Regular Article Photon correlation spectroscopy of brain mitochondrial populations: Application to traumatic brain injury Jonathan Lifshitz a, * , Paul A. Janmey b , Tracy K. McIntosh a,c a Traumatic Brain Injury Laboratory, Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA 19104, USA b Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA c VA Medical Center, Philadelphia, PA 19104, USA Received 17 February 2005; revised 29 August 2005; accepted 12 October 2005 Available online 14 November 2005 Abstract Mitochondrial dysfunction and pathology that contribute to a host of neurodegenerative diseases are deduced from changes in ultrastructure, routinely examined by a host of optical techniques. We adapted the technique of photon correlation spectroscopy (PCS) to evaluate calcium- induced structural alterations in isolated viable cortical and hippocampal mitochondria. In detecting calcium-induced reductions in light intensity, PCS was more sensitive than absorbance across varying calcium concentrations. Mitochondrial populations encompass a broad distribution of sizes, confirmed by ultrastructural profiles, both which remain unaffected by calcium exposure. Cortical and hippocampal populations show fractional calcium-induced reductions in light scatter compared to subsequent maximal alamethicin-induced reductions. Although reductions in light scatter (refractive index) have been interpreted as mitochondrial swelling, PCS quantification of the mean mitochondrial radius demonstrates that mitochondrial size is unaffected by calcium exposure, but not alamethicin. Likewise, the population distribution histograms remain stable with calcium exposure, but shift to larger radii after alamethicin exposure. Furthermore, hippocampal mitochondrial populations from a neurodegenerative model of traumatic brain injury, lateral fluid percussion, demonstrate greater calcium-induced reductions in scatter intensity, which are associated with an initial population of large mitochondria becoming smaller. The disparate responses to calcium and subsequent alamethicin of mitochondria at 3 and 24 h after injury attest to an acute disruption of membrane permeability in mitochondria from injured brain. PCS provides quantitative indices of refractive index and size in isolated mitochondrial populations, aiding the evaluation of mitochondria in degenerative diseases. D 2005 Elsevier Inc. All rights reserved. Keywords: Head injury; Mitochondrial swelling; Photon correlation spectroscopy; Dynamic light scatter; Rat Introduction Biochemical evaluation of isolated central nervous system (CNS) mitochondria has revealed inherent differences com- pared to heart and liver mitochondria (Kristian et al., 2000; Berman et al., 2000), as well as regional differences within the brain (Friberg et al., 1999; Brustovetsky et al., 2003). As a central component of calcium regulation within the normal cell, mitochondria are normally exposed to high calcium concentra- tions as the calcium uniporter moves cytosolic calcium into the mitochondrial matrix (Carafoli, 2002). However, matrix calcium concentrations in excess of the mitochondrial calci- um-buffering capacity may result in the rapid release of calcium through the highly-regulated, non-specific mitochon- drial permeability transition (MPT) pore with resultant osmotic swelling of the mitochondrial membranes (Szabo et al., 1992; Zoratti and Szabo, 1995). Moreover, isolated brain mitochon- dria demonstrate a dampened calcium-induced swelling re- sponse compared to liver or heart mitochondria, such that only a small fraction of the population is responsive to calcium (Kristian et al., 2000, 2002). The diverse function of mitochondria necessitate a heterogeneity within brain mito- chondrial populations, which can be categorized by their varying sensitivities to calcium, CsA, pH, metabolic substrate and traumatic or ischemic insults (Kristian et al., 2000; Brustovetsky and Dubinsky, 2000; Kristian et al., 2001, 2002; Lifshitz et al., 2003). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.10.002 * Corresponding author. Department of Anatomy and Neurobiology, Virginia Commonwealth University, P.O. Box 980709, Richmond, VA 23298-0709, USA. Fax: +1 804 828 6293. E-mail address: jlifshitz@vcu.edu (J. Lifshitz). Experimental Neurology 197 (2006) 318 – 329 www.elsevier.com/locate/yexnr