Nothobranchius furzeri, the Turquoise Killifish: A Model of Age-Related Osteoporosis?

Abstract

Introduction: Osteoporosis is a frequent age-related disease, which affects millions of people worldwide. Despite significant progress in the treatment of the disease, a high number of patients still are underdiagnosed and undertreated. Therefore, novel animal models for the investigation of the disease are necessary. Nothobranchius furzeri is the shortest-lived vertebrate (with a lifespan of 3-7 months) that can be kept in captivity. Although it is an established model for aging research, studies on bone are lacking. The aim of this study was therefore to characterize N. furzeri as a potential model for age-related osteoporosis.

Materials and methods: Bone properties of aging N. furzeri were investigated in male and female fish of the Gona Re Zhou strain, which were between 8 and 20 weeks old. Micro-computed tomography (Scanco Medical µCT35) was performed to determine the bone properties of the vertebral bodies. Bone structure and remodeling were investigated by different histological staining techniques and histomorphometry. The chemical composition of fish vertebrae and intervertebral discs was analyzed by Raman microspectroscopy.

Results: Osteoblasts, mono- and multinucleated osteoclasts but no osteocytes could be observed in the vertebral area of N. furzeri. Histomorphometric evaluations revealed a significant decrease of the number of osteoblasts/bone perimeter and for osteoid volume/bone volume (BV) a trend toward a decrease in old male N. furzeri. Comparing male and female fish, males showed higher BV densities and cortical thickness. The relative values of the bone volume density of 20-week-old male N. furzeri were significantly lower than 10-week-old ones. The mineral to matrix ratio increased with age in male and female fish. In the intervertebral discs, proteoglycans in relation to the organic matrix were significantly lower in older female fish.

Conclusion: Our finding of a lack of osteocytes is in agreement with the fact that N. furzeri belongs to the evolutionarily advanced teleost fish. Furthermore, not only age-specific but also sex-specific differences were visible in the bone properties of N. furzeri, which can be taken into consideration for the study of gender aspects of age-related musculoskeletal diseases.

Keywords: Accelerated aging; Bone histomorphometry; Killifish; Micro-computed tomography; Nothobranchius furzeri; Osteoblast; Osteoclast; Osteoporosis.

Fig. 1

a 15-week-old N. furzeri (top: male, bottom: female). The female is smaller and displays less coloration in contrast to the male one. b Two male N. furzeri, 15 weeks old with vertebral deformities.

Fig. 2

a HE-stained vertebral body of a 10-week-old male N. furzeri, original magnification, ×200. b HE-stained vertebral body of a 10-week-old male N. furzeri, original magnification, ×400, intervertebral ligament (rectangle), sagittal section. c Von Kossa/Toluidine blue-stained vertebral bodies of 8-week-old male N. furzeri, original magnification, ×400, osteoid (black arrows), bone (brown/black), ligament (bright blue), osteoblasts (blue, white arrows). d Von Kossa/toluidine blue-stained vertebral bodies of an 8-week-old male N. furzeri, original magnification, ×200. Bone tissue (brown to black), trabeculae (arrows), and sagittal section. e TRAP and toluidine blue-stained vertebral body of 12-week-old female N. furzeri, original magnification, ×200, TRAP-positive osteoclasts are indicated by arrows. TRAP, tartrate-resistant acid phosphatase.

Fig. 3

a Toluidine blue-stained jaw of a 12-week-old female N. furzeri, original magnification, ×100. b Toluidine blue-stained jaw of a 12-week-old female N. furzeri, original magnification, ×200, teeth (black arrows). c Toluidine blue-stained ribs of a 12-week-old male N. furzeri, original magnification, ×100. d Toluidine blue-stained rib of a 12-week-old male N. furzeri, original magnification, ×400.

Fig. 4

Comparison of (a) Ct. Th (***p < 0.0001), (b) density of BV (***p = 0.0001), and (c) height of vertebrae in precaudal versus caudal vertebrae within individual male and female N. furzeri (male 20 weeks of age n = 3, female 20 weeks of age n = 3, male 15 weeks of age n = 3, female 15 weeks of age n = 3, male 10 weeks of age n = 3).

Fig. 5

a Ct. Th, (b) density of BV, and (c) height of vertebral bodies of 15- and 20-week-old male and female N. furzeri (male 15 weeks of age n = 3, male 20 weeks of age n = 3, female 15 weeks of age n = 3, and female 20 weeks of age n = 3). Age-related and sex-related differences were assessed with Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

Relative (a) Ct. Th, (b) density of bone volume, and (c) height of vertebral bodies (in relation to body weight) of 15- and 20-week-old male and female N. furzeri (male 15 weeks of age n = 3, male 20 weeks of age n = 3, female 15 weeks of age n = 3, female 20 weeks of age n = 3). Age-related and sex-related differences were assessed with Bonferroni post hoc test. **p < 0.01, ***p < 0.001.

Fig. 7

Density of BV in N. furzeri with and without vertebral deformities. a 15-week-old males: healthy and with vertebral deformities. b 15-week-old females: healthy and with vertebral deformities.

Fig. 8

a, b N.Ob/B.Pm and OV/BV in vertebrae of 8- (male n = 3, female n = 3), 12- (male n = 3, female n = 3), and 18-week (male n = 3, female n = 3)-old male and female N. furzeri. Age-related and sex-related differences were assessed with Bonferroni post hoc test. *p < 0.05.

Fig. 9

a Mineral to matrix ratio (v₂PO₄/amide III), (b) proteoglycan in relation to organic matrix (GAG/amide III), (c) mineral maturity/crystallinity, and (d) cartilages proteoglycans in relation to organic matrix (GAG/amide III) in vertebrae of 5- (female n = 3, male n = 3), 10- (female n = 3, male n = 3), and 20–week (female n = 3, male n = 3)-old male and female N. furzeri. Age-related and sex-related differences were assessed by Bonferroni post hoc test. **p < 0.01, *p < 0.05, (f) = significant differences in females, (m) = significant differences in males.