OL linage evaluation.

<p>Immature OL (O1+ cells) and OL precursor (PDGFR-α+ cells) were evaluated by immunostaining in transverse ON sections. In the diabetic ON from eyes that received a sham treatment, a significant increase in O1(+) and PDGFR-α (+) area was observed, with the presence of disorganized and hypertrophic cells. In the right panel, the area occupied by glial cells (measured as total optical density (OD)) is shown. Ischemic conditioning significantly prevented these alterations and a clear decrease in O1- and PDGFR-α-immunoreactivity, with cells aligned parallel to axon bundles were found. Data are mean ± SEM (n = 6 nerves/group); * p<0.05, ** p<0.01 versus age-matched controls, a p<0.01 versus diabetic ON from sham-treated eyes, by Tukey’s test. Scale bar = 50 µm. D+P: diabetes+pulses.</p

Astrocyte analysis at the distal ON portion.

<p>Representative GFAP-immunostaining in transverse sections from a control (A) and diabetic ONs from an eye without (B) or with (C) ischemic conditioning are shown. In the ON from the diabetes+sham group, the area occupied by astrocytes (measured as total optical density (OD)) was significantly increased (D). Ischemic conditioning prevented these alterations. In panel E, a horizontal section through the optic chiasm (containing part of the ON and the optic tract) from a representative diabetic rat is shown. Note a clear decrease of GFAP-immunoreactivity in ON and the optic tract from the conditioned eye as compared with the contralateral (sham-treated) pathway. The inset is a schematic representation of the horizontal section. Data are mean ± SEM (n = 6 nerves/group); ** p<0.01 versus age-matched controls, a p<0.01 versus diabetic ON from sham-treated eyes, by Tukey’s test. Scale bar: A = 200 µm, E = 600 µm. D+P: diabetes+pulses; OT: optic tract.</p

Morphometric analysis of the myelinated distal ON portion.

<p>Light micrographs of semithin transverse sections at the distal ON portion from a control rat (A) and a 6-week diabetic rat without (B) or with ischemic conditioning (C) are shown in the upper panel. D-F: Size-frequency histograms showing the axon area distribution at the distal ON portion. In the diabetes+sham group, a significant reduction in the total axon number (G) and in the mean axon area (H), particularly affecting large axons (I), was found. The application of ischemic pulses prevented these alterations. No differences in the ON transversal area were found among groups. Data are mean ± SEM (n = 6 nerves/group); * p<0.05, ** p<0.01 versus age-matched controls, b p<0.05, a p<0.01 versus diabetic ON from sham-treated eyes, by Tukey’s test. Scale bar = 25 µm. D+P: diabetes+pulses.</p

Anterograde transport of CTB from the to the SC.

<p>Shown are four coronal sections (from rostral to caudal region) of the SC from a representative control animal (A) or a diabetic animal (B) that received weekly ischemic pulses in one eye and a sham treatment in the contralateral eye. C-E: dorsal view of retinotopic SC map reconstructions from a control (non-diabetic, C) and a diabetic animal submitted to a sham procedure in one eye and ischemia pulses in the contralateral eye are shown. A clear decrease in the CTB-staining pattern in the superficial layers of the SC was observed in the SC that received retinal terminals from a sham-treated eye (D), as compared with the contralateral SC (that received input from the conditioned eye, E). Shown are images representative of five animals per group. Scale bar = 1 mm.</p

Ultrastructural analysis of myelinated axons.

<p>The distal ON portion from control (A) and diabetic ON from eyes without (B) or with ischemic conditioning (C) were evaluated. Clear signs of axon degeneration and myelin disorganization (B), and a significant decrease in the mean axon area (D) were found in the diabetic distal ON. The application of ischemia pulses (C–D) prevented these alterations. Panel E: Calculation of the axon-myelin ratio. Scatter plot displays the ratio between the myelin thickness and the respective axon diameter. The slopes were: 0.22±0.03 in control (continuous line) rats, 0.33±0.06 in diabetic (dotted line) and 0.21±0.05 in diabetes+pulses group (dashed line). Data are mean ± SEM (n = 4 nerves/group); ** p<0.01 versus age-matched controls. b: p<0.05 versus diabetic ON from sham-treated eyes, by Tukey’s test. The levels of MBP were examined in the distal portion of the ON from a control (F), a diabetic (G) and a diabetic ON from an eye submitted to ischemia pulses (H). In diabetic ON from eyes with a sham procedure, a slight disorganization of MBP-immunostaining was observed as compared with control and diabetic+ischemia pulses groups was observed. Scale bar: C = 2 µm; H = 50 µm. D+P: diabetes+pulses.</p

Average body weight and blood glucose concentration assessed at different time points.

<p>The injection of STZ induced a significant decrease in body weight and an increase in blood glucose levels. Ischemia pulses in STZ-injected rats did not change these parameters. Data are mean ± SEM (n = 12 animals/group).</p>**<p>p<0.01 vs. aged-matched control animals, by Tukeýs test.</p

Retinal histology examination after 10 weeks of ocular hypertension.

<p>Upper panel: Representative photomicrographs of retinal sections stained with hematoxylin and eosin from a vehicle-injected eye, and a hypertensive eye without or with pulses of ischemia. Note the diminution of GCL cells in the eye injected with CS without ischemia pulses. The application of ischemia pulses preserved this parameter. The other retinal layers showed a normal appearance in all groups. Middle panel: Immunohistochemical detection of NeuN-positive neurons in the GCL from a vehicle-injected eye, a hypertensive eye without or with ischemia pulses. A strong NeuN-immunostaining (red) was confined to ganglion cells in the GCL. The number of NeuN positive ganglion cells was lower in hypertensive eyes without ischemia pulses than in vehicle- injected eyes, whereas the application of ischemia pulses in CS-injected eyes increased NeuN-immunostaining. A similar profile was observed for cell nuclei counterstained with DAPI (blue). Lower Panel: cell count in the GCL evaluated by H&E staining, NeuN immunostaining, and DAPI labeling. By all these methods, a significant decrease of the number of cells in the GCL was observed in CS- injected eyes without ischemia pulses as compared with vehicle-injected eyes (sham), whereas ischemia pulses significantly preserved this parameter in CS-injected eyes. Scale bar: Upper panel  =  50 µm; Middle panel  =  50 µm. Data are the mean ± SEM (n = 5 animals per group). * p<0.05, ** p<0.01 vehicle injected eyes without ischemia pulses (sham), a: p<0.05, b: p<0.01 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Electroretinographic preservation in hypertensive eyes induced by the application of brief ischemia pulses.

<p>ERGs were registered after 10 weeks of treatment with vehicle or CS. CS induced a significant decrease in ERG a- and b-wave amplitude, as compared with vehicle-injected eyes. In hypertensive eyes submitted to ischemia pulses, a significant reversion of these alterations was observed. The lower panel shows representative scotopic ERG traces from eyes injected with vehicle or CS without or with ischemia pulses. Data are the mean ± SEM (n = 10 animals per group); **p<0.01 versus vehicle injected eyes without ischemia pulses (sham); a: p<0.05, versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p

Flash VEPs in eyes injected with vehicle or CS without or with ischemia pulses.

<p>Animals were weekly injected with vehicle or CS for 10 weeks. Ischemia pulses were applied in one eye, while the contralateral eye was submitted to a sham procedure. Left panel shows average amplitudes of VEP N2-P2 component amplitude and right panel shows representative VEP traces. A significant reduction in flash VEP N2-P2 amplitude component was observed in eyes injected with CS for 10 weeks without ischemia pulses. The application of weekly ischemia pulses significantly abrogated the effect of ocular hypertension. No changes between vehicle injected eyes without or with ischemia pulses were observed. Data are mean ± SEM (n = 10 eyes/group), **p<0.01 versus vehicle injected eyes without ischemia pulses (sham), a: p<0.05 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p

ONH sections from a control or a CS-treated eye without or with ischemia pulses.

<p>(A) Healthy, intact control optic nerve. Note the homogeneity of the staining. In vehicle-injected eyes, individual axons were generally uniform in shape, rounded and packed together tightly to form the fibers of the healthy nerve. In CS-treated eye without ischemia pulses (B) a less stained area indicates a nerve alteration. Disease in individual axons was characterized by axonal distention and distortion that resulted in a departure from the circular morphology of normal axons. In contrast, a conserved structure of the ONH was observed in the CS-treated eye with ischemia pulses (C). Toluidine blue. (D) Number of axons in eyes injected with vehicle or CS without or with ischemia pulses. A significant decrease in the axon number was observed in CS- injected eyes without ischemia pulses as compared with vehicle-injected eyes (sham), whereas ischemia pulses significantly preserved this parameter. Scale bar: 10 µm. Data are mean ± SEM (n = 5 eyes/group) *p<0.05 vehicle injected eyes without ischemia pulses (sham), a: p<0.05 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p