Barotraumatic orbital emphysema of rhinogenic origin in a breath-hold diver: a case report.

Orbital emphysema is a well-recognized complication of fractures involving the orbit. Commonly, it occurs when high pressure develops in nasal cavity as during nose blowing, coughing or Valsalva's maneuver and usually occurs in the subcutaneous tissues. We report the case of a young breath-hold diver who developed spontaneous, non compressive orbital emphysema during underwater fishing, with a maximal depth of 25-30 meters in the Sardinian sea. He was otherwise healthy, without previous cranio-facial trauma and nasosinusal diseases or surgery were not present in the history. When he was referred to our attention the patient presented right eyelid ptosis but diplopia and vision impairment were absent. Computer tomography scans showed subcutaneous air in the right upper eyelid and around the eyeball, particularly near the orbit's roof but optic nerve area, intraconal, was free of air. A dehiscence in lamina papyracea was evident. In our opinion, this has been the point of air entry into the orbit. A supportive therapy was advised and two weeks later the emphysema was recovered completely and the subject was symptoms free. The literature has been revised and to our knowledge no previous cases of barotraumatic orbital emphysema, in a breath-hold diver, are referred.

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats.

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified beta-actin mRNA. The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Immunological evidence for increased oxidative stress in diabetic rats.

The role of oxidative stress in aging and diabetes mellitus is currently under discussion. We previously showed age-dependent accumulations of fluorescent protein adducts with lipoperoxidative aldehydes, (malondialdehyde (MDA), and hydroxynonenal (HNE)) in rat skin collagen with diabetic BB rats exhibiting faster accumulation. Modified proteins have been shown to be immunogenic: antibody titres against rat serum albumin modified by MDA and HNE (MDA-RSA and HNE-RSA) or oxidized by reactive oxygen species were measured by ELISA as markers of oxidative damage in BB diabetic and non-diabetic rats. Each tested antibody titre was significantly higher in the diabetic than in the non-diabetic rats. A significant correlation existed between anti-MDA-RSA and anti-HNE-RSA antibody titers. Only the anti-HNE-RSA antibody titre increased significantly with age (p=0.052) in diabetic animals, while no titres increased significantly in non-diabetic animals. A major factor which correlated with the development of these antibodies was diabetes duration: this was significant (p=0.032) for anti-HNE-RSA antibody titre and slightly significant (p=0.05) for anti-MDA-RSA antibody titre. Thus, chronic hyperglycaemia is probably fundamental in the increase of oxidative stress. There is correlation between anti-aldehyde-RSA antibody titres and the corresponding aldehyde-related collagen-linked fluorescence: modified collagen may play a part in the observed immune response. Our data indicate a stronger immune response of diabetic rats against proteins modified by lipoperoxidative aldehydes and oxygen free radicals, and they support the hypothesis of increased oxidative damage in diabetes.

Good glycaemic control reduces oxidation and glycation end-products in collagen of diabetic rats.

Blood glucose control plays a prominent role in the aetiology of diabetic complications. Recent data support the hypothesis that non-enzymatic pathways (glycation and oxidation) are involved in the pathogenesis of tissue damage in diabetes mellitus. In this study the level of pentosidine, a marker of glycation, and the intensity of collagen-linked fluorescence glycation (370/440 and 335/385 nm) and oxidation-related (356/460 and 390/460 nm), have been examined in spontaneously diabetic rats with good and poor glycaemic control. Pentosidine increased dramatically in rats with poor control, and slightly in those with good control. At the end of the study, after 6 months of diabetes, pentosidine levels were 13 +/- 5 and 2.1 +/- 0.5 pmol/mg collagen, respectively (control rats: 1.1 +/- 0.1 pmol/mg collagen). A similar pattern was observed for both glycation or oxidation-related fluorescence. The group of rats with poor control always showed elevated average values when compared to rats with good control, with a relative increase of over 200%. The results emphasize the role of good glycaemic control in preventing the growth of glycation or oxidation end-products in collagen. On comparison between the general mean level of all glycated haemoglobin and the mean pentosidine level of the three groups, a very good exponential correlation was found (r = 0.993, p < 0.001). The fluorescence values presented a less strong relationship, but a correlation with glycaemic control was still present. If the post-translational modifications of proteins play a leading role in the pathogenesis of complications it is possible to conclude that strict glycaemic control, obtained by accurate insulin therapy can prevent them by inhibiting the non-enzymatic modification of proteins and delaying their accumulation in collagen. The therapeutic implications are obvious.

Plasma advanced glycosylation end-products in maintenance haemodialysis patients.

BACKGROUND: Pentosidine is a useful marker of advanced glycation end-products (AGE) which form cross-links between proteins and have been found elevated in plasma and tissues of uraemic and haemodialysed subjects. The origin and fate of these molecules are not clearly understood, but they might play a role in the cardiovascular complications of end stage renal failure. The aim of this study was to evaluate the effect of different types of substitutive therapy on the removal of pentosidine. METHODS: Pentosidine was measured by a two-step HPLC methodology. Its concentration was evaluated in plasma before and after dialysis session, in 24-h urine, and in dialysate of subjects treated with three types of chronic substitutive therapy: bicarbonate haemodialysis, acetate-free biofiltration, and haemofiltration. Pentosidine levels were compared among the three therapy modalities and correlated with clinical and biochemical parameters. RESULTS: Plasma pentosidine level was extremely high (23.7 +/- 2.0 pmol/mg protein) in the patients treated with the different dialysis modalities. The dialysis session had no significant effect on its plasma concentration, but haemofiltration seemed to be the most efficient method (300-2000 nmol of pentosidine removed per session versus 250-700 nmol per session with the two other approaches). An interesting correlation was found between pentosidine and blood urea nitrogen (r = 0.58, P < 0.01) and pentosidine with uric acid (r = 0.48, P < 0.05). CONCLUSIONS: These results suggest that none of the methodology showed a good removal of pentosidine, but among them haemofiltration has the best efficiency. The statistical relationships between pentosidine and urea and uric acid respectively might provide insight into the origin of pentosidine. The accumulation of reactive AGE in uraemic patients may be implicated in the organ and tissue damage observed in uraemia.

Evidence for the Maillard reaction in rat lung collagen and its relationship with solubility and age.

This study investigated age-related changes in collagen solubility and collagen-linked fluorescence, and their relationship with the Maillard reaction. As a result of the collagen purification of rat lung samples, we obtained two pools of collagen with different degrees of solubility. The relative distribution of collagen between these two fractions was time-dependent, and the proportion of the smaller and less soluble fraction increased with time (r = 0.73, P < 0.0001). In this fraction, the intensity of fluorescence at Exc 335 nm/Em 385 nm, and the total amount of pentosidine increased with age (r = 0.66, P < 0.002, and r = 0.69, P < 0.01, respectively). The mean values for fluorescence and pentosidine per milligram of collagen were, respectively, six and ten times greater in the less soluble fraction. In this fraction the pentosidine per milligram of collagen increased with age (r = 0.59, P < 0.03). Our results demonstrated the presence of pentosidine in rat lung collagen. Moreover, its accumulation in the less soluble fraction suggested a relationship between Maillard reaction products, physico-chemical changes in collagen solubility, and the ageing process in rat lungs.

Relationships between glycation and oxidation related fluorescences in rat collagen during aging. An in vivo and in vitro study.

BACKGROUND: Glycation and oxidation are spontaneous chemical modifications of body proteins. Usually these reactions have been studied separately by assessing their fluorescent final products. Glycation of protein and its related fluorescence increases during aging, whereas the level of the fluorescence related to protein adducts from lipoperoxidation side products is unknown. Moreover, no data on the fluorescence, at different wavelengths, connected to the two reactions in the same sample are available. Nevertheless recent in vitro studies support the possibility of an interaction between the two spontaneous reactions. EXPERIMENTAL DESIGN: In this study, we evaluated the modification of proteins due to glycation and to lipoperoxidation side products, by measuring their specific fluorescence levels in the collagen of 65 healthy Wistar rats during the aging process. The relationships among the fluorescence at different wavelengths were also reported. The fluorescence pattern of insoluble collagen was characterized by a tridimensional study after the incubation of insoluble collagen with probable precursors of protein glycation (ribose) and oxidation (malondialdehyde and hydroxynonenal); the maximum peaks of fluorescence were recognized and compared. RESULTS: An increase of all fluorescence intensities was observed in rat collagen during aging: the glycation-related ones (y370/440 = 28.3 e0.08x, r = 0.808, p < 0.01; y335/385 = 66.7 e0.06x, r = 0.798, p < 0.01) and the hydroxynonenal adduct-related (y356/460 = 44.3 e0.06x, r = 0.810, p < 0.01) were exponential, whereas that derived from MDA-adduct was almost linear (y390/460 = 17.7 + 4.1x, r = 0.661, p < 0.01). A different accumulation rate might explain this result. Significant correlation coefficients were found within the age-adjusted fluorescence intensities of both reactions, suggesting a close relationship between glycation and oxidation, besides a mutual influence due to the broad spectrum area. The in vitro study confirmed a good specificity of collagen fluorescence after incubation with a reducing sugar (ribose 0.5 M for 6 hours) for protein glycation, and after incubation with malondialdehyde (0.1 mM for 3 hours) for lipoperoxidation adducts; surprisingly enough hydroxynonenal (0.5 mM for 3 hours) significantly increased the fluorescence related to pentosidine-like products (335 nm excitation/385 nm emission) suggesting that this compound might be the precursor of products with a fluorescence similar to pentosidine or of pentosidine itself. CONCLUSIONS: The in vivo results of this study confirm that nonenzymatic reactions, glycation and oxidation, significantly modify collagen fluorescence during aging and can play a role in tissue damage related to age. The close relationships among fluorescences may be due to a reciprocal interconnection rather than to a parallel increase of both reactions during aging; this hypothesis is supported by the in vitro findings of this study.