Oxidative stress following traumatic brain injury in rats

BACKGROUND: Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. METHODS: We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. RESULTS: No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. CONCLUSIONS: We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.     

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.     

Poisoning and overdose

The differential diagnosis of all patients with altered mental status must include drug toxicity. In particular, intentional or unintentional overdosing and/or poisoning are common emergency department presenting complaints. A comprehensive approach to managing these patients must incorporate aggressive information gathering, a careful physical examination looking for toxic syndromes, and diagnostic testing. Proper decontamination is the key to effective management, as is the use of specific antidotes when indicated.     

T cell stimulation in vivo by lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8(+) T cells, and, at high doses, stimulates naive (CD44(lo)) cells as well as memory (CD44(hi)) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44(hi) subset of CD8(+) cells. In contrast with B cells, proliferative responses of CD44(hi) CD8(+) cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44(hi) CD8(+) cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (alpha, beta) interferon (IFN-I). Similar selective stimulation of CD44(hi) CD8(+) cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory-phenotype CD8(+) cells found in normal animals.     

Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.     

Adrenal incidentaloma: proposal for a correct treatment

BACKGROUND: The incidence of unsuspected adrenal masses (incidentalomas) based on CT-scan results to be higher than in the past. The aim for our study was to establish some guidelines for an appropriate management. METHODS: From 1986 to 1995, 61 patients with no history or clinical findings suggestive of adrenal mass or adrenal hyperfunction were discovered by radiologic examination to have an incidentaloma larger than 1 cm. In each patient basal biochemical evaluations were obtained to exclude the presence of adrenal cortical or medullary dysfunction. There were 28 men and 33 (54.1%) women, with a mean age of 53 years (range 16-74). 19 patients underwent CT-guided fine-needle biopsy to exclude metastatic tumors. Furthermore in 29 patients 75-Se-selenomethyl-norcholesterol was performed and 17 were studied by MRI. RESULTS: At CT-scan mean lesion diameter was 5.48 +/- 3.76 cm (range 2-23); 32 adrenal masses were right sided and 3 (4.9%) were bilateral. 17 patients had concordant scintigraphic imaging pattern, 6 bilateral uptake and 6 had discordant imaging. CT-guided FNAB showed malignancy in 9. Adrenalectomy was performed in 45 patients according to a score calculated by 4 parameters: age of the patients, size of the mass, scintigraphic pattern, MR imaging. Twenty-four had a score greater than 9 and in the remaining 21 patients in spite of a score lower than 10 adrenalectomy was performed based on: 1) increased size at CT scan follow-up (15 pts); 2) either suspected primitive malignant neoplasm at CT-guided FNAB or history of malignancy (6 pts); 3) elevated 24-hour dopamine (4 pts). In 12 (26.7%) patients a malignant tumor was found. There were not any statistically significant differences (p > 0.05) between the age of the patients with malignant neoplasms and those with benign masses, and between the size of the masses, which were 7.58 +/- 5.93 cm (range 2-23) and 5.03 +/- 2.81 cm (range 3-17) respectively. The difference in scores between the patients with malignant masses (12.17 +/- 2.95) and those with benign ones (9.09 +/- 1.33) was statistically significant (p < 0.01). CONCLUSIONS: Since adrenal incidentaloma have a malignancy rate higher than the other adrenal tumors, it is crucial to outlinesome criteria to sort out the patients at risk for whom adrenalectomy is to be warranted. Based on our results we believe that patients with a score > 9 should undergo adrenalectomy.     

Duplication of the cervical esophagus in children

True duplication of the cervical esophagus is rare. An 18-month-old girl presented with a large asymptomatic neck mass, which was found to be an intramural duplication of the esophagus. The evaluation and treatment of this patient is presented as well as a review of the six previously reported cases.     

The microstructure of ultra-high molecular weight polyethylene used in total joint replacements

The microstructure of ultra-high molecular weight polyethylene (UHMWPE) has been studied using a range of techniques. Both the unprocessed base powder and ram-extruded polymer have been examined using optical microscopy, scanning and transmission electron microscopy and small-angle light scattering. By examining the microstructure of samples compression moulded at a range of temperatures, techniques have been developed to assess the degree of consolidation of the processed polymer. The raw polymer is a powder with a particle size in the range 50-250 microm. These particles are themselves agglomerates of much finer particles typically 0.5-1 microm in size. It has been suggested that these sub-micron particles may be the origin of the sub-micron wear debris found in tissues around total joint replacements. However, examination of the ram-extruded polymer, from which implants are machined, shows a different structure from the powder, with no evidence of retention of the 0.5-1 microm structure seen in the powder in the processed material. It thus appears that the similarity in size between the sub-micron wear debris particles and the fine structure seen in the unprocessed UHMWPE resin is coincidental. Processed UHMWPE does show a 'memory' of the grain boundaries between powder particles and the degree of consolidation can be assessed by observing the distinctiveness of these boundaries.