Abnormal centrifugal axons in streptozotocin-diabetic rat retinas

Abstract

Purpose: To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina.

Methods: Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein.

Results: Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas.

Conclusions: Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.

Figure 1

Specific hybridization of histidine decarboxylase mRNA in diabetic retinas. (a) Some retinas had labeled cells in the lower third of the INL; occasionally, a single cell in the GCL was labeled (arrow). (b) In other retinas, clusters of labeled cells were found in the INL (arrow). (c) Section incubated with the sense probe. No specific hybridization was found in any layer of the retina. (d) Labeled mast cells in the choroid and sclera. Scale bar, (c) 20 μm; (d) 100 μm.

Figure 2

(a) Tracing of a single histamine-IR axon. This axon supplied terminal branches to the IPL (red), GCL (green), and NFL (blue) that covered approximately 12 mm² of the retina. The primary axon emerged from the optic disc (OD) in the nerve fiber layer and ran to the peripheral retina. The axonal branches (arrows) ran through the GCL into the IPL where they terminated. The majority of terminals in the IPL were supplied by this branch (*). (b) A section of the wholemount rotated 90° as though it were viewed from the top to the bottom. This manipulation of the data set compresses all the branches into two dimensions. Typically, most terminals in the IPL arose from a single branch (*) that gradually ascends from the NFL to the outer half the IPL. Other branches (arrows) ran orthogonally to the border of the INL and IPL but did not contribute many terminal branches there. Scale bar, (a) 1.0 mm; (b) 30 μm.

Figure 3

Histamine-IR terminal branches in the IPL of the normal rat retina. (a) Distribution of varicosities at the end of a terminal branch. The varicosities were large and closely spaced. Branches often ended in a small swelling. (b) Contact with a retinal blood vessel. A labeled axon is seen passing below a large blood vessel (*). A single optical section (b, inset) shows that these terminals were in the same focal plane as the surface of the blood vessel. Scale bars, (b) 10 μm; (b, inset) 5 μm.

Figure 4

Specialized centrifugal axon terminals in the IPL. (a) Clusters of histamine-IR terminals were found in the IPL. They are formed by several overlapping varicose branches. No cell bodies or blood vessels were detected within these clusters. (b) Histamine-IR spines in the peripheral retina. These spines ended either with (arrowhead, inset) or without (arrow, inset) a small swelling. Spines were identified only on branches in the IPL. Scale bar, (a) 10 μm; (b, inset) 2 μm.

Figure 5

Histamine-IR axons in the normal optic nerve. This axon bifurcated and crossed the central retina artery (*). Widely spaced, small varicosities were present on most of these axons. Most branches ran toward the retina, but a few ran at oblique angles (arrow). Labeled axons were not restricted to any region of the optic nerve. Scale bar, 25 μm.

Figure 6

Axonal swellings in the IPL of diabetic retinas. (a) Spherical swelling on a histamine-IR branch. Inset: a single optical section through the center of this swelling showing no histamine immunore-activity in the center; only the outer surface was labeled (arrowhead). In some cases, other irregularly shaped structures (arrow) were found on branches within 10 μm of a spherical swelling. (b) Other large swellings on histamine-IR branches that were ovoid in shape also labeled only at the edges (arrowhead, inset). (c) Phosphorylated neurofilament-IR axon (arrow) with two large swellings in proximity to each other. Their centers were intensely immunoreactive. Scale bar, 10 μm.