.2021 Feb 4;11(1):3094.

doi: 10.1038/s41598-021-82672-9.

Temperature and species-dependent regulation of browning in retrobulbar fat

Fatemeh Rajaii¹, Dong Won Kim², Jianbo Pan³, Nicholas R Mahoney³, Charles G Eberhart^{3 4}, Jiang Qian³, Seth Blackshaw^{5 6 7 8 9}

Affiliations

¹ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. frajaii1@jhmi.edu.
² Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁴ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁵ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁶ Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁷ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁸ Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁹ Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.

PMID:33542375
PMCID: PMC7862600
DOI: 10.1038/s41598-021-82672-9

Temperature and species-dependent regulation of browning in retrobulbar fat

Fatemeh Rajaii et al. Sci Rep.2021.

.2021 Feb 4;11(1):3094.

doi: 10.1038/s41598-021-82672-9.

Authors

Fatemeh Rajaii¹, Dong Won Kim², Jianbo Pan³, Nicholas R Mahoney³, Charles G Eberhart^{3 4}, Jiang Qian³, Seth Blackshaw^{5 6 7 8 9}

Affiliations

¹ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. frajaii1@jhmi.edu.
² Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁴ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁵ Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁶ Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁷ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁸ Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.
⁹ Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. sblack@jhmi.edu.

PMID:33542375
PMCID: PMC7862600
DOI: 10.1038/s41598-021-82672-9

Abstract

Retrobulbar fat deposits surround the posterior retina and optic nerve head, but their function and origin are obscure. We report that mouse retrobulbar fat is a neural crest-derived tissue histologically and transcriptionally resembles interscapular brown fat. In contrast, human retrobulbar fat closely resembles white adipose tissue. Retrobulbar fat is also brown in other rodents, which are typically housed at temperatures below thermoneutrality, but is white in larger animals. We show that retrobulbar fat in mice housed at thermoneutral temperature show reduced expression of the brown fat marker Ucp1, and histological properties intermediate between white and brown fat. We conclude that retrobulbar fat can potentially serve as a site of active thermogenesis, that this capability is both temperature and species-dependent, and that this may facilitate regulation of intraocular temperature.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
Murine retrobulbar orbital fat derives fromWnt1-positive precursors. (A) Schematic illustrating adipose depots included in this study. (B) Immunohistochemistry of cryosection of orbital soft tissue from a Cre-negative,*Sun1-GFP* mouse reveals absent GFP staining of orbital fat. (C) Immunohistochemistry of cryosection of orbital soft tissue fromWnt1:Cre;Sun1-GFP mice reveals the presence of nuclear GFP signal in orbital fat. Scale bars = 50 μm.

**Figure 2**
Transcriptomic analysis of murine fat depots reveals significant similarity between orbital fat and interscapular brown fat. (A) Principal component analysis of transcriptome data from mouse brown fat (BF), epididymal fat (EF), inguinal fat (IF) and orbital fat (OF) demonstrates that BF and OF segregate more closely to each other than to EF and IF. (B) Sample-wise correlation of transcriptome data from BF, EF, IF and OF demonstrates that transcriptome of OF is more closely correlated with that of BF than with the transcriptomes of EF and IF. (C) Heat map of transcriptome data from mouse BF, EF, IF and OF demonstrates more significant shared gene expression between OF and BF. There is less significant overlap between OF and either EF or IF. Heatmap was created using Matlab 2018. (D) qPCR validates transcriptome data, demonstrating thatCxcl12 transcript variant 2 is enriched in orbital fat (OF) compared to brown fat (BF), epididymal fat (EF), and inguinal fat (IF). ** indicatesp < 0.0001 by one-way Anova. (E) qPCR demonstrates that brown fat markersUcp1,*Cidea*, andGdf5 are enriched in murine BF and OF compared to EF and IF. ** indicatesp < 0.0001 by one-way Anova. * indicatesp = 0.006 by one-way Anova.

**Figure 3**
Histologic analysis of orbital fat from different species reveals that brown fat is present in rodent species, but not the other mammals examined. (A) H&E stained section of CD1 mouse orbital fat demonstrates a mixture of cells with small lipid vacuoles interspersed with cells with large, single lipid vacuoles. (B) H&E stained section of hamster orbital fat reveals the presence of adipose cells containing multiple small lipid vacuoles. (C) H&E stained section of cat orbital adipose tissue reveals cells with large lipid vacuoles. (D) H&E stained section of dog orbital fat reveals cells with large lipid vacuoles. (E) H&E stained section of lamb orbital fat reveals cells with large lipid vacuoles. (F) H&E stained section of goat orbital adipose tissue reveals cells with large lipid vacuoles. Scale bars = 50 μm.

**Figure 4**
Mouse orbital fat resembles interscapular brown fat after thermoneutral adaptation.(A) H&E section of interscapular brown fat (BF) from CD1 mice housed at 30 °C for 4 weeks demonstrates increased proportion of adipocytes with large lipid droplets characteristic of white adipose tissue.(B) H&E section of orbital fat (OF) from CD1 mouse housed at 30 °C for 4 weeks demonstrates increased proportion of adipocytes with large lipid droplets characteristic of white adipose tissue.(C) H&E section of OF from C57BL/6 mouse housed at 30 °C for 4 weeks demonstrates increased proportion of adipocytes with large lipid droplets characteristic of white adipose tissue. (**D-I**) smFISH demonstrates decreasedUcp1 expression in interscapular brown fat of mice adapted to thermoneutral temperatures (30 °C) compared to those housed near room temperature (22 °C).E Higher magnification of D.F Higher magnification of E. (H) Higher magnification of G. (I) Higher magnification of H. (**J-O**) smFISH demonstrates decreasedUcp1 expression in orbital fat of mice adapted to thermoneutral temperatures (30 °C) compared to those housed near room temperature (22 °C). (K) Higher magnification of J. (L) Higher magnification of K. (N) Higher magnification of M.O Higher magnification of N. (**P-U**) SmfISH demonstrates decreasedGdf5 andCxcl12 expression in orbital fat of mice adapted to thermoneutral temperatures (30 °C) compared to those housed near room temperature (22 °C).Q Higher magnification of P. (R) Higher magnification of Q. (T) Higher magnification of S. (U) Higher magnification of T. Scale bars = 50 μm.

**Figure 5**
Human orbital adipose tissue is histologically similar to white fat, but expresses white, beige, and brown fat markers. (A) H&E stained section of human orbital fat reveals cells with large lipid vacuoles. Scale bar = 50 μm. (B) Comparison of transcriptome data from human orbital fat to publicly available data from various human samples.

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Temperature and species-dependent regulation of browning in retrobulbar fat

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Temperature and species-dependent regulation of browning in retrobulbar fat

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Conflict of interest statement

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