Visualization of axonal degeneration after optic nerve lesion in rat by immunohistochemical labelling for myelin basic protein (MBP).

Immunohistochemical staining of myelin basic protein (MBP) was followed during axonal degeneration of rat retinal fibers within the first 3 weeks after injury. Wallerian degeneration of rat retinal fibers was elicited by unilateral transection or crush injury of optic nerve. MBP-labelled fibers in central retinal pathways and visual nuclei showed sequential changes of the myelin sheath, such as swelling at 1-2 days post lesion (dpi), granular staining at 4-8 dpi, and granular debris formation at 21 dpi. Consequently, immunostaining for MBP could be used to identify early stages of degenerating myelin and persisting myelin debris which is known to contain neurite growth inhibitors.

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) after deafferentation or ischemic infarction in rat visual system: features of reactive and damaged astrocytes.

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) is standard for visualization of reactive astrocytes in tissue sections, whereas various forms of astrocytic damage remain to be described in detail. In this study we tested differences in GFAP labeling in reactive astrocytes and in glial cells damaged by ischemia and edema. Studies were performed in the anatomically well defined visual system of rat. Basic staining patterns for GFAP were established in subcortical visual nuclei and visual cortex. In the first model, deafferentation of visual centers was performed by unilateral optic nerve lesion, and characteristic changes of GFAP labeling in reactive astrocytes were studied at 0.5, 1, 1.5, 2, 4, 8 and 21 days after lesion. Initial changes were seen in the deafferented superior colliculus at 1 day after deafferentation with a diffuse increase and stellate types of reactive cells formed at 2-8 days. In the second model, small ischemic infarcts were produced in the visual cortex of rats using the method of photochemically-induced thrombosis. GFAP labeling with a polyclonal antiserum was massively enhanced in the infarct at 4 hr. Characteristic morphological changes in damaged astrocytes were seen which were also identified in experiments with simulated global ischemia. In the surround of the infarct, swelling of astrocytes also caused increased labeling. At 3-4 days infarction typical reactive astrocytes surrounded the lesioned area. In conclusion, these immunohistochemical studies on GFAP in rat visual system allow for the following classifications. (a) Normal astrocytes vary in labeling at different anatomical localizations. (b) Reactive astrocytes show enhanced labeling and larger cell-size within an interval of 1-2 days after lesion. (c) Astrocytes damaged by ischemia reveal increased labeling of disintegrating cellular elements within hours after a lesion. (d) Swollen astrocytes undergo enhanced labeling in areas with vasogenic edema.

Carbonic acid buffer species measured in real time with an intracellular microelectrode array.

Carbonic acid buffer anions, HCO3- and CO3(2-), play an instrumental role in a host of vital processes in animal cells and tissues. Yet study of carbonic acid buffer species is hampered because no means are available to simultaneously monitor them at a cellular level in a rapid and dynamic fashion. An ion-selective cocktail, previously reported to measure changes in bicarbonate activity (alpha HCO3-), was instead shown to be principally selective for alpha CO3(2-). Ion-selective micropipettes (ISMs) based on this exchanger and consisting of a 3:1:6 (volume) mixture of tri-n-octylpropylammonium chloride, 1-octanol, and trifluoroacetyl-p-butylbenzene showed no significant interference from bicarbonate, chloride, phosphate, ascorbate, lactate, glutamate, acetate, or hydroxyl ions at concentrations expected in vivo. Intracellular and triple-barrel ISMs, consisting of a CO3(2-) sensitive, pH-sensitive, and reference barrel, were fabricated. Skeletal muscle cells (n = 17) were penetrated in vivo and showed values of 74 +/- 7 mV for membrane potential, 6.94 +/- 0.09 pHi, and 11 +/- 5 microM intracellular alpha CO3(2-), from which intracellular alpha HCO3- of 25 +/- 10 mM and CO2 tension of 120 +/- 55 Torr were calculated. All ion measurements reached a new steady state in 9 +/- 2 s after cell penetration. Thus measurements of intracellular alpha CO3(2-) and pH and associated levels of alpha HCO3(2-) and CO2 tension can be determined in biological tissues and cells with a spatial and temporal resolution previously unattainable.