Euro Neuropharmacology 2018: PET imaging of ischemia-induced impairment of mitochondrial complex I function in living brain - Hideo Tsukada - Hamamtsu Photonics K K

The mitochondrial respiratory chain which is a major site of ATP production in eukaryotes. This organelle plays a key role in the center of the apoptotic signaling pathway. It has five complexes, mitochondrial complex I is the first enzyme of the respiratory electron transport chain. MC-I take two electrons from NADH and transfer them to ubiquinone in the inner-mitochondrial membrane. Mitochondrial complex I uses the energy released and move four protons across the membrane, creating charge separation across the membrane. Mitochondrial dysfunction contributes pathophysiology of acute neurologic disorders and neurodegenerative diseases. Mitochondria considered the main intracellular source of reactive oxygen species in cells and the main target of ROS-mediated damage. Ischemia cause mitochondrial alterations which favors ROS production when the oxygen concentration is re-established by reperfusion. After reperfusion from the middle cerebral artery (MCA) occlusion (MCAO) for 3 hours (3-hour MCAO), postischemic hyperperfusion was observed in the neocortical area of monkeym(living) brain. When ischemic tissue is reoxygenated, electron transport through the respiratory chain is impaired. Burst of ROS generation, because of depletion of ADP during ischemia during the first minutes of reoxygenation. MC-I function in the living brain using positron emission tomography (PET) and synthesized several novel PET probes, and assured that 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one was most preferable for MC-I imaging in the living brain of rat. and monkey. PET imaging of rat indicates high uptake and long retention in the brain. PET probes for MC-I applicable for the imaging of neuronal damage in living brain. Studies suggest that 18F-BCPP-EF could be useful to detect ischemic neuronal damage at the subacute phase 7 days after ischemic at which time unexpectedly higher 18F-fluoro-2-deoxy-d-glucose (FDG) uptake was observed in the damaged area than in the contralateral intact area. The FDG-PET is a unique technique for quantitative imaging of the regional cerebral metabolic rate of glucose (rCMRglc) in living brain, that is based on the assumption that rCMRglc reflects energy metabolism via oxidative phosphorylation. The unexpectedly high uptake of 18F-FDG in ischemia-damaged areas suggested that 18F-FDG taken up into not only normal tissues but also inflammatory regions with microglial activation. Recent PET research indicates neurodegenerative disorders showed neuroinflammation with microglial activation, PET probes to image MC-I activity would be favorable for more accurate assessment of neurodegenerative damage using PET. Recent Study validated that 18F-BCPP-EF, a novel PET probe for MC-I activity, as a particular marker of ischemia-induced neuronal death without being disturbed by inflammation. The binding properties of 18F-BCPP-EF were assessed in an ischemic brain model of living monkey by using high-resolution PET, and its cerebral uptake was compared with the regional cerebral blood flow (rCBF), regional cerebral metabolism of oxygen (rCMRO2), 11C-flumazenil (11C-FMZ) binding to the central-type benzodiazepine receptor (CBR), 11C-PBR28 binding to the translocator protein, and regional cerebral blood flow (rCMRglc) measured with 18F-FDG at Day-7/8 after 3-hour MCAO ischemic in the monkey brain. Animals were maintained in accordance with the recommendations of the US National Institutes of Health and the guidelines of the Central Research Laboratory, Hamamatsu Photonics. The research studies were approved by the Ethical Committee of the Central Research Laboratory, Hamamatsu Photonics. Seven male Cynomolgus monkeys (Macaca fascicularis) at the age between 4.5 and 5.3 years old with body weights which is ranging from 4.5 to 5.8 kg were used for the PET measurements. Magnetic resonance (MR) images (MRIs) of the monkeys were obtained with a 3.0-T MR imager (Signa Excite HDxt 3.0T; GE Healthcare Japan, Tokyo, Japan) using a 3D-Spoiled Gradient Echo sequence (176 slices with a 256 × 256 image matrix, slice thickness/spacing of 1.4/0.7 mm, echo time: 3.4 to 3.6 ms, repetition time: 7.7 to 8.0 ms, inversion time: 400 ms, and flip angle, 15°) under pentobarbital anesthesia. MR imaging have not conducted after ischemic insult because MRI system located outside the radiation regulated area. Japanese low for the radiation did not allow us to bring out the animals injected even positron emitters with short half-lives from radiation regulated area. Isoflurane and pancronium were purchased from Dainippon Pharmaceutical respectively. Rabbit anti-Iba1 polyclonal antibody was from Wako Pure Chemical Industry. Mouse anti-NeuN monoclonal antibody and EnVision were obtained from Millipore and DAKO respectively. Precursors of 18F-BCPP-EF, 11C-FMZ, and 11C-PBR28, and their corresponding cold compounds, were obtained from NARD Institute. Mannose triflate and Kryptofix222 were obtained from ABX and Merck respectively.

Immunohistochemical assessments were performed with brains sampled at end of the week after ischemic using rabbit anti-Iba1 polyclonal antibody and mouse anti-NeuN monoclonal antibody. Brains were perfused transcardially with saline, then 4% paraformaldehyde in 0.1 mol/L sodium phosphate (pH 7.4) using anesthesia with overdose sodium pentobarbital. The brains were removed and postfixed overnight at 4°C in 4% paraformaldehyde, then frozen in dry ice powder and sliced into 20-μm-thick coronal sections with a cryostat. The sections were mounted on slide glass, and incubated in phosphate buffer saline containing 0.1% Triton X-100 containing 5% normal goat serum for 30 minutes and reacted with antibodies at 4°C overnight with rabbit anti-Iba1 polyclonal antibody or mouse anti-NeuN monoclonal antibody. The slides were then washed in phosphate buffer saline containing 0.1% Triton X-100 and incubated with EnVision plus reagents for rabbit or mouse (DAKO) for 30 minutes at room temperature. The sections treated with 0.02% 3,3′-diaminobenzidine and 0.006% H2O2 in 50 mmol/L Tris-HCl buffer (pH 7.6). The slides finally counterstained with hematoxylin. The brain slides scanned with NanoZoomer. The current results showed that the ischemic-induced infarct areas (ROIInfarct), described as region with rCMRO2 of <40% of intact side at the end of the week. Studies proved that lower VT of 18F-BCPP-EF than that in ROIPBR. The inclusion of ROIInfarct in ROIPBR, result suggested that inflammatory region includes infarct and peri-infarct regions, which region might be curable remaining high rCMRO2. In hyperperfused areas, decreased rCMRO2 was observed that reperfusion-related ROS production may in part contribute to neuronal damage of reperfusion injury. Mitochondria considered the main intracellular source of ROS and the main target of oxyradical-mediated damage. MC-I exhibits lower activity compared with the other respiratory chain complexes, as it is a limiting factor in the regulation of oxidative phosphorylation. As the decrease in the MC-I activity induced in ischemic/reperfused brain should be associated with a decline in mitochondrial respiration. These factors could reasonably explain the current result of a good correlation between VT of 18F-BCPP-EF and rCMRO2, which is a conventional indicator of mitochondrial respiration.

NOTE: This work is partly presented at 10th World Congress on Neuropharmacology August 28-29, 2018 Paris, France.

Author(s): Hideo Tsukada

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