Evaluating the anti-neuropathic effects of the thymol-loaded nanofibrous scaffold in a rat model of spinal cord injury

Abstract

Background: Spinal cord injury (SCI) is a debilitating condition characterized by partial or complete loss of motor and sensory function caused by mechanical trauma to the spinal cord. Novel therapeutic approaches are continuously explored to enhance spinal cord regeneration and functional recovery.

Purpose: In this study, we investigated the efficacy of the poly(vinyl alcohol) and chitosan (PVA/CS) scaffold loaded with different thymol concentrations (5, 10, and 15 wt%) in a rat compression model for SCI treatment compare to other (e.g., thymol and scaffold) control groups.

Results and discussion: The thymol-loaded scaffold exhibited a smooth surface and a three-dimensional nanofibrous structure with nanoscale diameter. The conducted analyses verified the successful incorporation of thymol into the scaffold and its high water absorption, porosity, and wettability attributes. Behavioral assessment of functional recovery showed improving sensory and locomotor impairment. Furthermore, histopathological examinations indicated the regenerative potential of the thymol-loaded nanofiber scaffold, by neuronal survival.

Conclusion: Therefore, these findings suggest that the thymol-loaded nanofibrous scaffolds have promising pharmacological activities for alleviating neuropathic pain and addressing complications induced by SCI.

Keywords: chitosan; motor activity; neuropathic pain; poly(vinyl alcohol); spinal cord injury; thymol-scaffold.

FIGURE 1

Experimental design was conducted using a rat compression model of SCI to evaluate the therapeutic potential of poly(vinyl alcohol)/chitosan (PVA/CS) scaffolds loaded with varying concentrations of thymol (5, 10, and 15 wt%). The timeline of the experiment included scaffold implantation immediately post-injury, followed by behavioral assessments (e.g., locomotor and sensory function) at regular intervals. Histopathological analyses were performed at the endpoint to evaluate overall tissue regeneration. The design aimed to compare the efficacy of thymol-loaded scaffolds against controls in promoting functional recovery and reducing neuropathic pain associated with SCI.

FIGURE 2

SEM image of blank PVA/CS scaffold (A) and the scaffold containing 5 wt% (B), 10 wt% (C), and 15 wt% (D) thymol with corresponding diameter distribution histograms.

FIGURE 3

FT-IR spectrum of thymol, blank PVA/CS scaffold, and thymol-loaded PVA/CS scaffold.

FIGURE 4

(A) The water absorption capacity of the scaffolds at different immersion times, (B) The resultant images of the water contact angle of the scaffolds: blank PVA/CS scaffold (a) and the scaffolds containing 5 wt% (b), 10 wt% (c), and 15 wt% (d) thymol with the quantitative values.

FIGURE 5

DSC thermograms of (A) pure thymol, (B) blank PVA/CS scaffold, and (C) thymol-loaded scaffold.

FIGURE 6

(A) Free radical scavenging activity of thymol-loaded and blank PVA/CS scaffolds at different concentrations. (B) Determination of IC₅₀ values and sigmoidal curve fitting for representing radical scavenging activity of the scaffolds.

FIGURE 7

Investigating the effect of PVA/CS scaffold loaded with thymol on weight changes following SCI in rats. Data are presented as mean ± SEM (n = 6). ( ^p < 0.05 and ^ ^ ^p < 0.001) vs. TH group, ***p < 0.001, **p < 0.01, *p < 0.05) vs. SCI group and (+++ p < 0.001) vs. Sham group.

FIGURE 8

Investigating the effect of PVA/CS scaffold loaded with thymol on motor performance in the BBB test following SCI in rats. Data are presented as mean ± SEM (n = 6). ( ^ ^ ^p < 0.001) vs. TH group, (***p < 0.001, **p < 0.01, *p < 0.05) vs. SCI group and (+++p < 0.001) vs Sham group (SF: blank PVA/CS scaffold, TH: free thymol, SF + TH: scaffolds containing thymol).

FIGURE 9

Investigating the effect of PVA/CS scaffold loaded with thymol on mechanical pain tolerance threshold following SCI in rats. Data are presented as mean ± SEM (n = 6). ***p < 0.001, **p < 0.01, *p < 0.05) vs. SCI group and (+++ p < 0.001) vs. Sham group (SF: blank PVA/CS scaffold, TH: free thymol, SF + TH: scaffolds containing thymol).

FIGURE 10

Investigating the effect of PVA/CS scaffold loaded with thymol on cold (A) and thermal (B) pain tolerance threshold following SCI in rats. Data are presented as mean ± SEM (n = 6). (***p < 0.001, **p < 0.01, *p < 0.05) vs SCI group and (+++ p < 0.001) vs Sham group. (SF: blank PVA/CS scaffold, TH: free thymol, SF + TH: scaffolds containing thymol).

FIGURE 11

(A) Investigating the effect of PVA/CS scaffold loaded with thymol on changes in the number of motor neurons in the ventral horn of the spinal cord in hematoxylin-eosin staining of the transverse sections prepared from the spinal cord with 40x magnification following SCI in rats. (B) Data are presented as mean ± SEM (n = 3). (^p < 0.05) vs. TH group, (***p < 0.001, **p < 0.01, *p < 0.05) vs. SCI group and (+++ p < 0.001) vs. Sham group (SF: blank PVA/CS scaffold, TH: free thymol, SF + TH: scaffolds containing thymol).

FIGURE 12

Illustration of rat SCI and being treated with thymol-loaded nanofibrous scaffold. The integration of the thymol-loaded PVA/CS nanofibrous scaffold into the injured spinal cord. The scaffold, characterized by its three-dimensional nanofibrous structure and high porosity, is shown to release thymol at the injury site. Thymol exerts anti-inflammatory, antioxidant, and neuroprotective effects, reducing secondary damage and promoting a regenerative microenvironment. Additionally, the sustained release of thymol enhances functional recovery by alleviating neuropathic pain and improving locomotor and sensory functions.