A rat tail model was used to evaluate the kinetics of mRNA expression in the intervertebral disc anulus and nucleus following a single loading event and to establish a quantitative relationship between mechanical loading and mRNA levels. To aid in interpretation of results, a hypothetical model unique to each gene and disc region (i.e., anulus vs. nucleus), represents the general shape of the transient mRNA levels anticipated (). This model has mRNA levels that are altered from control levels at time t1
following an event, reach a maximum amplitude (Amax
) at time tmax
and recover to a final amplitude (A2, i.e., the new baseline gene expression level) at time t2
. A remodeling or injury response involves an upregulation following loading (Amax
≥0) that never returns to baseline levels (A2
>0). In the context of mechanical stimulation, the amplitudes are a specific function of both magnitude and frequency of compression joint forces,13,19
and are relative to control levels (i.e., = 0; homeostasis), >0 (i.e., upregulation), or <0 (i.e., downregulation) provided measurements are taken at times beyond the half-life of the mRNA being measured.
Figure 5 Hypothetical model for kinetic relationships between mRNA levels and mechanical stimulation specific to gene, disc region, and loading condition. Times t1, tmax, and t2 represented when relative mRNA expression level was significantly upregulated, reached (more ...)
Kinetic patterns of mRNA expression were observed over a 3-day period following a single loading event consistent with hypothesis 1. The transient responses were generally consistent with the hypothetical model although it is impossible to define precise timing or magnitudes based on the current methodologies and the chosen study design. The maximum measured mRNA levels were for proteases and their inhibitors which were the genes with low basal expression levels, consistent with hypothesis 2. Further, the majority of genes reached maximum observed levels 24 h following cessation of loading consistent with the hypothesis, but maximum mRNA levels were also observed 8 and 72 h following loading. Finally, mRNA levels returned to baseline levels within 72 h for some, but not all, genes in partial support of hypothesis 3.
Patterns of mRNA levels were consistent with turnover/repair in the nucleus and remodeling and/or injury in the anulus.1
In the nucleus, all mRNA levels returned to control levels (A2
= 0) representative of a turnover/repair response consistent with our hypothesis. In the anulus, however, cells exhibited mRNA levels that remained significantly upregulated for six genes (of nine measured) even 72 h after loading (A2
> 0). This continued upregulation of the anulus may represent adaptation to new steady state baseline mRNA levels consistent with a remodeling and/or injury responses where anulus cells and tissue physicochemical patterns were permanently modified. A second explanation for the continued upregulation of mRNA levels in the anulus regions is that the mRNA is not degraded before 72 h, suggestive of relatively slow metabolism. It is possible the extended changes in gene expression of the anulus could also represent a prolonged response associated with a cascade of events where initial mechanically induced changes in gene expression lead to subsequent changes (e.g., exposure to matrix breakdown products), which further influence gene expression. The mRNA levels for a few genes were not significantly altered in response to loading (MMP-2 in the anulus and nucleus and collagen-I and collagen-II in the nucleus) indicating a homeostasis response and/or lack of mechano-sensitivity of these genes under these cyclic compression loading conditions.
Based on the relative changes in gene expression, the short-term mRNA response to mechanical perturbation in vivo on important matrix proteins was suggestive of a strategy that first involved inhibiting tissue breakdown prior to accumulating structural proteins and enzymes. Due to the high baseline abundance of mRNA for collagens and aggrecan, significant upregulation of mRNA for these genes requires more mRNA and thus occurred later. The intermediate mRNA response involved increased aggrecan and matrix degrading enzymes mRNA in response to loading. The long-term mRNA response was associated with repair or anabolic remodeling modification of the matrix represented by collagen metabolism in the anulus. Results therefore suggest that attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur and underscores the fact that MMPs and TIMPs have greater potency in altering aggrecan and collagen levels than protein synthesis alone.
The mRNA levels for aggrecan and ADAMTS-4 in both regions of the disc had maximum levels in 24-h measurements while collagens and MMP-13 (collagenase-3) in the anulus fibrosus had maximum levels in 72-h measurements. This finding complements the understanding that aggrecan turnover occurs more quickly than collagen turnover in intervertebral disc22
Differences between anulus and nucleus responses may also be attributed to the distinct cell populations and different physical signals in each region,3
and it is notable that in the nucleus collagen mRNA patterns were different with no significant alterations in collagen mRNA levels and MMP-13 reaching maximal levels at 24 h.
The mRNA levels of greatest abundance in the disc coded for genes of structural proteins, i.e., aggrecan followed closely by collagen-II in the nucleus and collagen-II followed by aggrecan and then collagen-I in the anulus fibrosus (). It is noteworthy that resident collagen-II and ADAMTS-4 mRNA levels were most and least abundant, respectively, in the anulus similar to that reported for articular cartilage.21
Thus, the 20-fold increase in relative mRNA expression for ADAMTS-4 in the anulus requires substantially less mRNA to be expressed than does the 10-fold increase in collagen-II mRNA observed in this study.
The kinetic mRNA response for a gene depends on several factors. First, genes with greater basal levels of mRNA require more time for significant mRNA changes to accumulate in order to achieve similar relative changes in mRNA levels. Second, cell signaling pathways and their stimuli vary for different proteins and thus kinetics are likely to vary between genes. Third, the minimum time for gene levels to return to baseline levels must be several half-lives greater than the half-life of the mRNA for that gene. Findings provided strong justification for taking measurements of mRNA levels associated with aggrecan metabolism at time points approximately 24 h past mechanical stimulation. While the 24-h time point also coincided with significantly upregulated mRNA levels for genes associated with collagen catabolism, and is therefore a reasonable time point for making such measurements, the maximum levels of collagen-I and II mRNA occurred at substantially later times and had not returned to baseline levels 72 h after mechanical stimulation.
The mRNA kinetics may also be affected by alterations in cell pressurization and strain which vary in vivo with load duration, magnitude, and frequency. It is likely that gene expression profiles change immediately after the initiation of loading, and, in a prior study on load duration effects, 30 min of cyclic loading was sufficient to induce measurable changes in mRNA expression levels in vivo.24
The single loading event used in this study was of 1.5-h duration which was chosen since it corresponded to a time when disc creep reached steady state24
so that alterations in cell strain due to viscoelasticity would not confound interpretation of the mRNA kinetic results. The magnitude and frequency were chosen since measurable changes in mRNA levels for several anulus and nucleus genes were previously observed,13
and these levels were predicted to be on the high end of physiological loading.24
The disc height change and water loss in rat caudal discs under 1-Hz cyclic compression was found to depend most strongly on peak load magnitudes and not average load values.25
The distinct anulus and nucleus responses may be associated with different biomechanical signals within each disc region, and it was reported that water redistribution from nucleus to anulus occurs with an initial disc bulge followed by a reduction in disc volume over time under 1-Hz compression loading.25
However, cells from the anulus and nucleus also respond to mechanical stimulation distinctly.3
Due to the fact that gene expression was measured far beyond the expected half-life of the genes in question,26,27
we are able to make a distinction between homeostasis (i.e., maintenance of mRNA at baseline levels), downregulation (i.e., a decrease or complete termination of mRNA production) and upregulation (i.e., an increase in mRNA production). No significant downregulation was observed following loading for any of the genes and results indicate cyclic compression had a significant stimulatory response on 14 of 18 genes measured (9 per disc region) and support the known stimulatory effects of cyclic loading on cartilaginous tissues.28,29
We may also conclude that levels of collagen-I and collagen-II in the nucleus and MMP-2 mRNA in both nucleus and anulus were unaffected by loading under these cyclic compression conditions. The finding for MMP-2 is consistent with Hsieh and Lotz who reported no change in total MMP-2 but an increase in the percentage active enzyme with compression loading on mouse tail intervertebral discs.30
Collagen-I and collagen-II are mechanically sensitive and were upregulated in the nucleus under different loading conditions13
; it is also noteworthy that collagen-I in the NP had a nonsignificant decrease in mRNA levels immediately following loading.
Levels of mRNA coding for nine genes were measured at four time points using 49 animals in order to evaluate kinetics of matrix protein metabolism in two disc regions following a single loading event. While clear kinetic patterns were established, the determination of important times when mRNA levels were upregulated, peaked, and returned to baseline is not quantitative since time cannot be resolved beyond the four points measured. For example, the prolonged anulus mRNA response suggests that longer durations than 72 h may be necessary to definitively identity a new steady state. Based on the small tissue size of rat caudal discs and limited mRNA, a trade-off was necessary between the number of time points and the number of genes being evaluated. Measurements were therefore focused on genes coding for matrix proteins, proteases, and TIMPs; future evaluation of mRNA for other matrix molecules, cytokines, and transcription factors are important for a more thorough understanding of short-term matrix remodeling. Levels of MMP-2, 3, and 13 were all slightly elevated in the sham group (and only significant for MMP-13 in the annulus) similar to what has been previously seen, which may indicate a catabolic response to surgery and/or anesthesia. However, these effects were small in comparison to those seen 24 h after loading, and given the low basal levels of these genes represent a minor effect. The sham group assessed effects of surgical intervention, and, while potential effects of anesthesia cannot be completely eliminated since only a single 0-h recovery sham group was included, it is clear that surgical interventions were very small compared to effects of loading.
In conclusion, this study evaluated kinetics of mRNA expression in the disc in response to mechanical compression. Patterns of mRNA kinetics were consistent with turnover/repair in the nucleus and remodeling and/or injury in the anulus. The magnitude and frequency of loading used in this study is predicted to be on the high end of physiological loading for the rat24
which would therefore be expected to induce these types of gene expression alterations. The kinetic results also supported a strategy of mRNA expression that first involved inhibition of tissue breakdown (through expression of TIMPs) followed by synthesis of aggrecan and matrix degrading enzymes, and then collagens. Put in the context of disc repair and regeneration, these findings might also suggest that biological strategies that prevent degeneration through anti-catabolic pathways such as upregulation of TIMPs or downregulation of MMPs may be more easily achievable and more effective than strategies that focus only on increasing structural proteins (aggrecan and collagen) alone. Results demonstrated that resident mRNA levels in control discs were most abundant for structural matrix proteins and least abundant for proteases. Results provide justification for taking measurements of mRNA levels associated with aggrecan metabolism at time points approximately 24 h after stimulation, but for collagen metabolism at later times. This improved understanding of the quantitative relationship between loading and gene expression may help elucidate the mechanisms involved with injury, may lead to more effective rehabilitation treatments, and may help optimize tissue engineering approaches for intervertebral disc repair.