Chuan Pu Lee

Mitochondrial dysfunction after experimental and human brain injury and its possible reversal with a selective N-type calcium channel antagonist (SNX-111).

We have recently demonstrated in a rat model that traumatic brain injury induces perturbation of cellular calcium homeostasis with an overload of cytosolic calcium and excessive calcium adsorbed on the mitochondrial membrane, consequently the mitochondrial respiratory chain-linked oxidative phosphorylation. was impaired. We report the effect of a selective N-type calcium channel blocker, SNX-111 on mitochondrial dysfunction induced by a controlled cortical impact. Intravenous administration of SNX-111 at varying times post injury was made. The concentration titration profile revealed SNX-111 at 4 mg kg -1 to be optimal, and the time window to be administration at 4 h post-injury, in line with that reported on the effect of SNX-l11 in experimental stroke. Under optimal conditions, SNX-111 significantly improved the mitochondrial respiratory chain-linked functions, such as the electron transfer activities with both succinate and NAO-linked substrates, and the accompanied energy coupling capacities measured ...

Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI‐induced formation of cortical lesion is determined by the expression levels of copper‐zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD‐linked bioenergetic capacity in brain mitochondria of wild‐type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild‐type mice. Inclusion of the calcium‐chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.

P/O ratios reassessed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD-linked substrates.

The efficiency of ATP synthesis coupled to cell respiration, commonly referred to as the P/O ratio, has been the subject of extensive studies for more than 50 years. The general conclusion from these studies is that respiring mitochondria can convert external ADP to ATP at a maximal P/O ratio of 3 for NAD‐linked substrates and 2 for succinate. However, in recent years the validity of these “integral” values has been questioned on both mechanistic and thermodynamic grounds, and a mechanistic P/O ratio of 2.5 for NAD‐linked substrates and 1.5 for succinate have been concluded on the basis of experiments with isolated mitochondria. These values have been widely adopted in the scientific literature, including several recent textbooks. In this paper we report that under optimal conditions with respect to preparation and assay procedures, the P/O ratios obtained with isolated rat liver mitochondria consistently exceed 2.5 with NAD‐linked substrates and 1.5 with succinate. These results, although not excluding “nonintegral” P/O ratios due to various energy‐dissipating side reactions, warrant caution in accepting the reported lower values and, in general, in referring to mechanistic considerations unless the underlying molecular mechanisms are understood.—Lee, C. P., Gu, Q., Xiong, Y., Mitchell, R. A., Ernster, L. P/O ratios reassessed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD‐linked substrates. FASEB J. 10, 345‐350 (1996)

Erythropoietin improves brain mitochondrial function in rats after traumatic brain injury.

Abstract Mitochondria play a central role in cellular energetics, calcium homeostasis and apoptosis. Our previous study demonstrates traumatic brain injury induces brain mitochondrial dysfunction after injury. Preservation and/or restoration of mitochondrial function may be one of the strategies for neuroprotection. Erythropoietin, a hormone for erythropoiesis, also provides tissue protection against traumatic brain injury and stroke. The present study was undertaken to evaluate the effect of erythropoietin on traumatic brain injury-induced brain mitochondrial dysfunction. Traumatic brain injury decreased rates of respiration at the active state (state 3), increased that at the resting state (state 4) and consequently decreased respiratory control index (state 3/state 4 ratio) and the efficiency of ATP synthesis (the amount of ADP phosphorylated by inorganic phosphate divided by the amount of oxygen consumed during state 3 respiration). Erythropoietin administered intraperitoneally 30 minutes post-injury at 1000 U/kg partially improved mitochondrial function at day 1 post-injury. However, erythropoietin-induced improvement was not sustained at day 7 post-injury. Erythropoietin at 2000 or 5000 U/kg restored states 3 and 4 examined at day 1 post-injury to the sham levels. Consequently, the energy coupling capacities, such as respiratory control index and/or the efficiency of ATP synthesis, were also improved. The beneficial effect of erythropoietin at these doses persisted for at least 7 days post-injury. The beneficial effect of erythropoietin on brain mitochondrial function was observed with a wide therapeutic window from 5 minutes to 6 hours post-injury. Our data, for the first time, demonstrate that erythropoietin treatment restores brain mitochondrial function after traumatic brain injury, which will enhance cellular energy generation and reduce oxidative stress, strongly supporting erythropoietin as a promising agent for the therapeutic treatment of traumatic brain injury.

Polarographic Assays of Mitochondrial Functions

Publisher Summary This chapter describes a typical protocol for measuring respiratory rates and its accompanied oxidative phosphorylation, which are catalyzed by isolated mitochondria. The polarographic technique for measuring mitochondrial oxidative changes requires the following four basic components: an oxygen electrode, a closed reaction vessel, a constant voltage source, and a recorder. The reaction vessel can be constructed of glass, Plexiglas, or polycarbonate, and the design and the size of the vessel vary, depending on the requirements of the system under investigation. The temperature of the reaction chamber is constantly maintained with a water jacket. Constant stirring of the contents of the reaction chamber is accomplished by a disk-shaped, plastic-encased magnet located at the bottom of the reaction chamber. It is imperative that closed reaction vessels are utilized so that air bubbles are not trapped and back diffusion of oxygen is reduced to a minimum. To facilitate this, the bottom of the plug is uneven to ensure that no air bubbles are trapped.