Horseradish peroxidase histochemistry. A comparison between various methods used for identifying neurons labeled by retrograde axonal transport

Horseradish (HRP) histochemistry is widely used in neuroanatomical and neuropathological research. In this study the sensitivity of 6 methods commonly used for demonstration of HRP were compared mainly by observation on retrogradely labeled hypoglossal neurons found after injection of HRP into the tongue of adult mice. For this purpose groups of equivalent sections were obtained from the brainstem of each mouse. In one series, 3 diaminobenzidine techniques (Graham-Karnovsky; Malmgren-Olsson; Streit-Reubi), and Mesulam's tetramethyl benzidine procedure were compared. In a second series the groups of sections were incubated with DAB (Graham-Karnovsky; Malmgren-Olsson), p-phenylene diamine (Hanker) or TMB (Mesulam; Hardy-Heimer).All the new methods (Streit-Reubi; Malmgren-Olsson; Hanker, Mesulam; Hardy-Heimer) revealed many more labeled neurons per section than the original Graham-Karnovsky technique and Mesulam's TMB procedure was in this regard superior to all of the other tested methods. Particularly in sections from the tongue, disturbing precipitates were often obtained with the TMB methods and they are not as well suited as the new DAB procedures for observations on fine details at the light microscopical level. The new DAB methods (Streit-Reubi; Malmgren-Olsson), apart from their high sensitivity, can very well be used for electron microscopy also.Mesulam's TMB procedure should therefore be the method of choice in studies on retrogradely labeled neurons if highest possible sensitivity is needed. For studies on fine details in such neurons, i.e. when highest possible resolution is required, any one of the new DAB procedures should be chosen. Due to their lower tendency to form artefactual precipitates, they can be recommended for other HRP applications as well, such as vascular permeability studies, observations on vasogenic edema and perineurial permeability.     

Effects of intraluminal pressure on regional blood flow in obstructed and unobstructed small intestines in the rat

Small bowel circulation in the rat was studied with the microsphere technique before and after 10 minutes of elevated intraintestinal pressure. A distal small bowel loop consisting of 10 vascular arcades was used. The specimens were inflated with nitrogen gas. Regional blood flow in both obstructed and unobstructed small bowels was significantly diminished by an intraintestinal pressure of 40 mm Hg. A pressure of 20 mm Hg impaired circulation only in the obstructed small intestine, whereas the unobstructed intestine did not show a similar decrease. These findings suggest that moderate intraluminal pressures can imply a potential hazard to the vulnerable capillary bed of an obstructed intestine. Hence, it is suggested that intraoperative decompression should be performed to avoid this postoperative threat to bowel viability.     

External Fixation for Redislocated Colles' Fractures

Rereduction was carried out in 5% of all treated Colles' fractures and they were externally fixated with a one-bar Hoffmann apparatus. The results of 57 of the first 69 patients treated with a follow-up time of at least 1 year are presented. Using Frykman's criteria, 18 patients were judged as “excellent”, 25 as “good” and 14 patients were judged as “unsatisfactory”. The anatomical analysis showed no increase of deformity during the fixation. The final mean results were a 3.0 mm radial shortening and the articular plane of the radius at a right angle to the long axis. Among the first patients, five cases of pin-loosening were seen, but there were no adverse effects in the final result. There were no pin-tract infections. The results justify using the method as a routine when a redislocated Colles' fracture is rere-duced.     

Multiple Antigen Recognition by Individual Human Monoclonal Antibodies of Rheumatic Disease Patient Origin

We tested by Western blot several thousand antibody-secreting human cell lines immortalized by hybridoma fusion or Epstein-Barr virus transformation of peripheral blood lymphocytes from patients with systemic lupus erythematosus or mixed connective tissue disease. The blots utilized total human Jurkat cell extract as the antigen. More than 20% of these established lines produced antibodies which recognized multiple bands on the blots, frequently 50 bands or more. Experiments were performed to rule out the possibility of the bands being the result of mixed cell populations or nonspecific antibody-antigen binding. Cloning of these cell lines failed to alter the Western blot patterns produced, indicating that the populations were monoclonal. Antibody eluted from a number of the different single blot bands showed the same ability to reproduce the multiple band pattern, thus revealing the presence of only one antibody. Western blots performed in the presence of specific and nonspecific inhibitors demonstrated the ability of the antibody to specifically recognize and bind to certain antigens. Binding did not result from indiscriminate sticking of IgM molecules to the nitrocellulose paper. The patterns of multiple antigen recognition were not due to antigen degradation. Additionaly, enzyme linked immunosorbant assays revealed binding of the monoclonal antibodies to specific antigens, and the antibodies failed to recognize commonly crossreactive antigens such as DNA, histone, poly-L-lysine, glycophorin, and serum glycoproteins. The patterns of multiple antigen binding to a large number of polypeptides are therefore due to single antibodies, and the binding is specific.     

Regional adipose tissue cellularity in relation to metabolism in young and middle-aged women

Body composition and total number of fat cells were investigated in 31 randomly selected women 52 yr of age and in 13 young women (mean age 22 yr) whose body weights were within ±10% of the ideal weight. Two of the 52-yr-old women were obviously obese and excluded. Regional determinations of adipose tissue thickness, fat cell size, and number were also determined. Middleaged women had more body fat (BF) but a lower body cell mass (BCM) than the younger group. The increased BF in the middle-aged women was exclusively explained by larger fat cells, since the younger women had a significantly higher total number of fat cells. This increase was also found when differences in height, body weight, and body fat were matched out. Local fat cell number was also increased in the younger group. Local fat cell size was increased in all regions investigated in the middle-aged women, but the increase was particularly pronounced in the abdominal region. The highest degrees of correlation between fat cell sizes of different regions were found between the epigastric and hypogastric regions and between femoral and gluteal regions. Furthermore, the plasma insulin levels correlated with the fat cell sizes of the abdominal region but not with those of the femoral or gluteal regions.In conclusion, the data might indicate that the fat cells of the abdominal region are more sensitive to nutritional and/or hormonal factors than those of other regions. This may in turn indicate the existence of different fat cell populations.