IDENTIFICATION OF PHAGOCYTIC GLIAL-CELLS AFTER LESION-INDUCED ANTEROGRADE DEGENERATION USING DOUBLE-FLUORESCENCE LABELING - COMBINATION OF AXONAL TRACING AND LECTIN OR IMMUNOSTAINING
I. Bechmann et R. Nitsch, IDENTIFICATION OF PHAGOCYTIC GLIAL-CELLS AFTER LESION-INDUCED ANTEROGRADE DEGENERATION USING DOUBLE-FLUORESCENCE LABELING - COMBINATION OF AXONAL TRACING AND LECTIN OR IMMUNOSTAINING, HISTOCHEM C, 107(5), 1997, pp. 391-397
Retrograde and anterograde degeneration have been reported to be suffi
cient stimuli to activate glial cells, which, in turn, are involved in
phagocytosis of degenerating material. Here we describe a double-fluo
rescence technique which allows for direct and simultaneous visualizat
ion of both labeled incorporated axonal debris and incorporating glial
cells in the course of anterograde degeneration. Stereotaxic applicat
ion of small crystals of biotinylated and tetramethylrhodamine (TRITC)
-conjugated dextran amine Mini Ruby into the medial entorhinal cortex
resulted in a stable rhodamine fluorescence confined to fibers and ter
minals in the middle molecular layer of the dentate gyrus, the stratum
lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Su
bsequent stereotaxic lesion of the entorhinal cortex induced transform
ation of rhodamine-fluorescent fibers and terminals into small granule
s. Incorporation of these granules by microglial cells [labeled by flu
orescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolec
tin B-4] or astrocytes (labeled by FITC-coupled glial fibrillary acidi
c protein antibodies) resulted in phagocytosis-dependent labeling of t
hese non-neuronal cells, which could be identified by double-fluoresce
nce microscopy. Electron microscopical analysis revealed that, followi
ng lesion, the tracer remained confined to entorhinal axons which were
found to be incorporated by glial cells. Our data show that TRITC- an
d biotin-conjugated dextran amines are versatile tracers leading to Ph
aseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced
phagocytosis of anterogradely degenerating axons by immunocytochemical
ly identified glial cells can be directly observed by this technique o
n the light and electron microscopical levels.