Ly and phenotypically normal along most of its length. Only a segment of the muscle fiber contains a clonal expansion of deletion-containing mtDNA molecules that compromise mitochondrial electron transport. The segmental nature of this accumulation necessitates a focal approach as homogenization would disrupt the link between genotype and phenotype, diluting the signal from the ETS abnormal region with normal tissue. To examine the cellular response to deletion mutation accumulation, we combined the selectivity of laser capture micro-dissection with the transcriptional analysis 256373-96-3 capabilities of high density oligonucleotide arrays to identify genes expressed in muscle fibers containing mtDNA deletion mutations and concomitant ETS abnormalities. We found that cells affected by intracellular expansions of deletion mutations upregulated genes involved in mitochondrial biogenesis. We hypothesize that this response results in a positive feedback loop wherein mtDNA deletion mutation accumulation results in mitochondrial dysfunction, resulting biogenesis and so enhancing deletion mutation accumulation. We tested this hypothesis by pharmacological intervention with b-Guanidinopropionic acid (bGPA), a compound known to induce mitochondrial biogenesis [22]. We found that pharmacological intervention, in old rats, led to an increase in the incidence of ETS abnormalities. These findings suggest that pharmaceutical or environmental interventions that drive or inhibit mtDNA replication could affect mtDNA deletion mutation accumulation and ETS abnormality abundance.Animal Care and Use Committees at the University of Wisconsin and University of Alberta.Animals, Tissue Preparation and HistochemistryVastus lateralis muscle was dissected from 33-month old male Fischer 344 x Brown Norway F1 hybrid rats purchased from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN). The muscle was bisected at the midbelly, embedded in optimal cutting media (Miles Inc., Elkhart, IN) and flash frozen in liquid nitrogen. Using a cryostat, 200 10micron-thick serial transverse cryosections were cut from the frozen tissue blocks and placed on Probe-on Plus slides. At 60micron intervals, cross-sections were 1081537 stained for COX and the (��)-Hexaconazole site subsequent slide for SDH as previously described [16]. Individual muscle fibers 
were followed throughout the 200 microns of tissue and those that possessed regions lacking COX and were hyperreactive for SDH activities were identified and images recording their phenotype and location obtained. All the ETS abnormal muscle fibers were exhaustively spatially identified throughout the muscle sections.Laser Capture MicrodissectionHistological sections were dehydrated through a series of ethanol and active 1 DEPC water mixes (30 , 50 , 60 , 70 , 80 , 95 , 100 and 100 ) at 4uC. Ethanol was subsequently removed with one 50 xylene:ethanol mix and 
two 100 xylene washes. All ETS abnormal fiber regions (840 abnormal fibers) within the tissue were micro-dissected from the sections using a PixCell II laser capture microscope (Arcturus). For each cell, the laser capture film was resected to isolate that portion of the film containing the specific cell material. The resected bits of film containing the cell were placed into RNA isolation solution. An equivalent number of ETS normal fiber sections were also micro-dissected and served as a control.Gene Expression ProfilingRNA isolation was performed using the MagneSil total R.Ly and phenotypically normal along most of its length. Only a segment of the muscle fiber contains a clonal expansion of deletion-containing mtDNA molecules that compromise mitochondrial electron transport. The segmental nature of this accumulation necessitates a focal approach as homogenization would disrupt the link between genotype and phenotype, diluting the signal from the ETS abnormal region with normal tissue. To examine the cellular response to deletion mutation accumulation, we combined the selectivity of laser capture micro-dissection with the transcriptional analysis capabilities of high density oligonucleotide arrays to identify genes expressed in muscle fibers containing mtDNA deletion mutations and concomitant ETS abnormalities. We found that cells affected by intracellular expansions of deletion mutations upregulated genes involved in mitochondrial biogenesis. We hypothesize that this response results in a positive feedback loop wherein mtDNA deletion mutation accumulation results in mitochondrial dysfunction, resulting biogenesis and so enhancing deletion mutation accumulation. We tested this hypothesis by pharmacological intervention with b-Guanidinopropionic acid (bGPA), a compound known to induce mitochondrial biogenesis [22]. We found that pharmacological intervention, in old rats, led to an increase in the incidence of ETS abnormalities. These findings suggest that pharmaceutical or environmental interventions that drive or inhibit mtDNA replication could affect mtDNA deletion mutation accumulation and ETS abnormality abundance.Animal Care and Use Committees at the University of Wisconsin and University of Alberta.Animals, Tissue Preparation and HistochemistryVastus lateralis muscle was dissected from 33-month old male Fischer 344 x Brown Norway F1 hybrid rats purchased from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN). The muscle was bisected at the midbelly, embedded in optimal cutting media (Miles Inc., Elkhart, IN) and flash frozen in liquid nitrogen. Using a cryostat, 200 10micron-thick serial transverse cryosections were cut from the frozen tissue blocks and placed on Probe-on Plus slides. At 60micron intervals, cross-sections were 1081537 stained for COX and the subsequent slide for SDH as previously described [16]. Individual muscle fibers were followed throughout the 200 microns of tissue and those that possessed regions lacking COX and were hyperreactive for SDH activities were identified and images recording their phenotype and location obtained. All the ETS abnormal muscle fibers were exhaustively spatially identified throughout the muscle sections.Laser Capture MicrodissectionHistological sections were dehydrated through a series of ethanol and active 1 DEPC water mixes (30 , 50 , 60 , 70 , 80 , 95 , 100 and 100 ) at 4uC. Ethanol was subsequently removed with one 50 xylene:ethanol mix and two 100 xylene washes. All ETS abnormal fiber regions (840 abnormal fibers) within the tissue were micro-dissected from the sections using a PixCell II laser capture microscope (Arcturus). For each cell, the laser capture film was resected to isolate that portion of the film containing the specific cell material. The resected bits of film containing the cell were placed into RNA isolation solution. An equivalent number of ETS normal fiber sections were also micro-dissected and served as a control.Gene Expression ProfilingRNA isolation was performed using the MagneSil total R.
