Immunohistochemical methods for semiquantitative analysis of collagen content in human peripheral nerve

Methods are described for the semiquantitative analysis of the connective tissue components of human peripheral nerve using light microscopy. General histological preservation was assessed using haematoxylin and eosin staining and the distribution of collagen type IV was investigated using immunohistochemistry. Several techniques were investigated to establish the one giving optimum structural preservation, immunobinding and greatest contrast for image analysis. Frozen sections were unsuitable for this tissue and paraffin wax sections were therefore used. Alcohol fixation was rejected due to poor preservation of the endoneurium, although immunobinding was excellent. Ice-cold formalin fixation for 24 h was found to be adequate for structural preservation and antibody binding, provided that a protease step was introduced. Trypsin was found to be superior to pepsin for exposing collagen type IV epitopes. Of the detection systems investigated indirect immunofluorescence was not suitable due to considerable autofluorescence of the nerve. The avidin-biotin method provided the greatest contrast, and was therefore the detection method of choice for image analysis. The optimum techniques for image analysis were then used on control human sural nerve to ascertain the best comparative method for collagen type IV in the perineurium. A method of semiquantitative analysis is described which takes into account the fact that there is a close linear relationship between collagen content per unit of perineurium and perineurial perimeter as fascicle size increases in peripheral nerve. This means that data from 2 different sample groups can easily be compared, provided that a range of fascicle sizes is analysed in each case.     

Review article: antibiotic-induced Clostridium difficile infection

The great majority of cases of Clostridium difficile infection are hospital-acquired, and the reported incidence in England and Wales has increased sixfold between 1990 and 1993, with at least 17 patients dying in a recent large nosocomial outbreak. C. difficile infection accounts for an average 3-week increased length of stay in hospital. Acquisition of a toxigenic strain of Clostridium difficile may be followed by asymptomatic carriage, diarrhoea, colitis or pseudomembranous colitis. Antibiotic treatment and older age are major risk factors for the development of symptomatic disease, but less well-defined differences in strain virulence and host susceptibility are also probably important. Accurate data on the relative risks of different antibiotics to induce symptomatic C. difficile infection are scarce, but third-generation cephalosporins are frequently implicated. New kits are becoming available for the laboratory diagnosis of C. difficile infection but many of these lack sensitivity. Oral metronidazole or vancomycin are the main treatment options but avoidance of further antibiotics should also be encouraged where possible. The role of environmental C. difficile spores, which are highly resistant to conventional disinfectants, needs to be defined. Proven strategies for the prevention of C. difficile infection are required, in particular protocols to ensure that cross-infection does not occur.     

Pre-operative nutritional support in children with end-stage liver disease accepted for liver transplantation: An approach to management

Pre-operative nutritional support was studied in 28 children with end-stage liver disease awaiting orthotopic liver transplantation. Nasogastric supplemental administration of a standard semi-elemental enteral nutritional formula was compared with a similar formula enriched with branched chain amino acids, and with a group receiving oral nutrition only. The duration of treatment in all groups was similar (mean 90 days). Energy intakes in the supplemented groups were 120-150% of recommended daily intakes (RDI), whereas ad libitum intakes in the oral group ranged 58-100% RDI. A significant improvement in mean Z-score for body weight (denoting catch-up) was noted only in those children who received nasogastric supplements enriched with branched-chain amino acids. The standard enterally-fed group maintained their body weight and Z-scores did not change significantly. In contrast, body weight Z-scores in those fed orally declined significantly. Nutritional supportive therapy of malnourished children with end-stage liver disease can minimize or improve nutritional status in children awaiting liver transplantation. The use of nutritional formulae rich in branched-chain amino acids may have nutritional advantages in children with chronic liver disease which require further study and evaluation.     

PRIMARY DEMYELINATION IN THE CENTRAL NERVOUS SYSTEM OF CATS

Primary demyelinating lesions have been observed in the central nervous system of 16 (approximately 7%) of a total of 235 clinically normal cats. The size of the lesions varied from small perivascular lesions in white matter to a large lesion occupying the diameter of an optic nerve. Intracytoplasmic inclusions consisting of tubular structures were common to all lesions examined by electron microscopy. The features of these feline lesions are briefly compared with those seen in multiple sclerosis.     

Embryotoxic Effects of Sodium Azide Infusions in the Syrian Hamster

Embryotoxic Effects of Sodium Azide Infusions in the SyrianHamster. SANA, T. R., FERM, V. H., SMITH, R. P., KRUSZYNA, R.,KRUSZYNA, H., AND WILCOX, D. E. (1990). Fundam. Appl. Toxicol.15, 754–759. Pairs of osmotic minipumps containing 400mg/ml (6.15 M) sodium azide in distilled water were subcutaneouslyimplanted in timed pregnancy Syrian golden hamsters. The totaldelivered dose was calculated as 6 X 10^–2 mmol kg^–1hr^–1 at the maximal pumping rate. Most dams exhibitedobvious signs of toxicity during the period of pump implantationwhich was Days 7 through 9 of gestation. After removal of thepumps the dams were euthanized on Day 13 of gestation, and theuteri were removed for counting of the number of living, malformed,and resorbed fetuses. This dose rate resulted in a significantlyincreased incidence of resorptions of embryos over that in acontrol group implanted with pumps delivering only distilledwater. The incidence of gross malformations exclusively in theform of encephaloceles was not different between control andazide-infused groups. The extent of nitrosylation of circulatinghemoglobin was followed with time and found to involve onlyabout 0.1% of the total blood pigment. Thus, this commerciallyimportant and widely distributed chemical with high acute toxicityis not considered to be teratogenic in hamsters, and it producesembryotoxicity only at dose rates that result in toxic signsin the dams.