Effects of different tidal volumes in pulmonary and extrapulmonary lung injury with or without intraabdominal hypertension

We hypothesized that: (1) intraabdominal hypertension increases pulmonary inflammatory and fibrogenic responses in acute lung injury (ALI); (2) in the presence of intraabdominal hypertension, higher tidal volume reduces lung damage in extrapulmonary ALI, but not in pulmonary ALI. Wistar rats were randomly allocated to receive Escherichia coli lipopolysaccharide intratracheally (pulmonary ALI) or intraperitoneally (extrapulmonary ALI). After 24 h, animals were randomized into subgroups without or with intraabdominal hypertension (15 mmHg) and ventilated with positive end expiratory pressure = 5 cmH(2)O and tidal volume of 6 or 10 ml/kg during 1 h. Lung and chest wall mechanics, arterial blood gases, lung and distal organ histology, and interleukin (IL)-1 beta, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions in lung tissue were analyzed. With intraabdominal hypertension, (1) chest-wall static elastance increased, and PCIII, IL-1 beta, IL-6, and caspase-3 expressions were more pronounced than in animals with normal intraabdominal pressure in both ALI groups; (2) in extrapulmonary ALI, higher tidal volume was associated with decreased atelectasis, and lower IL-6 and caspase-3 expressions; (3) in pulmonary ALI, higher tidal volume led to higher IL-6 expression; and (4) in pulmonary ALI, liver, kidney, and villi cell apoptosis was increased, but not affected by tidal volume. Intraabdominal hypertension increased inflammation and fibrogenesis in the lung independent of ALI etiology. In extrapulmonary ALI associated with intraabdominal hypertension, higher tidal volume improved lung morphometry with lower inflammation in lung tissue. Conversely, in pulmonary ALI associated with intraabdominal hypertension, higher tidal volume increased IL-6 expression.

Phenylephrine-induced hypertension during transient middle cerebral artery occlusion alleviates ischemic brain injury in spontaneously hypertensive rats

Arterial hypertension is a major risk factor for ischemic stroke. However, the management of preexisting hypertension is still controversial in the treatment of acute stroke in hypertensive patients. The present study evaluates the influence of preserving hypertension during focal cerebral ischemia on stroke outcome in a rat model of chronic hypertension, the spontaneously hypertensive rats (SHR). Focal cerebral ischemia was induced by transient (1 h) occlusion of the middle cerebral artery, during which mean arterial blood pressure was maintained at normotension (110-120 mm Hg, group 1, n=6) or hypertension (160-170 mm Hg, group 2, n=6) using phenylephrine. T2-, diffusion- and perfusion-weighted MRI were performed serially at five different time points: before and during ischemia, and at 1, 4 and 7 days after ischemia. Lesion volume and brain edema were estimated from apparent diffusion coefficient maps and T2-weighted images. Regional cerebral blood flow (rCBF) was measured within and outside the perfusion deficient lesion and in the corresponding regions of the contralesional hemisphere. Neurological deficits were evaluated after reperfusion. Infarct volume, edema, and neurological deficits were significantly reduced in group 2 vs. group 1. In addition, higher values and rapid restoration of rCBF were observed in group 2, while rCBF in both hemispheres was significantly decreased in group 1. Maintaining preexisting hypertension alleviates ischemic brain injury in SHR by increasing collateral circulation to the ischemic region and allowing rapid restoration of rCBF. The data suggest that maintaining preexisting hypertension is a valuable approach to managing hypertensive patients suffering from acute ischemic stroke. Published by Elsevier B.V.

Infrared (810 nm) Low-Level Laser Therapy in Experimental Model of Strain-Induced Skeletal Muscle Injury in Rats: Effects on Functional Outcomes

Muscle strains are among the most prevalent causes for athletes absence from sport activities. Low-level laser therapy (LLLT) has recently emerged as a potential contender to nonsteroidal anti-inflammatory drugs in muscle strain treatment. In this work we investigated effects of LLLT and diclofenac on functional outcomes in the acute stage after muscle strain injury in rats. Muscle strain was induced by overloading the tibialis anterior muscle of rats during anesthesia. The injured groups received either no treatment, or a single treatment with diclofenac 30 min prior to injury, or LLLT (810 nm, 100 mW) with doses of 1, 3, 6 or 9 J, at 1 h after injury. Functional outcome measures included a walking index and assessment of electrically induced muscle performance. All treatments (except 9 J LLLT) significantly improved the walking index 12 h postinjury compared with the untreated group. The 3 J group also showed a significantly better walking index than the drug group. All treatments significantly improved muscle performance at 6 and 12 h. LLLT dose of 3 J was as effective as the pharmacological agent in improving functional outcomes in the early phase after a muscle strain injury in rats.

N-Acetylcysteine protects the peritoneum from the injury induced by hypertonic dialysis solution

Background: Oxidative stress has been implicated in the development of peritoneal damage. The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) in a rat peritoneal infusion model. Methods: Eighteen male Wistar rats were divided in 3 groups: (i) control group; (ii) HDS group, receiving peritoneal dialysis solution (PDS); and (iii) HDS+NAC group, receiving PDS and oral NAC. Six weeks later they were evaluated for dialysate to plasma urea ratio (D/P), ratio of glucose concentration in peritoneal fluid (G1/G0), thiobarbituric acid reactive substances in plasma and urine and histology of peritoneal membrane. Results: The HDS+NAC group presented a lower increase in solute transport (D/P 0.51 +/- 0.1, and G1/GO 0.35 +/- 0.06) in comparison with the HDS group (D/P 0.67 +/- 0.1; p=0.03, and G1/G0 0.27 +/- 0.07; p=0.01). The HDS+NAC group showed lower thiobarbituric acid reactive substance concentrations compared with the HDS group. In the treated group, the peritoneal membrane presented lower thickness. Conclusions: Functional and histological peritoneal changes were significantly reduced by the treatment with NAC.

Tricuspid Valve Injury After Heart Transplantation Due to Endomyocardial Biopsy: An Analysis of 3550 Biopsies

Introduction. Tricuspid regurgitation (TR) is the most commonly valvular dysfunction found after heart transplantation (HTx). It may be related to endomyocardial biopsy (EMB) performed for allograft rejection surveillance. Objective. This investigation evaluated the presence of tricuspid valve tissue fragments obtained during routine EMB performed after HTx and its possible effect on short-term and long-term hemodynamic status. Method. This single-center review included prospectively collected and retrospectively analyzed data. From 1985 to 2010, 417 patients underwent 3550 EMB after HTx. All myocardial specimens were reviewed to identify the presence of tricuspid valve tissue by 2 observers initially and in doubtful cases by a third observer. The echocardiographic and hemodynamic parameters were only considered for valvular functional damage analysis in cases of tricuspid tissue inadvertently removed during EMB. Results. The 417 HTx patients to 3550 EMB, including 17,550 myocardial specimens. Tricuspid valve tissue was observed in 12 (2.9%) patients corresponding to 0.07% of the removed fragments. The echocardiographic and hemodynamic parameters of these patients before versus after the biopsy showed increased TR in 2 cases (2/12; 16.7%) quantified as moderate without progression in the long term. Only the right atrial pressure showed a significant increase (P = .0420) after tricuspid injury; however, the worsening of the functional class was not significant enough in any of the subjects. Thus, surgical intervention was not required. Conclusions. Histological evidence of chordal tissue in EMB specimens is a real-world problem of relatively low frequency. Traumatic tricuspid valve injury due to EMB rarely leads to severe valvular regurgitation; only a minority of patients develop significant clinical symptoms. Hemodynamic and echocardiographic alterations are also less often observed in most patients.

RESULTS OF ULNAR NERVE NEUROTIZATION TO BICEPS BRACHII MUSCLE IN BRACHIAL PLEXUS INJURY

Objective: To evaluate the factors influencing the results of ulnar nerve neurotization at the motor branch of the brachii biceps muscle, aiming at the restoration of elbow flexion in patients with brachial plexus injury. Methods: 19 patients, with 18 men and 1 woman, mean age 28.7 years. Eight patients had injury to roots C5-C6 and 11, to roots C5-C6-C7. The average time interval between injury and surgery was 7.5 months. Four patients had cervical fractures associated with brachial plexus injury. The postoperative follow-up was 15.7 months. Results: Eight patients recovered elbow flexion strength MRC grade 4; two, MRC grade 3 and nine, MRC <3. There was no impairment of the previous ulnar nerve function. Conclusion: The surgical results of ulnar nerve neurotization at the motor branch of brachii biceps muscle are dependent on the interval between brachial plexus injury and surgical treatment, the presence of associated fractures of the cervical spine and occipital condyle, residual function of the C8-T1 roots after the injury and the involvement of the C7 root. Signs of reinnervation manifested up to 3 months after surgery showed better results in the long term. Level of Evidence: IV, Case Series.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

Near-Term Fetal Hypoxia-Ischemia in Rabbits MRI Can Predict Muscle Tone Abnormalities and Deep Brain Injury

Background and Purpose-The pattern of antenatal brain injury varies with gestational age at the time of insult. Deep brain nuclei are often injured at older gestational ages. Having previously shown postnatal hypertonia after preterm fetal rabbit hypoxia-ischemia, the objective of this study was to investigate the causal relationship between the dynamic regional pattern of brain injury on MRI and the evolution of muscle tone in the near-term rabbit fetus. Methods-Serial MRI was performed on New Zealand white rabbit fetuses to determine equipotency of fetal hypoxia-ischemia during uterine ischemia comparing 29 days gestation (E29, 92% gestation) with E22 and E25. E29 postnatal kits at 4, 24, and 72 hours after hypoxia-ischemia underwent T2- and diffusion-weighted imaging. Quantitative assessments of tone were made serially using a torque apparatus in addition to clinical assessments. Results-Based on the brain apparent diffusion coefficient, 32 minutes of uterine ischemia was selected for E29 fetuses. At E30, 58% of the survivors manifested hind limb hypotonia. By E32, 71% of the hypotonic kits developed dystonic hypertonia. Marked and persistent apparent diffusion coefficient reduction in the basal ganglia, thalamus, and brain stem was predictive of these motor deficits. Conclusions-MRI observation of deep brain injury 6 to 24 hours after near-term hypoxia-ischemia predicts dystonic hypertonia postnatally. Torque-displacement measurements indicate that motor deficits in rabbits progressed from initial hypotonia to hypertonia, similar to human cerebral palsy, but in a compressed timeframe. The presence of deep brain injury and quantitative shift from hypo-to hypertonia may identify patients at risk for developing cerebral palsy. (Stroke. 2012;43:2757-2763.)

Proliferative Activity in Ischemia/Reperfusion Injury in Hepatectomized Mice: Effect of N-Acetylcysteine

Background. Dysfunction of the liver after transplantation may be related to the graft size and ischemia/reperfusion (I/R) injury. N-Acetylcysteine (NAC) exerts beneficial effects on livers undergoing ischemia reperfusion. We sought to evaluate NAC modulation on reduced livers associated with I/R injury. Methods. Male C57BL/6 mice of 8 weeks of age were divided into groups: 50% hepatectomy (G-Hep); NAC (G-Hep + NAC [150 mg/kg]) via vena cava 15 minutes before hepatectomy; ischemia (G-Hep + IR); NAC with hepatectomy (G-IR + Hep + Nac); and IR using 30 minutes selective hepatic occlusion and reperfusion for 24 hours. After 24 hours, the remaining liver was removed, for staining with hematoxylin and eosin or labeling by proliferating cell nuclear antigen. Blood was collected for biochemical evaluations. Significance was considered for P <= .05. Results. Aspartate aminotransferase was high in all studied groups reflecting the hepatectomy and intervention. injuries. However, when assessing alanine aminotransferase, which depicts liver function, induction of IR promoted a greater increase than hepatectomy (P = .0003). NAC decreased ALT activity in all groups, even in association with I/R (P < .05), reflecting a modulation of the injury. Necrosis resulting from IR was mitigated by NAC. The experimental model of 50% reduced live promoted regeneration of the hepatic remnant, which was accentuated by NAC, according to the total number of hepatocytes and PCNA values. Conclusion. NAC preserved the remnant liver in mice and stimulates regeneration even after IR injury.

N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation

Campos R, Shimizu MH, Volpini RA, de Bragan a AC, Andrade L, Lopes FD, Olivo C, Canale D, Seguro AC. N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation. Am J Physiol Lung Cell Mol Physiol 302: L640-L650, 2012. First published January 20, 2012; doi: 10.1152/ajplung.00097.2011.-Sepsis is a common cause of acute kidney injury (AKI) and acute lung injury. Oxidative stress plays as important role in such injury. The aim of this study was to evaluate the effects that the potent antioxidant N-acetylcysteine (NAC) has on renal and pulmonary function in rats with sepsis. Rats, treated or not with NAC (4.8 g/l in drinking water), underwent cecal ligation and puncture (CLP) 2 days after the initiation of NAC treatment, which was maintained throughout the study. At 24 h post-CLP, renal and pulmonary function were studied in four groups: control, control + NAC, CLP, and CLP + NAC. All animals were submitted to low-tidal-volume mechanical ventilation. We evaluated respiratory mechanics, the sodium cotransporters Na-K-2Cl (NKCC1) and the alpha-subunit of the epithelial sodium channel (alpha-ENaC), polymorphonuclear neutrophils, the edema index, oxidative stress (plasma thiobarbituric acid reactive substances and lung tissue 8-isoprostane), and glomerular filtration rate. The CLP rats developed AKI, which was ameliorated in the CLP + NAC rats. Sepsis-induced alterations in respiratory mechanics were also ameliorated by NAC. Edema indexes were lower in the CLP + NAC group, as was the wet-to-dry lung weight ratio. In CLP + NAC rats, alpha-ENaC expression was upregulated, whereas that of NKCC1 was downregulated, although the difference was not significant. In the CLP + NAC group, oxidative stress was significantly lower and survival rates were significantly higher than in the CLP group. The protective effects of NAC (against kidney and lung injury) are likely attributable to the decrease in oxidative stress, suggesting that NAC can be useful in the treatment of sepsis.

Translation and Cross-cultural Adaptation of the Lower Extremity Functional Scale Into a Brazilian Portuguese Version and Validation on Patients With Knee Injuries

STUDY DESIGN: Clinical measurement. OBJECTIVE: To translate and culturally adapt the Lower Extremity Functional Scale (LEFS) into a Brazilian Portuguese version, and to test the construct and content validity and reliability of this version in patients with knee injuries. BACKGROUND: There is no Brazilian Portuguese version of an instrument to assess the function of the lower extremity after orthopaedic injury. METHODS: The translation of the original English version of the LEFS into a Brazilian Portuguese version was accomplished using standard guidelines and tested in 31 patients with knee injuries. Subsequently, 87 patients with a variety of knee disorders completed the Brazilian Portuguese LEES, the Medical Outcomes Study 36-Item Short-Form Health Survey, the Western Ontario and McMaster Universities Osteoarthritis Index, and the International Knee Documentation Committee Subjective Knee Evaluation Form and a visual analog scale for pain. All patients were retested within 2 days to determine reliability of these measures. Validation was assessed by determining the level of association between the Brazilian Portuguese LEFS and the other outcome measures. Reliability was documented by calculating internal consistency, test-retest reliability, and standard error of measurement. RESULTS: The Brazilian Portuguese LEES had a high level of association with the physical component of the Medical Outcomes Study 36-Item Short-Form Health Survey (r = 0.82), the Western Ontario and McMaster Universities Osteoarthritis Index (r = 0.87), the International Knee Documentation Committee Subjective Knee Evaluation Form (r = 0.82), and the pain visual analog scale (r = -0.60) (all, P<.05). The Brazilian Portuguese LEES had a low level of association with the mental component of the Medical Outcomes Study 36-Item Short-Form Health Survey (r = 0.38, P<.05). The internal consistency (Cronbach alpha = .952) and test-retest reliability (intraclass correlation coefficient = 0.957) of the Brazilian Portuguese version of the LEES were high. The standard error of measurement was low (3.6) and the agreement was considered high, demonstrated by the small differences between test and retest and the narrow limit of agreement, as observed in Bland-Altman and survival-agreement plots. CONCLUSION: The translation of the LEFS into a Brazilian Portuguese version was successful in preserving the semantic and measurement properties of the original version and was shown to be valid and reliable in a Brazilian population with knee injuries. J Ort hop Sports Phys Ther 2012;42(11):932-939, Epub 9 October 2012. doi:10.2519/jospt.2012.4101

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-kappa B and upregulating endothelial nitric oxide synthase

de Souza ACCP, Volpini RA, Shimizu MH, Sanches TR, Camara NOS, Semedo P, Rodrigues CE, Seguro AC, Andrade L. Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting nuclear factor-kappa B and upregulating endothelial nitric oxide synthase. Am J Physiol Renal Physiol 302: F1045-F1054, 2012. First published January 11, 2012; doi:10.1152/ajprenal.00148.2011.-The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-kappa B activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP + EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-L-arginine methyl ester (L-NAME) simultaneously with EPO administration (CLP + EPO + L-NAME). A fifth group (CLP + EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP + EPO rats presented significantly higher inulin clearance than did CLP and CLP + EPO + L-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP + EPO rats; and inulin clearance was significantly higher in CLP + EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP + EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-alpha activation, NF-kappa B activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-kappa B downregulation.

Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury

de Araujo CC, Silva JD, Samary CS, Guimaraes IH, Marques PS, Oliveira GP, do Carmo LGRR, Goldenberg RC, Bakker-Abreu I, Diaz BL, Rocha NN, Capelozzi VL, Pelosi P, Rocco PRM. Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury. J Appl Physiol 112: 1206-1214, 2012. First published January 19, 2012; doi:10.1152/japplphysiol.01061.2011.-Physical activity modulates inflammation and immune response in both normal and pathologic conditions. We investigated whether regular and moderate exercise before the induction of experimental sepsis reduces the risk of lung and distal organ injury and survival. One hundred twenty-four BALB/c mice were randomly assigned to two groups: sedentary (S) and trained (T). Animals in T group ran on a motorized treadmill, at moderate intensity, 5% grade, 30 min/day, 3 times a week for 8 wk. Cardiac adaptation to exercise was evaluated using echocardiography. Systolic volume and left ventricular mass were increased in T compared with S group. Both T and S groups were further randomized either to sepsis induced by cecal ligation and puncture surgery (CLP) or sham operation (control). After 24 h, lung mechanics and histology, the degree of cell apoptosis in lung, heart, kidney, liver, and small intestine villi, and interleukin (IL)-6, KC (IL-8 murine functional homolog), IL-1 beta, IL-10, and number of cells in bronchoalveolar lavage (BALF) and peritoneal lavage (PLF) fluids as well as plasma were measured. In CLP, T compared with S groups showed: 1) improvement in survival; 2) reduced lung static elastance, alveolar collapse, collagen and elastic fiber content, number of neutrophils in BALF, PLF, and plasma, as well as lung and distal organ cell apoptosis; and 3) increased IL-10 in BALF and plasma, with reduced IL-6, KC, and IL-1 beta in PLF. In conclusion, regular and moderate exercise before the induction of sepsis reduced the risk of lung and distal organ damage, thus increasing survival.

Kidney injury and cell therapy: Preclinical study

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst (R). Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 X 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

Relationship between ozone, meteorological conditions, gas exchange and leaf injury in Nicotiana tabacum Bel-W3 in a sub-tropical region

The city of Sao Paulo is located in a subtropical region whose climate exhibits few defined seasons as well as frequent oscillations in temperature and rainfall throughout the year. In addition to interfering with physiological processes, these peculiar climatic dynamics influence the formation of O-3 and its influx into leaves, causing species used as bioindicators in temperate climates to be ineffective here. This study evaluated gas exchange variations in CO2 and H2O and leaf injuries induced by O-3 in Nicotiana tabacum Bel-W3 in relation to oscillations in environmental conditions. Plants were exposed to an O-3-polluted environment for fifteen periods of fourteen days each throughout 2008. Gas exchange and O-3 were higher during the summer and winter but were highly variable in all seasons. Severe injuries occurred during the winter and spring, with significant variation in this parameter being observed throughout the year. An analysis of biotic and abiotic variables revealed complex relationships among them, with great importance of meteorological factors in plant responses. We conclude that under unstable climatic conditions, the relationship between O-3 flux and injury is weak, and the qualitative character of biomonitoring is further confirmed. (c) 2012 Elsevier Ltd. All rights reserved.