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TETRAZOLIUM STUDIES ON THE RETINA: II. SUBSTRATE DEPENDENT PATTERNS
1 Howe Laboratory of Ophthalmology, Harvard University Medical School, and Massachusetts Eye and Ear Infirmary, Boston 14, Massachusetts
Unsectioned retinas were incubated in a medium consisting of buffer, blue tetrazolium and one of the following substrates: lactate, lactate-DPN, diphosphopyridine nucleotide (DPN), DPNH, and succinate. Control retinas incubated without substrate showed only an occasional reduction of tetrazolium and this was so slight as to be inconsequential.
With lactate as the sole substrate no appreciable reduction of tetrazolium occurred at neutral pH but when DPN was also added a constant pattern of precipitation occurred that was qualitatively the same in all species. This pattern, associated with lactate-DPN substrate, was characterized by precipitation of tetrazolium in ganglion cells, Müller's fibers, and other glial cells. It was particularly obvious in human retinas and most particularly in pathologic retinas which were accompanied by gliosis. Also characteristic was the absence of activity in the ellipsoids of the rods and cones.
The lactate-DPN activity was somewhat enhanced by incubation anaerobically or with cyanide. It was completely inhibited by p-chloromercuribenzoate or by borate. It was not affected by prior blinding of the animal with iodoacetate, and section of the optic nerve affected it only by abolishing the activity in the ganglion cells.
When DPNH was used as a substrate the reduction of tetrazolium followed the same pattern. The inference is that lactate contributes the electron to DPNH and that tetrazolium reduction is effected through diaphorase. On the other hand lactate activity occurs at an alkaline pH (8.8-9.2) without the addition of DPN.
In contrast to the lactate-DPN system, succinate substrates showed predominant activity in the ellipsoids of the rods and cones and in inner portion of the retina unassociated with glial cells or ganglion cells. It was approximately equal in all species. The succinate activity was greatly decreased in pathologic retinas associated with gliosis. It was inhibited by cyanide and malonate but not by borate. Section of the optic nerve caused no appreciable alteration but prior blinding of the animal with iodoacetic acid showed a loss of activity from the rods and cones and a significant increase in activity in the inner retinal layers.
It is concluded that tetrazolium precipitation in the retina with lactate-DPN as a substrate is most marked in the glial cells and ganglion cells whereas that with succinate as a substrate is most marked in the nerve fibers and in the ellipsoids of the rods and cones.
Submitted on March 9, 1959
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