Finding the fifth intercostal space for chest drain insertion: guidelines and ultrasound.

OBJECTIVES: International guidelines exist for chest drain insertion and recommend identifying the fifth intercostal space or above, around the midaxillary line. In a recent study, applying these guidelines in cadavers risked insertion in the 6th intercostal space or below in 80% of cases. However, there are limitations of cadaveric studies and this investigation uses ultrasound to determine the intercostal space identified when applying these guidelines in healthy adult volunteers. METHODS: On each side of the chest wall in 31 volunteers, the position for drain insertion was identified using the European Trauma Course method, Advanced Trauma Life Support (ATLS) method, British Thoracic Society's 'safe triangle' and the 'traditional' method of palpation. Ultrasound imaging was used to determine the relationship of the skin marks with the underlying intercostal spaces. RESULTS: Five methods were assessed on 60 sides. In contrast to the cadaveric study, 94% of skin marks lay over a safe intercostal space. However, the range of intercostal spaces found spanned the second to the seventh space. In 44% of women, the inferior boundary of the 'safe triangle' and the ATLS guidelines located the sixth intercostal space or below. CONCLUSIONS: Current guidelines often identify a safe site for chest drain insertion, although the same site is not reproducibly found. In addition, women appear to be at risk of subdiaphragmatic drain insertion when the nipple is used to identify the fifth intercostal space. Real-time ultrasonography can be used to confirm the intercostal space during this procedure, although a safe guideline is still needed for circumstances in which ultrasound is not possible.

Guidelines for chest drain insertion may not prevent damage to abdominal viscera.

OBJECTIVES: Guidelines to improve the ease and safety of chest drain insertion recommend using the fifth intercostal space, around the midaxillary line (MAL). This study aimed to assess whether compliance with published guidelines reliably ensured such placement and avoided the potentially serious complications of subdiaphragmatic insertion and peripheral nerve injury. METHODS: Three international guidelines were assessed by identifying the intercostal space for chest drain insertion using 16 cadavers (32 sides) at a point 1 cm anterior to MAL. The European Trauma Course method was compared with the British Thoracic Society's 'safe triangle' and the ATLS course technique. RESULTS: The level most commonly found was the sixth intercostal space (43%; 41 of 96 sides). Overall the sixth space or below was found in 83% of insertions (80 of 96 sides). In the fifth intercostal space, the long thoracic nerve ran posterior to the marker placed in all cases and the lateral cutaneous branches of intercostal nerves arose anteriorly to the marker in all but one case. CONCLUSIONS: The results suggest these guidelines may result in insertion of chest drains below the fifth intercostal space, potentially risking injury to subdiaphragmatic structures. Peripheral nerves of the lateral thoracic wall appear safe from incisions 1 cm anterior to MAL.

Increase in transglutaminase and its extracellular products in response to an inflammatory stimulus by lipopolysaccharide.

Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. epsilon (gamma-Glutamyl)lysine and [3H]putrescine-labelled gamma-glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its gamma-glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.

epsilon(gamma-Glutamyl)lysine crosslinks are concentrated in a non-collagenous microfibrillar fraction of cartilage.

Fractions of rib cartilage were obtained by homogenization and extracted with 4 M guanidinium chloride, and the washed residue was digested with purified collagenase. Differential centrifugation of the insoluble residue from this digestion yielded a non-collagenous fraction that earlier work had shown to contain microfibrils. This material contains a much higher concentration of epsilon(gamma-glutamyl)lysine than the ribs or any of the other cartilage fractions. This transglutaminase-derived crosslink may be a common component of extracellular matrix microfibrils.

Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis.

The dermis of the sea cucumber Cucumaria frondosa is a mutable collagenous tissue composed of collagen fibrils, microfibrils, proteoglycans, and other soluble and insoluble components. A major constituent of the dermis is a network of 10-14 nm microfibrils which surrounds and penetrates bundles of collagen fibrils. These microfibrils, which are morphologically very similar to the fibrillin microfibrils of vertebrates, were found to be insoluble in protein denaturants, including chaotropic agents and ionic and nonionic detergents, regardless of the reduction of disulfide bonds. The microfibrils are covalently crosslinked by epsilon-(gamma-glutamyl)lysine at a concentration of 3.725 nmol/mg dry weight of purified insoluble material. The network is susceptible to proteolysis by trypsin, chymotrypsin, and pancreatic elastase, but not by bacterial collagenase. Amino acid compositional analysis of the network shows it to be composed of 25% ASX and GLX residues. Comparison with the proteins in the SwissProt database gives the network protein a high probability of being related to the mammalian protein fibrillin. The network is glycosylated: approximately 7% of the mass is constituted by neutral and amino sugars. The intact microfibrillar network cross-reacted with a well-characterized antiserum to mammalian fibrillin.

Increase in epsilon(gamma-glutamyl)lysine crosslinks in atherosclerotic aortas.

Portions of aortas from normal and atherosclerotic rabbits and from human autopsy subjects were washed and separated into layers which were subjected to exhaustive proteolytic digestion. The digests were assayed for epsilon(gamma-glutamyl)lysine crosslinks by a two-stage high performance liquid chromatography (HPLC) procedure. Crosslink concentrations in intima-media from rabbits where more than 15% of the aorta lumen surface was lesioned are greater than in normal aortas or aortas with less than 15% of the surface lesioned. Higher crosslink concentrations occur in fibrolipid plaques from human aortas than in intima-media layers of equal thickness from non-lesioned areas of the same aortas. Much of the crosslink in fibrolipid plaques occurs in the proteins which float at d < 1.18 g/ml. Non-lesioned areas of intima-media from aortas with fatty streaks or plaques have higher crosslink concentrations than intima-media from aortas with no lesions. In normal and lesioned intimas thinner than 0.2 mm, the concentration of the crosslink is lower than in the subjacent media. These findings indicate that increased epsilon(gamma-glutamyl)lysine crosslinking occurs in the atherosclerotic aorta and is associated principally with smooth muscle cells. It is suggested that the crosslinked products may be involved in retention of lipoproteins and the increase in collagen production.

Effect of putrescine on tissue transglutaminase activity in wounds: decreased breaking strength and increased matrix fucoprotein solubility.

Topical application of putrescine, a transglutaminase inhibitor, for 3 days directly to rat skin wounds produced a significant average decrease of 48 percent in wound breaking strength in test animals from 8 pairs studied between day 5 and day 10 after wounding. No external or systemic toxic effects of putrescine were seen with localized topical application of 50 mM putrescine for 3 days in any of the test rats (n = 12), and no systemic toxicity was seen in rabbits (n = 4) after topical exposure to 50 mM putrescine for 3 weeks. Quantitation of tritiated fucose incorporation in rat wound explants from 10 pairs of rats revealed that a significant overall decrease in radiolabeled glycoprotein production of 23 percent occurred when putrescine was present; in addition, the fraction of tritiated glycoprotein which was soluble in buffer was significantly increased, while that in the buffer-insoluble fraction decreased. This study suggests that putrescine inhibits tissue transglutaminase-mediated cross-linking of fucoprotein in the extracellular wound matrix and supports a role for this process in the generation of incisional wound strength.

Increased epsilon(gamma-glutamyl)lysine crosslinking associated with increased protein synthesis in the inner layers of healing rat skin wounds.

Three days after biopsy wounds were made in the dorsal skin of rats the animals were killed and explants of wounded and unwounded skin were incubated for 7 h with either [3H]glutamine or [3H]lysine. Both incubated and fresh control explants were then dissected into three layers which were homogenized, extracted, digested and then assayed for epsilon (gamma-glutamyl)lysine. The concentration of epsilon(gamma-glutamyl)lysine was greater in all three wounded layers than in the corresponding unwounded layers. The concentration in the wounded middle (dermal) layer and in the unwounded middle layer of younger rats was greater than in the unwounded outer (keratinized) layer, which has previously been shown to contain epsilon(gamma-glutamyl)lysine crosslinks. The incorporation of label from both [3H]glutamine and [3H]lysine into buffer-insoluble protein of the middle and inner (muscle) layers was much greater in the wounded explants than in the unwounded. Except for [3H]lysine in the inner layer there was also an increase in the fraction of incorporated label which was converted to epsilon(gamma-glutamyl)lysine. These results show that increased protein biosynthesis during repair in the wounded explants is associated with increased formation of epsilon(gamma-glutamyl)lysine. In addition, they indicate that the crosslink is involved in some process in the middle and inner layers which is distinct from its known function in keratinization of the epidermis.

Increased transglutaminase in the aortas of cholesterol-fed rabbits: occurrence of buffer soluble and insoluble forms and an inhibitor.

Rabbits were fed for 10-12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl-sucrose-dithiothreitol-Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.

Lipoprotein binding of crosslinked type III collagen aminopropeptide and fractions of its antigen in blood.

When [125I] labelled bovine type III collagen aminopropeptide (PIIIP) is incubated with tissue transglutaminase (TGase) mixed with hyperlipemic rabbit plasma and subjected to ultracentrifugation the labelled fraction with density less than 1.2 g/ml is larger than when either lipoprotein or TGase is omitted. Chromatography of the fraction with density less than 1.2 g/ml shows the presence of peaks which are not present in the denser material. Since their elution positions indicate that they have higher molecular weights than PIIIP it is concluded that they consist of [125I]PIIIP which had been crosslinked by TGase and bound to lipoprotein. Low concentrations of similar low density, high molecular weight PIIIP antigens were found in normal human plasma and pooled sera from angiography subjects. In two out of seven infarct patients an unusually large fraction of the PIIIP antigen in the serum was found in a very high molecular weight peak containing low density material. It is speculated that this may arise from atherosclerotic lesions.

Transglutaminase-catalysed cross-linking: a potential mechanism for the interaction of fibrinogen, low density lipoprotein and arterial type III procollagen.

Bovine type III [3H]procollagen or its [125I]aminopropeptide were shown by chromatography under dissociating conditions to form very high molecular weight compounds with excess bovine fibrinogen after incubation with purified tissue transglutaminase, though none is formed with other major plasma proteins. Larger compounds of this type formed from fibrinogen or fibrin monomer can be separated by centrifugation and they are insoluble on washing with 1% SDS. Ultracentrifugation showed that a significant fraction of [3H]procollagen III forms a low density complex on incubation with transglutaminase plus excess IDL or LDL, but not HDL. SDS polyacrylamide gel electrophoresis showed that type III collagen [125I]aminopropeptide forms high molecular weight compounds after incubation with transglutaminase plus excess IDL or LDL but not with HDL. It is hypothesized that, in the presence of excessive concentrations of LDL and/or fibrinogen and of tissue transglutaminase, crosslinking reactions of the type demonstrated may interfere with normal injury-repair processes and stimulate the formation of atherosclerotic lesions in arteries.

Increased transglutaminase activity during skin wound healing in rats.

Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1-5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3-5 day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation.

Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.

Cartilage fucoproteins with sites for cross-linking by transglutaminase.

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of gamma-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15,000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.

Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen.

The aminopropeptide of type III collagen incorporates [3H]putrescine in the presence of liver transglutaminase, and the change in incorporation with concentration indicates one binding site on each of the Mr = 15,000 subunits of the peptide. At low concentrations the incorporation was comparable to that of dimethyl casein and much greater than actin or fibrinogen. Cleavage and Edman degradation of the aminopropeptide identified the major putrescine-binding site as glutamine in position 14. The surrounding amino acid sequence (Leu-Gly-Gln-Ser) shows homology with some synthetic peptide substrates of transglutaminase.

Components of increased labelling with putrescine and fucose during healing of skin wounds.

To study the glycoproteins and transglutaminase substrates involved in healing, wounds were made in the skin of anesthetized rats with a biopsy punch. Explants made 1-5 days later were incubated with [3H]-labelled putrescine, fucose or proline. As compared with unwounded skin there was an increased incorporation of label which was greatest at 3 days. Incubation for various times suggests that the incorporation of fucose and proline is dependent on protein synthesis, whereas putrescine is incorporated into preformed proteins. Putrescine and fucose label polypeptides with an Mr of about 45,000 before and 14,000 after reduction. These correspond in size with the aminopropeptide of type III collagen. Other labelled material of higher molecular weight is partly degraded to similar polypeptides on collagenase digestion. Much of the [3H]putrescine in the polypeptides is in the form of gamma-glutamyl putrescine. It is hypothesized that isopeptide linkage of the aminopropeptide III occurs in wound healing.

Increased glycosaminoglycan excretion after chymopapain injection of intervertebral discs.

The urinary excretion of glycosaminoglycans during the 24 hours after intradiscal injection of chymopapain was found to be greater than during the 24 hours before injection when comparison was made either on the basis of a complete 24-hour urine collection or by the use of a glycosaminoglycan/creatinine ratio. Each individual in a group of 14 patients showed this increase after injection. In contrast, no significant increase over the pre-injection levels was detected in serum glycosaminoglycans during the post-injection time periods for which blood samples were available.

[3H]Fucose incorporation by healing skin wounds and the effect of transglutaminase inhibitors.

Punch wounds (3 mm) were made in the skin of rats and the animals were killed after 1 or 3 days. Plugs (4 mm) of wounded and unwounded skin were incubated in vitro with [3H]fucose. The labelled plugs were homogenized and subjected to sequential extraction with buffered salt solutions, ethanol-ether, and 8 M urea - 50 mM dithiothreitol (DTT). Nondialysable counts in the extracts and insoluble residue were determined and the incorporation of label by wounded and unwounded skin plugs was compared. Wound plugs showed a greater total incorporation of [3H]fucose. In addition, a greater proportion of [3H]fucose was found in the urea-DTT extracts. The highest specific activity (disintegrations per minute [3H]fucose per milligram dry weight) was found in a finely dispersed precipitate, sedimenting at 10000 x g but not at 1000 x g. The transglutaminase inhibitors aminoacetonitrile and dansyl cadaverine were found to increase the extractability of a portion of the material which incorporated [3H]fucose without affecting the total incorporation. These results show that healing wounds have an increased biosynthetic capacity for an insoluble fucosylated glycoprotein fraction and they suggest that transglutaminase is necessary to make this fraction fully insoluble.