Folic Acid Modulates Matrix Metalloproteinase-9 Expression Following Spinal Cord Injury

Abstract

Background: Treatment of spinal cord injury (SCI) induced neuropathic pain (NP) proves to be extremely clinically challenging as the mechanism behind SCINP is poorly understood. Matrix metalloproteinase (MMP) is largely responsible for the early disruption of the blood spinal cord barrier. This system initiates macrophage infiltration and degradation of myelin, which plays a pivotal role in how NP occurs. In a recent study, we demonstrated that folic acid (FA) treatment to cSCI rats reduced NP and improved functional recovery by repressing MMP-2 expression. We hypothesize that MMP-2 expression is suppressed because FA actively methylates the DNA sequence that encodes for the MMP-2 protein. However, modulation of MMP-2 expression for alleviation of NP is only pertinent to the mid- to late-phase of injury. Therefore, we need to explore alternate therapeutic methods to target the early- to mid-phase of injury to wholly alleviate NP.

Purpose: Furthering our previous findings on inhibiting MMP-2 expression by FA in mid- and late- phase following cSCI in rats, we hypothesized that FA will methylate and suppress MMP-9 expression during the early- phase, day 1, 3, 7 post cSCI and mid- phase (day 18 post cSCI), in comparison with MMP-2 expression during mid- and the late-phase of cSCI.

Methods: Adult male Sprague Dawley rats (250-270g) underwent cSCI, using a NYU impactor, with 12.5 gm/cm injury. The spinal cord-injured animals were treated intraperitoneally (i.p.) with a standardized dose of FA (80 μg/kg body weight) on day 1, 2, 3, prior to cSCI, followed by daily injection up to 14 or 17 days post-cSCI in different experiments. Animals were euthanized on day 1, 3, 7 post cSCI (early- phase), day 18 post cSCI (mid- phase), and day 42 post cSCI (late-phase) and the epicenter region of injured spinal cord were harvested for MMP-9 and MMP-2 expression analysis by Western blots technique.

Results: i) During early-phase on day 1, 3, and 7, the quantitation displayed no statistical significance in MMP-9 expression, between water- and FA- injected rats. ii) On day 18 post-cSCI, FA significantly modulates the expression of MMP-9 (p = 0.043) iii) Comparing results with MMP-2 expression and inhibition, FA significantly modulates the expression of MMP-2 on day 18 post cSCI (FA- and water-injected rats (p = 0.003). iv) In addition, FA significantly modulates the expression of MMP-2 on day 42 post-cSCI comparing FA- and water- injected rat groups (p = 0.034).

Conclusion: We report that FA administration results in alleviating cSCI-induced NP by inhibiting MMP-9 in the proposed mid- phase of cSCI. However, FA administration resulted in MMP-2 decline during both mid- through late- phase following cSCI. Our study elucidates a new phase of cSCI, the mid-phase. We conclude that further investigation on discovering and quantifying the nature of the mid- phase of SCI injury is needed.

Keywords: Demethylation; Folic acid (FA); MMP-2; MMP-9; Matrix metalloproteinses (MMPs).

Fig. 1:

Expression of MMP-9 in early phase of SCI. A: Western blots of MMP-9 on Day 1, Day 3 and Day 7 in Folic acid treated and non-treated animals. B: The normalized intensity values expressed as mean ± SE. The quantitation displayed no statistical significance, for days 1, 3, and 7, in MMP-9 expression between water- and FA- injected rats (p>0.05).

Fig. 2:

Expression of MMP-9 in mid phase of SCI. A: Western blots of MMP-9 on Day 18 in Folic acid treated and non-treated animals. B: The normalized intensity values expressed as mean ± SE. The quantitation of data displayed statistical difference between FA- and water- injected rat groups (*p<0.05)

Fig. 3:

Expression of MMP-2 in mid phase and late phase of SCI A: Western blots of MMP-2 on day 18 from FA treated and non-treated animals. B: The normalized intensity values of blots represented in A, expressed as mean ± SE C: Western blots of MMP-2 on day 42 from FA treated and non-treated animals. D: The normalized intensity values of blots represented in C, expressed as mean ± SE. The quantitation of data displayed statistical difference between FA- and water- injected rat groups (*p<0.05)