Alterations in the vimentin cytoskeleton in response to single impact load in an in vitro model of cartilage damage in the rat

Abstract

Background: Animal models have provided much information on molecular and cellular changes in joint disease, particularly OA. However there are limitations to in vivo work and single tissue in vitro studies can provide more specific information on individual events. The rat is a commonly used laboratory species but at the current time only in vivo models of rat OA are available to study. The purpose of this study was to investigate the damage that single impact load (SIL) of 0.16J causes in a rat cartilage in vitro model and assess whether this load alters the arrangement of vimentin.

Methods: Rat cartilage was single impact loaded (200 g from 8 cm) and cultured for up to 48 hours (n = 72 joints). Histological changes were measured using a semi-quantitative modified Mankin score. Immunolocalisation was used to identify changes in vimentin distribution.

Results: SIL caused damage in 32/36 cartilage samples. Damage included surface fibrillation, fissures, fragmentation, changes in cellularity and loss of proteoglycan. SIL caused a statistically significant increase in modified Mankin score and chondrocyte clusters over time. SIL caused vimentin disassembly (as evidenced by collapse of vimentin around the nucleus).

Conclusion: This study describes a model of SIL damage to rat cartilage. SIL causes changes in histological/chemical parameters which have been measured using a semi-quantitative modified Mankin score. Single impact load also causes changes in the pattern of vimentin immunoreactivity, indicating vimentin dissassembley. Using a semi-quantitative scoring system the disassembly was shown to be statistically significant in SIL damaged cartilage. The changes described in this paper suggest that this novel single tissue rat model of joint damage is a possible candidate model to replace in vivo models.

Figure 1

Photomicrographs to show the representative appearance of the vimentin cytoskeleton within each of the four scoring categories. Chondrocytes have been stained with monoclonal anti-vimentin antibody and visualized with an immunofluorescent secondary antibody (green). Counter stained with DAPi nuclear stain (blue). Chondrocytes with a normal cytoskeletal appearance i.e. lattice throughout the cell were scored as 0 (A), cells with a slight increase in immunofluorescent intensity of stain around the nucleus were scored as 1 (B), with a moderate intensity around the nucleus 2 (C) and with the stain entirely around the nucleus 3 (D).

Figure 2

2a Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This is a control section i.e. has not received single impact load and it is has not been cultured i.e. represents a control section, T = 0. Stained with toluidine blue. There is no damage to the cartilage; the articular surface is smooth and flat. There are no micro-fractures or fragments and there is no loss of proteoglycan. Scale bar = 10 μm. 2b Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. T = 0. Stained with toluidine blue. There is marked surface damage to the cartilage including lamination. Scale bar = 10 μm. 2c Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. T = 0. Stained with toluidine blue. There is marked surface damage to the cartilage including the formation of a fragment discreet from the parent cartilage. Scale bar = 10 μm. 2d Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. This cartilage has been cultured for 48 hours i.e. T = 48 h. Stained with toluidine blue. There is a fissure at the articular surface and marked proteoglycan loss. Scale bar = 10 μm

Figure 3

Graph to show the modified Mankin score in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. There is a significant difference between control and single impact loaded cartilage at t = 8, 24 and 48 hours in culture (*).

Figure 4

Graph to show the modified Mankin score minus the proteoglycan component score in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. The SIL cartilage is significantly increased relative to the control in all sections.

Figure 5

Graph to show the numbers of chondrocyte clusters in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. There is a significant difference between control and single impact loaded cartilage at t= 4, 8, 24 and 48 hours in culture (*).

Figure 6

Histological sections of cartilage obtained from the distal femur of a rat at 23 weeks of age. 4a. This cartilage has been not been impacted i.e. is a control section but has been cultured for 48 hours. Few chondrocyte clusters (defined as cell groupings of 4+ nuclei, not arranged perpendicular to the joint surface) are present. 4b This cartilage has been subjected to single impact load of 200 g from 8 cm and cultured for 48 hours. There are significantly more chondrocyte clusters in this section than in the control section. Both sections are stained with H&E. Scale bar = 15 μm.

Figure 7

Graph to show vimentin scoring in cartilage sections in control and single impact loaded (SIL) cartilage over 48 hours in culture. Vimentin scoring is shown in arbitrary units, with a high score representing increased disassembly of the vimentin. At all time points there is a statistically significant increase in vimentin score in SIL cartilage, * shows statistical significance between the control and the SIL cartilage at each time point.

Figure 8

Photomicrographs of the cartilage from the distal femur of a rat. The photomicrographs illustrate the immunoreactivity of ERK and pERK within the cartilage. Sections 6a and 6b have been immunohistochemically labeled with anti-ERK antisera and visualized with an immunofluorescent secondary antibody (green), sections 6c and d have been immunohistochemically labeled with anti-pERK antisera and visualized with an immunofluorescent secondary antibody. Sections 6a and 6c represent t= 0 and sections 6 b and d represent t = 1 hour. In Figure 6a (ERK, t = 0) there is immunoreactiviey tht is not ssignificantly different from Figure 6b (ERK, t = 1 hour) i.e. there has been no change in ERK immunoreactivity levels. In contrast in Figure 6c (pERK, t = 0) there is no immunoreactivity i.e. no activation of pERK, whilst in Figure 6d (pERK, t = 1 hour) strong immunoreactivity is seen. Scale bar = 13 μm