HYPERBARIC OXYGEN THERAPY PROTECTS AGAINST MITOCHONDRIAL DYSFUNCTION AND DELAYS ONSET OF MOTOR NEURON DISEASE IN WOBBLER MICE K. R. DAVE, a R. PRADO, a R. BUSTO, a A. P. RAVAL, a W. G. BRADLEY, a,b D. TORBATI c AND M. A. PE ´ REZ-PINZO ´ N a,b * a Department of Neurology, D4-5, University of Miami School of Med- icine, P.O. Box 016960, Miami, FL 33101, USA b Department of Neuroscience, University of Miami School of Medicine, Miami, FL 33101, USA c Division of Critical Care Medicine, Miami Children’s Hospital, Miami, FL 33155, USA Abstract—The Wobbler mouse is a model of human motor neuron disease. Recently we reported the impairment of mito- chondrial complex IV in Wobbler mouse CNS, including motor cortex and spinal cord. The present study was designed to test the effect of hyperbaric oxygen therapy (HBOT) on (1) mito- chondrial functions in young Wobbler mice, and (2) the onset and progression of the disease with aging. HBOT was carried out at 2 atmospheres absolute (2 ATA) oxygen for 1 h/day for 30 days. Control groups consisted of both untreated Wobbler mice and non-diseased Wobbler mice. The rate of respiration for complex IV in mitochondria isolated from motor cortex was improved by 40% (P<0.05) after HBOT. The onset and progres- sion of the disease in the Wobbler mice was studied using litters of pups from proven heterozygous breeding pairs, which were treated from birth with 2 ATA HBOT for 1 h/day 6 days a week for the animals’ lifetime. A “blinded” observer examined the onset and progression of the Wobbler phenotype, including walking capabilities ranging from normal walking to jaw walking (unable to use forepaws), and the paw condition (from normal to curled wrists and forelimb fixed to the chest). These data indi- cate that the onset of disease in untreated Wobbler mice aver- aged 364.3 days in terms of walking and 405.7 days in terms of paw condition. HBOT significantly delayed (P<0.001 for both paw condition and walking) the onset of disease to 598.2 days (in terms of walking) and 637.6 days (in terms of paw condi- tion). Our data suggest that HBOT significantly ameliorates mitochondrial dysfunction in the motor cortex and spinal cord and greatly delays the onset of the disease in an animal model of motor neuron disease. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: amyotrophic lateral sclerosis, neurodegeneration, mitochondrial dysfunction, hyperbaric oxygen therapy, motor system diseases, spinal muscular atrophy. Motor system diseases (MSD) such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are diseases that have been recognized for nearly 150 years. ALS is age dependent (onset in middle life) and results in degeneration of both upper and lower motor neurons of the cortex and brain stem (Brownell et al., 1970). SMA has an early age of onset (fetal period to several months of life), is inherited in an autosomal recessive pattern and affects the anterior horn cells of the spinal cord (Soler-Botija et al., 2002). The pathological mechanisms of most types of MSD remain uncertain, and currently there is no treatment that can significantly delay the progression of the disease. Riluzole, an antiglutamate agent, is the only drug currently known to have any proven effect on the progression of ALS, slowing it by 20%. Wobbler mice have been used as models of MSD (Duchen and Strich, 1968; Pioro and Mit- sumoto, 1995; Pioro et al., 1998). There are a number of theories on the etiology of MSD (Bradley and Krasin, 1982; Lange et al., 1983; Bradley, 1996), including oxidative damage (Browne and Beal, 1994). This is supported by observations indicating muta- tion of the Cu, Zn superoxide dismutase (SOD-1) gene in approximately 20% of patients with familial ALS (Siddique et al., 1996). Mitochondria are the major intracellular source for free radical generation (Cadenas and Davies, 2000; Loschen et al., 1971; Raha and Robinson, 2001). Mitochondrial abnormalities have been demonstrated in skeletal muscle, hepatocytes, motor cortex and spinal cord of patients with ALS, SMA, as well as in animal models (Atsumi, 1981; Browne et al., 1998; Fujita et al., 1996; Hirano et al., 1984; Nakano et al., 1987; Okamoto et al., 1980; Salviati et al., 2002; Sasaki and Iwata, 1996; Swerd- low et al., 1998, 2000; Vielhaber et al., 1999; Wiedemann et al., 1998; Xu et al., 2001). We have recently reported mitochondrial respiration dysfunction at the levels of com- plex I, III and/or IV in the brain, motor cortex and/or spinal cord of Wobbler mice (Xu et al., 2001; Dave et al., unpub- lished observations) and of complex IV in the brain and spinal cord of the SOD-1 transgenic mouse (Kirkinezos et al., 2001). Mitochondrial dysfunction generates reactive oxygen species, which then promotes oxidative stress. There is indirect evidence for oxidative damage in ALS patients. For example, the levels of 8-hydroxy-guanosine, an indicator of oxidative damage to nuclear and mitochon- drial DNA (Ferrante et al., 1997), and protein carbonyl *Correspondence to: M. A. Pe ´ rez-Pinzo ´ n, Department of Neurology, D4 –5, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA. Tel: +1-305-243-7698; fax: +1-305-243-5830 (M. A. Pe ´ rez-Pinzo ´n). Abbreviations: ALS, amyotrophic lateral sclerosis; CO, carbon monoxide; EDTA, ethylenediaminetetraacetic acid; HBO, hyperbaric oxygenation; HBOT, hyperbaric oxygen therapy; HSP-72, 72-kDa heat-shock protein; MSD, motor system disease; RCI, respiratory control index; SMA, spinal muscular atrophy; SOD-1, Cu, Zn super- oxide dismutase; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; TNF-, tumor necrosis factor . Neuroscience 120 (2003) 113–120 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00244-6 113