Folic Acid Modulates Matrix Metalloproteinase-2 Expression, Alleviates Neuropathic Pain, and Improves Functional Recovery in Spinal Cord-Injured Rats

Abstract

Background: The molecular underpinnings of spinal cord injury (SCI) associated with neuropathic pain (NP) are unknown. Recent studies have demonstrated that matrix metalloproteinases (MMPs) such as MMP2 play a critical role in inducing NP following SCI. Promoter methylation of MMPs is known to suppress their transcription and reduce NP. In this context, it has been shown in rodents that folic acid (FA), an FDA approved dietary supplement and key methyl donor in the central nervous system (CNS), increases axonal regeneration and repair of injured CNS in part via methylation. Purpose: Based on above observations, in this study, we test whether FA could decrease MMP2 expression and thereby decrease SCI-induced NP. Methods: Sprague-Dawley male rats weighing 250-270 g received contusion spinal cord injuries (cSCIs) with a custom spinal cord impactor device that drops a 10 g weight from a height of 12.5 mm. The injured rats received either i.p. injections of FA (80 µg/kg) or water (control) 3 days prior and 17 days post-cSCI (mid phase) or for 3 days pre-cSCI and 14 days post-cSCI ending on the 42nd day of cSCI (late phase). The functional neurological deficits due to cSCI were then assessed by Basso, Beattie, and Bresnahan (BBB) scores either on post-impaction days 0 through 18 post-cSCI (mid phase) or on days 0, 2, 7, 14, 21, 28, 35, and 42 (late phase). Baseline measurements were taken the day before starting treatments. Thermal hyperalgesia (TH) testing for pain was performed on 4 days pre-cSCI (baseline data) and on days 18, 21, 28, 35, and 42 post-cSCI. Following TH testing, animals were euthanized and spinal cords harvested for MMP-2 expression analysis. Result: The FA-treated groups showed higher BBB scores during mid phase (day 18) and in late phase (day 42) of injury compared to controls, suggesting enhanced functional recovery. There is a transient decline in TH in animals from the FA-treated group compared to controls when tested on days 18, 21, 28, and 35, indicative of a decrease in NP. However, when tested 25 days after stopping FA administration on day 42 of cSCI, no significant difference in TH was observed between FA-treated and control animals. Western blot analysis of the injured spinal cord from FA-treated animals showed significant decline in MMP2 expression compared to spinal cord samples from water-treated controls. Conclusion: Together, these data suggest that FA could alleviate NP and improve functional recovery post-SCI, possibly by reducing the expression of MMP2. Further studies will open up a novel and easy natural therapy, ideal for clinical translation with minimal side effects, for managing SCI-induced NP. Such studies might also throw light on a possible epigenetic mechanism in FA-induced recovery after SCI.

Folate action in nervous system development and disease

“…Folate enhances regrowth of sensory spinal axons in vivo after bilateral lesion of rat dorsal root ganglia (Iskandar et al, 2004). Intraperitoneal injections with folic acid in injured rats result in enhanced axon regeneration of damaged spinal cord and optic nerves as well as functional recovery following spinal cord contusion (Iskandar et al, 2004;Miranpuri et al, 2017). The mechanism underlying folate action in axon regeneration depends on FOLR1, and is correlated with changes in DNA methylation (Iskandar et al, 2010).…”

“…Matrix metalloproteinases, such as matrix metalloproteinase‐2 (MMP2), are upregulated upon spinal cord injury, and not only interfere with functional recovery but also contribute to the associated neuropathic pain (Kawasaki et al, ; Miranpuri et al, ). Folate treatment managed to reduce MMP2 expression through methylating its promoter, reducing neuropathic pain and increasing functional recovery after spinal cord contusion (Miranpuri et al, ).…”

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

“…Adult male Sprague Dawley rats (250-270g) underwent cSCI, using a NYU impactor, with 12.5 gm/cm injury as per protocol used in earlier studies and well established in our lab [12][13][14][15][16][17][18][19][20][21][22][23]. A total of 40 male rats were used to assure an adequate number of animals exhibiting NP behavior.…”

“…Briefly, the spinal cord-injured animals were treated intraperitoneally (IP) with a standardized dose of FA (80 μg/kg body weight) [13]. For all phases, rats were treated for 3 days prior to the cSCIs.…”

“…In addition, we have tested the role of folic acid (FA) in modulating MMP-2 expression. FA implementation in SCI injuries has demonstrated alleviation of NP and functional recovery improvement in SCI rodents [13]. To explore the benefits of FA in SCI recovery and NP alleviation, we have targeted MMP-9 regulation during the early stage and MMP-2 in late stage, as these are the times in which MMP-9 and MMP-2 are at peak expression, respectively.…”