (a) alpha-adrenergic inhibition did not block the secretory effect of ventilation at two times normal tidal volume; (b) indomethacin completely blocked the secretory action of two times tidal volume ventilation; (c) medium previously used to perfuse lungs ventilated at two times tidal volume, but not medium previously used to ventilate lungs at normal tidal volume, stimulated secretion by Clara cells when used to perfuse fresh lungs ventilated at tidal volume; (d) addition of prostacyclin to the fresh perfusate increased secretion by Clara cells of lungs ventilated at normal tidal volume. In anesthetized mechanically ventilated rats, sighs stimulated secretion by Clara cells; this increased secretion was inhibited by indomethacin but not by cholinergic blockade (bilateral vagotomy). These studies indicate that increased volume ventilation stimulates secretion by Clara cells in vivo and in vitro; they provide evidence that chemical nonadrenergic, noncholinergic mechanisms are involved in this secretion, and that prostaglandins may be the chemical messenger coupling the mechanico-secretory events.

Surfactant deficiency in rats without a decreased amount of extracellular surfactant.

Low volume ventilation without periodic large inflations leads to diminished alveolar stability and to the accumulation of increased amounts of airway disaturated phosphatidylcholine (DSPC) in large aggregates that sediment at 1,000 g; surfactant in this form lowers surface tension less rapidly than surfactant present in the 1,000-g supernatant fraction. These observations led to the present work in which we tested the notion that alveolar instability may develop in the presence of an undiminished quantity of total airway surfactant, if the amount of surfactant found in the 1,000-g supernatant fraction is diminished. Pulmonary compliance fell and the alveolar-arterial O2 gradient widened in normothermic rats during constant ventilation in the resting tidal volume range, and, in hyperthermic rats (approximately 39 degrees C) similarly ventilated but with the addition of periodic sighs. The total amount of airway DSPC was undiminished in each group, but in each less DSPC was present in the 1,000-g supernatant fraction compared with controls. Alveolar instability and hypoxemia also developed in hyperthermic rats during low volume ventilation without periodic sighs. Although the total amount of airway DSPC was decreased in these rats, enough remained to theoretically form a continuous monomolecular film over the entire alveolar surface at functional residual capacity; however, there was insufficient surfactant in the 1,000-g supernatant fraction to form such a continuous film. These findings demonstrate that the mode of ventilation, and moderate hyperthermia, may lead to decreased alveolar stability despite the presence of normal amounts of airway surfactant, and, by inference, indicate the extracellular form or state of surfactant has an important effect on alveolar stability.

Changes in sedimentation of surfactant in ventilated excised rat lungs. Physical alterations in surfactant associated with the development and reversal of atelectasis.

We ventilated excised rat lungs at a constant tidal volume (CTV); they developed areas of atelectasis which could be reversed by a large inflation (CTV + I) or prevented by the addition of positive end-expiratory pressure to the CTV. To explore the possibility that these modes of ventilation led to changes in surfactant, we lavaged the lungs and centrifuged the returns at 500 g; we measured the amount of disaturated phosphatidylcholine (DSPC) in the resultant pellet and supernatant fluid as a marker for surfactant. We found 16.9+/-1.5 (mean+/-SE), 38.0+/-2.4, 18.3+/-1.6, and 21.7+/-2.3% of the total lavage DSPC, in the pellet from freshly excised, CTV, CTV + I, and positive end-expiratory pressure to the CTV lungs, respectively. The total amount of lavage DSPC was the same in all groups. The ultrastructure of acellular material pelleted by sequential centrifugation of lavage returns at 500, 1,000, and 60,000 g was examined. We found mostly tubular myelin in the 500-g and 1,000-g pellets, but no tubular myelin in the 60,000-g pellet. Air inflation pressure-volume measurements from the degassed state revealed that the opening pressure and recoil pressures up to 75% of total lung capacity were significantly higher in the CTV than in the CTV + I lungs. There were no differences between these groups in air deflation or in saline inflation and deflation pressure-volume measurements. Our findings suggest that CTV leads to increases in the tubular myelin form of surfactant and that this leads to increased surface tension in alveoli which results in alveolar collapse.

Regulation of secretion in Clara cells: studies using the isolated perfused rat lung.

Previous studies from our laboratory indicated that both beta-adrenergic and cholinergic agents stimulate in vivo secretion by rat bronchiolar Clara cells. Those studies also provided support for an in-series beta-adrenergic-cholinergic stimulation of secretion. To further explore the regulation of secretion in Clara cells, and to do it in the absence of systemic influences, we have used the isolated ventilated perfused rat lung. We have again used morphometry and electron microscopy to assess secretion by measuring the volume density (fraction of cell volume) of the secretory granules of bronchiolar Clara cells. We found that in the isolated perfused lung, as in the intact animal, isoproterenol stimulated secretion in Clara cells and that this effect was blocked by the beta-adrenergic antagonist propranolol. Pilocarpine, unlike its action in the intact animal, did not stimulate secretion in the isolated lung; rather it inhibited the secretory effect of isoproterenol. Increased tidal-volume ventilation stimulated secretion; propranolol did not block this effect. Analogs of cyclic (c)AMP and of cGMP also stimulated secretion by Clara cells. These findings indicate that there are at least two mechanisms by which Clara cells can be stimulated to secrete. One seems to be beta-adrenergic-cAMP mediated but the triggering event is unknown. The other is initiated by increased tidal volume and cGMP may be involved in the intracellular mediation of this stimulatory event. Finally, we found evidence of beta-adrenergic (stimulatory) -cholinergic (inhibitory antagonism in the regulation of secretion in Clara cells.

Postnatal development of alveoli. Regulation and evidence for a critical period in rats.

In many species, including humans, pulmonary alveoli are formed after birth by septal subdivision of the large gas-exchange saccules present at birth. In rats septation occurs mainly between the 4th and 14th postnatal days (Burri, P. H. 1974. Anat. Rec. 180:77-98), but little is known about the regulation of this process. We found that dexamethasone (0.1 micrograms daily) given to rats from age 4 to 13 d markedly impaired saccule septation to at least age 60 d and also diminished the extent of the increase of alveolar surface area (Sa). Underfeeding from birth to age 14 d did not diminish saccule septation but did result in diminished Sa. We conclude dexamethasone-treated rats have a critical period during which the gas-exchange saccules present at birth must be subdivided. Since Sa increased in dexamethasone-treated rats without a change in alveolar size, and, the enlargement of Sa was diminished in underfed rat pups without a deficit of saccule septation, we postulate new alveoli were formed by means other than septation of the large gas-exchange saccules present at birth. Furthermore, these various means of forming alveoli, and hence of increasing Sa, were differently regulated: dexamethasone decreased the enlargement of Sa brought about by both septation of the gas-exchange saccules present at birth and by other, as yet unidentified, means of forming alveoli; underfeeding did not diminish Sa increases produced by saccule septation but did decrease the extent of Sa enlargement due to the other means of forming alveoli.

Rat lung Cu,Zn superoxide dismutase. Isolation and sequence of a full-length cDNA and studies of enzyme induction.

The synthesis of Cu,Zn SOD by rat lung increases spontaneously in the fetus in late gestation and during exposure of neonatal and adult rats to greater than 95% O2. To explore the regulation of these increases, we measured rat lung Cu,Zn SOD synthesis and activity. We also cloned and sequenced a rat lung Cu,Zn SOD cDNA that was used to measure Cu,Zn SOD mRNA concentration. We found that (a) under normal gestational and postgestational conditions the synthesis of this enzyme was regulated pretranslationally; (b) the increased synthesis that occurs under hyperoxia (greater than 95% O2), was pretranslationally mediated in otherwise unmanipulated neonatal rats but translationally controlled in hyperoxic adult rats; and (c) in lungs of rats made tolerant to greater than 95% O2 by allowing 24 h rest in air after an initial 48 h in greater than 95% O2, the increased Cu,Zn SOD synthesis that occurred during the second period of hyperoxia was regulated pretranslationally. We conclude Cu,Zn SOD gene expression in the lung is developmentally regulated under normal conditions and in response to an oxidant challenge. Tolerance, whether endogenous or induced, appears to require the accumulation of increased amounts of Cu,Zn SOD mRNA.

Pertussis toxin treatment alters manganese superoxide dismutase activity in lung. Evidence for lung oxygen toxicity in air-breathing rats.

Exposure of rats to hyperoxia or to treatment with endotoxin, increases lung manganese superoxide dismutase (MnSOD) gene expression. However, the paths by which these environmental signals are transduced into enhanced MnSOD gene expression are unknown. We now provide evidence that heterotrimeric G proteins are involved in the hyperoxia-induced increase in lung MnSOD gene expression but that pertussis toxin-sensitive G proteins are not involved in the endotoxin-induced elevation of lung MnSOD gene expression. We also show that treating rats with pertussis toxin decreased lung MnSOD activity approximately 50%. This decline in MnSOD activity occurred without a change in the lung activity of copper-zinc SOD, catalase, or glutathione peroxidase. In air-breathing rats, the pertussis toxin-induced decrease in MnSOD activity was associated with the development of lung edema, pleural effusion with a high concentration of protein, and biochemical evidence of lung oxygen toxicity. Compared to air-breathing rats, maintenance of pertussis toxin-treated rats under hypoxic or hyperoxic conditions respectively decreased or increased intrathoracic fluid. Endotoxin treatment elevated lung MnSOD activity and protected pertussis toxin-treated rats from an increase in intrathoracic fluid.

Dexamethasone implant in diabetic macular edema in real-life situations.

PURPOSE: To report outcome of eyes with recalcitrant and naive eyes with diabetic macular edema (DME) treated with intravitreal dexamethasone implants (Ozurdex) injection. METHODS: Retrospective multicenter data analysis of eyes with DME treated with Ozurdex implant and with minimum follow-up of at least one year after the first implant. Data collected included demographic details, history of presenting illness, past treatment history, clinical examination details including visual acuity at presentation, and follow-up with imaging and treatment details. Paired sample t-test was used to measure mean differences between pre- and post-implant values obtained at baseline and last follow-up. RESULTS: A total of 79 eyes (62 subjects) were included. Sixty-four eyes had been previously treated; 15 eyes were naive. Among the previously treated eyes, mean interval between first Ozurdex injection and any previous treatment was 7.69±8.2 months. In naive eyes, the visual acuity improved from baseline 0.58±0.25 to 0.44±0.33 logMAR at last follow-up (P=0.05). In eyes that had been previously treated, the improvement was from 0.65±0.34 at baseline to 0.48±0.35 logMAR (P=0.01). Mean treatment-free interval was 6.5±4.5 months. Nine eyes were steroid responder with controlled intraocular pressure (IOP), none showed any spike in IOP during the follow-up period. CONCLUSIONS: Ozurdex implant could be a good alternative for recalcitrant as well as naive eyes with DME. The visual gain after initial implant injection was fairly maintained, with additional treatment usually after 6 months in naive eyes. Ozurdex appeared safe even in steroid responders with good control of IOP with antiglaucoma medications.

Protein synthesis by attached pulmonary macrophages. Effect of phagocytosis.

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per microgram polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.