As shown previously, mineral coatings have a high protein-binding propensity and the capability to release proteins over a controllable time frame with high levels of protein biological activity [
18‐
20,
33‐
36]. In this study, we hypothesized a local sustained release of IL-10 from mineral coatings would reduce the prolonged inflammation that occurs after SCI. The in vitro release profile indicates an initial burst of IL-10 for 2 days, followed by a continuous release of IL-10 for at least 17 days, evidence that the coating has the ability to maintain protein delivery for a length of time through the initial inflammatory period post-SCI [
2,
3].
IL-10’s effect on inflammation
Normal wound healing in skin or muscle occurs over three defined stages: inflammation, proliferation, and remodeling. Throughout healing, macrophages exist on a continuum from M1 inflammatory macrophages to M2 anti-inflammatory macrophages dictated by the extracellular environment. In the inflammatory stage, M1 macrophages aid the innate immunity by inducing inflammation through the production of TNFα and IL-1β, while phagocytosing bacteria and debris [
1,
37]. As proliferation begins, M1 macrophages gradually change phenotype to a variety of M2 macrophage subsets with different functions. Generally, M2 macrophages attenuate the production of pro-inflammatory cytokines and reactive oxygen species (ROS), in addition to having tissue-repairing properties. The production of IL-10 is traditionally attributed to the M2b phenotype [
31,
38] and acts in tissue remodeling during the proliferative stage of muscle and skin wound healing [
8,
39]. During healing, M2c macrophages remain to aid in the remodeling phase [
39]. However, defined stages of healing are not present post-SCI [
1,
4]. Without proper termination of the inflammatory stage, the presence of activated macrophages (M1 and M2) may last for years [
1,
39].
Post-SCI, the poor transition of macrophages complicates healing [
39‐
41]. Binding of IL-10 to the IL-10 receptor activates the Jak/STAT pathway, which results in a reduction of pro-inflammatory cytokines released from macrophages/microglia [
12]. One mechanism by which IL-10 treatment is thought to improve functional recovery post-SCI is through macrophage deactivation: hindering the production of pro-inflammatory cytokines prevents further damage to the spinal cord by attenuating cell death [
42,
43] and blood–spinal cord barrier disruption [
44], in addition to reducing ROS and neurotoxic substance production. These benefits have been observed in previous studies where IL-10 induced nerve growth factor production in astrocytes and reduced astrogliosis post-TBI in rats [
6,
45].
In this study, the MCMs+IL-10 group observed a significant increase in IL-10 at both 24 h and 7 days post-SCI (Fig.
3a-b). In accordance with previous studies, the Systemic IL-10 treatment attenuated TNFα and IL-1β production at 24 h post-SCI, but failed to reach significance at 7 days post-SCI [
14,
46,
47]. The MCMs+IL-10 group did significantly attenuate the production of TNFα at 7 days post-SCI, proving the sustained release of IL-10 effective. However, there was no significant difference in IL-1β production at 7 days post-SCI (Fig.
3f). In agreement with the cytokine analysis, MCMs+IL-10 observed a significant decrease in the number of “M1” macrophages as compared to Controls at 7 days post-SCI, evaluated with flow cytometry (Fig.
6d). Although there was no difference in the expression of the M2c marker CD163 between any of the groups 7 days post-SCI, there was a significant increase in the “M2b” population (CD11b
+ CD45
HIGH CD68
− CD80
− CD86
+ CD163
−) in the MCM + IL-10 group (Fig.
6e). The sustained release of IL-10 is likely contributing to the decreased expression of M1 macrophage markers, but complete conversion to an M2c expressing CD163 was not observed at the 7-day time point. Immunohistochemistry analysis at 7 weeks post-SCI, however, did show a significant increase in the ratio of CD163:MARCO (M2c:M1) in MCMs+IL-10 as compared to Controls (Fig.
7c). Co-expression of MARCO and CD163 was observed, indicating that these macrophages lie between M1 and late stage M2 on the continuum but lean closer to M2 with the MCMs+IL-10 treatment as compared to Controls (Fig.
7). This is likely beneficial as M2c macrophages (CD163
+) are found in the later stages of wound healing [
1,
37,
39].
Using IL-10 to treat SCI
IL-10 treatment has been shown to improve functional recovery in Sprague-Dawley rats post-SCI [
13‐
15]. Here, the MCMs+IL-10 group had the highest functional score on day 28 and was the only group to reach significance when compared to the Controls (Fig.
8d). In addition, IL-10 treatment has previously reduced lesion size post-SCI [
14,
48]. We observed similar reductions in lesion size, with a significant reduction in infarct size with both the Systemic IL-10 treatment and the MCMs+IL-10 treatment as compared to Controls. However, there was no significant difference between MCMs+IL-10 and Systemic IL-10 in atrophy (
P = 0.9358; Fig.
12f) or infarct size (
P = 0.9791; Fig.
12g).
The MCMs+IL-10 group also expressed the greatest percent of spared axons in the reticulospinal and rubrospinal tracts, which was the only group significantly higher than the Controls. Though functional recovery was observed across all groups, the dorsal corticospinal tract presented with virtually no axons crossing the injury site in any of the five contused groups, unlike the reticulospinal and rubrospinal tracts. This data, in conjunction with the functional recovery assessed through BBB scores, suggests changes involving spared axons may have occurred within the spinal cord to permit functional recovery.
The number of axons spared in the rubrospinal and reticulospinal tracts suggests a plastic capability of the rat central nervous system (CNS) which allows for heavier reliance on these tracts for motor function. Both the rubrospinal and reticulospinal tracts are known to participate in motor function in healthy models, specifically through limb movement, though to different capacities. The rubrospinal tract maintains greater control of more precise movements and the reticulospinal tract expresses control of larger, gross locomotor movement and rhythm [
49‐
54]. It is possible that the CNS is utilizing the spared axons in these tracts, which already express an innate participation in locomotor function, to regain motor capabilities after injury. Animal models have shown the rubrospinal tract is capable of absorbing the role of the corticospinal tract (CST) in the event of injury [
55‐
59]. Although the rubrospinal tract is well developed in lower mammals (i.e., cats and rats), its importance in upper mammals appears to have diminished with the evolutionary development of the CST [
60]. This may hinder translation to human models, which do not express a prominent rubrospinal tract [
61].
Another possible explanation is that spontaneous axonal sprouting may have occurred and aided in improving functional recovery in the MCM+IL-10 group. Previous studies have shown the ability of various tracts within the spinal cord to express axonal sprouting [
55,
62‐
65]. The axon tracer used in this study, BDA, does not cross synapses. Thus if there was axonal sprouting and synapsing, it would not have been detected.
No significant difference was found between contused groups for conduction velocity or amplitude (Fig.
11c-d). With few axons extending through the injury site post-SCI, the CST likely created a very small signal in all the rats, regardless of treatment. The location of the rubrospinal and reticulospinal tracts, along with the placing of the recording electrode, may have impacted the results. The rubrospinal and reticulospinal tracts are located deep and laterally in the spinal cord. With the receiving electrode placed gently on the dorsal side of the spinal cord, voltage from action potentials in deeper tracts would be reduced due to distance.