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Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Gendron RL, Armstrong E, Paradis H, Haines L, Desjardins M, Short CE, Clow KA, Driedzic WR - Mol. Vis. (2011)

Bottom Line: The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known.We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space.This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, NL, A1B 3V6, Canada. rgendron@mun.ca

ABSTRACT

Purpose: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt.

Methods: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes.

Results: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry.

Conclusions: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

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Quantitation of staining by a mouse anti-Tbdn antibody (A) and by a mouse anti-ZO-1 antibody (B) on smelt eye tissues. Compared to fish maintained at 8–10 °C (warm), there was a significant increase (p<0.001; n=15 color intensity measurements per group) in Tbdn staining of rete-choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel A. There was no significant difference in the staining of Tbdn in the neural retina. Compared to fish maintained at 8–10 °C (warm), there was a significant decrease (p<0.001; n=15 color intensity measurements per group) in ZO-1 staining of rete- choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel B. There was no significant difference in the staining of ZO-1 in the cornea.
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f6: Quantitation of staining by a mouse anti-Tbdn antibody (A) and by a mouse anti-ZO-1 antibody (B) on smelt eye tissues. Compared to fish maintained at 8–10 °C (warm), there was a significant increase (p<0.001; n=15 color intensity measurements per group) in Tbdn staining of rete-choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel A. There was no significant difference in the staining of Tbdn in the neural retina. Compared to fish maintained at 8–10 °C (warm), there was a significant decrease (p<0.001; n=15 color intensity measurements per group) in ZO-1 staining of rete- choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel B. There was no significant difference in the staining of ZO-1 in the cornea.

Mentions: Tbdn and ZO-1 proteins show reciprocal regulation in cold-adapted smelt rete and choroidal blood vessels. Compared to warm fish maintained at 8–10 °C (A: warm specimen/Tbdn stain; C: warm specimen/ZO-1 stain), the endothelial linings of the choroidal (c) and rete (r) blood vessels of cold fish maintained at 0.5 °C show a higher expression level of Tbdn protein, but a lower level of ZO-1 protein in these regions (B: cold specimen/Tbdn stain; D: cold specimen/ZO-1 stain). The arrows indicate choroidal and rete blood vessel endothelia. E: Sections were also incubated with a control IgG and showed no staining of the blood vessels. Positive staining for Tbdn (OE5) and ZO-1 appears as bright red staining. The dark brown or black color in all panels is intrinsic due to pigmentation from the pigments cells of the choroidal vasculature and/or retinal tissue. The immunohistochemical results shown here are representative of three smelt in each of the warm and cold groups and are quantitated in Figure 6. The scale bar in the lower right corner of the figure indicates 100 μm.


Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Gendron RL, Armstrong E, Paradis H, Haines L, Desjardins M, Short CE, Clow KA, Driedzic WR - Mol. Vis. (2011)

Quantitation of staining by a mouse anti-Tbdn antibody (A) and by a mouse anti-ZO-1 antibody (B) on smelt eye tissues. Compared to fish maintained at 8–10 °C (warm), there was a significant increase (p<0.001; n=15 color intensity measurements per group) in Tbdn staining of rete-choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel A. There was no significant difference in the staining of Tbdn in the neural retina. Compared to fish maintained at 8–10 °C (warm), there was a significant decrease (p<0.001; n=15 color intensity measurements per group) in ZO-1 staining of rete- choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel B. There was no significant difference in the staining of ZO-1 in the cornea.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198499&req=5

f6: Quantitation of staining by a mouse anti-Tbdn antibody (A) and by a mouse anti-ZO-1 antibody (B) on smelt eye tissues. Compared to fish maintained at 8–10 °C (warm), there was a significant increase (p<0.001; n=15 color intensity measurements per group) in Tbdn staining of rete-choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel A. There was no significant difference in the staining of Tbdn in the neural retina. Compared to fish maintained at 8–10 °C (warm), there was a significant decrease (p<0.001; n=15 color intensity measurements per group) in ZO-1 staining of rete- choroidal blood vessels in the cold-adapted smelt (maintained at 0.5 °C [cold]), as shown in panel B. There was no significant difference in the staining of ZO-1 in the cornea.
Mentions: Tbdn and ZO-1 proteins show reciprocal regulation in cold-adapted smelt rete and choroidal blood vessels. Compared to warm fish maintained at 8–10 °C (A: warm specimen/Tbdn stain; C: warm specimen/ZO-1 stain), the endothelial linings of the choroidal (c) and rete (r) blood vessels of cold fish maintained at 0.5 °C show a higher expression level of Tbdn protein, but a lower level of ZO-1 protein in these regions (B: cold specimen/Tbdn stain; D: cold specimen/ZO-1 stain). The arrows indicate choroidal and rete blood vessel endothelia. E: Sections were also incubated with a control IgG and showed no staining of the blood vessels. Positive staining for Tbdn (OE5) and ZO-1 appears as bright red staining. The dark brown or black color in all panels is intrinsic due to pigmentation from the pigments cells of the choroidal vasculature and/or retinal tissue. The immunohistochemical results shown here are representative of three smelt in each of the warm and cold groups and are quantitated in Figure 6. The scale bar in the lower right corner of the figure indicates 100 μm.

Bottom Line: The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known.We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space.This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, NL, A1B 3V6, Canada. rgendron@mun.ca

ABSTRACT

Purpose: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt.

Methods: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes.

Results: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry.

Conclusions: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

Show MeSH
Related in: MedlinePlus