Adipose-derived stem cells modulate neuroinflammation and improve functional recovery in chronic constriction injury of the rat sciatic nerve

Abstract

Introduction: Compressive neuropathy, a common chronic traumatic injury of peripheral nerves, leads to variable impairment in sensory and motor function. Clinical symptoms persist in a significant portion of patients despite decompression, with muscle atrophy and persistent neuropathic pain affecting 10%-25% of cases. Excessive inflammation and immune cell infiltration in the injured nerve hinder axon regeneration and functional recovery. Although adipose-derived stem cells (ASCs) have demonstrated neural regeneration and immunomodulatory potential, their specific effects on compressive neuropathy are still unclear.

Methods: We conducted modified CCI models on adult male Sprague-Dawley rats to induce irreversible neuropathic pain and muscle atrophy in the sciatic nerve. Intraneural ASC injection and nerve decompression were performed. Behavioral analysis, muscle examination, electrophysiological evaluation, and immunofluorescent examination of the injured nerve and associated DRG were conducted to explore axon regeneration, neuroinflammation, and the modulation of inflammatory gene expression. Transplanted ASCs were tracked to investigate potential beneficial mechanisms on the local nerve and DRG.

Results: Persistent neuropathic pain was induced by chronic constriction of the rat sciatic nerve. Local ASC treatment has demonstrated robust beneficial outcomes, including the alleviation of mechanical allodynia, improvement of gait, regeneration of muscle fibers, and electrophysiological recovery. In addition, locally transplanted ASCs facilitated axon remyelination, alleviated neuroinflammation, and reduced inflammatory cell infiltration of the injured nerve and associated dorsal root ganglion (DRG). Trafficking of the transplanted ASC preserved viability and phenotype less than 7 days but contributed to robust immunomodulatory regulation of inflammatory gene expression in both the injured nerve and DRG.

Discussion: Locally transplanted ASC on compressed nerve improve sensory and motor recoveries from irreversible chronic constriction injury of rat sciatic nerve via alleviation of both local and remote neuroinflammation, suggesting the promising role of adjuvant ASC therapies for clinical compressive neuropathy.

Keywords: adipose derived stem cells; chronic constriction injury; compressive neuropathy; immunomodulation; neuroinflammation; neuropathic pain; stem cell therapy.

Figure 1

Stem cell therapy promoted sensory and motor functional recoveries from chronic constriction injury of the rodent model. (A) Treatment algorism of chronic constriction injury over the ipsilateral sciatic nerve. After 1 week of nerve constriction, one group received nerve release alone plus intraneural PBS injection (as the control group) and the other group received nerve release combined with intraneural stem cells injection (as ASC group). (B) Mechanical allodynia was evaluated by von Frey test over the hind paw of rats. The mechanical withdrawal forces (g) indicate the forces that induced paw withdrawal by the von Frey test. After treatment, the ASC group showed increased mechanical withdrawal forces compared with the control group. (C) Dynamic gait analysis in the different groups. The ASC group revealed a significantly increased sciatic functional index (SFI)compared with the control group. (D, E) Gastrocnemius muscle recovery in the different treatment groups on post-treatment day 28 (L, left injured side; R, right uninjured side). Left gastrocnemius muscle weight demonstrated a significant increase in the ASC group compared with the control group (scale bar: 1 cm) (F, G) Histological analysis of left gastrocnemius muscle fiber demonstrated a decrease of muscle fiber surface area in the control group, with an increase in the ASC group on day 28 (Scale bar: 50 mm). (H) The electrophysical evaluation of gastrocnemius muscle re-innervation on day 28. The amplitude compound muscle action potential significantly increased in the ASC group compared with the control group, while the latency of nerve conduction was shorter in the ASC group than in the control group (data were presented with mean ± standard deviation, *indicated p < 0.05, **indicated p < 0.01, ****indicated p < 0.0001; n = 5 for each group).

Figure 2

Immunofluorescent staining of functional axon regeneration and inflammatory cell infiltration in the injured nerve at day 28. (A, B) Double staining of NF200/S100β revealed decreased regenerated axon density in the control group but increased density in the ASC group. (C, D) Inflammatory cell staining with CD68 revealed a significant increase in the control group but a decrease in the ASC group (scale bar: 50 μm; ****indicated p < 0.0001, ns indicated no significant difference; n = 4 for each group).

Figure 3

Immunofluorescent staining of neuroinflammatory signals in the injured sciatic nerve at day 28. A significant increase in the signal intensity of TNF-α (A, B) and IL-1β (C, D) was observed in the control group, as compared to the contralateral nerve as the baseline on day 28. In the ASC group, the signal intensity significantly decreased as compared to the control group (scale bar: 50 μm; ***indicated p < 0.001, ****indicated p < 0.0001, ns indicated no significant difference; n = 4 for each group).

Figure 4

Immunofluorescent staining of pain signals and inflammatory cell infiltration in the injured dorsal root ganglion (DRG) at day 28. A significant increase of signal intensity in IB4 and CGRP (A, B) was observed in the control group, as compared to the contralateral DRG as the baseline. In the ASC group, the signal intensity of both IB4 and CGRP significantly decreased as compared to the control group. (C, D) Inflammatory cell staining with CD68 revealed a significant increase in the control group but a decrease in the ASC group of injured DRG at day 28 (scale bar: 50 μm; **indicated p < 0.01, ****indicated p < 0.0001, ns indicated no significant difference; n = 3 for each group).

Figure 5

Immunofluorescent staining of neuroinflammatory signals in the injured dorsal root ganglion at day 28. (A, B) A significant increase of IL-1β signal was observed in the control group, as compared to contralateral uninjured DRG as a baseline. In the ASC group, the signal intensity significantly decreased as compared to the control group on day 28. (C, D) The signal intensity of TNF-α, the other kind of neuroinflammation marker, showed no significant difference between groups (scale bar: 50 μm; **indicated p < 0.01, ns indicated no significant difference; n = 3 for each group).

Figure 6

Trafficking for the terminal fate of transplanted ASC after local intraneural injection. (A, B) The signal intensity of ICG-bioluminescent labeling ASC was trackable and revealed a significant increase as compared to baseline (PBS injection) on day 3 but returned to baseline signal on day 7. (C) Immunofluorescent staining of the injured nerve demonstrated the colocalization of CD90⁺/CD105⁺ cells (arrow), indicating the maintenance of ASC phenotype at least on day 3 (scale bar: 50 μm; ****indicated p < 0.0001, ns indicated no significant difference; n = 3–5 for each group).

Figure 7

Quantitative PCR analysis of inflammatory gene in the injured nerve dorsal root ganglion at day 3. (A) Upregulation of IL-1β, TNF-α, CD68, CD11b, CD80, and CD86 expression in the injured control nerve, as compared to the contralateral uninjured nerve. In the ASC group, significant downregulation of the inflammatory gene expression on the injured nerve as compared to the control group. (B) Upregulation of TNF-α, IL-1β, substance P, and VIP expression in the injured control DRG as compared to the contralateral uninjured DRG. In the ASC group, significant downregulation of inflammatory gene expression on the injured DRG as compared to the control group (data were presented with mean ± standard deviation, *indicated p < 0.05; **indicated p < 0.01, ***indicated p < 0.001, ****indicated p < 0.00001, ns indicated no significant difference; n = 3–5 for each group).

Figure 8

Schematic summary of the therapeutic benefit of ASCs for the alleviation of neuropathic pain in chronic constriction injury of rats.