Use of a combination strategy to improve neuroprotection and neuroregeneration in a rat model of acute spinal cord injury

Abstract

Spinal cord injury is a very common pathological event that has devastating functional consequences in patients. In recent years, several research groups are trying to find an effective therapy that could be applied in clinical practice. In this study, we analyzed the combination of different strategies as a potential therapy for spinal cord injury. Immunization with neural derived peptides (INDP), inhibition of glial scar formation (dipyridyl: DPY), as well as the use of biocompatible matrix (fibrin glue: FG) impregnated with bone marrow mesenchymal stem cells (MSCs) were combined and then its beneficial effects were evaluated in the induction of neuroprotection and neuroregeneration after acute SCI. Sprague-Dawley female rats were subjected to a moderate spinal cord injury and then randomly allocated into five groups: 1) phosphate buffered saline; 2) DPY; 3) INDP + DPY; 4) DPY+ FG; 5) INDP + DPY + FG + MSCs. In all rats, intervention was performed 72 hours after spinal cord injury. Locomotor and sensibility recovery was assessed in all rats. At 60 days after treatment, histological examinations of the spinal cord (hematoxylin-eosin and Bielschowsky staining) were performed. Our results showed that the combination therapy (DPY+ INDP + FG + MSCs) was the best strategy to promote motor and sensibility recovery. In addition, significant increases in tissue preservation and axonal density were observed in the combination therapy group. Findings from this study suggest that the combination theapy (DPY+ INDP + FG + MSCs) exhibits potential effects on the protection and regeneration of neural tissue after acute spinal cord injury. All procedures were approved by the Animal Bioethics and Welfare Committee (approval No. 178544; CSNBTBIBAJ 090812960) on August 15, 2016.

Keywords: fibrin glue; glial scar; mesenchymal stem cells; neural derived peptides; neural regeneration; protective autoimmunity.

Figure 1

The combination therapy induced better locomotor recovery after spinal cord injury. The BBB score showed a significant improvement from 7 days after injury. To the third week, the diffence was markedly higher. ^aP < 0.05, DPY + INDP + FG + MSCs groups vs. the other groups; ^bP < 0.05, DPY, DPY + FG, and DPY + INDP groups vs. PBS group (two-way repeated measures analysis of variance). Each point represents the mean ± SD of seven rats. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 2

The combination strategy improves the nociceptive hypersensitivity after spinal cord injury. The mechanical withdrawal threshold was increased in the DPY + INDP + FG + MSC and DPY + INDP groups compared with PBS, DPY, and DPY+ FG groups (^cP < 0.001, ^dP < 0.0001, vs. PBS, DPY and DPY + FG groups; one-way analysis of variance followed by Tukey’s test). Evaluation was performed 60 days after spinal cord injury. The bars represent the mean ± SD of seven rats. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 3

Representative hematoxylin and eosin stained cross-sections of the injured spinal cord at different distance sites (both rostral and caudal) from the epicenter at 60 days after spinal cord injury. The preservation of tissue was improved in animals treated with the combination therapy compared with the PBS group. Arrows depict preserved tissue. Scale bar: 500 μm. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 4

The combination therapy increases the amount of spared tissue. Quantitative analysis demonstrated a significant increase in spared tissue at both, rostral and caudal sites from the epicenter of injury. DPY + INDP + FG + MSC (combination strategy) and DPY + INDP groups presented a higher amount of preserved tissue as compared to PBS group (^aP < 0.05, ^eP < 0.001, vs. PBS group and ^fP < 0.05, vs. DPY + INDP group; one-way repeated measures analysis of variance). Each point represents the mean ± SD of seven rats. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 5

Bielschowsky’s silver stain shows cross-sections of the injured spinal cord at different distance sites (both rostral and caudal) from the epicenter at 60 days after spinal cord injury. The figure shows a higher density of axonal fibers in rats treated with DPY + INDP + FG + MSC or DPY + INDP. Scale bar: 500 μm. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 6

Bielschowsky silver staining of nearby areas to central canal at 60 days after spinal cord injury. Representative microphotographs of sham-operated rats (A) and experimental groups: B) PBS, C) DPY, D) DPY +INDP, E) DPY + FG and F) DPY + INDP + FG + MSC. Scale bar: 50 μm. CC: Central canal; PBS: phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.

Figure 7

Spinal cord injury rats treated with the combination therapy show a higher axonal density. The percentage with respect to the axonal density observed in the spinal cord of sham-operated rats is presented. The percentage of axonal density of rats treated with the combination therapy (DPY + INDP + FG + MSC) was significantly increased compared with the other groups: ^eP < 0.001, vs. PBS group; ^gP < 0.05, vs. DPY and DPY + FG groups; ^fP =0.05, vs. DPY + INDP group (one-way repeated measures analysis of variance). Rats in the DPY + INDP group also showed a significant increase in the percentage of axonal density compared with rats in the other groups: ^hP < 0.05, vs. DPY, DPY + FG, and PBS groups (one-way repeated measures analysis of variance). Rats in the DPY + FG group showed an increase in axonal density compared to rats in the DPY and PBS groups: ⁱP < 0.05 (one-way repeated measures analysis of variance). Each point represents the mean ± SD of seven rats. PBS: Phosphate buffered saline; DPY: dipyridyl; INDP: immunization with neural derived peptides; FG: fibrin glue; MSC: mesenchymal stem cells.