Figure 3.

Ultrastructural colocalization of Nr1 on P2 axons correlates with injury. (A) Immunolocalization of Nr1 protein in P2 rat optic nerve (RON) (all ultra-micrographs are cross-sections). Left: Axons surround a glial process (‘gp'), boxed area shown at high magnification below. Typical axon features are microtubules, vesicular-tubular inclusions, diameter <0.4 μm, and the absence of glial ensheathment. Three gold-particles (arrows) are localized to the axolemma of two neighboring axons. Right: Axons proximal and distal to a glial process have gold particles aligned with the axolemma. (B) Blind quantification of gold-particle density in axons, astrocytes, extracellular space (ECS), peri-neural collagen, and glial nuclei. The data show significantly higher staining density in axons versus all other groups (**P<0.01; n=7 nerve sections). (C) Nr1 immunolocalization after 90 minutes oxygen–glucose deprivation (OGD)/60 minutes recovery. Axon injury is variable with some axons showing axoplasmic swelling and/or flocculent debris, swollen mitochondria and microtubule loss. Gold particles are localized to the axolemma of badly damaged axon, boxed area shown at high magnification to the right. (D) Similar images showing two gold particles localized to the axolemma of a damaged axon. (E) Blinded viability scoring of control, showing little pathology and no difference between Nr1(+) axons and Nr1(−) axons (n=400 axons analyzed from 8 sections). (F) A similar analysis after OGD/recovery. Note that viability scores are shifted to lower values for both Nr1(+) axons and Nr1(−) axons, but this effect is greatest in Nr1(+) axons (n=398 axons analyzed).