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.     

Nitric oxide synthesis in the in vivo allograft response: a possible regulatory mechanism.

Activated macrophages are known to oxidatively metabolize L-arginine to nitric oxide and citrulline. We have recently shown that nitric oxide is a potent inhibitory molecule in the in vitro rat mixed-splenocyte culture, resulting in inhibition of proliferation and cytolytic T-cell induction. We undertook this study using the sponge matrix allograft model in the rat to determine whether nitric oxide plays a role in an in vivo allograft response. Our experiments showed that on day 6 after grafting, when cytolytic activity of allograft-infiltrating cells is first detected, allogeneic graft fluid contains higher levels of NO2-/NO3- (the stable endproducts of nitric oxide metabolism) than syngeneic graft fluid. Furthermore, evaluation of the supernatants of cultured graft-infiltrating cells revealed that allogeneic graft-infiltrating cells spontaneously produce higher amounts of nitric oxide than syngeneic graft-infiltrating cells. The nitric oxide production was inhibited in the presence of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of nitric oxide production. Most of the nitric oxide production was observed in the adherent macrophage fraction of the allograft-infiltrating cells. When allograft-infiltrating cells were cultured in the presence of NMA, donor-specific cytolytic activity was observed, whereas allograft-infiltrating cells cultured in the absence of NMA showed no cytolytic activity. These data show that nitric oxide production may play an important regulatory role in the allograft response.     

COMPARISON OF REFLEX MODIFICATION PROCEDURES AND AUDITORY BRAINSTEM RESPONSE IN HIGH-RISK NEONATES

This study compared the results of reflex modification (RM)--an objective technique for assessing brainstem sensorineural processing--with those of auditory brainstem response (ABR) for a group of high-risk infants at comparable postconceptional ages. For the RM procedure, an eyeblink-eliciting tap to the glabella was presented either alone or accompanied by a brief 90dB SPL tone. 37 high-risk infants were tested with both RM and ABR at a mean postconceptional age of 37.3 weeks. Seven had an increased brainstem conduction time ('failed ABR') and eight did not exhibit significant reflex augmentation ('failed RM'), seven of whom also failed the ABR. These data provide evidence that sensory stimuli which affect the neural mechanisms responsible for the organization of the startle response and auditory processing share essential neural components.     

Elemental composition and water content of myelinated axons and glial cells in rat central nervous system

The distribution of elements (e.g. Na, Cl, K) and water in CNS cells is unknown. Therefore, electron probe X-ray microanalysis (EPMA) was used to measure water content and concentrations (mmol/kg dry or wet weight) of Na, Mg, P, S, Cl, K and Ca in morphological compartments of myelinated axons and glial cells from rat optic nerve and cervical spinal cord white matter. Axons in both CNS regions exhibited similar water content ( 90%), and relatively high concentrations (wet and dry weight) of K with low Na and Ca levels. The K content of axons was related to diameter, i.e. small axons in spinal cord and optic nerve had significantly less (25–50%) K than larger diameter axons from the same CNS region. The elemental composition of spinal cord mitochondria was similar to corresponding axoplasm, whereas the water content (75%) of these organelles was substantially lower than that of axoplasm. In glial cell cytoplasm of both CNS areas, P and K (wet and dry weight) were the most abundant elements and water content was approximately 75%. CNS myelin had predominantly high P levels and the lowest water content (33–55%) of any compartment measured. The results of this study demonstrate that each morphological compartment of CNS axons and glia exhibits a characteristic elemental composition and water content which might be related to the structure and function of that neuronal region.