Table 1.
Antibodies.
Figure 1.
Representative images of Hsp27 and Hsp70 expression in rat brain after transient MCAO.
Hsp27 immunolabelling is restricted to blood vessels in sham brains (A, B), but is widely expressed by reactive astrocytes in ipsilateral grey and white matter regions at 1d following MCAO (C, D). Hsp70 immunolabelling is minimal in sham brains (E, F), but is exclusively associated with neurons in ipsilateral grey matter regions at 1d following MCAO (G). No unambiguous Hsp70 labelling is evident in white matter tracts (H). Note: grey matter, neocortex; white matter, corpus callosum; MCAO, middle cerebral artery occlusion. Scale bar = 50 µm.
Figure 2.
Temporal characterization of NF-L, Hsp27 and Hsp70 mRNAs in the retina during RGC injury.
Values (represented as mean ± SEM) are normalised for GAPDH and expressed relative to the control group. *P<0.05, **P<0.01 by one-way ANOVA followed by post-hoc Dunnett's Multiple Comparison Test (NMDA, ON crush, 2-VO) or by Student's unpaired t-test followed by modified Bonferroni correction (experimental glaucoma). NF-L, neurofilament light; 2VO, bilateral occlusion of the common carotid arteries.
Figure 3.
Characterization of Hsp27 and Hsp70 proteins in the retina and optic nerve during RGC injury.
A, B: Representative Hsp27, Hsp70 and actin immunoblots in control (cont) and injured (trt) samples. Single bands of the expected molecular weight are apparent. C-J: Levels of Hsp27 and Hsp70 proteins. Values (represented as mean ± SEM) are normalised for actin and expressed relative to controls. *P<0.05, **P<0.01 by Student's unpaired t-test.
Figure 4.
Representative images of Hsp27 and Hsp70 expression in control and 7d injured retinas.
In control retinas (A, B), Hsp27 is weakly associated with blood vessels and astrocytes (arrowhead), but negligible labelling is evident the other layers of the retina. In all four models of RGC injury (E–H), Hsp27 is upregulated in astrocytes (arrowhead). Following induction of ON crush, 2VO and experimental glaucoma, but not NMDA, Hsp27 is also associated with occassional cells in the GCL and their trailing processes in the IPL. In control retinas (C, D), Hsp70 is exclusively localised to photoreceptors in the ONL (arrowhead). No alteration to the pattern of Hsp70 is apparent in any of the four models of RGC injury (I–L). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer ONL, outer nuclear layer. Scale bar: A, C = 50 µm; B, D–L = 25 µm.
Figure 5.
Double labelling immunofluorescence of Hsp27 in the retina and optic chiasm 7d after ON crush.
In the retina, Hsp27 colocalises with the RGC marker χ-synuclein (arrows, A–C). In the optic chiasm, Hsp27 colocalises with the astrocytic marker vimentin (D–F), but not with the oligodendrocyte marker glutamine synthetase (arrows, G–I). GCL, ganglion cell layer; INL, inner nuclear layer; glut syn, glutamine synthetase. Scale bar = 25 µm.
Figure 6.
Representative images of np-NFH, Hsp27 and Hsp70 in the ON and OT after ON crush.
In the uninjured pathway (contralateral ON and corresponding ipsilateral OT), a pattern of uniform labelling of RGC axons by np-NFH is largely evident (A, C). In the injured pathway (ipsilateral ON and corresponding contralateral OT), numerous swollen, beaded np-NFH-labelled axons are apparent, indicative of Wallerian degeneration (B, D). In the uninjured ON and OT, Hsp27 is associated with a population of glial cells. One week after induction of ON crush, Hsp27 expression is markedly upregulated throughout the injured pathway. No expression of Hsp70 is readily apparent in the uninjured or injured ON or OT (I–L). ON, optic nerve; OT, optic tract; Np-NFH, non-phosphorylated neurofilament heavy chain. Scale bar = 50 µm.
Figure 7.
Representative images of parvalbumin, Hsp27 and Hsp70 expression in the LGN 7d after ON crush.
A–C: Parvalbumin labelling in the LGN. In the dorsal LGN (dLGN), parvalbumin immunoreactivity is fine and punctate (A, B). In the ventral LGN (vLGN), numerous labelled perikarya are present (A, C). Parvalbumin immunoreactivity is absent from the intergeniculate leaflet (IGL, A). D–G: Hsp27 labelling in the LGN. Hsp27 immunoreactivity is strikingly upregulated in the injured (contralateral) LGN (D, E) when compared to the uninjured (ipsilateral) side (F, G). H–K: Hsp70 labelling in the LGN. Hsp70 immunoreactivity is uniformly low throughout the injured (contralateral; H, I) and uninjured (ipsilateral; J, K) LGN. LGN, lateral geniculate nucleus. Scale bar: A, D, F, H, J = 250 µm; B, C, E, G, I, K = 50 µm.
Table 2.
Number of of Hsp27-positive cells in the lateral geniculate nucleus after ON crush.
Figure 8.
Representative images of calbindin, parvalbumin, Hsp27 and Hsp70 in the SC 7d after ON crush.
A–D: Calbindin and parvalbumin labelling in the SC. The characteristic trait of calbindin immunolabelling in the SC is a band of large cell bodies that are principally localised to the stratum opticum (A, B). The distinguishing feature of parvalbumin immunolabelling in the SC is a population of smaller sized cells that are chiefly localised to the stratum griseum superficiale. The stratum opticum contains parvalbumin-positive fibres, but only scattered cells (C, D). E–H: Hsp27 labelling in the SC. Hsp27 immunoreactivity is manifest in a tight band stretching across the injured (contralateral) SC (E, F), but is negligible in the uninjured (ipsilateral) SC (G, H). I–L: Hsp70 labelling in the SC. Hsp70 immunoreactivity is uniformly low throughout the injured (contralateral; I, J) and uninjured (ipsilateral; K, L) SC. SC, superior colliculus. Scale bar: A, C, E, G, I, K = 250 µm; B, D, F, H, J, L = 50 µm.
Table 3.
Number of of Hsp27-positive cells in the superior colliculus after ON crush.
Figure 9.
Spatial distribution and cellular localisation of Hsp27 in the SC 7d after ON crush.
In the contralateral (injured) SC, the majority of Hsp27 immunolabelling is localised to the stratum opticum, as delineated by comparison to calbindin (A–C), where the dashed line indicates the accepted boundary between the stratum griseum superficiale and stratum opticum. In contrast, both GFAP (D, E) and iba1 (F, G) are upregulated throughout the superficial layers of the SC on the contralateral (injured) side. Here, the dashed lines indicate the boundary of GFAP and iba1 upregulation. Double labelling immunofluorescence reveals that Hsp27 colocalises with the astrocytic marker GFAP (H), but not with the microglial marker iba1 (I), nor with the neuronal marker NeuN (J), nor with the proliferative marker PCNA (K). Scale bar: A–C = 100 µm; D–G = 250 µm; H–K = 50 µm.
Figure 10.
Representative images of Hsp27 and Hsp70 expression in the visual cortex 7d after ON crush.
Hsp27 and Hsp70 immunoreactivities are uniformly low throughout the injured (contralateral; A, C) and uninjured (ipsilateral; B, D) visual cortex. Scale bar = 100 µm.