A stereologic study of the plantar fat pad in young and aged rats

Abstract

Plantar fat pad (PFP) is a tissue structure that absorbs the initial impact of walking and running and ultimately bears body weight at standing. This study was designed to quantify the histomorphological changes of the PFP in aged rats. The most medial PFP was dissected from the hind feet of young rats (4 months old, n = 6) and aged rats (24 months old, n = 6). Histological structure and cellular senescence of PFP were analyzed stereologically and histomorphometrically. Immunohistochemistry of matrix metalloproteinase 9 (MMP9) was also performed on PFP tissue sections. Compared with young rats, the thickness of epidermis, dermis and septa of the PFP were significantly reduced in the aged rats. The total volume of adipose tissue in the PFP of aged rats was only about 65% of that in the young rats. The microvascular density and the number of fat pad units (FPU), a cluster of adipocytes enclosed by elastin septa, in the PFP were unchanged in the aged rats. In the aged rats, the number of adipocytes per FPU was reduced but the number of simple adipocyte clusters, without surrounding septa, was increased. The shift of the types of adipocyte clusters in the aged PFP was accompanied by degradation of elastin fibers and increased expression of MMP9. In conclusion, the PFP, particularly the elastic septa, degenerates significantly in aged rats and this may contribute to the pathology of PFP-related diseases.

Keywords: adipocytes; aging; fat pad; rats; stereology.

Fig. 1

Orthogonal intercept method for measuring epidermal thickness. A 100 × 100 μm grid was randomly overlaid on H&E-stained PFP sections. An orthogonal intercept was measured at the intersection of the grid and the epidermal–squamous junction.

Fig. 2

Histology of typical FPU and adipocyte clusters in the PFP. In a typical FPU, adipocyte clusters in the subcutaneous tissue are enclosed by dense fibrous tissue, which forms septa. In each of the FPUs, there are bundles of vascular and nerve fibers. An adipocyte cluster include more than three cells, but does not have a septal wall. A, adipocyte; MV, microvasculature; FPU, fat pad unit; C, adipocyte cluster; S, elastic septa.

Fig. 3

Stereology of PFP in young and aged rats. The thickness of epidermis, dermis and septa was reduced in the aged rats, as compared with that in young rats (*P < 0.05).

Fig. 4

Total volume of adipose tissue in the PFP of aged rats was decreased by approximately 34%, compared with young rats (A) The number of FPUs and the density of vascular bundles in the PFP were not significantly different between the aged and young rats (B and C; *P < 0.05).

Fig. 5

The number of adipocytes per FPU was significantly reduced in the aged PFP compared to the young ones (A). The number of adipocyte clusters (> three adipocytes), however, was significantly greater in the aged PFP than the young (B). The longest dimension of the adipocytes in the PFP was virtually the same between the aged and young rats (C). *P < 0.05; **P < 0.001.

Fig. 6

(A) Cellular senescence was detected primarily in the fibroblasts of the dermis. (B) Although aged rats showed a trend for higher senescent cell density in the PFP, compared with young rats, this is not statistically significant.

Fig. 7

Elastin staining. Elastin fibers had fewer branches and were more disorganized in the septal wall of the PFP (A) of aged rats, compared to the thicker and more organized fibers found in younger rats (B).

Fig. 8

Immunohistochemistry of MMP9. MMP9 was found primarily in the microvasculature in the FPUs (A) and at the dermal–epidermal junction (B) of aged rats. MMP9 was also found in the septa between adipocytes (arrowhead). There was no significant MMP9 staining at the corresponding locations in the PFP of young rats (C and D). A, adipocyte; DEJ, dermal–epidermal junction; MV, microvasculature.