Introduction

Immunosuppressive drugs are essential in prolonging allograft survival. FK506 is a macrolide lactane antibiotic that was introduced in 1989 by Starzl as an immunosuppressive agent.1 FK506 binds intracellular receptor FKB-12, immunophilin that is rotamase, and this complex interferes with intracellular calcium-dependent signal transduction pathways, processes that are central to T-cell activation.2,3,4 The FK506/FKB12 complex inhibits calcium- and calmodulin-dependent protein phosphatase, calcineurin, which that prevents the dephosphorylation of nuclear factor of activated T cell (NF-AT), leading to inactivation of IL-2 gene transcription and immunosuppression.5,6 In vitro, it has been shown that FK506 also inhibits lymphocyte migration, including T cells, in response to IL-2 or IL-8 chemotactic signaling.7

This study investigated the ability of immunosuppressant FK506 to reduce the free radical count in spinal cords after the experimental traumatic spinal cord injury (SCI) in rats.

Materials and methods

A total at 28 adult male Wistar albino rats weighing between 250 and 310 g were used in this experiment. The animal experimentation was conducted in conformity with the ‘Guide for the care and use of laboratory animals’ (National Institutes of Health publication No: 86–23). Anaesthesia was induced with (10 mg/kg) xylazine and (50 mg/kg) ketamine hydrochloride. C6-T2 laminectomy was performed. Dura mater was exposed and the extradural spinal cord at T1 was compressed for 1 min with an aneurysm clip (closing force 70 g), which caused complete paraplegia that remained unchanged during the course of the study. After the injury, the paraspinal musculature and subcutaneous tissues were closed with absorbable suture material. The paraplegic animals were then divided into four groups. In the first group (G1) (n=8), the SCI animals received a daily subcutaneous injection of FK506 treatment of 1 mg/kg in 3 days. In group 2 (G2) (n=8), FK506 was administered 1 mg/kg once per day for 3 days started after the intraperitoneal methylprednisolone administration following the SCI. In group 3 (G3) (n=6), each animal received two doses of methylprednisolone 30 mg/kg. Rats in the control group (G4) (n=6) received no medication after the SCI. The first dosage of FK506 and methylprednisolone was administered within 30 min of surgery; the final and the second dosage of MP was administered 24 h later via intraperitoneally. The animals were housed in a temperature-controlled room (26–28°C) for 3 days, one per cage. Bladders were manually expressed three times per day. All animals were killed by decapitation 72 h after spinal cord injury. Injured segment (1 cm) of spinal cord was dissected and stored at −20°C for assays at malondialdehyde (MDA). In order to evaluate the histopathological changes in the animals that received FK506 and MP, we studied another eight rats (n=4 each group) that were subjected to the same procedure in G1 and G3.

Sources of supplies and equipment

FK506 (Prograf, 5 mg/ml) was obtained from Fujisawa Ireland Co. Kerry (Ireland). Methylprednisolone sodium succinate (Prednol-L 20 mg) was obtained from Mustafa Nevzat Co. (Istanbul, Turkey). The aneurysm clip was purchased from Aesculap AG (FE 751 K with a closing force at 70 g, Tuttlingen, Germany).

Measurement of tissue MDA levels

Tissue homogenates were prepared in a ratio of 1 g of wet tissue to 9 ml of 1.15%. KCl by using a Teflon homogeniser.

Procedure: In all, 0.2 ml of 10% (w/v) tissue homogenate was added to 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH, and 1.5 ml of 0.8% aqueous solution of TBA. The mixture was made up to 4.0 ml with distilled water, and then heated at 95°C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15:1 v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbency at 532 nm was measured. 1,1,1,3-Tetraethoxy propane was used as the standard. The results were expressed as nmol MDA/g wet tissue.8

Histopathology

For histopathologic examination, sections obtained from formalin-fixed, paraffin-embedded tissue samples were used. The samples were stained using haematoxylin and eosin to evaluate tissue changes after SCI.

Statistical analysis

Data were expressed as the mean±standard error of the mean (Table 1). Statistical differences between the four groups and triple comparison for G1–G2–G3 were compared using the Kruskal–Wallis variance analysis. A probability value of less than 0.05 was considered statistically significant. In the comparison of groups, two-by-two Mann–Whitney u-test was used (in case the P-value was <0.05).

Table 1 Mean tissue MDA levels (nmol/g wet tissue) of each group were identified
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Results

Figure 1 shows the effect of SCI with and without the drug administration on MDA levels in spinal cord homogenate, measured 72 h after the trauma. Although comparison between the four groups showed statistical difference (P=0.01), the intergroup comparison between G1–G2–G3 revealed no difference (P=0.547). For G1–G2, G1–G3, G2–G3, the MDA values were not different statistically (P=0.528, 0.30 and 0.560, respectively). For G1–G4, G2–G4 and G3–G4, the MDA values were significantly different (P=0.01, 0.07 and 0.06, respectively). FK506, FK506 with MP and MP administration (G1, G2, G3) attenuated the decrease in the MDA level noted in the trauma-only group (G4).

Figure 1
figure 1

Bar graph showing MDA levels (nmol/g wet tissue) in spinal cord homogenate between the four groups following the compression injury. Statistically there was no difference found between the G1–G2–G3 (P=0.547, Kruskal–Wallis variance analysis)

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Histopathologic examinations of G1 and G3 were carried out. In G3, there were signs of tissue degeneration and necrosis that was evident in the posterior and central regions of the spinal cord, with severe oedema where the necrotic regions also included polymorphonuclear leucocytes (Figure 2). In G1, histopathological changes in the spinal cord were similar to those observed in G3, but the oedema pattern was relatively less and no signs of inflammatory cells in the necrotic regions were determined (Figure 3).

Figure 2
figure 2

In G3, necrotic white matter of the spinal cord tissue with oedema and polymorphonuclear leucocyte infiltration were observed. H&E, original magnification × 200

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Figure 3
figure 3

Necrotic regions of G1 display relatively less oedema than G3 and no polymorphonuclear leucocytes were examined. H&E, original magnification × 100

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Discussion

The central nervous system possesses high levels of polyunsaturated lipids and metabolic rate, and, on the other hand, low antioxidant enzyme activity when compared to other tissues. This makes the free radical formation hazardous.9,10,11 In SCI, both primary and secondary injuries are involved in the physiopathology. Activation of endogenous substances prompted the secondary injury. There have been a number of studies performed in an attempt to identify the pathophysiological mechanisms occurring after traumatic SCI.12,13,14,15 Lipid peroxidation is one of the main pathological mechanisms involved in secondary damage after a SCI. The primary autodestructive event is started by the hydrolysis of fatty acids from membrane phospholipids, leading to cellular damage.16 Reduction of lipid peroxidation with methylprednisolone treatment in SCI seemed to be the main factor in the improvement of the outcome.17,18,19 Another key factor in the secondary injury mechanism was Ca+2 ion. Following the trauma or ischaemia, Ca+2 influx plays an important role in the pathogenesis of neural injury.20,21 In contused spinal cords, Happel et al22 showed that time promotes encouraged increases in calcium content in the total tissue. Banik et al23 reported that perfusion onto the spinal cord with high concentrate Ca+2-included solutions causes similar biochemical and histopathological changes, mimicking trauma.

There are different mechanisms discussed in the neuroprotective effect of FK 506. In T lymphocytes, FK506 binds immunophilin FKB12 that inhibits the calcineurin. This action involves the immunosuppresive effect of the drug.24,25 Calcineurin is reported to regulate calcium channel activity that is proposed for calcium in neurodegeneration.26,27 Ca+2 influx activates a Ca+2-sensitive protein phosphatase, calcineurin, which dephosphorylates the channel, thereby inactivating it.28 Another well-known effect of FK506 is reducing the superoxide radical formation in neutrophils dose dependently.29 The inhibition of dephosphorylation of nitric oxide synthase reduces the formation of nitric oxide (NO) and NO-dependent free radical formation that leads to cellular damage.30,31 Calcineurin also regulates nitric oxide synthase (NOS) activity in a Ca+2-dependent manner.32 Phosphorylated NOS is inactive and its dephosphorylation by calcineurin elicits the formation of nitric oxide, which can then react with superoxide radicals to form highly toxic peroxynitrite radicals.30,32 Toung et al33 reported that FK506 failed to reduce NO production in vivo during brain ischaemia, which indicates a neuroprotective mechanism of the drug, rather than reducing NO. However, Dowson et al34 suggested that FK506 shows its cerebroprotective actions by inhibiting NO production, indirectly preventing the calcineurin-mediated dephosphorylation of NOS.30 In the presented study, no statistical difference was observed in measured MDA levels between the glucocorticoid and glucocorticoid with FK506-combined and FK506-received groups. These results show that administration of FK506 is able to diminish the production of LP in injured spinal cords. This result might be postulated as FK506 could decrease the free radical formation following the traumatic SCI by preventing the calcineurin-mediated dephosphorylation of NOS. However, the calcineurin level in the spinal cord is low according to the data by the measurement of the enzyme-immunoassay method, whereas the putamen, caudate nucleus and hippocampus have high levels.34 In this study, measured LP levels between G1–G2–G3 indicated no statistical difference. It may be implicated that FK506 is effective as glucocorticoids in the inhibition of SCI, inducing LP to diminish neural tissue damage.

Bavetta et al35 examined the effects of FK506 and GPI 1046 on the responses to partial transection of ascending spinal dorsal column axons in rats. In their study, they also compared the effect of MP and concluded that the combination of FK506 and MP is more significantly effective than MP alone in protecting axons but not more effective than FK506 alone. FK506 is considered to improve neural damage by inhibition of neuronal calcineurin activity, and the inhibitory effects for calcineurin on inflammatory cells may contribute to improvement.36 Following the SCI, inflammatory reaction with polymorphonuclear leucocytes and other inflammatory cells causes cytokine-induced macrophage migration that increases vascular permeability, favouring oedema.37 In the presented study, a histopathological absence of polymorphonuclear leucocytes and relatively less oedema formation in G1 indicated the effectiveness of FK506 on the inflammatory cells, eosinophils, and neutrophils.38,39,40 As a conclusion, in SCI, FK506 could be used to inhibit the inflammatory reaction, decreasing the formation of free radicals and lipid peroxidation, which is valuable in neuroprotection and in maintaining the cord function.