Discussion
After SCI, a significant number of patients develop ISS due to noradrenergic and GC overproduction, which decreases the number of immune cells in the blood and impairs T-cell activation, thus generating an immunosuppressive phenomenon. This alteration increases the susceptibility to develop various pathologies that compromise the stability and life of SCI patients16,43,44.
Several studies have elucidated that, in addition to the important role of T-cells in response to any infectious disease, they also participate in processes related to neuroprotection and neuroregeneration after injury18,19,45. Nevertheless, these cells decrease after SCI because of ISS, as reported by Riegger and coworkers3 . In the first 24 h, T-lymphocytes and other cells such as monocytes and B-lymphocytes decrease dramatically up to 60%, and this suppression continues until day 14. This correlates with the release of adrenocorticotropic hormone (ACTH), GCs, and NE, leading to splenic atrophy, leukocyte apoptosis, and immune depletion with decreased T- and B-cells after SCI3,9,46.
In addition, the administration of MP—a GC approved for the treatment of SCI—amplifies immunosuppression and, at higher doses, induces kidney damage without having any proven benefit in improving motor or sensory function24 . That is why several studies are still being carried out aimed at finding the best therapeutic strategy. Therefore, our goal in the present study was to determine whether Vitamin E, Zn, Se, and Cu supplementation preserves the number and proliferative function of T-lymphocytes—the main cells in the immune response—after acute SCI. These micronutrients are involved in the protection of immune cells through antioxidant mechanisms47 .
In addition to their antioxidant effects, these supplements can improve the activation and proliferation of T-cells25,48,49. The proliferative effect that we observed in supplemented animals could be explained by the production of IL-2, which could be induced by the components of the supplement26 . This cytokine is the main inducer of T-lymphocyte activation and proliferation. Supplementation helped to optimize T-lymphocyte response; this was observed in both the SI and lymphocyte count, which were similar between the supplemented and sham groups. These results suggest that the immunosuppression phenomenon induced by SCI could be circumvented by supplementation with trace elements and Vitamin E. In addition, the use of this therapeutic approach as an adjunctive treatment together with or even instead of MP could be contemplated. In any case, it will be important to first explore this supplementation strategy alone and the synergy when combining it with other therapies, as well as its pharmacokinetic, pharmacodynamic, and safety aspects, among others.
The beneficial effects of the supplementation therapy could be explained by the individual action of each one of the supplements. For example, different studies have reported an increase in lymphocyte proliferation after a supplementation period of 8 weeks with Vitamin E in old rodents. These same authors conducted other studies in which supplementation with Vitamin E using doses of 50 and 200 mg/kg increased the mitogenic responsiveness of T-lymphocytes by increasing the IL-2 production capacity from naïve T-cells, thereby promoting communication between immune cells46,50,51. In the present work, the recovery of T-cell-proliferative function was observed in a shorter time—after 2 weeks of supplementation—compared with previous investigations (8 weeks). This difference in time response could be due to the age-associated deterioration of T-cell-mediated immune response. Our study was performed in young animals, while the previously mentioned studies were done in old ones. Due to the age-related decline in T-cell-mediated immune response, lymphocytes from old rodents might need more stimulation time compared with those from young animals50 .
On the contrary, Zn supplementation can restore thymulin activity, which is involved in the differentiation, maturation, and enhancement of T-lymphocyte activities, thus increasing the number of T-cells and improving phagocytic activity and antigenic presentation to T-lymphocytes49,52,53. The supplementation with Se, an immunostimulant, can boost cellular immunity, improve T-lymphocyte count54 , and modify the expression of high-affinity IL2-R30,55,56. Finally, Cu is capable of enhancing the proliferation, differentiation, and activity of T-lymphocytes and improving the production of IL-246-56.
Together, the above observations support the results of our present investigation and suggest that a combined strategy of these supplements could improve T-cell function in SCI patients. This is the first time that this supplementation strategy has been used after acute SCI. The present results reveal that the supplementation strategy was able to maintain the number and functional capacity of T-lymphocytes with values quite similar to those presented by non-injured animals. This is a relevant finding as it implies that this supplementation therapy could prevent infectious complications, one of the main problems in SCI patients.
On the contrary, to verify the homogeneity of the injury among SCI animals, the BBB scale was performed. We noticed that supplemented animals also presented a better motor recovery compared with the non-supplemented ones. This is also an important finding, as it means that the supplementation therapy itself could enhance neuroprotection after SCI in addition to improving T-cell function restoration. This topic should be deeply investigated in future studies. By now, it is important to highlight that the motor recovery observed in supplemented animals could be the result of the antioxidant effect exerted by the supplements. In line with this, it has been reported that Vitamin E is a fat-soluble antioxidant that stops the production of peroxyl radicals and the oxidation of polyunsaturated fatty acids (PUFAs). In addition, Vitamin E improves bladder and motor recovery in SCI models57,58.
Zinc is capable of up- or down-regulating the immune response as, in addition to enhancing the activity of T-lymphocytes, it has anti-inflammatory effects. This alternative function results from the inhibition of the inducible nitric oxide synthase, decreasing the inflammatory effects caused by nitric oxide and, therefore, acute neurodegeneration secondary to neural injury59 . Zinc has also a direct modulatory effect on oxidative indicators such as glutathione peroxidase and malondialdehyde, thus further promoting antioxidant effects. Finally, research on the topic has reported clinical improvement and significant motor neuron preservation after SCI and Zn therapy60 . Thus, Zn is considered a predictor of clinical recovery after SCI as its levels are directly proportional to neurological impairment, especially in the first 24 h61,62.
Similarly, and through the action of different enzymes (glutathione peroxidases, thioredoxin reductases, and methionine sulfoxide reductase63,64), Se plays an important role in the homeostasis of the central nervous system65 . These enzymes participate as anti-inflammatory factors, regulating redox reactions that are essential during the secondary phase of SCI66,67. Furthermore, previous studies have proven that Se supplementation induces faster improvement in bladder control68 .
Last, Cu is used in various physiological mechanisms as a coenzyme to preserve homeostasis69 , protecting tissues against lipid peroxidation and oxidative reactions in several degenerative diseases. In a similar way to Vitamin E and Zn, it has been shown that the administration of copper sulfate in rats with SCI induces better neurological recovery, as well as a reduction in malondialdehyde and myeloperoxidase and an increase in superoxide dismutase and glutathione70 .
The hypotheses of the neuroprotective effect induced by antioxidants administered immediately after injury may be supported by previous studies in which antioxidant therapies combined with other drugs have shown increased protection of spinal cord tissue associated with enhanced motor recovery by preventing neural and glial apoptosis71,72. This topic should be investigated in detail in future studies.
According to our results and previous studies on the subject, supplementation with trace elements and vitamin E could be part of a therapy for patients with acute SCI, helping to re-establish T-cell function, avoiding complications, and promoting better motor recovery. Establishing the therapeutic window for this promising therapy should be the next step for research.
This study is the first approach to the evaluation of the immunological and neuroprotective effects induced by this supplementation therapy. The function of other cells, including B-lymphocytes, neutrophils, natural killer cells, and even the effect on a specific response or morphological recovery should be the aim of future investigations.