UV SERS at well ordered Pd sphere segment void (SSV) nanostructures.

Ultraviolet laser excited surface-enhanced Raman scattering was obtained for the first time at the well ordered palladium sphere segment void (SSV) nanostructures, using adenine as the probe molecule, and the UV-SERS enhancement is found to be correlated well with the plasmon absorption of Pd SSVs in the UV region.

Successful management of both early and delayed-onset neurological deficit following extent II thoracoabdominal aneurysm repair.

INTRODUCTION: Delayed-onset paraplegia is an uncommon but devastating complication of thoracoabdominal aneurysm repair. REPORT: We report the successful use of repeat cerebrospinal fluid drainage in the management of both immediate- and delayed-onset (21 days) paraplegia in the same patient undergoing open Type II thoracoabdominal aneurysm repair. DISCUSSION: Few studies have looked specifically at preventing delayed onset of symptoms. We advocate continued attention to blood pressure management and hydration for the duration of hospital stay and recommend repeat CSF drainage if symptoms occur.

A prospective audit of cost of sedation, analgesia and neuromuscular blockade in a large British ICU.

Bottom-up costs of sedative, analgesic and neuromuscular blocking drugs used in the intensive care unit have not been reported. We performed a prospective audit of the cost of these drugs using a bottom-up approach by prospectively recording the daily amount of drugs administered to patients over a 3-month period. Of 172 admissions, complete data were collected for 155 (92%). Propofol and alfentanil were the drugs most commonly used, being administered to 136 (88%) and 106 (68%) patients, respectively. The total cost was 14,070 pounds sterling, which was 81% of the pharmacy figure (based on central purchasing). Ninety-four per cent of the cost was for drugs administered to the 50% of patients who stayed in the intensive care unit longer than 48 h. The median (interquartile range [range]) cost per day was 9.30 pounds sterling (3.60-20.10 [0-61.20]). This represents less than 1% of reported total daily cost of intensive care per patient.

Advances in the Raman depth profiling of polymer laminates.

The use of Raman microspectroscopy to depth profile multi-layered polymer laminates is becoming increasingly popular. However, the results are generally degraded by aberrations introduced by the change in refractive index at the air/sample interface. Recent research has suggested that the use of an immersion oil and suitable objective can reduce this effect. This study evaluates this proposal by comparing depth profiling results on a multi-layer poly(styrene)/poly(methylmethacrylate) (PS/PMMA) laminate polymer from both dry metallurgical objectives and immersion objectives (used in combination with an oil of suitable refractive index). The immersion technique enabled successful depth profiling to the full working distance of the objective (100 microm), showing clear and distinct variations in 11 different layers within the laminate; a dry metallurgical objective used for comparison achieved poor resolution of only two layers. This is the first demonstration of depth profiling within a polymer laminate to this depth. The depth profiling results are compared to results obtained by sectioning the PS/PMMA sample after setting it in resin.

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts.

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.

The development of the external urethral sphincter in humans.

OBJECTIVE: To assess the hypothesis that during fetal development, the external urethral sphincter changes from a concentric sphincter of undifferentiated muscle fibres to a transient ring of striated muscle which regresses caudo-cranially in the posterior urethra during the first year of life, when the sphincter assumes its omega-shaped configuration. MATERIALS AND METHODS: The anatomy and development of the external urinary sphincter was assessed in human males and females during fetal life. Plastic-embedded sections (transverse, sagittal and frontal planes; 300-700 microm) of the pelvis of 31 females and 31 males (9 weeks of gestation to newborn) were stained with azure II/methylene blue/basic fuchsin and viewed at x 4-80. The sections of interest were taken from the bladder neck to the perineum. The sections of the membranous urethra were reconstructed three-dimensionally using a computer program. RESULTS: In both male and female an omega-shaped external sphincter was apparent in all specimens at > 10 weeks of gestation. In the early fetal period (ninth week), there was undifferentiated mesenchyme; in this period the mesenchyme was more dense in the anterior part and loose in the posterior part of the urethra. In females, there was a close connection between the urethra and the anterior wall of the vagina. CONCLUSION: The omega-shaped configuration of the external urethral sphincter was recognisable from 10 weeks of gestation in both sexes. There was no suggestion of a change from a cylindrical to an omega-shaped sphincter in the fetal period to birth. Also, a transient 'tail' posterior to the sphincter was not apparent. The rectovesical septum was well developed in neonates. There is no reason to assume that the development of the septum leads to an apoptosis of muscle cells in the posterior part of the external sphincter in males after birth. The anatomical development of the external sphincter does not explain transient outlet obstruction during fetal life. The function of the muscle may change during development because of neuronal maturation.

Relationship of glycosaminoglycan and matrix changes to vascular smooth muscle cell phenotype modulation in rabbit arteries after acute injury.

PURPOSE: The phenotype of vascular smooth muscle cells (SMCs) is altered in several arterial pathologies, including the neointima formed after acute arterial injury. This study examined the time course of this phenotypic change in relation to changes in the amount and distribution of matrix glycosaminoglycans. METHODS: The immunochemical staining of heparan sulphates (HS) and chondroitin sulphates (CS) in the extracellular matrix of the arterial wall was examined at early points after balloon catheter injury of the rabbit carotid artery. SMC phenotype was assessed by means of ultrastructural morphometry of the cytoplasmic volume fraction of myofilaments. The proportions of cell and matrix components in the media were analyzed with similar morphometric techniques. RESULTS: HS and CS were shown in close association with SMCs of the uninjured arterial media as well as being more widespread within the matrix. Within 6 hours after arterial injury, there was loss of the regular pericellular distribution of both HS and CS, which was associated with a significant expansion in the extracellular space. This preceded the change in ultrastructural phenotype of the SMCs. The glycosaminoglycan loss was most exaggerated at 4 days, after which time the HS and CS reappeared around the medial SMCs. SMCs of the recovering media were able to rapidly replace their glycosaminoglycans, whereas SMCs of the developing neointima failed to produce HS as readily as they produced CS. CONCLUSIONS: These studies indicate that changes in glycosaminoglycans of the extracellular matrix precede changes in SMC phenotype after acute arterial injury. In the recovering arterial media, SMCs replace their matrix glycosaminoglycans rapidly, whereas the newly established neointima fails to produce similar amounts of heparan sulphates.

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: association with vascular morphogenesis.

The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.

Matrix metalloproteinase can facilitate the heparanase-induced promotion of phenotype change in vascular smooth muscle cells.

Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMPI1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions.

Total urogenital sinus mobilization: expanded applications.

OBJECTIVE: To report further applications of total urogenital sinus mobilization, earlier described as an easier method to correct a cloaca. PATIENTS AND METHODS: Seven children (six girls and one boy, mean age 4 years, range 3 months to 10.5 years) underwent surgery and were followed for a mean of 1 year; their diagnoses included persistent cloaca and congenital adrenal hyperplasia (CAH) in two each, and a urogenital sinus (UGS), bladder exstrophy and penile agenesis in one each. The UGS is approached through a posterior sagittal incision and dissected circumferentially to the retropubic space, allowing the UGS to descend. It is then excised and separate openings of the vagina and urethra created. This technique is applicable to a UGS of

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo.

PURPOSE: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS: Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.

Electric cautery lowers the contamination threshold for infection of laparotomies.

BACKGROUND: Interplay between wound resistance factors and bacterial innoculum determines the risk of surgical infection. Since cautery causes more damage than the scalpel, our hypothesis is that lower numbers of bacteria are required to infect wounds made by electric cautery than to infect wounds made with a scalpel. METHODS: Abdominal fascia was incised in 375 rats by cold knife, cutting current, or coagulation current. Wounds were innoculated with increasing numbers of bacteria and histologically scored at 7 days for necrosis, inflammation, and abscess. RESULTS: Coagulation current causes more inflammation, necrosis, and abscesses than the scalpel at all bacterial levels. Electric cutting current is intermediate, causing more damage than the scalpel only after contamination reached 10(5). Above this threshold most wounds were infected in all groups. CONCLUSIONS: Electric coagulation current should be used only when the need for meticulous hemostasis outweighs the considerably increased risk of infection. Electric cutting current is less destructive but also less hemostatic; indications for its use are difficult to identify.

Acute neurologic decompression illness in pigs: lesions of the spinal cord and brain.

A detailed histopathologic description of central nervous system lesions from a porcine model of neurologic decompression illness is presented. Pigs were dived in a dry chamber to 200 feet of seawater for 24 min before the start of decompression. Of 120 pigs, 40 (33.3%) were functionally unaffected and 80 (66.6%) developed neurologic decompression illness; 16 died, 64 survived. Petechial hemorrhages were grossly visible in the spinal cord of 73% of the survivors, 63% of the fatalities, and 3% of the clinically unaffected pigs. The thoracic part of the cord was most commonly involved. Histologic cord lesions were found in 75 (63%) pigs: 83% of decompression illness survivors, 81% of the fatalities, and 23% of those clinically unaffected. Morphologically, hemorrhagic lesions were the most common (54%). Other common findings included spongiosis (48%), axonal swelling and loss (39%), and myelin degeneration (35%). White matter hemorrhages in the spinal cord were generally more numerous and extensive than those affecting the gray matter; however, gray matter hemorrhage was associated with increasing disease severity. Brain lesions were present in 23% of pigs and were most frequent in fatalities. Cerebellar and brain stem hemorrhages were the most common brain lesions; the molecular layer of the cerebellum appeared particularly susceptible. Pigs were chosen because of their cardiovascular and gas exchange similarities to humans. The clinical and histopathologic features of the pig model were compared with previous accounts in animals and humans; the model was judged analogous to severe human decompression illness. The finding of occult brain and cord lesions in clinically unaffected pigs is discussed. The model provides a useful tool for the study of dysbaric lesions of the central nervous system. Its noninvasive nature may facilitate the study of nervous system injury and repair processes.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide.

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 microM inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited approximately 80% by 5 microM LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited approximately 70% by 0.3 microM and 5 microM LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality.

alpha 1-Adrenoceptors on rabbit aortic smooth muscle cells in culture and in experimental intimal thickening.

1. This study has defined alpha 1-adrenoceptors and their reactivity in rabbit aorta, following removal of the endothelium and formation of a myointimal thickening, and also in smooth muscle cells (SMC) in cell culture which had undergone serial passaging and changes in phenotype. 2. [3H]-prazosin binding to SMC from control aorta, vessels 2 weeks after endothelial denudation and sub-cultured SMC (passage 3-6) was specific (displaceable with 10 mumol/L phentolamine), and of high affinity to a single class of sites (KD range: 71-114 pmol/L). The maximum binding density (Bmax) of alpha 1-adrenoceptors on SMC from the neointima (11,105 +/- 771 sites/cell) was not significantly different to that of control medial SMC (14,014 +/- 2472 sites/cell). However, SMC cultured to passage 6, showed a 2-fold increase in Bmax (30,227 +/- 4349 sites/cell). 3. The production of inositol phosphates (IP1, IP2 and IP3) by SMC following 10 mumol/L phenylephrine was assayed. Both freshly-dispersed aortic SMC and sub-cultured SMC were stimulated to produce increased inositol phosphates by the addition of phenylephrine which was completely inhibited by pre-incubation with 10 mumol/L phentolamine, suggesting that the stimulation was via alpha 1-adrenoceptors. 4. Maximal contractile responses of isolated thoracic and abdominal aortic rings to KCl (100 mmol/L), 5-HT and phenylephrine were unchanged two weeks after endothelial denudation. However, phenylephrine was significantly less potent (2.7-fold) in both areas of the aorta, while the potency of 5-HT was significantly enhanced (2.7-fold) after endothelial denudation only in the abdominal aorta. 5. The decreased sensitivity of the rabbit aorta to alpha 1-adrenoceptor agonists following endothelial denudation and the formation of a myointimal thickening is not due to changes in affinity or density of alpha 1-adrenoceptors. However multiple passaging of SMC in culture leads to an increase in alpha 1-adrenoceptor density. This change can be related to the altered cytodifferentiation of irreversible synthetic state SMC which are similar to those in atherosclerotic lesions.