Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 17;21(6):2063.
doi: 10.3390/ijms21062063.

Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation

Mooud Amirkavei  1   2 , Marja Pitkänen  1 , Ossi Kaikkonen  1 , Kai Kaarniranta  3 , Helder André  2 , Ari Koskelainen  1
Affiliations

Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation

Mooud Amirkavei et al. Int J Mol Sci. .

Abstract

The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.

Keywords: age-related macular degeneration (AMD); heat shock protein 70 (HSP70); immunohistology; mouse; retinal pigment epithelium (RPE); transpupillary laser-induced heating.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Whole-tissue protein extracts from laser-treated eyecups and non-lasered controls (C) across different laser powers. (A) SDS-PAGE stain-free gel to assess protein hydrolysis. (B) Western blot analysis for nitrotyrosine and actin as a loading control. The protein oxidative status was determined by relative densitometry (RD) in percentage of controls.
Figure 2
Figure 2
(A) heat shock protein (HSP) 70 expression in retinal pigment epithelium (RPE) flatmount across different laser powers. Panels (a) represent actin staining by phalloidin (green); panels (b) HSP70 staining (red). (B) Semi-quantitative analysis of HSP70 fluorescence ratio of laser irradiation for different powers. * p < 0.05, determined by one-way analysis of variance (ANOVA). Scale bar: 100 µm.
Figure 3
Figure 3
(A) Immunostaining of RPE flatmount for different laser powers. Panels (a) illustrate Hoechst (Hst) staining for nuclei (blue); panels (b) actin staining by phalloidin (green). White circles highlight the laser-induced area after irradiation. (B) Semi-quantitative analysis of laser-induced area after irradiation over different powers. * p < 0.05, determined by one-way ANOVA. Scale bar: 100 µm.
Figure 4
Figure 4
Hematoxylin and eosin staining of paraffin-embedded retinal cross sections of laser irradiations at different powers. Scale bar: 25 µm.
Figure 5
Figure 5
(A) Tunel staining of RPE flatmount for laser irradiations at different powers. Panels (a) displays actin staining by phalloidin (green), panels (b) represent Tunel staining (magenta). (B) Semi-quantitative analysis for Tunel fluorescence ratio of laser irradiation for different powers. * p < 0.05, determined by one-way ANOVA. Scale bar: 100 µm.
Figure 6
Figure 6
Immunostaining of RPE flatmount irradiated with different laser powers. Panels (a) represent actin staining by phalloidin (green), panels (b) HSP70 staining (red), panels (c) Bestrophin1 (BEST1) staining (yellow). Scale bar: 100 µm.
Figure 7
Figure 7
Probability analysis to estimate HSP70 expression threshold and ED50 damage threshold for 10 min duration of the 1064 nm laser to the mouse eye. Dashed line represents ED50. Zero represents no damage and no HSP70 expression, while 1 represents HSP70 expression and damage. 52 mW, n = 5; 64 mW, n = 10; 70 mW, n = 9; 76 mW, n = 7; and 82 mW, n = 5.
Figure 8
Figure 8
(A) The retinal laser treatment system: the output of the fiber-coupled laser system (FCL, λ = 1064 nm) is delivered through a speckle reduction device (SRD). The beam emitted from the speckle reduction device fiber is projected onto an adjustable iris (AI) by a lens (L1). The light passing through the iris is projected as a top hat spot onto the fundus by a second lens (L2) and a direct fundus lens (FL). Light scattered back from the eye is reflected towards a camera system (CS) by a beam splitter (BS). (B) Fundus image of mouse eye. The solid line circle represents the laser spot and the dashed-line shows the optic nerve. (C) Laser-induced spot in RPE flatmount is shown with solid circle, as identified by actin staining (green). Scale bar: 500 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Mayer M.P., Bukau B. Hsp70 chaperones: Cellular functions and molecular mechanism. Cell. Mol. Life Sci. 2005;62:670–684. doi: 10.1007/s00018-004-4464-6. - DOI - PMC - PubMed
    1. Yenari M.A., Liu J., Zheng Z., Vexler Z.S., Lee J.E., Giffard R.G. Antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection. Ann. N. Y. Acad. Sci. 2005;1053:74–83. doi: 10.1196/annals.1344.007. - DOI - PubMed
    1. Lanneau D., Brunet M., Frisan E., Solary E., Fontenay M., Garrido C. Heat shock proteins: Essential proteins for apoptosis regulation: Apoptosis Review Series. J. Cell. Mol. Med. 2008;12:743–761. doi: 10.1111/j.1582-4934.2008.00273.x. - DOI - PMC - PubMed
    1. Richter K., Haslbeck M., Buchner J. The Heat Shock Response: Life on the Verge of Death. Mol. Cell. 2010;40:253–266. doi: 10.1016/j.molcel.2010.10.006. - DOI - PubMed
    1. Penke B., Bogár F., Crul T., Sántha M., Tóth M.E., Vígh L. Heat shock proteins and autophagy pathways in neuroprotection: From molecular bases to pharmacological interventions. Int. J. Mol. Sci. 2018;19:325. doi: 10.3390/ijms19010325. - DOI - PMC - PubMed

LinkOut - more resources