Table 1 summarizes the mean weight and blood glucose levels after injection of vehicle or STZ. At 3, 6, and 15 weeks postinjection, significant weight loss and increased blood glucose levels were observed in STZ-treated rats compared with vehicle-injected rats.

The active anterograde transport of RGC projections to the SC at different time points after diabetes induction was analyzed using CTB. In control animals, CTB labeled the entire retinotopic projection to the stratum zonale and the stratum griseum superficiale, the most superficial layers of the SC (Figure 1). No alterations were found in CTB labeling 3 weeks after diabetes induction, whereas 6 weeks after diabetes onset, a clear reduction in CTB staining was observed in the central and lateral regions of the SC, extending in the rostral to caudal direction. In the lateral region, areas with virtually no CTB staining were also observed (Figure 1). A similar staining profile was observed after 15 weeks of diabetes (Figure 1).

Figure 2 shows the analysis of GCL cell density. The number of cells in the GCL significantly decreased after 15 (but not 3 or 6) weeks of diabetes induction (Figure 2F). At this time point, a significant increase in the number of TUNEL⁺ cells in the GCL was observed in diabetic rats (Figure 2G). At all the assessed time points, no other morphologic alterations or differences in total retina and retinal layer thicknesses were observed (see Supplemental Table S1 at http://ajp.amjpathol.org).

Histologic analysis of the SC at different time points after diabetes induction revealed no substantial alterations in its cytoarchitecture (Figure 3). No significant differences in the number of neurons were found in the SC of diabetic animals (Figure 3B), apoptotic cells were absent (data not shown), and no signs of reactive astrocytes were evident (Figure 3, C–G).

The ON was morphometrically evaluated at different regions (Figure 4). Six weeks after diabetes induction, no changes were observed in transverse sections obtained from the intraorbital myelinated ON portions (Figure 4, A and F). In contrast, clear alterations were evident at the distal region (ie, intracranial ON, near the optic chiasm) (Figure 4, B and G). Light microscopic morphometry of semithin sections of the distal ON revealed significant decreases in the total number of axons (Figure 4D) and the mean axon area (Figure 4E) compared with age-matched controls. The myelinated axon area frequency histograms revealed a clear shift to a smaller-sized area (Figure 4, C and H), with a significant decrease in large fiber number (Figure 4I). No significant morphometric differences were observed in the proximal myelinated portion (data not shown). For all the ON regions analyzed, no differences in the total area were found (data not shown). The proximal region (ie, the intraorbital ON, close to the ON head), named the glial lamina, contains unmyelinated axons surrounded by a meshwork of astrocytes (GFAP immunoreactive). Beyond this area, axons become myelinated toward the chiasm (Figure 5, A–D). Immunostaining of GFAP in transverse ON sections was examined at different portions (Figure 5). No signs of glial reactivity were observed in the intraorbital ON portion (at the glial lamina and the myelinated region) in diabetic animals (Figure 5, E, F, I, and J). In the distal portion, diabetic ON astrocytes showed a reactive morphology with an increased number of processes (Figure 5K). Measurement of the GFAP⁺ area revealed a significant increase in GFAP levels compared with controls (Figure 5, G and H). In all the sections analyzed, the number of nuclei remained unchanged (Figure 5L).

Figure 6 shows MBP immunostaining in horizontal sections obtained at the optic chiasm level. Slight disorganization of myelin, with some demyelinated zones, was observed in the diabetic distal ON and optic tract (Figure 6D). Double-labeling analysis revealed that zones of demyelination were occupied by GFAP⁺ cells (Figure 6F). To analyze oligodendrocyte (OL) lineage, immature OL (O1⁺ cells) and OL progenitors (PDGFR-α⁺ cells) were examined. In the diabetic ON, a marked increase in O1 immunoreactivity was found (Figure 7). O1⁺ cells showed hypertrophic somas and a high number of processes (Figure 7). Similarly, a marked increase in PDGFR-α immunolabeling, with a high number of hypertrophic cells, was observed in the distal ON (Figure 7).

At the ultrastructural level, alterations and loss of larger axons were observed in the distal ON from animals that were diabetic for 6 weeks. In these fibers, myelin was highly disorganized, and frequent lamellar membranous bodies were observed (Figure 8). Measurements of electron micrographs demonstrated a significant decrease in the number of axons and the mean axon area in the diabetic ON. The mean ± SEM axon area was 1.28 ± 0.02 μm² in control rats and 1.20 ± 0.04 μm² in diabetic rats. In contrast, analysis of the g-ratio (the ratio of the inner axonal diameter to the total outer diameter) revealed no clear differences between groups. The mean ± SEM g-ratio was 0.70 ± 0.04 in control rats and 0.72 ± 0.05 in diabetic rats.

No differences in microglial cell morphology assessed by Griffonia simplicifolia I staining in the distal ON were observed between control and diabetic animals. Moreover, no increase in CD11b+ or ED1+ cells (markers of reactive microglia) were observed in the diabetic ON (see Supplemental Figure S1 at http://ajp.amjpathol.org).

CTB is a reliable anterograde tracer that binds specifically to surface receptors of neurons and is actively taken up and transported by the axons.^24,25 CTB has been extensively used to analyze the communication between the retina and its synaptic targets. The present results demonstrate a deficit in CTB anterograde transport from the retina to the SC, its primary projection site, at an early stage of diabetes (ie, 6 weeks after injection of STZ). At this time point, morphologic studies indicated significant alterations in the distal portion of the ON but no substantial alterations in the retina or SC. After 15 weeks of diabetes, a significant decrease in cell number and an increase in TUNEL⁺ cell number in the GCL were observed, but no changes in the SC were evident.

The anterograde axonal transport system is responsible for transporting proteins associated with axonal structure and synaptic transmitter function to the axon and its terminals. In the opposite direction is the retrograde axonal transport system, which carries neurotrophic factors that influence steady-state activities in the cell body. It was previously reported as a progressive deficit in the retrograde axonal transport, mainly mediated by large axons, that is evident 1 month after STZ injection and does not involve RGC loss.¹⁸ Taken together, these results support that early diabetes provokes a “misconnection” between the retina and the SC, which precedes RGC death.

To identify the locus for the transport disruption along the visual pathway, representative intraorbital (unmyelinated and myelinated) and intracranial ON sections were analyzed. In diabetic rats, the distal (intracranial) portion of the ON degenerated at a time point in which the proximal portions remained unchanged. In animals that had been diabetic for 6 weeks, a large increase in astrocyte reactivity (as shown by GFAP immunoreactivity) occurred in the distal (but not the intraorbital) portion of the ON, which coincided with significant axon loss, making larger axons significantly more vulnerable than smaller axons. In agreement, Zhang et al²⁶ described a higher susceptibility of ON large axons in STZ-treated rats. Furthermore, a preferential loss of ON large axons was demonstrated in experimental models of glaucoma.^27,28 Increases in astrocyte reactivity have been observed in many neurologic disorders and acute nervous system injuries and involve increases in gene expression and redistribution of intermediate filaments, and occasionally increases in proliferation (reviewed by Escartin and Bonvento²⁹). Although microglial cell activation was described in the diabetic retina at different stages of the disease,^30,31 no changes in the microglia morphologic appearance (as shown by Griffonia simplicifolia I staining) or signs of microglial reactivity were observed in the distal portion of the ON, at least at 6 weeks of diabetes onset.

Structural analyses of peripheral nerves in humans and animals with diabetes have shown that the main lesions involve axonal atrophy and axoglial dysfunction.³² Morphologic studies of ON in autopsy materials demonstrated gliosis, demyelination, focal infarction, and vascular lesions, especially significant in elderly patients with a long history of diabetes.³³ Ino-ue et al³⁴ showed a reduction in mean axon size and myelinated fiber atrophy at 3 months of diabetes induction in rats, whereas in the present study, changes in the axoglial structure of the distal ON were observed at an earlier stage of the disease. Notably, these changes were demonstrated by the appearance of a marked decrease in corneal sensitivity, possibly associated with peripheral neuropathy in rats with 5 weeks of diabetes.³⁵

At the ON head, RGC axons pass unmyelinated into the nerve. MBP is an important constituent of peripheral and central nervous system myelin sheaths and plays a major role in myelin membrane formation and structure.^36,37 Thus, MBP is a reliable index of OL differentiation and myelin formation. In that sense, myelin disarrangement shown by alterations and disorganization of MBP immunoreactivity and profound myelin alterations at the ultrastructural level were observed in the distal portion of the diabetic ON. In myelin abnormalities and demyelination in the central nervous system, OL remyelination is expected.³⁸ After demyelination, a remyelination process occurs through activation of OL progenitor cells in the lesion. New OLs then are formed, and insulating function recovers.³⁹ However, the remyelination process progressively becomes limited. OL progenitor cells in the lesion, for unknown reasons, gradually lose the ability to form myelinating OLs.⁴⁰ In animals with 6 weeks of diabetes, progenitor (PDGFR-α⁺) and immature (O1⁺) OLs showed an altered morphologic appearance, with hypertrophic somas and multiple processes. The role of these cells in progression of the disease needs to be further examined. It is possible that these reactive cells contribute to a remyelination state but that the axon loss could provoke a fail to complete the differentiation to mature OLs, resulting in aberrant cells that accumulate adjacent to the lesion sites, similar to that seen in chronic models of white matter injury.⁴¹ In an experimental model of glaucoma, it was described that OL alteration is not only an early event but also a central mechanism through which the axons are damaged, leading to the subsequent death of RGCs.⁴² Although DR and glaucoma differ in several aspects (such as causes, risk factors, and retinal cell types involved, among many others), based on the present results, it is tempting to speculate that a similar sequence of events described in glaucoma could occur in experimental diabetes. However, whether changes in GFAP, O1, and PDGFR-α levels are cause or consequence of the decrease in axon number observed in the diabetic ON cannot be determined, and, in that sense, a more detailed temporal course of these alterations will be examined in the near future. The present results suggest a progression of RGC degeneration in experimental diabetes, where axon dysfunction or degeneration preceded neuronal loss. In fact, neuronal compartments often crucially depend on one another for survival, and molecular defects in one compartment can trigger cellular degeneration in distant parts of the neuron.⁴³ Evidence of axon degeneration occurring separate from and before somal degeneration has been observed in many neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, and glaucoma.^23,44–46 However, the fact that axoglial alterations at the distal portion of the ON seems to be the first structural alteration in the diabetic visual pathway does not imply that the initial neuronal event is axonal. Instead, a putative sublethal impairment of RGCs could render them unable to support their axons, resulting in progressive dying back of the axon toward the cell body.⁴³ Alternatively, RGC death in experimental diabetes can occur independently from distal axon degeneration. On the other hand, there is a possibility that diabetes causes stress to cells that results in lower efficiency in their mitochondria functioning. Neurons, particularly RGCs (documented by large numbers of their axons in the globe), are more dependent on their mitochondria than are dividing cells. If this were the case, RGC axonal transport (which requires ATP) would be affected before the occurrence of RGC death signs, and, in turn, the interaction between ON axons and glia could be altered. In this scenario, ON glial complications could be secondary rather than being a primary cause for ON alterations. Further studies will clarify whether either compartment dies secondarily to the other or whether both are directly affected by diabetes.

Alterations in the distal ON portion may be involved in the deficit of anterograde transport to the SC and in decreased propagation of nerve impulse and visual evoked potentials,¹⁶ contributing to visual disorders observed in early diabetes. In fact, abnormal color vision and contrast sensitivity have been observed before vasculopathy,⁴⁷ indicating the early presence of neuropathy. In agreement, a growing body of evidence suggests that neural and glial complications may precede vascular changes in DR.^3,48,49 In summary, these data indicate, for the first time, that in experimental diabetes, axonal alterations at the distal portion of the ON precede RGC loss. Therefore, early vision loss in diabetes could be abated by interventions that preserve distal axonal function before loss of the neuronal substrate.

d30e443Supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica; the University of Buenos Aires; CONICET, Argentina; and Sigma Xi, The Scientific Research Society.

d30e458Supplemental material for this article can be found at http://ajp.amjpathol.org or at doi: 10.1016/j.ajpath.2011.09.018.

Supplemental Figure S1

Griffonia simplicifolia I–positive cells in normal and diabetic distal ONs. A: In transverse normal sections, weak-stained Griffonia simplicifolia I–positive cells were observed. Note the fine and ramified processes and the rod-shaped soma characteristic from resting microglia (a detail is shown in the inset). B: In diabetic animals, no clear differences were found in the morphologic appearance of microglial cells present in the distal portion of the ON. Shown are images representative of five animals per group. Scale bars: 50 μm (B); 25 μm (inset).

We thank Juana Pasquini for helpful discussion of the manuscript.