Immunosuppressant neurotoxicity in rat brain models: oxidative stress and cellular metabolism

Abstract

Coadministration of the calcineurin inhibitor cyclosporine (CsA) and the mTOR inhibitors sirolimus (SRL) or everolimus (RAD) increases the efficacy of immunosuppression after organ transplantation. Neurotoxicity of CsA is a major clinical problem. Our goal was to assess the effects of CsA, SRL, and RAD on brain cell metabolism. The studies included the comparison of immunosuppressant-mediated effects on glucose metabolism, energy production, and reactive oxygen species (ROS) formation in perfused rat brain slices, primary rat astrocytes, and C6 glioma cells. In brain slices and astrocytes, CsA inhibited Krebs cycle metabolism, while activating anaerobic glycolysis, most likely to compensate for the inhibition of mitochondrial energy production. SRL and RAD inhibited cytosolic glycolysis but did not cause changes in mitochondrial energy production. CsA + SRL inhibited Krebs cycle and glycolysis, thus reducing the ability of the cell to compensate for the negative effects of CsA on mitochondrial nucleoside triphosphate synthesis. In contrast to SRL at the concentrations tested, RAD reduced the CsA-induced ROS formation and antagonized CsA-induced effects on glucose and energy metabolism. Surprisingly, in C6 cells, SRL and RAD exposure resulted in high ROS concentrations without significant impairment of cell metabolism. Our results suggested that SRL enhances CsA-induced ROS formation and negative metabolic effects in brain cells, while RAD seems to antagonize the CsA effects. However, the three models showed different metabolic responses when challenged with the study drugs. In contrast to SRL, RAD enhances ROS formation in C6 glioma cells but has only minor effects on normal rat brain tissue.

Figure 1

Representative fluorescence recordings of brain slices treated with immunosuppressants and hydrogen peroxide (H₂O₂). Increase in fluorescence intensity, as measured by the angle α =α2-α1 (with α2: emission angle and α2>α1) correlates with the level of DCF in the cell and therefore with the ROS production. H₂O₂ was added after 90 seconds to verify loading of the cells with DCFH-dA, leading to a faster increase in fluorescence intensity.

Figure 2

(A) Formation of ROS in rat brain slices after perfusion with CsA, SRL, RAD alone or combinations of CsA with SRL or RAD (CSA: 500µg/L, SRL, RAD: 100 µg/L) for 3 hours or with 1 mM H₂O₂ for 30 minutes. One-way ANOVA confirmed significant differences among treatments (p<0.0001). (B) ROS-formation in primary astrocytes and C6 glioma cells after incubations with the same concentrations of the immunosuppressants for 3.5 hours or with 0.1 mM H₂O₂ for 30 minutes. All data presented as means ± standard deviations of three to six experiments. *p< 0.05, **p<0.005 versus controls. One-way ANOVA in combination with Tukey’s post-hoc test confirmed a significant difference between SRL and RAD versus all other treatment groups (p<0.0001). In the primary astrocytes model, SRL versus CsA+SRL group was significantly lower with p<0.05, n=5.

Figure 3

Representative (A) ¹H–NMR and (B) ³¹P-NMR spectra of water-soluble metabolites in rat brain slices from vehicle controls and rat brain slices exposed to the study drugs and their combinations. Brain slices were perfused with 500 µg/L CsA in combination with either 100 µg/L SRL or 100 µg/L RAD for 3 hours. Changes in metabolite concentrations are marked by arrows. Abbreviations: Ala: alanine, Asp: aspartate, Cholines: choline-containing phospholipids, Cr: creatine, GABA: γ-amino butyric acid, Gln: glutamine, Glc: glucose, Glu: glutamate, GPC: glycerophosphocholine, GPE: glycerophosphoethanolamine, Lac: lactate, myo-Ins: myo-inositol, NAA: N-acetyl-aspartate, NAD(H): nicotineamide adenine dinucleotides, NDP: nucleoside diphosphates, NTP: nucleoside triphosphates, PC: phosphocholine, PCr: phosphocreatine, PE: phosphoethanolamine, Pi: inorganic phosphate, Tau: taurine, Val/Leu/Isoleu: valine/leucine/isoleucine.

Figure 3

Representative (A) ¹H–NMR and (B) ³¹P-NMR spectra of water-soluble metabolites in rat brain slices from vehicle controls and rat brain slices exposed to the study drugs and their combinations. Brain slices were perfused with 500 µg/L CsA in combination with either 100 µg/L SRL or 100 µg/L RAD for 3 hours. Changes in metabolite concentrations are marked by arrows. Abbreviations: Ala: alanine, Asp: aspartate, Cholines: choline-containing phospholipids, Cr: creatine, GABA: γ-amino butyric acid, Gln: glutamine, Glc: glucose, Glu: glutamate, GPC: glycerophosphocholine, GPE: glycerophosphoethanolamine, Lac: lactate, myo-Ins: myo-inositol, NAA: N-acetyl-aspartate, NAD(H): nicotineamide adenine dinucleotides, NDP: nucleoside diphosphates, NTP: nucleoside triphosphates, PC: phosphocholine, PCr: phosphocreatine, PE: phosphoethanolamine, Pi: inorganic phosphate, Tau: taurine, Val/Leu/Isoleu: valine/leucine/isoleucine.

Figure 4

Representative ¹³C-NMR spectra of water-soluble metabolites from control and immunosuppressant-treated C6 glioma cells. The cells were incubated with 5mM [1-¹³C]glucose for 3 hours. Treatment with immunosuppressants occurred at the same time (500 µg/L CsA, 100 µg/L SRL or 100 µg/L RAD alone or in combination with CsA for 3 hours and addition of 1 mM H₂O₂ for 30 minutes). Changes in metabolites are marked by arrows. Abbreviations: Ala: alanine, Asp: aspartate, Gln: glutamine, Glc: glucose, Glu: glutamate, Lac: lactate, myo-Ins: myo-inositol (natural abundance).

Figure 5

Changes in volume- and osmoregulators after incubation with immunosuppressants as calculated from ¹H-NMR spectra. A) After perfusion of rat brain slices with immunosuppressants (500 µg/L CsA, 100 µg/L SRL or 100 µg/L RAD alone or combinations with CsA) for 3 hours or with 1 mM H₂O₂ for 30 minutes. B) After incubation of primary astrocytes with immunosuppressants (the same concentration as in rat brain slices) for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. C) After incubation of C6 cells with the same concentrations of the immunosuppressants for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. All values are given as µmol/g (tissue for brain slices and protein for astrocytes and C6 cells) and are means ± standard deviations of three to six experiments. One-way ANOVA with Tukey’s post-hoc analysis was performed and the significance between groups is presented within the graphs. Significance levels compared with controls are presented: *p<0.05, **p<0.005, ***p<0.001. Abbreviations: GABA: γ-aminobutyric acid, Hypotau: hypotaurine, Ins: myo-inositol, NAA: N-acetylaspartic acid, Tau: taurine.

Figure 5

Changes in volume- and osmoregulators after incubation with immunosuppressants as calculated from ¹H-NMR spectra. A) After perfusion of rat brain slices with immunosuppressants (500 µg/L CsA, 100 µg/L SRL or 100 µg/L RAD alone or combinations with CsA) for 3 hours or with 1 mM H₂O₂ for 30 minutes. B) After incubation of primary astrocytes with immunosuppressants (the same concentration as in rat brain slices) for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. C) After incubation of C6 cells with the same concentrations of the immunosuppressants for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. All values are given as µmol/g (tissue for brain slices and protein for astrocytes and C6 cells) and are means ± standard deviations of three to six experiments. One-way ANOVA with Tukey’s post-hoc analysis was performed and the significance between groups is presented within the graphs. Significance levels compared with controls are presented: *p<0.05, **p<0.005, ***p<0.001. Abbreviations: GABA: γ-aminobutyric acid, Hypotau: hypotaurine, Ins: myo-inositol, NAA: N-acetylaspartic acid, Tau: taurine.

Figure 5

Changes in volume- and osmoregulators after incubation with immunosuppressants as calculated from ¹H-NMR spectra. A) After perfusion of rat brain slices with immunosuppressants (500 µg/L CsA, 100 µg/L SRL or 100 µg/L RAD alone or combinations with CsA) for 3 hours or with 1 mM H₂O₂ for 30 minutes. B) After incubation of primary astrocytes with immunosuppressants (the same concentration as in rat brain slices) for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. C) After incubation of C6 cells with the same concentrations of the immunosuppressants for 3 hours or with 0.1 mM H₂O₂ for 30 minutes. All values are given as µmol/g (tissue for brain slices and protein for astrocytes and C6 cells) and are means ± standard deviations of three to six experiments. One-way ANOVA with Tukey’s post-hoc analysis was performed and the significance between groups is presented within the graphs. Significance levels compared with controls are presented: *p<0.05, **p<0.005, ***p<0.001. Abbreviations: GABA: γ-aminobutyric acid, Hypotau: hypotaurine, Ins: myo-inositol, NAA: N-acetylaspartic acid, Tau: taurine.