High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography.

Ultrasensitive bright field in situ hybridization assays using enzyme metallography (EnzMet) have been developed and validated, but little is known regarding the applicability of EnzMet for immunophenotypic detection of protein via IHC. Superior resolution via discrete metallographic deposits offers the potential for enhancing high-resolution immunophenotyping. Using high-complexity tissue microarrays (TMAs), 88 common solid tumors were evaluated by automated EnzMet (Nanoprobes and Ventana). Targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. Results were compared with conventional IHC diaminobenzidine (DAB) immunostaining. There was full concordance between the EnzMet and conventional IHC results. Furthermore, the EnzMet reaction products did not appreciably diffuse, were dense and sharply defined, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, allowing more tissue to be screened in an efficient manner. This preliminary study shows the utility of using enzyme metallography for high-resolution immunophenotyping in TMAs.     

Adding Bupivacaine to High-potassium Cardioplegia Improves Function and Reduces Cellular Damage of Rat Isolated Hearts after Prolonged, Cold Storage

Background: Bupivacaine retards myocardial acidosis during ischemia. The authors measured function of rat isolated hearts after prolonged storage to determine whether bupivacaine improves cardiac protection compared with standard cardioplegia alone.

Methods: After measuring cardiac function on a Langendorff apparatus, hearts were perfused with cardioplegia alone (controls), cardioplegia containing 500 [mu]m bupivacaine, or cardioplegia containing 2 mm lidocaine; were stored at 4[degrees]C for 12 h; and were then reperfused. Heart rate and left ventricular developed pressures were measured for 60 min. Maximum positive rate of change in ventricular pressure, oxygen consumption, and lactate dehydrogenase release were also measured.

Results: All bupivacaine-treated, four of five lidocaine-treated, and no control hearts beat throughout the 60-min recovery period. Mean values of heart rate, left ventricular developed pressure, maximum positive rate of change in ventricular pressure, rate-pressure product, and efficiency in bupivacaine-treated hearts exceeded those of the control group (P < 0.001 at 60 min for all). Mean values of the lidocaine group were intermediate. Oxygen consumption of the control group exceeded the other groups early in recovery, but not at later times. Lactate dehydrogenase release from the bupivacaine group was less than that from the control group (P < 0.001) but did not differ from baseline.     

The nuts and bolts of hemisection treatment: managing congenitally missing mandibular second premolars.

When you treat the congenital absence of mandibular second premolars by extracting the residual deciduous second molar and closing the space, you run the risk of flattening facial fullness, especially in patients with nonextraction features--diminished facial fullness, absence of crowding, and reduced tooth size. This challenge is heightened by the increased likelihood that these features will occur in patients who have agenesis. This article describes a new approach that has been shown to minimize such a loss of anchorage. The technique is called hemisection and involves buccolingual sectioning of the second deciduous molar, followed by removal of its distal half. As necessary, the mesial half is also removed, and space closure can be completed. The advantages, philosophy, and technique are discussed in detail.     

Minimally Invasive Coronary Artery Bypass Grafting: A New Method Using an Anterior Mediastinotomy

The benefit of internal mammary artery (IMA) grafting as a long-lasting intervention for coronary artery disease is well recognized. However, largely because they are less invasive, catheter based alternatives are frequently chosen, particularly to treat single or double vessel disease. To retain the advantages of the IMA graft, and to offset the invasiveness of conventional coronary artery bypass grafting, we developed a new minimally invasive method using an anterior mediastinotomy for treating left anterior descending (LAD) or right coronary artery disease, or both. Feasibility studies using 16 pigs and a human cadaver led to approval by the Institutional Review Board for use of this procedure to treat six patients (four men, two women; mean age, 63.8 ± 13.6 [SD] yrs) who granted informed consent. Pedicle dissection of the IMA, using video assisted thoracoscopy if necessary, was made through a 2-to 3-inch horizontal anterior mediastinotomy. The underlying LAD artery was grafted during femoral vessel cardiopulmonary bypass, with cooling to 30°C, induced ventricular fibrillation, and left ventricular venting if required. Transesophageal echocardiography performed after bypass showed that two patients maintained normal wall motion and four had improvement from the original impairment. One patient suffered a recurrence of angina 4 weeks after the procedure; recatheterization showed an acutely angled IMA, subsequently corrected by balloon angioplasty. The results of follow-up dobutamine echocardiographic stress tests were negative in all patients. With this minimally invasive approach, the procedure should provide the benefits of IMA grafting with shorter hospital stay, more rapid recovery, and less overall cost.     

Hypoplasia of the cerebellar vermis in neurogenetic syndromes

There are conflicting reports on the relationship between cerebellar vermal lobule hypoplasia and autism. Using quantitative magnetic resonance image analysis, we measured the cerebellar vermis in 125 normal individuals with a broad age range and 102 patients with a variety of neurogenetic abnormalities. We conclude that hypoplasia of cerebellar vermal lobules VI and VII is a nonspecific finding that even occurs in several conditions without autistic behavior. This suggests that it is not a specific neuroanatomical marker for autism, nor is cerebellar dys- genesis likely to be solely responsible for clinical autistic behaviors.     

Pediatric cerebellar hemorrhages

Cerebellar hemorrhage is a devastating condition with morbidity and mortality related not only to the etiology of the hemorrhage, but also to the timing of the intervention. Sixteen consecutive pediatric patients with acute cerebellar hemorrhages are presented: 6 had vascular abnormalities, 3 had tumors, and 2 had hemorrhages of unknown etiology. Thirteen of the 16 patients survived with only 1 of the 13 having persistent vegetative state as a neurologic outcome. Six of 8 patients presenting in a moribund condition had good outcomes, and 3 of 4 patients presenting with fixed and dilated pupils also had good outcomes. Thus, in contrast to adults, rapid evaluation by CT scanning, followed by the judicious use of ventricular drainage and prompt surgical treatment, have resulted in favorable outcomes in pediatric patients despite their poor clinical presentations. None of the neonates having cerebellar hemorrhages required surgical intervention; their courses could be followed clinically and with transfontanel ultrasound.Presented as a poster at the 14th Congress of the European Society for Paediatric Neurosurgery, Lyon, France, 21–23 September 1994, and the XXII Annual Meeting of the International Society for Pediatric Neurosurgery, Birmingham, UK, 25–28 September 1994     

Glycosaminoglycan-sulfate as plasma membrane component of pig spermatozoa.

The effect of specific glycosaminoglycan-hydrolyzing enzymes on the ruthenium red staining of pig spermatozoa was studied. Washed spermatozoa were incubated at 35 degrees C in buffer or with neuraminidase 0.5 units/ml, heparinase 0.2 mg/ml, or chondroitinase ABC 2.0 units/ml. After incubation sperm cells were washed, stained with ruthenium red and studied under the electron microscope. Anionic sites in the surface of untreated spermatozoa follow regularly the plasma membrane, but present are numerous processes constituting what has been defined as the glycocalyx. Neuraminidase did not affect the distribution of ruthenium red on the surface of the spermatozoa, but eliminated almost completely the processes of the glycocalyx. Heparinase caused loss of the ruthenium red-stained sites on the membrane surface of pig spermatozoa with less influence on the dense processes of the glycocalyx. A similar loss of ruthenium red-stained sites was observed with nitrous acid treatment. A striking effect of treatment with chondroitinase ABC was the production of a typical acrosome reaction.