A large share of climate impacts of beef and dairy can be attributed to ecosystem services other than food production.

Domesticated ruminants supply nutrient-dense foods but at a large environmental cost. However, many ruminant production systems are multi-functional, providing ecosystem services (ES) other than direct provision of food. When quantifying the climate impact of ruminant products using life cycle assessment (LCA), provisioning ES (i.e. beef and milk) are generally considered the only valuable outputs and other ES provided are ignored, which risks overlooking positive contributions associated with ruminant production. Non-provisioning ES can be included in LCA by economic allocation, using compensatory payments (through agri-environmental schemes) as a proxy for the economic value of ES. For example, farmers can receive payments for maintenance of pastures, which supports e.g. pollination. However, the association between different payment schemes, the ES provided, and livestock production is not always straightforward and it can be difficult to determine which payment schemes to include in the allocation. This study examined how accounting for ES in quantification of climate impact for beef and milk production on Swedish farms was affected by different ways of coupling ES to livestock production through payment schemes. Quantification was done using LCA, attributing the climate impact to beef, milk, and other ES by economic allocation. This resulted in <1-48% and 11-31% of climate impacts being allocated to other ES, instead of beef and milk, respectively, affecting suckler farms most. The results were influenced by which payment schemes, representing different ES, that were included; when only payments directly related to livestock rearing were included, the difference in the climate impact was still large between farm types, while the difference decreased considerably when all environmental schemes were included. While emissions do not disappear, ES-corrected climate impact can potentially be useful as part of consumer communication or in decision-making, reducing the risk of overlooking ES provided by ruminant production in a simpler way than using separate indicators.

Effects of 17ß-estradiol on proliferation, cell viability and intracellular redox status in native human lens epithelial cells.

PURPOSE: The purpose of this study was to examine the effects of 17ß-estradiol on proliferation, cell death and redox status in cultured human lens epithelial cells (HLECs). METHODS: HLECs were exposed to 17ß-estradiol after which cell viability was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and the number of mitotic and apoptotic cell nuclei was determined after staining with Hoechst 33342. Apoptosis was also determined by measuring caspase-3 activity and propidium iodide was used to determine the proportion of non-viable cells. Pro- and antioxidative effects of 17ß-estradiol was investigated by measuring peroxides, superoxides and glutathione, using dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HET), and monochlorobimane (MCB), respectively. Effects on mitochondrial membrane potential were determined using 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The ability of 17ß-estradiol to prevent reactive oxygen species (ROS)-production in HLECs after exposure to 25 µM H2O2 for 24h was also measured. RESULTS: This study demonstrates increased mitotic activity in HLECs exposed to physiologic concentrations of 17ß-estradiol (1 nM). Pharmacological concentrations of 17ß-estradiol caused increased number of apoptotic cell nuclei and caspase-3 activation. Physiologic concentrations of 17ß-estradiol (0.1-10 nM) stabilized the mitochondrial membrane potential. Similar or slightly higher concentrations of 17ß-estradiol (0.01-1 µM) protected against H2O2-induced oxidative stress as evident by decreased levels of peroxides and superoxides. CONCLUSIONS: The present study demonstrates mitogenic and anti-oxidative effects of 17ß-estradiol at physiologic concentrations, whereas pharmacological levels induced oxidative stress and acted pro-apoptotic in cultured lens cells.

Acute effects of the sigma-2 receptor agonist siramesine on lysosomal and extra-lysosomal proteolytic systems in lens epithelial cells.

PURPOSE: The aim of the present study was to examine the effects of the sigma-2 receptor agonist, siramesine, on morphology, growth, cell death, lysosomal function, and effects on extra-lysosomal proteolytic systems in human lens epithelial cells. METHODS: Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and Magic Red (RR-cresyl violet). Nuclear morphology was studied using Hoechst 33342 and propidium iodide. Enzymatic activities in living cells or cell lysates were studied using fluorogenic substrates. RESULTS: Siramesine at low concentrations increased the cytoplasmic proteolytic activity of the proteasome and the calpain system. Effects were also observed with respect to lysosomal morphology, acidity and function. In addition, activation of caspase-3 and the appearance of nuclei with an apoptotic morphology was found. CONCLUSIONS: Siramesine at low concentrations affects lens epithelial cells with perturbations of the major proteolytic systems and lysosomal morphology, resulting in caspase activation and cell death. Siramesine may be a possible substance for the treatment or prevention of posterior capsular opacification (PCO).

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume.

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle-van't Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15+/-0.01 using the new method, which was significantly smaller than the estimate of 0.37+/-0.02 obtained using the conventional method (p<0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.

Effects of dexamethasone on human lens epithelial cells in culture.

PURPOSE: Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells. METHODS: Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486. RESULTS: Low doses of dexamethasone (0.1 microM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 microM, 10 microM, and 100 microM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 microM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 microM) than with dexamethasone alone. CONCLUSIONS: Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract.

Incidence of ventricular fibrillation during left coronary arteriography in pigs: comparison of a solution of the nonionic dimer iodixanol with solutions of five different nonionic monomers.

BACKGROUND: Solutions of iodine contrast media (CM) used for selective coronary arteriography (CA) should have minimal propensity to cause ventricular fibrillation (VF). Commonly used CM for CA are nonionic monomers or dimers. PURPOSE: To compare VF propensity of ready-to-use solutions of one nonionic dimer, iodixanol, and five nonionic monomers, iobitridol, iopamidol, iomeprol, iopromide, and ioversol. MATERIAL AND METHODS: Twenty milliliters of each CM was injected into the left coronary artery (LCA) through an inflated balloon catheter (0.5 ml/s) in 14 pigs; the longest period of injection was 40 s. If VF occurred before 40 s, the injection was stopped and the heart was defibrillated. After VF, there was a delay of 40 min before the next injection. Hemodynamic parameters and vector electrocardiography (VECG) were monitored. A CM with a lower frequency of VF and a longer period between start of injection and start of VF was considered to have a lower VF propensity. RESULTS: Following 14 injections, each of the five nonionic monomers caused 14 VF, whereas iodixanol caused three VF (P<0.01). When VF occurred after iodixanol, it occurred later than after the other CM (P<0.001). Iodixanol caused less prolongation in QRS time (P<0.01) and QTc time (P<0.05) than the other CM. Prolongations in QRS and QTc times caused by CM parallel the VF propensities of the CM. CONCLUSION: Ready-to-use solutions of the dimer iodixanol have lower VF propensity than solutions of the five monomeric CM. This is related to the fact that the solutions of the dimer iodixanol have lower osmolality, higher viscosity, and higher concentrations of NaCl and CaCl2 than solutions of the five monomers.

Intracellular effects of NSAIDs/ASA in oxidatively stressed human lens epithelial cells in culture.

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H2O2 in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H2O2-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H2O2-exposed HLECs in culture.

The proteasome and intracellular redox status: implications for apoptotic regulation in lens epithelial cells.

PURPOSE: This study aimed to investigate redox regulation of the proteasome as well as the effect of proteasome inhibition on intracellular oxidative status and apoptosis. METHODS: Oxidative stress was induced in cultured human lens epithelial cells (HLECs) and intact mouse lenses by 100 microM H2O2. HLECs were also exposed to the reduced and the oxidized forms of glutathione (GSH/GSSG) and the reducing agent dithiotreitol (DTT). The chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured using synthetic fluorogenic substrates. Superoxide as well as peroxide production, mitochondrial membrane potential, and the level of GSH was measured in HLECs after proteasome inhibition by MG-132 or lactacystin. Apoptosis was determined by measuring caspase-3 activation and by studying apoptotic nuclei after staining with Hoechst 33342. RESULTS: All three peptidase activities of the proteasome were inhibited by 100 microM H2O2 and by the oxidized form of glutathione (GSSG), whereas the reduced form (GSH) stimulated chymotrypsin-like and peptidylglutamyl peptidase activities in HLECs lysates. Intact mouse lenses exposed to 100 microM H2O2 exhibited loss of transparency and trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH levels. Inhibition of the proteasome in cultured HLECs caused significant increase in apoptosis and disturbed intracellular redox balance. Simultaneous addition of exogenous GSH completely abolished the increased apoptosis seen after MG-132 treatment. CONCLUSIONS: This study supports the hypothesis that intracellular proteolytic and oxidative regulatory systems are tightly coupled. The current data also indicate that apoptosis by proteasome inhibition is mediated through oxidative mechanisms.

The iodinated radiographic contrast medium iohexol mimics the vasodilator effect seen with small increases in extracellular K+ in the isolated rabbit central ear artery.

PURPOSE: To investigate whether iodinated radiographic contrast media (IRCM) mimic the hyperpolarizing and vasodilator effects of K+ by comparing the vasodilator effect of a transient rise in extracellular K+ with that of the IRCM iohexol. MATERIAL AND METHODS: Immersed rabbit central ear arterial rings with and without endothelium and pre-contracted with phenylephrine (PE) were used to investigate the dependency of the endothelium in K+-induced vasodilatation. Perfused rabbit central ear arteries, pre-contracted with PE, were used to study the effects of bolus administrations of the IRCM iohexol or KCl on arterial tone under conditions that mimic those employed during clinical arteriography. RESULTS: A small rise in K+ caused an endothelium-independent and ouabain-sensitive relaxation of PE-constricted rabbit central ear artery rings. The relaxation was not changed in the presence of barium. The IRCM iohexol and KCl, injected as boluses into perfused PE-constricted rabbit ear arteries, caused transient decreases in perfusion pressure. Iohexol- and K+-induced pressure decreases were significantly reduced in the presence of 10 microM ouabain alone or in combination with 30 microM barium. Neither iohexol- nor K+-induced pressure decrease was significantly changed in the presence of barium alone compared to controls. CONCLUSION: The vasodilator effect of IRCM mimics the vasodilator effect seen upon small increase in extracellular K+. Under the experimental conditions employed in the present study, a considerable part of the IRCM-induced vasodilatation appears to be due to activation of Na+/K+-ATPase in the smooth muscle cells.

Potential protective effects of NSAIDs/ASA in oxidatively stressed human lens epithelial cells and intact mouse lenses in culture.

PURPOSE: To study possible toxic effects of indomethacin, diclofenac, and celecoxib (NSAIDs) and acetylsalicylic acid (ASA) as well as potentially protective effects of these substances in oxidatively stressed human lens epithelial cells (HLEC) and in intact mouse lenses in culture. METHODS: HLEC and mouse lenses were incubated with NSAIDs or ASA alone or in the presence of H2O2. To study apoptosis the cells were then either stained with Hoechst 33342 or assayed for caspase-3 activity. Mouse lenses were studied with respect to lens transparency. RESULTS: Low concentrations of NSAIDs/ASA caused a significant protection against H2O2-induced apoptosis in HLEC whereas higher concentrations were toxic. CONCLUSION: The protective effects of NSAIDs/ASA against oxidative damage are confined to a relatively small therapeutic window.

Iodinated radiographic contrast media possess antioxidant properties in vitro.

PURPOSE: To study potential properties of iodinated radiographic contrast media (IRCM) for intravascular use in in vitro free radical generating reactions. MATERIAL AND METHODS: Superoxide (*O2-) and hydroxyl (*OH) radicals were generated in xanthine oxidase and Fenton reactions. *O2- was assayed by the nitroblue tetrazolium (NBT) method, whereas *OH was assayed by an aromatic hydroxylation (2-hydroxybenzoic acid) method. Total antioxidant status (TAS) of test substances was determined by a colorimetric assay. Finally, acetyl-cholinesterase (AChE) activity was measured in the absence and presence of IRCM. RESULTS: High concentrations (>50 mM) of IRCM inhibited *O2- production, ionic more than non-ionic IRCM. Medium concentrations (25-50 mM) of IRCM reduced *OH production, and both types of IRCM were equally potent. Low concentrations (<25 mM) of non-ionic IRCM displayed higher antioxidant capacity than their ionic counterparts when tested in the TAS assay. Visipaque 320 (iodixanol) was found to have the highest TAS value, followed by Omnipaque 350 (iohexol), Hexabrix 320 (ioxaglate), and Urografin 370 (diatrizoate). CONCLUSION: IRCM have in vitro antioxidant properties in concentrations relevant for their clinical application. These properties may therefore be of potential importance when evaluating IRCM effects in vivo, particularly those concerning cardiovascular and renal function.

A new model for assessing proteolysis in the intact mouse lens in organ culture.

PURPOSE: To develop a new method to investigate proteolysis in the intact lens in organ culture. METHODS: Intact mouse lenses were assayed at regular intervals for proteolytic activity using fluorogenic peptide substrates +/- addition of ionomycin. Specific inhibitors were used to determine the activity of calpains, the proteasome and acid lysosomal enzymes. RESULTS: Significant levels of proteolytic activity were present in the intact lens. Proteolysis was stimulated by ionomycin. Preincubation with an inhibitor to the proteasome significantly decreased proteolysis whereas inhibitors of calpain and acid lysosomal enzymes did not. CONCLUSION: This study indicates that in the intact mouse lens in culture, the proteasome is an important protease. Its activity is at least partially regulated by calcium.

Interferon-gamma down-regulates claudin-1 and impairs the epithelial barrier function in primary cultured human thyrocytes.

OBJECTIVE: Proinflammatory cytokines are known to affect the follicular epithelium in autoimmune thyroid disease. Here we investigated the effect of interferon-gamma (IFN-gamma) on the barrier function of primary cultured human thyrocytes. DESIGN: Graves' thyroid follicle segments were cultured as a tight and polarised monolayer on the filter of a bicameral chamber, thereby allowing the in vivo epithelial characteristics to be maintained. METHODS: Transepithelial electrical resistance was measured with a Millicell ERS ohmmeter. The tight junction proteins claudin-1 and occludin were analysed by immunofluorescence and Western blotting. Cell morphology was studied by transmission electron microscopy. RESULTS: Thyrotrophin (TSH; 1 mU/ml) promoted the development of a tight epithelium monitored as a persistent increase in the transepithelial resistance to about 800 omega x cm2. IFN-gamma (100 U/ml), on the other hand, decreased the resistance to 60-150 omega x cm2 after 48 h. In IFN-gamma-treated cells the expression of claudin-1, but not that of occludin, was decreased along with a diminished intracellular and cell surface immunostaining. In addition, claudin-1 was disrupted at cell-cell contacts. IFN-gamma also caused profound cell shape changes and a multilayered cellular organisation, without ultrastructural or biochemical (caspase-3 activity) signs of cytotoxicity. TSH was unable to counteract the effects of IFN-gamma. CONCLUSIONS: IFN-gamma destroys the barrier function of filter-cultured human thyroid epithelial cells. The loss of barrier involves down-regulation and an altered distribution of claudin-1. This novel effect of IFN-gamma on target cells in thyroid autoimmunity might be of pathophysiological relevance to the exposure of hidden autoantigens.

Cardioprotective effects of the MR contrast agent MnDPDP and its metabolite MnPLED upon reperfusion of the ischemic porcine myocardium.

PURPOSE: To evaluate whether manganese dipyridoxyl diphosphate (MnDPDP) or its metabolite manganese dipyridoxyl ethyldiamine (MnPLED) reduces post-ischemic myocardial injury. MATERIAL AND METHODS: Left anterior descending artery (LAD) in anesthetized pigs was occluded (30 min) followed by reperfusion (120 min) during hemodynamic monitoring and infarct assessment. Three micromol/kg MnDPDP, 1 micromol/kg MnPLED (or a mixture of both) or saline was injected i.v. 10 min before reperfusion followed by infusion of either 3 micromol/kg/h MnDPDP, 1 micromol/kg/h MnPLED (or a mixture of both) or saline. The plasma concentrations of MnDPDP, MnPLED and other metabolites (e.g., ZnDPDP and ZnPLED) were analyzed. RESULTS: Femoral blood flow was reduced by 60% during early reperfusion in controls, whereas only 23 and 31% reductions were seen in animals treated with MnDPDP and MnPLED. During that time, +LV/dP and -LV/dP (maximum rate of left ventricular isovolumic contraction and relaxation, respectively), systolic pressure and diastolic pressure fell significantly less in animals treated with MnDPDP or MnPLED. Three out of 5 control animals experienced ventricular fibrillation (VF) during reperfusion, whereas VF was not seen in any of the pigs treated with MnPLED or/and MnDPDP. The infarct sizes in saline- and MnPLED-treated animals were 39+/-6 and 16+/-5%, respectively, of the occluded areas. MnDPDP did not reduce the infarct size. A mixture of MnDPDP and MnPLED significantly reduced infarct size (10+/-4%). When reperfusion started and throughout reperfusion, almost all injected MnDPDP was present as Zn-metabolites. CONCLUSION: MnPLED seems to reduce reperfusion-induced cardiac dysfunction and infarct size in pigs. MnDPDP does not reduce infarct size in the pig, probably because of the rapid exchange of Mn2+ for Zn2+ taking place in the pig.

A theoretical model of intracellular devitrification.

Devitrification of the intracellular solution can cause significant damage during warming of cells cryopreserved by freezing or vitrification. Whereas previous theoretical investigations of devitrification have not considered the effect of cell dehydration on intracellular ice formation, a new model which couples membrane-limited water transport equations, classical nucleation theory, and diffusion-limited crystal growth theory is presented. The model was used to explore the role of cell dehydration in devitrification of human keratinocytes frozen in the presence of glycerol. Numerical simulations demonstrated that water transport during cooling affects subsequent intracellular ice formation during warming, correctly predicting observations that critical warming rate increases with increasing cooling rate. However, for cells with a membrane transport activation energy less than approximately 50 kJ/mol, devitrification was also affected by cell dehydration during warming, leading to a reversal of the relationship between cooling rate and critical warming rate. Thus, for low warming rates (less than 10 degrees C/min for keratinocytes), the size and total volume fraction of intracellular ice crystals forming during warming decreased with decreasing warming rate, and the critical warming rate decreased with increasing cooling rate. The effects of water transport on the kinetics of intracellular nucleation and crystal growth were elucidated by comparison of simulations of cell warming with simulations of devitrification in H(2)O-NaCl-glycerol droplets of constant size and composition. These studies showed that the rate of intracellular nucleation was less sensitive to cell dehydration than was the crystal growth rate. The theoretical methods presented may be of use for the design and optimization of freeze-thaw protocols.

Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium-dependent vasodilation in the femoral artery of developing piglets.

Mechanisms mediating endothelium-dependent vasodilation were investigated in femoral artery rings from <2-day-old (newborn) and 2-week-old piglets. Based on previous results we hypothesized an age difference in the relative contribution of nitric oxide(NO)-cyclic 3',5'-guanosine monophosphate (cGMP) and K+ channel-activation to acetylcholine (ACh)-induced vasodilation. Changes in vascular tone were studied in organ baths in the absence or presence of NO synthase(NOS) inhibition or K+ channel blockade and the intra-arterial accumulation of cGMP in response to ACh was measured with radioimmunoassay (RIA). In control experiments, relaxant responses to ACh were equal in the two age groups. In the presence of the NOS-inhibitors N G-monomethyl-L-arginine acetate (L-NMMA; 100 microM) or NG-nitro-L-arginine (L-NOARG; 1-100 microM), however, relaxation was significantly more reduced in femoral artery rings from 2-week-old than from newborn, with lower pD2 values in the older age group. Inhibition of large (BKCa) conductance calcium-sensitive K+ channels with tetraethylammonium chloride (TEA; 1 mM), gave a significant rightward shift in the concentration-response curves to ACh which was of the same magnitude in both age groups. The ACh-induced vasodilation was abolished in both age groups by high K+ (20 mM) in combination with L-NOARG (100 microM). The relative increase in cGMP levels after addition of ACh (10 nM) was significantly larger in rings from newborn compared with 2-week-old piglets (12- vs. four-fold). In summary, sensitivity to NOS inhibition increased with age while the effect of K+ channel blockade with TEA was the same in femoral artery rings from newborn to 2-week-old piglets. Lower sensitivity to NOS inhibition and a larger increase in cGMP in response to ACh could indicate a higher efficacy of the NO/cGMP pathway in this vessel in the newborn piglet.

Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia.

Caspase-3 is a major effector protease in several apoptotic pathways, but its role in hypoxic-ischemic (HI) brain injury is incompletely understood. Cerebral HI was induced in 7-day-old rats by unilateral carotid artery ligation and exposure to 7.7% oxygen for 55 min. Caspase-3-like activity was significantly increased at 1 h (208%), peaked at 24 h (2,563%) and was still increased 6 days after HI (169%) in the ipsilateral cerebral cortex. Concomitantly, cleavage of the caspase-3 proform (31/33 kD) was detected on immunoblots, producing 29- and 17-kD fragments. Furthermore, significant degradation of the endogenous caspase-3 substrates inhibitor of caspase-activated DNase (DNA fragmentation factor 45), poly(ADP-ribose) polymerase and fodrin occurred. In conclusion, caspase-3 is activated extensively in the immature brain after HI. The subsequent cleavage of proteins involved in cellular homeostasis and repair may contribute to the process of brain injury.

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."