Abstract View

Introduction: Mitochondrial complex I (MC-1) function is essential for regulating neurotransmission and supporting brain function, in which its dysregulation has been implicated in multiple neurological conditions.^1,2,3, [¹⁸F]BCPP-EF is an  MC-1 radioligand and  is currently under clinical trials.^4–6 Currently, there is no standardized or optimized protocol for [¹⁸F]BCPP-EF autoradiography (ARG), making it difficult to interpret and compare results across experiments and to establish the reliability of findings for broader applications of this tracer. Herein, this work looks to investigate the effects of incubation buffer composition and additives on [¹⁸F]BCPP-EF binding in rodent brain sections to determine the optimal conditions for [¹⁸F]BCPP ARG.

Methods: [¹⁸F]BCPP-EF was synthesized on the GE Tracerlab FX2N automated synthesis module from the corresponding tosylate precursor using [¹⁸F]KF/K₂₂₂/K₂CO₃ in DMSO at 100°C for 10min (Fig. 1a), affording [¹⁸F]BCPP-EF with high radiochemical purity (RCP) of 99.9±0.1% (n=2) and molar activity of 33.65Gbq/µmol (d.c. E.O.B). Thin-section ARG assays were performed using fresh-frozen postmortem mouse and rat brain sections (n_brain=1 n_slice=2-5). Non-specific binding was determined through homologous blocking using 10µM BCPP-EF standard. Incubation conditions included 50 mM Tris-HCl buffer (pH 7.4), Tris-HCl supplemented with 2% vitamin C (Tris + VitC), Tris-HCl supplemented with 2% VitC and 5% ethanol (Tris-HCl + VitC + EtOH), standard binding buffer (BB) (51.4 mM Tris, 140 mM NaCl, 1.48 mM MgCl₂·6H₂O, 5.37 mM KCl, 1.53 mM CaCl₂·2H₂O), and binding buffer supplemented with 2% VitC (BB + VitC).  Radioactivity signals were quantified using calibrated digital autoradiography.

Results: In mouse brain tissues, Tris-HCl based incubations resulted in higher specific binding (Tris HCl only: 1.01 ± 0.35µCi/g (n=5); Tris-HCl + VitC: 0.95 ± 0.10µCi/g (n_slice=4); Tris-HCl + VitC + EtOH: 1.10 ± 0.13µCi/g (n=4)) compared to incubation using binding buffer (BB: 0.22 ± 0.06µCi/g (n=3); BB + VitC: 0.20 ± 0.06µCi (n=5)) (ig. 1c). A similar trend was also observed in rat brain tissue when comparing specific binding values in Tris-HCl based buffers (Tris-HCl: 0.19 ± 0.05µCi/g (n_slice=2);  Tris-HCl + VitC: 0.33 ± 0.03µCi/g (n_slice=2); Tris-HCl + VitC + EtOH: 0.18 ± 0.02µCi/g(n_slice=2)) compared to binding buffer-based incubations (BB: 0.02 ± 0.01µCi/g (n_slice=2); BB + VitC: 0.15 ± 0.01µCi(n_slice=2)) (Fig. 1d). However, rat brain showed an increase in specific binding with the addition of VitC in both Tris-HCl and BB, while the ethanol resulted in an apparent decrease in specific binding.

Conclusion: This preliminary optimization study demonstrated that incubation buffer composition can substantially influence [¹⁸F]BCPP-EF binding signals in ARG. Tris-HCl conditions produced higher apparent specific binding than standard binding buffer in both rodent sections, with the addition of vitamin C in Tris-HCl showing enhanced binding in rat sections. This data supports the need for further optimization and standardization of [¹⁸F]BCPP-EF ARG protocols before broader application of this tracer to mitochondrial dysfunction diseases.

References

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