%PDF-1.3 1 0 obj << /Type /Catalog /Outlines 2 0 R /Pages 3 0 R >> endobj 2 0 obj << /Type /Outlines /Count 0 >> endobj 3 0 obj << /Type /Pages /Kids [6 0 R 158 0 R 221 0 R 266 0 R 268 0 R ] /Count 5 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R >> /XObject << /I1 12 0 R /I2 13 0 R /I3 180 0 R /I4 225 0 R >> >> /MediaBox [0.000 0.000 612.000 792.000] >> endobj 4 0 obj [/PDF /Text /ImageC ] endobj 5 0 obj << /Creator (DOMPDF) /CreationDate (D:20180724083653+00'00') /ModDate (D:20180724083653+00'00') /Title (Characterization of Striatal Neuronal Loss and Atrophy in the R6/2 Mouse Model of Huntingtons Disease PLOS Currents Huntington Disease) >> endobj 6 0 obj << /Type /Page /Parent 3 0 R /Annots [ 14 0 R 16 0 R 18 0 R 20 0 R 22 0 R 24 0 R 26 0 R 28 0 R 30 0 R 32 0 R 34 0 R 36 0 R 38 0 R 40 0 R 42 0 R 44 0 R 46 0 R 48 0 R 50 0 R 52 0 R 54 0 R 56 0 R 58 0 R 60 0 R 62 0 R 64 0 R 66 0 R 68 0 R 70 0 R 72 0 R 74 0 R 76 0 R 78 0 R 80 0 R 82 0 R 84 0 R 86 0 R 88 0 R 90 0 R 92 0 R 94 0 R 96 0 R 98 0 R 100 0 R 102 0 R 104 0 R 106 0 R 108 0 R 110 0 R 112 0 R 114 0 R 116 0 R 118 0 R 120 0 R 122 0 R 124 0 R 126 0 R 128 0 R 130 0 R 132 0 R 134 0 R 136 0 R 138 0 R 140 0 R 142 0 R 144 0 R 146 0 R 148 0 R 150 0 R 152 0 R 154 0 R 156 0 R ] /Contents 7 0 R >> endobj 7 0 obj << /Length 29372 >> stream q 375.000 0 0 39.000 222.000 738.000 cm /I2 Do Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Characterization of Striatal Neuronal Loss and Atrophy in the )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(R6/2 Mouse Model of Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(January 6, 2014)] TJ ET BT 83.513 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 88.388 675.088 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Lindsay Dodds)] TJ ET 0.271 0.267 0.267 rg BT 90.737 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 96.158 663.247 Td /F1 9.8 Tf [(Jianfang Chen)] TJ ET 0.271 0.267 0.267 rg BT 159.026 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 164.447 663.247 Td /F1 9.8 Tf [(Kiersten Berggren)] TJ ET 0.271 0.267 0.267 rg BT 243.022 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 248.443 663.247 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Dodds L, Chen J, Berggren K, Fox J. Characterization of Striatal Neuronal Loss and Atrophy in the R6/2 Mouse Model of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntingtons Disease. PLOS Currents Huntington Disease. 2014 Jan 6 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.hd.48727b68b39b82d5fe350f753984bcf9.)] TJ ET q 15.000 31.315 577.500 593.837 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Striatal neuronal degeneration and loss is an important feature of human Huntingtons disease \(HD\). R6/2 HD mice recapitulate )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(many features of human HD including striatal atrophy. While striatal neuronal atrophy and loss is reported in R6/2 HD mice the )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(degree of neuronal loss and the characteristics of cell body atrophy are unclear. We used stereological approaches to estimate )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(whole striatal neuronal numbers and characterize changes in striatal neuronal size distribution. R6/2 HD mice had ~126000 )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(fewer neurons per striatum \(~12% decline\) at 12 weeks of age than wild-type litter-mates; differences were not present at 5 )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(weeks. Analysis of striatal neuronal numbers per cell body size category revealed declines in neuron numbers in the size )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(ranges 550-1050 m3 suggesting that larger striatal neurons are more susceptible to atrophy or loss in late stages of disease. )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(R6/2 HD mice have a striatal neuronal loss phenotype. As striatal neuronal loss in human HD is dramatic, neuronal loss in R6/2 )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(striatum provides an important late-stage outcome measure for study of disease modifying interventions)] TJ ET BT 26.250 446.636 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(This research was funded in part by NIH grants 5P20RR015640-10 and NIH R01 NS079450-01. Lindsay Dodds was supported )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(by an INBRE undergraduate student summer fellowship \(NIH P20RR016474 and NIH P20GM103432\).)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Human Huntingtons disease \(HD\) is characterized by slowly progressive brain atrophy primarily affecting striatum and cerebral )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(cortex )] TJ ET 0.267 0.267 0.267 rg BT 55.510 355.322 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 60.328 353.815 Td /F1 9.8 Tf [(that develops in the pre-clinical phase of the disease process )] TJ ET 0.267 0.267 0.267 rg BT 326.416 355.322 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 331.234 353.815 Td /F1 9.8 Tf [( . There is considerable evidence that atrophy of striatum )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(results from a combination of neuronal degeneration and loss. Vonsattel et al )] TJ ET 0.267 0.267 0.267 rg BT 360.626 343.418 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 365.445 341.910 Td /F1 9.8 Tf [( reported loss of 50% of caudate neurons in )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(neuropathology grade I and 95% in grade IV human disease. Neurons within the striatum comprise ~95% medium spiny )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(neurons which degenerate early in HD and remaining interneurons which are less susceptible to degeneration.)] TJ ET BT 26.250 298.696 Td /F1 9.8 Tf [(Numerous genetic mouse models of HD exist and these have proven invaluable for studying disease mechanisms and )] TJ ET BT 26.250 286.791 Td /F1 9.8 Tf [(therapies )] TJ ET 0.267 0.267 0.267 rg BT 69.062 288.299 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 73.881 286.791 Td /F1 9.8 Tf [( . R6/2 mice express exon 1 of the human huntingtin gene containing an expanded CAG repeat )] TJ ET 0.267 0.267 0.267 rg BT 486.862 288.299 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 491.680 286.791 Td /F1 9.8 Tf [( . These mice )] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(experience rapid disease progression with symptoms beginning around 6 weeks of age, large behavioral declines by 12 weeks )] TJ ET 0.267 0.267 0.267 rg BT 574.658 276.394 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 26.250 262.982 Td /F1 9.8 Tf [(, and death as early as 10 weeks of age in some colonies \(e.g. )] TJ ET 0.267 0.267 0.267 rg BT 298.850 264.489 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 303.669 262.982 Td /F1 9.8 Tf [( \). R6/2 mice recapitulate many features of human HD including )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(progressive brain atrophy involving neostriatum and cerebral cortex, weight loss, motor dysfunction and accumulation of mutant )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(huntingtin aggregates in brain neurons. While these mice express only a fragment of human mutant huntingtin evidence has )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(been provided that this fragment occurs endogenously in mouse HD )] TJ ET 0.267 0.267 0.267 rg BT 322.679 228.775 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 327.498 227.268 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 207.863 Td /F1 9.8 Tf [(YAC128 HD mice express full-length human mutant huntingtin from a yeast artificial chromosome. They have a slowly )] TJ ET BT 26.250 195.958 Td /F1 9.8 Tf [(progressive HD-like disease process. Slow )] TJ ET 0.267 0.267 0.267 rg BT 213.713 197.465 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 218.532 195.958 Td /F1 9.8 Tf [( and colleagues reported a 15% decrease in striatal volume at 9 months of age, )] TJ ET BT 26.250 184.053 Td /F1 9.8 Tf [(and a 15-18% reduction in striatal neuronal estimates at 12 months of age in these mice. Knock-in models of HD also exhibit )] TJ ET BT 26.250 172.149 Td /F1 9.8 Tf [(late-onset neurodegeneration. For example, in the CAG140 mouse HD model 38% and 40% reductions in striatal volume and )] TJ ET BT 26.250 160.244 Td /F1 9.8 Tf [(striatal neuron estimates are reported, respectively, in 20-26-month-old animals )] TJ ET 0.267 0.267 0.267 rg BT 371.420 161.751 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 376.238 160.244 Td /F1 9.8 Tf [( . While fragment mouse models of HD \(e.g. )] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(R6/2\) are genetically less accurate than full-length models their rapid disease progression and HD-like phenotype has resulted )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(in their continued use.)] TJ ET BT 26.250 117.030 Td /F1 9.8 Tf [(Several approaches have been used to demonstrate striatal neuronal degeneration in HD models. R6/2 HD mice have )] TJ ET BT 26.250 105.125 Td /F1 9.8 Tf [(increased activity of neuronal caspase 1 and 3 and changes in Bcl-2 family members supportive of activation of striatal neuronal )] TJ ET BT 26.250 93.220 Td /F1 9.8 Tf [(apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 70.691 94.727 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 80.328 93.220 Td /F1 9.8 Tf [( . One study reported significant mitochondrial abnormalities in neurons of R6/2 mice )] TJ ET 0.267 0.267 0.267 rg BT 447.727 94.727 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 457.365 93.220 Td /F1 9.8 Tf [( . Findings from TUNEL )] TJ ET BT 26.250 81.315 Td /F1 9.8 Tf [(labeling for DNA nicks have however been variable )] TJ ET 0.267 0.267 0.267 rg BT 249.506 82.823 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 259.143 85.204 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 261.552 82.823 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 271.190 81.315 Td /F1 9.8 Tf [( . Several studies have demonstrated the presence of neuronal cell )] TJ ET BT 26.250 69.411 Td /F1 9.8 Tf [(body atrophy in the striatum of R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 223.493 70.918 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 233.130 73.299 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 235.539 70.918 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 245.177 69.411 Td /F1 9.8 Tf [( . However, we are aware of only one study that has estimated striatal )] TJ ET BT 26.250 57.506 Td /F1 9.8 Tf [(neuronal numbers in R6/2 mice as a means to quantify neurodegeneration. Stack et al )] TJ ET 0.267 0.267 0.267 rg BT 400.728 59.013 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 410.365 57.506 Td /F1 9.8 Tf [( estimated neuronal numbers in the )] TJ ET BT 26.250 45.601 Td /F1 9.8 Tf [(anterior 2/3 of the striatum, to the level of the anterior commissure, and found significantly fewer neurons at 12-weeks of age in )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Characterization of Striatal Neuronal Loss and Atrophy in the )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(R6/2 Mouse Model of Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(January 6, 2014)] TJ ET BT 83.513 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 88.388 675.088 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Lindsay Dodds)] TJ ET 0.271 0.267 0.267 rg BT 90.737 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 96.158 663.247 Td /F1 9.8 Tf [(Jianfang Chen)] TJ ET 0.271 0.267 0.267 rg BT 159.026 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 164.447 663.247 Td /F1 9.8 Tf [(Kiersten Berggren)] TJ ET 0.271 0.267 0.267 rg BT 243.022 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 248.443 663.247 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Dodds L, Chen J, Berggren K, Fox J. Characterization of Striatal Neuronal Loss and Atrophy in the R6/2 Mouse Model of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntingtons Disease. PLOS Currents Huntington Disease. 2014 Jan 6 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.hd.48727b68b39b82d5fe350f753984bcf9.)] TJ ET q 15.000 31.315 577.500 593.837 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Striatal neuronal degeneration and loss is an important feature of human Huntingtons disease \(HD\). R6/2 HD mice recapitulate )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(many features of human HD including striatal atrophy. While striatal neuronal atrophy and loss is reported in R6/2 HD mice the )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(degree of neuronal loss and the characteristics of cell body atrophy are unclear. We used stereological approaches to estimate )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(whole striatal neuronal numbers and characterize changes in striatal neuronal size distribution. R6/2 HD mice had ~126000 )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(fewer neurons per striatum \(~12% decline\) at 12 weeks of age than wild-type litter-mates; differences were not present at 5 )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(weeks. Analysis of striatal neuronal numbers per cell body size category revealed declines in neuron numbers in the size )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(ranges 550-1050 m3 suggesting that larger striatal neurons are more susceptible to atrophy or loss in late stages of disease. )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(R6/2 HD mice have a striatal neuronal loss phenotype. As striatal neuronal loss in human HD is dramatic, neuronal loss in R6/2 )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(striatum provides an important late-stage outcome measure for study of disease modifying interventions)] TJ ET BT 26.250 446.636 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(This research was funded in part by NIH grants 5P20RR015640-10 and NIH R01 NS079450-01. Lindsay Dodds was supported )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(by an INBRE undergraduate student summer fellowship \(NIH P20RR016474 and NIH P20GM103432\).)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Human Huntingtons disease \(HD\) is characterized by slowly progressive brain atrophy primarily affecting striatum and cerebral )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(cortex )] TJ ET 0.267 0.267 0.267 rg BT 55.510 355.322 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 60.328 353.815 Td /F1 9.8 Tf [(that develops in the pre-clinical phase of the disease process )] TJ ET 0.267 0.267 0.267 rg BT 326.416 355.322 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 331.234 353.815 Td /F1 9.8 Tf [( . There is considerable evidence that atrophy of striatum )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(results from a combination of neuronal degeneration and loss. Vonsattel et al )] TJ ET 0.267 0.267 0.267 rg BT 360.626 343.418 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 365.445 341.910 Td /F1 9.8 Tf [( reported loss of 50% of caudate neurons in )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(neuropathology grade I and 95% in grade IV human disease. Neurons within the striatum comprise ~95% medium spiny )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(neurons which degenerate early in HD and remaining interneurons which are less susceptible to degeneration.)] TJ ET BT 26.250 298.696 Td /F1 9.8 Tf [(Numerous genetic mouse models of HD exist and these have proven invaluable for studying disease mechanisms and )] TJ ET BT 26.250 286.791 Td /F1 9.8 Tf [(therapies )] TJ ET 0.267 0.267 0.267 rg BT 69.062 288.299 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 73.881 286.791 Td /F1 9.8 Tf [( . R6/2 mice express exon 1 of the human huntingtin gene containing an expanded CAG repeat )] TJ ET 0.267 0.267 0.267 rg BT 486.862 288.299 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 491.680 286.791 Td /F1 9.8 Tf [( . These mice )] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(experience rapid disease progression with symptoms beginning around 6 weeks of age, large behavioral declines by 12 weeks )] TJ ET 0.267 0.267 0.267 rg BT 574.658 276.394 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 26.250 262.982 Td /F1 9.8 Tf [(, and death as early as 10 weeks of age in some colonies \(e.g. )] TJ ET 0.267 0.267 0.267 rg BT 298.850 264.489 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 303.669 262.982 Td /F1 9.8 Tf [( \). R6/2 mice recapitulate many features of human HD including )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(progressive brain atrophy involving neostriatum and cerebral cortex, weight loss, motor dysfunction and accumulation of mutant )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(huntingtin aggregates in brain neurons. While these mice express only a fragment of human mutant huntingtin evidence has )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(been provided that this fragment occurs endogenously in mouse HD )] TJ ET 0.267 0.267 0.267 rg BT 322.679 228.775 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 327.498 227.268 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 207.863 Td /F1 9.8 Tf [(YAC128 HD mice express full-length human mutant huntingtin from a yeast artificial chromosome. They have a slowly )] TJ ET BT 26.250 195.958 Td /F1 9.8 Tf [(progressive HD-like disease process. Slow )] TJ ET 0.267 0.267 0.267 rg BT 213.713 197.465 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 218.532 195.958 Td /F1 9.8 Tf [( and colleagues reported a 15% decrease in striatal volume at 9 months of age, )] TJ ET BT 26.250 184.053 Td /F1 9.8 Tf [(and a 15-18% reduction in striatal neuronal estimates at 12 months of age in these mice. Knock-in models of HD also exhibit )] TJ ET BT 26.250 172.149 Td /F1 9.8 Tf [(late-onset neurodegeneration. For example, in the CAG140 mouse HD model 38% and 40% reductions in striatal volume and )] TJ ET BT 26.250 160.244 Td /F1 9.8 Tf [(striatal neuron estimates are reported, respectively, in 20-26-month-old animals )] TJ ET 0.267 0.267 0.267 rg BT 371.420 161.751 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 376.238 160.244 Td /F1 9.8 Tf [( . While fragment mouse models of HD \(e.g. )] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(R6/2\) are genetically less accurate than full-length models their rapid disease progression and HD-like phenotype has resulted )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(in their continued use.)] TJ ET BT 26.250 117.030 Td /F1 9.8 Tf [(Several approaches have been used to demonstrate striatal neuronal degeneration in HD models. R6/2 HD mice have )] TJ ET BT 26.250 105.125 Td /F1 9.8 Tf [(increased activity of neuronal caspase 1 and 3 and changes in Bcl-2 family members supportive of activation of striatal neuronal )] TJ ET BT 26.250 93.220 Td /F1 9.8 Tf [(apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 70.691 94.727 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 80.328 93.220 Td /F1 9.8 Tf [( . One study reported significant mitochondrial abnormalities in neurons of R6/2 mice )] TJ ET 0.267 0.267 0.267 rg BT 447.727 94.727 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 457.365 93.220 Td /F1 9.8 Tf [( . Findings from TUNEL )] TJ ET BT 26.250 81.315 Td /F1 9.8 Tf [(labeling for DNA nicks have however been variable )] TJ ET 0.267 0.267 0.267 rg BT 249.506 82.823 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 259.143 85.204 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 261.552 82.823 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 271.190 81.315 Td /F1 9.8 Tf [( . Several studies have demonstrated the presence of neuronal cell )] TJ ET BT 26.250 69.411 Td /F1 9.8 Tf [(body atrophy in the striatum of R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 223.493 70.918 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 233.130 73.299 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 235.539 70.918 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 245.177 69.411 Td /F1 9.8 Tf [( . However, we are aware of only one study that has estimated striatal )] TJ ET BT 26.250 57.506 Td /F1 9.8 Tf [(neuronal numbers in R6/2 mice as a means to quantify neurodegeneration. Stack et al )] TJ ET 0.267 0.267 0.267 rg BT 400.728 59.013 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 410.365 57.506 Td /F1 9.8 Tf [( estimated neuronal numbers in the )] TJ ET BT 26.250 45.601 Td /F1 9.8 Tf [(anterior 2/3 of the striatum, to the level of the anterior commissure, and found significantly fewer neurons at 12-weeks of age in )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Characterization of Striatal Neuronal Loss and Atrophy in the )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(R6/2 Mouse Model of Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(January 6, 2014)] TJ ET BT 83.513 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 88.388 675.088 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Lindsay Dodds)] TJ ET 0.271 0.267 0.267 rg BT 90.737 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 96.158 663.247 Td /F1 9.8 Tf [(Jianfang Chen)] TJ ET 0.271 0.267 0.267 rg BT 159.026 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 164.447 663.247 Td /F1 9.8 Tf [(Kiersten Berggren)] TJ ET 0.271 0.267 0.267 rg BT 243.022 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 248.443 663.247 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Dodds L, Chen J, Berggren K, Fox J. Characterization of Striatal Neuronal Loss and Atrophy in the R6/2 Mouse Model of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntingtons Disease. PLOS Currents Huntington Disease. 2014 Jan 6 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.hd.48727b68b39b82d5fe350f753984bcf9.)] TJ ET q 15.000 31.315 577.500 593.837 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Striatal neuronal degeneration and loss is an important feature of human Huntingtons disease \(HD\). R6/2 HD mice recapitulate )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(many features of human HD including striatal atrophy. While striatal neuronal atrophy and loss is reported in R6/2 HD mice the )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(degree of neuronal loss and the characteristics of cell body atrophy are unclear. We used stereological approaches to estimate )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(whole striatal neuronal numbers and characterize changes in striatal neuronal size distribution. R6/2 HD mice had ~126000 )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(fewer neurons per striatum \(~12% decline\) at 12 weeks of age than wild-type litter-mates; differences were not present at 5 )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(weeks. Analysis of striatal neuronal numbers per cell body size category revealed declines in neuron numbers in the size )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(ranges 550-1050 m3 suggesting that larger striatal neurons are more susceptible to atrophy or loss in late stages of disease. )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(R6/2 HD mice have a striatal neuronal loss phenotype. As striatal neuronal loss in human HD is dramatic, neuronal loss in R6/2 )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(striatum provides an important late-stage outcome measure for study of disease modifying interventions)] TJ ET BT 26.250 446.636 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(This research was funded in part by NIH grants 5P20RR015640-10 and NIH R01 NS079450-01. Lindsay Dodds was supported )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(by an INBRE undergraduate student summer fellowship \(NIH P20RR016474 and NIH P20GM103432\).)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Human Huntingtons disease \(HD\) is characterized by slowly progressive brain atrophy primarily affecting striatum and cerebral )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(cortex )] TJ ET 0.267 0.267 0.267 rg BT 55.510 355.322 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 60.328 353.815 Td /F1 9.8 Tf [(that develops in the pre-clinical phase of the disease process )] TJ ET 0.267 0.267 0.267 rg BT 326.416 355.322 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 331.234 353.815 Td /F1 9.8 Tf [( . There is considerable evidence that atrophy of striatum )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(results from a combination of neuronal degeneration and loss. Vonsattel et al )] TJ ET 0.267 0.267 0.267 rg BT 360.626 343.418 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 365.445 341.910 Td /F1 9.8 Tf [( reported loss of 50% of caudate neurons in )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(neuropathology grade I and 95% in grade IV human disease. Neurons within the striatum comprise ~95% medium spiny )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(neurons which degenerate early in HD and remaining interneurons which are less susceptible to degeneration.)] TJ ET BT 26.250 298.696 Td /F1 9.8 Tf [(Numerous genetic mouse models of HD exist and these have proven invaluable for studying disease mechanisms and )] TJ ET BT 26.250 286.791 Td /F1 9.8 Tf [(therapies )] TJ ET 0.267 0.267 0.267 rg BT 69.062 288.299 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 73.881 286.791 Td /F1 9.8 Tf [( . R6/2 mice express exon 1 of the human huntingtin gene containing an expanded CAG repeat )] TJ ET 0.267 0.267 0.267 rg BT 486.862 288.299 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 491.680 286.791 Td /F1 9.8 Tf [( . These mice )] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(experience rapid disease progression with symptoms beginning around 6 weeks of age, large behavioral declines by 12 weeks )] TJ ET 0.267 0.267 0.267 rg BT 574.658 276.394 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 26.250 262.982 Td /F1 9.8 Tf [(, and death as early as 10 weeks of age in some colonies \(e.g. )] TJ ET 0.267 0.267 0.267 rg BT 298.850 264.489 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 303.669 262.982 Td /F1 9.8 Tf [( \). R6/2 mice recapitulate many features of human HD including )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(progressive brain atrophy involving neostriatum and cerebral cortex, weight loss, motor dysfunction and accumulation of mutant )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(huntingtin aggregates in brain neurons. While these mice express only a fragment of human mutant huntingtin evidence has )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(been provided that this fragment occurs endogenously in mouse HD )] TJ ET 0.267 0.267 0.267 rg BT 322.679 228.775 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 327.498 227.268 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 207.863 Td /F1 9.8 Tf [(YAC128 HD mice express full-length human mutant huntingtin from a yeast artificial chromosome. They have a slowly )] TJ ET BT 26.250 195.958 Td /F1 9.8 Tf [(progressive HD-like disease process. Slow )] TJ ET 0.267 0.267 0.267 rg BT 213.713 197.465 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 218.532 195.958 Td /F1 9.8 Tf [( and colleagues reported a 15% decrease in striatal volume at 9 months of age, )] TJ ET BT 26.250 184.053 Td /F1 9.8 Tf [(and a 15-18% reduction in striatal neuronal estimates at 12 months of age in these mice. Knock-in models of HD also exhibit )] TJ ET BT 26.250 172.149 Td /F1 9.8 Tf [(late-onset neurodegeneration. For example, in the CAG140 mouse HD model 38% and 40% reductions in striatal volume and )] TJ ET BT 26.250 160.244 Td /F1 9.8 Tf [(striatal neuron estimates are reported, respectively, in 20-26-month-old animals )] TJ ET 0.267 0.267 0.267 rg BT 371.420 161.751 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 376.238 160.244 Td /F1 9.8 Tf [( . While fragment mouse models of HD \(e.g. )] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(R6/2\) are genetically less accurate than full-length models their rapid disease progression and HD-like phenotype has resulted )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(in their continued use.)] TJ ET BT 26.250 117.030 Td /F1 9.8 Tf [(Several approaches have been used to demonstrate striatal neuronal degeneration in HD models. R6/2 HD mice have )] TJ ET BT 26.250 105.125 Td /F1 9.8 Tf [(increased activity of neuronal caspase 1 and 3 and changes in Bcl-2 family members supportive of activation of striatal neuronal )] TJ ET BT 26.250 93.220 Td /F1 9.8 Tf [(apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 70.691 94.727 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 80.328 93.220 Td /F1 9.8 Tf [( . One study reported significant mitochondrial abnormalities in neurons of R6/2 mice )] TJ ET 0.267 0.267 0.267 rg BT 447.727 94.727 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 457.365 93.220 Td /F1 9.8 Tf [( . Findings from TUNEL )] TJ ET BT 26.250 81.315 Td /F1 9.8 Tf [(labeling for DNA nicks have however been variable )] TJ ET 0.267 0.267 0.267 rg BT 249.506 82.823 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 259.143 85.204 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 261.552 82.823 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 271.190 81.315 Td /F1 9.8 Tf [( . Several studies have demonstrated the presence of neuronal cell )] TJ ET BT 26.250 69.411 Td /F1 9.8 Tf [(body atrophy in the striatum of R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 223.493 70.918 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 233.130 73.299 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 235.539 70.918 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 245.177 69.411 Td /F1 9.8 Tf [( . However, we are aware of only one study that has estimated striatal )] TJ ET BT 26.250 57.506 Td /F1 9.8 Tf [(neuronal numbers in R6/2 mice as a means to quantify neurodegeneration. Stack et al )] TJ ET 0.267 0.267 0.267 rg BT 400.728 59.013 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 410.365 57.506 Td /F1 9.8 Tf [( estimated neuronal numbers in the )] TJ ET BT 26.250 45.601 Td /F1 9.8 Tf [(anterior 2/3 of the striatum, to the level of the anterior commissure, and found significantly fewer neurons at 12-weeks of age in )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 12 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4223>> stream xە8=,8C#h!hGv#0=j$q1uaNĥT*J&a_Zaa0 a0 a0 H^UUUUX!0 3SY|^G0 ÌÔ{]wa_>"OeYaG!8e}a6̖{4M&d:"qʲ\QUUMS<ϛNld2l6Z a<=2B(2vK7M\.GW0 rrWWavߖe9C?Eq8Gqy~~}_qdϽ(neYʒGjK4EQNjͷm{X?|7m۶mqP:Ȳp8Kmu]lNzuZ,NTeY"jlUUQW\,aRiVbȗLkHkEsjd.L6=Iו P]1t7Sc2uIuMM; K5>A$Ǒ$I׋nY 0_Hat;aq䃰U$nR ,zO7bT mhhԫܻu1F*qm'P}ܙ<ϕALO6Lw3nB.T4co{߽.lV $laԀIu!gwChxJ]Ne (+M"d-Eqc=P ! 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The purpose of the current study was to complete a detailed stereologic analysis )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(of the entire striatum, in particular characterizing neuronal numbers and size changes with disease progression.)] TJ ET BT 26.250 718.969 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(All procedures were pre-approved by the University of Wyoming Animal Care and Use Committee \(IACUC\). R6/2 HD mice were )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(bred and maintained as previously described )] TJ ET 0.267 0.267 0.267 rg BT 222.420 688.617 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 227.239 687.110 Td /F1 9.8 Tf [( . CAG repeat sizes were determined from genomic DNA by Laragen Inc. \(Culver )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(City, CA\) and averaged 181 with a standard deviation of 6. Mice were sacrificed by deep anesthesia then intra-cardiac perfusion )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(with 4% paraformaldehyde and processed for stereology as described )] TJ ET 0.267 0.267 0.267 rg BT 330.801 664.808 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 340.438 663.300 Td /F1 9.8 Tf [( . In brief, brains were sectioned coronally to include )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(the entire striatum at 40 m thickness; every 12)] TJ ET BT 231.088 655.284 Td /F1 8.7 Tf [(th)] TJ ET BT 238.316 651.396 Td /F1 9.8 Tf [( section was mounted and stained using the thionin method. We used the )] TJ ET BT 26.250 639.491 Td /F1 9.8 Tf [(unbiased optical dissector method to estimate striatal neuronal numbers )] TJ ET 0.267 0.267 0.267 rg BT 339.469 640.998 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 349.106 639.491 Td /F1 9.8 Tf [( . We used a counting frame size of 40 x 40 m and )] TJ ET BT 26.250 627.586 Td /F1 9.8 Tf [(a grid size of 500 x 500 m. During the optical dissector procedure we also estimated striatal neuronal cell body volume using )] TJ ET BT 26.250 615.681 Td /F1 9.8 Tf [(the 2D nucleator with five isotropic uniform random sections per cell. All slides were coded so the operator was unaware of the )] TJ ET BT 26.250 603.777 Td /F1 9.8 Tf [(genotype. The numbers of mice per group were: wild-type 5-weeks n=15; HD 5-weeks n=13; wild-type 12 weeks n=9; and HD )] TJ ET BT 26.250 591.872 Td /F1 9.8 Tf [(12 weeks n=10. The average number of neurons / mouse analyzed was 384 for 5-week mice \(range: 245-521\) and 359 for 12-)] TJ ET BT 26.250 579.967 Td /F1 9.8 Tf [(week mice \(range: 272-436\). For each mouse, neurons were binned into 50 m)] TJ ET BT 368.192 583.855 Td /F1 8.7 Tf [(3)] TJ ET BT 373.011 579.967 Td /F1 9.8 Tf [( cell body size categories using Microsoft excel )] TJ ET BT 26.250 568.062 Td /F1 9.8 Tf [(software. The estimated total number of striatal neurons in each size category in wild-type and HD mice was then analyzed )] TJ ET BT 26.250 556.158 Td /F1 9.8 Tf [(using the students t-test in SAS software version 9.2 \(Cary, NC, USA\).)] TJ ET BT 26.250 519.555 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 499.601 Td /F1 9.8 Tf [(R6/2 HD mice had 12.7% fewer striatal neurons at 12-weeks of age \(p=0.035\) \()] TJ ET BT 367.919 499.601 Td /F4 9.8 Tf [(Fig. 1A)] TJ ET BT 400.426 499.601 Td /F1 9.8 Tf [(\). Wild-type mice had a mean of 993891 )] TJ ET BT 26.250 487.696 Td /F1 9.8 Tf [( 38483 \(SEM\) neurons; R6/2 HD mice had a mean of 868043 38483 \(SEM\). To determine if this difference was due to )] TJ ET BT 26.250 475.791 Td /F1 9.8 Tf [(neuronal loss or developmental effects we studied 5-week-old mice. As shown in )] TJ ET BT 376.850 475.791 Td /F4 9.8 Tf [(Fig. 1B)] TJ ET BT 409.357 475.791 Td /F1 9.8 Tf [( there were no differences at 5-weeks )] TJ ET BT 26.250 463.887 Td /F1 9.8 Tf [(of age indicating the presence of neuronal loss in R6/2 mouse striatum occurring between 5 and 12 weeks. Use of the nucleator )] TJ ET BT 26.250 451.982 Td /F1 9.8 Tf [(stereologic tool has been reported in numerous studies to characterize changes in striatal neuronal cell body volume in HD )] TJ ET BT 26.250 440.077 Td /F1 9.8 Tf [(mice; however, distribution of neuronal sizes and number estimates by cell size were not determined \(e.g. [12,13]\). We )] TJ ET BT 26.250 428.172 Td /F1 9.8 Tf [(analyzed neuronal population estimates on a cell body size basis \()] TJ ET BT 312.383 428.172 Td /F4 9.8 Tf [(Fig. 2)] TJ ET BT 337.850 428.172 Td /F1 9.8 Tf [(\). At 12 weeks, the most significant finding was a loss )] TJ ET BT 26.250 416.268 Td /F1 9.8 Tf [(of neurons in the size range 550-1050 m)] TJ ET BT 206.722 420.156 Td /F1 8.7 Tf [(3 )] TJ ET BT 213.951 416.268 Td /F1 9.8 Tf [(\()] TJ ET BT 217.197 416.268 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 249.704 416.268 Td /F1 9.8 Tf [(\). Within smaller neuron size categories \(200-500 m)] TJ ET BT 478.361 420.156 Td /F1 8.7 Tf [(3)] TJ ET BT 483.179 416.268 Td /F1 9.8 Tf [(\) neuronal population )] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(mean estimates were increased in R6/2 mice but this did not approach significance \()] TJ ET BT 389.857 404.363 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 422.363 404.363 Td /F1 9.8 Tf [(\). At 5 weeks, there were no )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(significant differences in neuron number estimates per size category \()] TJ ET BT 326.452 392.458 Td /F4 9.8 Tf [(Fig. 2B)] TJ ET BT 358.959 392.458 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 373.053 Td /F1 9.8 Tf [(While there is abundant biochemical and morphologic evidence to support neuronal degeneration in R6/2 mouse striatum )] TJ ET 0.267 0.267 0.267 rg BT 551.365 374.561 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 561.003 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 563.412 374.561 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 573.050 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 362.656 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 35.887 361.149 Td /F1 9.8 Tf [( , we are not aware of reports of morphologic evidence of late-stage neuronal death events in R6/2 striatal neurons. Our )] TJ ET BT 26.250 349.244 Td /F1 9.8 Tf [(findings, and those of others, suggest that while the process of neuronal degeneration is gradual, neuronal death and removal is )] TJ ET BT 26.250 337.339 Td /F1 9.8 Tf [(rapid. We estimated the number of striatal neurons in the process of death when sacrificed at 12-weeks of age. To do this we )] TJ ET BT 26.250 325.434 Td /F1 9.8 Tf [(assumed that neuron death in R6/2 mice begins at 60 days of age )] TJ ET 0.267 0.267 0.267 rg BT 313.495 326.942 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 323.132 325.434 Td /F1 9.8 Tf [( and that the process is linear across time \(a constant )] TJ ET BT 26.250 313.530 Td /F1 9.8 Tf [(number of neurons dying / day\). Based on this, we calculated that R6/2 HD mice lose ~6909 neurons / day / striatum. In one )] TJ ET BT 26.250 301.625 Td /F1 9.8 Tf [(rodent model of acute neuronal injury cell death was detected ~6 hours post-insult )] TJ ET 0.267 0.267 0.267 rg BT 382.574 303.132 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 392.211 301.625 Td /F1 9.8 Tf [( . In one study phagocytosis of apoptotic )] TJ ET BT 26.250 289.720 Td /F1 9.8 Tf [(neurons occurred within 1.5 hours of introduction of microglia into cultures )] TJ ET 0.267 0.267 0.267 rg BT 347.591 291.227 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 357.228 289.720 Td /F1 9.8 Tf [( . Based on these findings we assumed that in )] TJ ET BT 26.250 277.815 Td /F1 9.8 Tf [(R6/2 mice the process of morphologically detectable neuronal death and removal lasts 8 hours. From this we calculated that a )] TJ ET BT 26.250 265.911 Td /F1 9.8 Tf [(20 m section of striatum taken at the level of the anterior commissure of a 12 week R6/2 mouse would contain ~4 dying )] TJ ET BT 26.250 254.006 Td /F1 9.8 Tf [(neurons. Therefore, based on our estimates, the kinetics of neuronal degeneration, loss and removal underlies the difficulty of )] TJ ET BT 26.250 242.101 Td /F1 9.8 Tf [(identifying neuronal death morphologically. Stereologic approaches to quantifying neurons, as used here, can overcome these )] TJ ET BT 26.250 230.196 Td /F1 9.8 Tf [(difficulties.)] TJ ET 0.965 0.965 0.965 rg 26.250 -86.434 555.000 306.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 220.316 m 581.250 220.316 l 581.250 219.566 l 26.250 219.566 l f q 450.000 0 0 291.000 35.250 -80.434 cm /I3 Do Q q 35.250 -86.434 537.000 0.000 re W n Q Q q 15.000 -86.434 577.500 863.434 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(R6/2 mice compared to wild-type littermates. The purpose of the current study was to complete a detailed stereologic analysis )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(of the entire striatum, in particular characterizing neuronal numbers and size changes with disease progression.)] TJ ET BT 26.250 718.969 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(All procedures were pre-approved by the University of Wyoming Animal Care and Use Committee \(IACUC\). R6/2 HD mice were )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(bred and maintained as previously described )] TJ ET 0.267 0.267 0.267 rg BT 222.420 688.617 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 227.239 687.110 Td /F1 9.8 Tf [( . CAG repeat sizes were determined from genomic DNA by Laragen Inc. \(Culver )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(City, CA\) and averaged 181 with a standard deviation of 6. Mice were sacrificed by deep anesthesia then intra-cardiac perfusion )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(with 4% paraformaldehyde and processed for stereology as described )] TJ ET 0.267 0.267 0.267 rg BT 330.801 664.808 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 340.438 663.300 Td /F1 9.8 Tf [( . In brief, brains were sectioned coronally to include )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(the entire striatum at 40 m thickness; every 12)] TJ ET BT 231.088 655.284 Td /F1 8.7 Tf [(th)] TJ ET BT 238.316 651.396 Td /F1 9.8 Tf [( section was mounted and stained using the thionin method. We used the )] TJ ET BT 26.250 639.491 Td /F1 9.8 Tf [(unbiased optical dissector method to estimate striatal neuronal numbers )] TJ ET 0.267 0.267 0.267 rg BT 339.469 640.998 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 349.106 639.491 Td /F1 9.8 Tf [( . We used a counting frame size of 40 x 40 m and )] TJ ET BT 26.250 627.586 Td /F1 9.8 Tf [(a grid size of 500 x 500 m. During the optical dissector procedure we also estimated striatal neuronal cell body volume using )] TJ ET BT 26.250 615.681 Td /F1 9.8 Tf [(the 2D nucleator with five isotropic uniform random sections per cell. All slides were coded so the operator was unaware of the )] TJ ET BT 26.250 603.777 Td /F1 9.8 Tf [(genotype. The numbers of mice per group were: wild-type 5-weeks n=15; HD 5-weeks n=13; wild-type 12 weeks n=9; and HD )] TJ ET BT 26.250 591.872 Td /F1 9.8 Tf [(12 weeks n=10. The average number of neurons / mouse analyzed was 384 for 5-week mice \(range: 245-521\) and 359 for 12-)] TJ ET BT 26.250 579.967 Td /F1 9.8 Tf [(week mice \(range: 272-436\). For each mouse, neurons were binned into 50 m)] TJ ET BT 368.192 583.855 Td /F1 8.7 Tf [(3)] TJ ET BT 373.011 579.967 Td /F1 9.8 Tf [( cell body size categories using Microsoft excel )] TJ ET BT 26.250 568.062 Td /F1 9.8 Tf [(software. The estimated total number of striatal neurons in each size category in wild-type and HD mice was then analyzed )] TJ ET BT 26.250 556.158 Td /F1 9.8 Tf [(using the students t-test in SAS software version 9.2 \(Cary, NC, USA\).)] TJ ET BT 26.250 519.555 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 499.601 Td /F1 9.8 Tf [(R6/2 HD mice had 12.7% fewer striatal neurons at 12-weeks of age \(p=0.035\) \()] TJ ET BT 367.919 499.601 Td /F4 9.8 Tf [(Fig. 1A)] TJ ET BT 400.426 499.601 Td /F1 9.8 Tf [(\). Wild-type mice had a mean of 993891 )] TJ ET BT 26.250 487.696 Td /F1 9.8 Tf [( 38483 \(SEM\) neurons; R6/2 HD mice had a mean of 868043 38483 \(SEM\). To determine if this difference was due to )] TJ ET BT 26.250 475.791 Td /F1 9.8 Tf [(neuronal loss or developmental effects we studied 5-week-old mice. As shown in )] TJ ET BT 376.850 475.791 Td /F4 9.8 Tf [(Fig. 1B)] TJ ET BT 409.357 475.791 Td /F1 9.8 Tf [( there were no differences at 5-weeks )] TJ ET BT 26.250 463.887 Td /F1 9.8 Tf [(of age indicating the presence of neuronal loss in R6/2 mouse striatum occurring between 5 and 12 weeks. Use of the nucleator )] TJ ET BT 26.250 451.982 Td /F1 9.8 Tf [(stereologic tool has been reported in numerous studies to characterize changes in striatal neuronal cell body volume in HD )] TJ ET BT 26.250 440.077 Td /F1 9.8 Tf [(mice; however, distribution of neuronal sizes and number estimates by cell size were not determined \(e.g. [12,13]\). We )] TJ ET BT 26.250 428.172 Td /F1 9.8 Tf [(analyzed neuronal population estimates on a cell body size basis \()] TJ ET BT 312.383 428.172 Td /F4 9.8 Tf [(Fig. 2)] TJ ET BT 337.850 428.172 Td /F1 9.8 Tf [(\). At 12 weeks, the most significant finding was a loss )] TJ ET BT 26.250 416.268 Td /F1 9.8 Tf [(of neurons in the size range 550-1050 m)] TJ ET BT 206.722 420.156 Td /F1 8.7 Tf [(3 )] TJ ET BT 213.951 416.268 Td /F1 9.8 Tf [(\()] TJ ET BT 217.197 416.268 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 249.704 416.268 Td /F1 9.8 Tf [(\). Within smaller neuron size categories \(200-500 m)] TJ ET BT 478.361 420.156 Td /F1 8.7 Tf [(3)] TJ ET BT 483.179 416.268 Td /F1 9.8 Tf [(\) neuronal population )] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(mean estimates were increased in R6/2 mice but this did not approach significance \()] TJ ET BT 389.857 404.363 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 422.363 404.363 Td /F1 9.8 Tf [(\). At 5 weeks, there were no )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(significant differences in neuron number estimates per size category \()] TJ ET BT 326.452 392.458 Td /F4 9.8 Tf [(Fig. 2B)] TJ ET BT 358.959 392.458 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 373.053 Td /F1 9.8 Tf [(While there is abundant biochemical and morphologic evidence to support neuronal degeneration in R6/2 mouse striatum )] TJ ET 0.267 0.267 0.267 rg BT 551.365 374.561 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 561.003 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 563.412 374.561 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 573.050 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 362.656 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 35.887 361.149 Td /F1 9.8 Tf [( , we are not aware of reports of morphologic evidence of late-stage neuronal death events in R6/2 striatal neurons. Our )] TJ ET BT 26.250 349.244 Td /F1 9.8 Tf [(findings, and those of others, suggest that while the process of neuronal degeneration is gradual, neuronal death and removal is )] TJ ET BT 26.250 337.339 Td /F1 9.8 Tf [(rapid. We estimated the number of striatal neurons in the process of death when sacrificed at 12-weeks of age. To do this we )] TJ ET BT 26.250 325.434 Td /F1 9.8 Tf [(assumed that neuron death in R6/2 mice begins at 60 days of age )] TJ ET 0.267 0.267 0.267 rg BT 313.495 326.942 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 323.132 325.434 Td /F1 9.8 Tf [( and that the process is linear across time \(a constant )] TJ ET BT 26.250 313.530 Td /F1 9.8 Tf [(number of neurons dying / day\). Based on this, we calculated that R6/2 HD mice lose ~6909 neurons / day / striatum. In one )] TJ ET BT 26.250 301.625 Td /F1 9.8 Tf [(rodent model of acute neuronal injury cell death was detected ~6 hours post-insult )] TJ ET 0.267 0.267 0.267 rg BT 382.574 303.132 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 392.211 301.625 Td /F1 9.8 Tf [( . In one study phagocytosis of apoptotic )] TJ ET BT 26.250 289.720 Td /F1 9.8 Tf [(neurons occurred within 1.5 hours of introduction of microglia into cultures )] TJ ET 0.267 0.267 0.267 rg BT 347.591 291.227 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 357.228 289.720 Td /F1 9.8 Tf [( . Based on these findings we assumed that in )] TJ ET BT 26.250 277.815 Td /F1 9.8 Tf [(R6/2 mice the process of morphologically detectable neuronal death and removal lasts 8 hours. From this we calculated that a )] TJ ET BT 26.250 265.911 Td /F1 9.8 Tf [(20 m section of striatum taken at the level of the anterior commissure of a 12 week R6/2 mouse would contain ~4 dying )] TJ ET BT 26.250 254.006 Td /F1 9.8 Tf [(neurons. Therefore, based on our estimates, the kinetics of neuronal degeneration, loss and removal underlies the difficulty of )] TJ ET BT 26.250 242.101 Td /F1 9.8 Tf [(identifying neuronal death morphologically. Stereologic approaches to quantifying neurons, as used here, can overcome these )] TJ ET BT 26.250 230.196 Td /F1 9.8 Tf [(difficulties.)] TJ ET 0.965 0.965 0.965 rg 26.250 -86.434 555.000 306.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 220.316 m 581.250 220.316 l 581.250 219.566 l 26.250 219.566 l f q 450.000 0 0 291.000 35.250 -80.434 cm /I3 Do Q q 35.250 -86.434 537.000 0.000 re W n Q Q q 15.000 -86.434 577.500 863.434 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(R6/2 mice compared to wild-type littermates. The purpose of the current study was to complete a detailed stereologic analysis )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(of the entire striatum, in particular characterizing neuronal numbers and size changes with disease progression.)] TJ ET BT 26.250 718.969 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(All procedures were pre-approved by the University of Wyoming Animal Care and Use Committee \(IACUC\). R6/2 HD mice were )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(bred and maintained as previously described )] TJ ET 0.267 0.267 0.267 rg BT 222.420 688.617 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 227.239 687.110 Td /F1 9.8 Tf [( . CAG repeat sizes were determined from genomic DNA by Laragen Inc. \(Culver )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(City, CA\) and averaged 181 with a standard deviation of 6. Mice were sacrificed by deep anesthesia then intra-cardiac perfusion )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(with 4% paraformaldehyde and processed for stereology as described )] TJ ET 0.267 0.267 0.267 rg BT 330.801 664.808 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 340.438 663.300 Td /F1 9.8 Tf [( . In brief, brains were sectioned coronally to include )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(the entire striatum at 40 m thickness; every 12)] TJ ET BT 231.088 655.284 Td /F1 8.7 Tf [(th)] TJ ET BT 238.316 651.396 Td /F1 9.8 Tf [( section was mounted and stained using the thionin method. We used the )] TJ ET BT 26.250 639.491 Td /F1 9.8 Tf [(unbiased optical dissector method to estimate striatal neuronal numbers )] TJ ET 0.267 0.267 0.267 rg BT 339.469 640.998 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 349.106 639.491 Td /F1 9.8 Tf [( . We used a counting frame size of 40 x 40 m and )] TJ ET BT 26.250 627.586 Td /F1 9.8 Tf [(a grid size of 500 x 500 m. During the optical dissector procedure we also estimated striatal neuronal cell body volume using )] TJ ET BT 26.250 615.681 Td /F1 9.8 Tf [(the 2D nucleator with five isotropic uniform random sections per cell. All slides were coded so the operator was unaware of the )] TJ ET BT 26.250 603.777 Td /F1 9.8 Tf [(genotype. The numbers of mice per group were: wild-type 5-weeks n=15; HD 5-weeks n=13; wild-type 12 weeks n=9; and HD )] TJ ET BT 26.250 591.872 Td /F1 9.8 Tf [(12 weeks n=10. The average number of neurons / mouse analyzed was 384 for 5-week mice \(range: 245-521\) and 359 for 12-)] TJ ET BT 26.250 579.967 Td /F1 9.8 Tf [(week mice \(range: 272-436\). For each mouse, neurons were binned into 50 m)] TJ ET BT 368.192 583.855 Td /F1 8.7 Tf [(3)] TJ ET BT 373.011 579.967 Td /F1 9.8 Tf [( cell body size categories using Microsoft excel )] TJ ET BT 26.250 568.062 Td /F1 9.8 Tf [(software. The estimated total number of striatal neurons in each size category in wild-type and HD mice was then analyzed )] TJ ET BT 26.250 556.158 Td /F1 9.8 Tf [(using the students t-test in SAS software version 9.2 \(Cary, NC, USA\).)] TJ ET BT 26.250 519.555 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 499.601 Td /F1 9.8 Tf [(R6/2 HD mice had 12.7% fewer striatal neurons at 12-weeks of age \(p=0.035\) \()] TJ ET BT 367.919 499.601 Td /F4 9.8 Tf [(Fig. 1A)] TJ ET BT 400.426 499.601 Td /F1 9.8 Tf [(\). Wild-type mice had a mean of 993891 )] TJ ET BT 26.250 487.696 Td /F1 9.8 Tf [( 38483 \(SEM\) neurons; R6/2 HD mice had a mean of 868043 38483 \(SEM\). To determine if this difference was due to )] TJ ET BT 26.250 475.791 Td /F1 9.8 Tf [(neuronal loss or developmental effects we studied 5-week-old mice. As shown in )] TJ ET BT 376.850 475.791 Td /F4 9.8 Tf [(Fig. 1B)] TJ ET BT 409.357 475.791 Td /F1 9.8 Tf [( there were no differences at 5-weeks )] TJ ET BT 26.250 463.887 Td /F1 9.8 Tf [(of age indicating the presence of neuronal loss in R6/2 mouse striatum occurring between 5 and 12 weeks. Use of the nucleator )] TJ ET BT 26.250 451.982 Td /F1 9.8 Tf [(stereologic tool has been reported in numerous studies to characterize changes in striatal neuronal cell body volume in HD )] TJ ET BT 26.250 440.077 Td /F1 9.8 Tf [(mice; however, distribution of neuronal sizes and number estimates by cell size were not determined \(e.g. [12,13]\). We )] TJ ET BT 26.250 428.172 Td /F1 9.8 Tf [(analyzed neuronal population estimates on a cell body size basis \()] TJ ET BT 312.383 428.172 Td /F4 9.8 Tf [(Fig. 2)] TJ ET BT 337.850 428.172 Td /F1 9.8 Tf [(\). At 12 weeks, the most significant finding was a loss )] TJ ET BT 26.250 416.268 Td /F1 9.8 Tf [(of neurons in the size range 550-1050 m)] TJ ET BT 206.722 420.156 Td /F1 8.7 Tf [(3 )] TJ ET BT 213.951 416.268 Td /F1 9.8 Tf [(\()] TJ ET BT 217.197 416.268 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 249.704 416.268 Td /F1 9.8 Tf [(\). Within smaller neuron size categories \(200-500 m)] TJ ET BT 478.361 420.156 Td /F1 8.7 Tf [(3)] TJ ET BT 483.179 416.268 Td /F1 9.8 Tf [(\) neuronal population )] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(mean estimates were increased in R6/2 mice but this did not approach significance \()] TJ ET BT 389.857 404.363 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 422.363 404.363 Td /F1 9.8 Tf [(\). At 5 weeks, there were no )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(significant differences in neuron number estimates per size category \()] TJ ET BT 326.452 392.458 Td /F4 9.8 Tf [(Fig. 2B)] TJ ET BT 358.959 392.458 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 373.053 Td /F1 9.8 Tf [(While there is abundant biochemical and morphologic evidence to support neuronal degeneration in R6/2 mouse striatum )] TJ ET 0.267 0.267 0.267 rg BT 551.365 374.561 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 561.003 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 563.412 374.561 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 573.050 376.942 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 362.656 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 35.887 361.149 Td /F1 9.8 Tf [( , we are not aware of reports of morphologic evidence of late-stage neuronal death events in R6/2 striatal neurons. Our )] TJ ET BT 26.250 349.244 Td /F1 9.8 Tf [(findings, and those of others, suggest that while the process of neuronal degeneration is gradual, neuronal death and removal is )] TJ ET BT 26.250 337.339 Td /F1 9.8 Tf [(rapid. We estimated the number of striatal neurons in the process of death when sacrificed at 12-weeks of age. To do this we )] TJ ET BT 26.250 325.434 Td /F1 9.8 Tf [(assumed that neuron death in R6/2 mice begins at 60 days of age )] TJ ET 0.267 0.267 0.267 rg BT 313.495 326.942 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 323.132 325.434 Td /F1 9.8 Tf [( and that the process is linear across time \(a constant )] TJ ET BT 26.250 313.530 Td /F1 9.8 Tf [(number of neurons dying / day\). Based on this, we calculated that R6/2 HD mice lose ~6909 neurons / day / striatum. In one )] TJ ET BT 26.250 301.625 Td /F1 9.8 Tf [(rodent model of acute neuronal injury cell death was detected ~6 hours post-insult )] TJ ET 0.267 0.267 0.267 rg BT 382.574 303.132 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 392.211 301.625 Td /F1 9.8 Tf [( . In one study phagocytosis of apoptotic )] TJ ET BT 26.250 289.720 Td /F1 9.8 Tf [(neurons occurred within 1.5 hours of introduction of microglia into cultures )] TJ ET 0.267 0.267 0.267 rg BT 347.591 291.227 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 357.228 289.720 Td /F1 9.8 Tf [( . Based on these findings we assumed that in )] TJ ET BT 26.250 277.815 Td /F1 9.8 Tf [(R6/2 mice the process of morphologically detectable neuronal death and removal lasts 8 hours. From this we calculated that a )] TJ ET BT 26.250 265.911 Td /F1 9.8 Tf [(20 m section of striatum taken at the level of the anterior commissure of a 12 week R6/2 mouse would contain ~4 dying )] TJ ET BT 26.250 254.006 Td /F1 9.8 Tf [(neurons. Therefore, based on our estimates, the kinetics of neuronal degeneration, loss and removal underlies the difficulty of )] TJ ET BT 26.250 242.101 Td /F1 9.8 Tf [(identifying neuronal death morphologically. Stereologic approaches to quantifying neurons, as used here, can overcome these )] TJ ET BT 26.250 230.196 Td /F1 9.8 Tf [(difficulties.)] TJ ET 0.965 0.965 0.965 rg 26.250 -86.434 555.000 306.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 220.316 m 581.250 220.316 l 581.250 219.566 l 26.250 219.566 l f q 450.000 0 0 291.000 35.250 -80.434 cm /I3 Do Q q 35.250 -86.434 537.000 0.000 re W n Q Q q 450.000 0 0 291.000 35.250 -80.434 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 160 0 obj << /Type /Annot /Subtype /Link /A 161 0 R /Border [0 0 0] /H /I /Rect [ 222.4200 687.8154 227.2387 696.6337 ] >> endobj 161 0 obj << /Type /Action >> endobj 162 0 obj << /Type /Annot /Subtype /Link /A 163 0 R /Border [0 0 0] /H /I /Rect [ 330.8010 664.0059 340.4383 672.8243 ] >> endobj 163 0 obj << /Type /Action >> endobj 164 0 obj << /Type /Annot /Subtype /Link /A 165 0 R /Border [0 0 0] /H /I /Rect [ 339.4688 640.1964 349.1061 649.0147 ] >> endobj 165 0 obj << /Type /Action >> endobj 166 0 obj << /Type /Annot /Subtype /Link /A 167 0 R /Border [0 0 0] /H /I /Rect [ 551.3655 373.7589 561.0028 382.5773 ] >> endobj 167 0 obj << /Type /Action >> endobj 168 0 obj << /Type /Annot /Subtype /Link /A 169 0 R /Border [0 0 0] /H /I /Rect [ 563.4122 373.7589 573.0495 382.5773 ] >> endobj 169 0 obj << /Type /Action >> endobj 170 0 obj << /Type /Annot /Subtype /Link /A 171 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 361.8542 35.8873 370.6725 ] >> endobj 171 0 obj << /Type /Action >> endobj 172 0 obj << /Type /Annot /Subtype /Link /A 173 0 R /Border [0 0 0] /H /I /Rect [ 313.4948 326.1399 323.1321 334.9583 ] >> endobj 173 0 obj << /Type /Action >> endobj 174 0 obj << /Type /Annot /Subtype /Link /A 175 0 R /Border [0 0 0] /H /I /Rect [ 382.5735 302.3304 392.2108 311.1488 ] >> endobj 175 0 obj << /Type /Action >> endobj 176 0 obj << /Type /Annot /Subtype /Link /A 177 0 R /Border [0 0 0] /H /I /Rect [ 347.5905 290.4257 357.2278 299.2440 ] >> endobj 177 0 obj << /Type /Action >> endobj 178 0 obj << /Type /Annot /Subtype /Link /A 179 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -80.4345 485.2500 210.5655 ] >> endobj 179 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2013/11/Fig1.jpg) >> endobj 180 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 388 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 44816>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?K(?*W(?_V)-((J? 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P-value: *=p<0.05 )] TJ ET Q 0.965 0.965 0.965 rg 26.250 307.773 555.000 383.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 691.636 m 581.250 691.636 l 581.250 690.886 l 26.250 690.886 l f 26.250 307.773 m 581.250 307.773 l 581.250 308.523 l 26.250 308.523 l f q 450.000 0 0 290.250 35.250 391.636 cm /I4 Do Q q 35.250 319.023 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 376.113 Td /F4 9.8 Tf [(Fig. 2: Striatal neuronal atrophy and loss in R6/2 HD mice.)] TJ ET BT 35.250 356.743 Td /F1 9.8 Tf [(Striatal neuronal atrophy and loss in R6/2 HD mice. )] TJ ET BT 259.588 356.743 Td /F4 9.8 Tf [(A.)] TJ ET BT 269.338 356.743 Td /F1 9.8 Tf [( R6/2 HD mice have fewer neurons in the cell body size range 550-)] TJ ET BT 35.250 343.006 Td /F1 9.8 Tf [(1050 m)] TJ ET BT 73.187 348.726 Td /F1 8.7 Tf [(3 )] TJ ET BT 80.415 343.006 Td /F1 9.8 Tf [(at 12 weeks of age. )] TJ ET BT 167.678 343.006 Td /F4 9.8 Tf [(B. )] TJ ET BT 180.138 343.006 Td /F1 9.8 Tf [(There are no significant differences in neuronal number estimates per cell body size )] TJ ET BT 35.250 329.270 Td /F1 9.8 Tf [(category in 5-week-old mice. Round circles = R6/2 HD; closed circles=wild-type. P-values: *=p<0.05, **=p<0.01)] TJ ET Q BT 26.250 273.551 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 253.597 Td /F1 9.8 Tf [(Numerous studies have documented a reduction in striatal neuronal cell body size in R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 455.962 255.104 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 465.599 257.485 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 468.008 255.104 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 477.646 253.597 Td /F1 9.8 Tf [( . Here we took a new )] TJ ET BT 26.250 241.692 Td /F1 9.8 Tf [(approach to analysis by determining estimates of neuronal numbers in specific size categories. Our analysis cannot clearly )] TJ ET BT 26.250 229.788 Td /F1 9.8 Tf [(reveal in what population neuronal loss occurs in R6/2 striatum as decreased numbers of neurons in the size range 550-1050 )] TJ ET BT 26.250 217.883 Td /F1 9.8 Tf [(m)] TJ ET BT 39.793 221.771 Td /F1 8.7 Tf [(3)] TJ ET BT 44.611 217.883 Td /F1 9.8 Tf [( \()] TJ ET BT 50.569 217.883 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 83.075 217.883 Td /F1 9.8 Tf [(\) could result from atrophy, loss or a combination. However, the results do suggest that larger medium spiny )] TJ ET BT 26.250 205.978 Td /F1 9.8 Tf [(neurons are more vulnerable to degeneration in R6/2 HD.)] TJ ET BT 26.250 186.573 Td /F1 9.8 Tf [(Taken together, our findings corroborate and extend those reported previously )] TJ ET 0.267 0.267 0.267 rg BT 366.047 188.081 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 375.685 186.573 Td /F1 9.8 Tf [( . Our analysis of striatal neuron number within )] TJ ET BT 26.250 174.669 Td /F1 9.8 Tf [(whole striatum reveals a 12% loss of neurons in R6/2 mice which is less than the 26% loss reported in an analysis based on )] TJ ET BT 26.250 162.764 Td /F1 9.8 Tf [(examination of the anterior striatum to the level of the anterior commissure, which is about 2/3 of the striatum )] TJ ET 0.267 0.267 0.267 rg BT 497.702 164.271 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 507.339 162.764 Td /F1 9.8 Tf [( . This )] TJ ET BT 26.250 150.859 Td /F1 9.8 Tf [(discrepancy could be for a number of reasons that includes differences in neuronal loss in whole striatum versus anterior )] TJ ET BT 26.250 138.954 Td /F1 9.8 Tf [(striatum and differences in the respective R6/2 mouse models including CAG size and background genetic effects due to the )] TJ ET BT 26.250 127.050 Td /F1 9.8 Tf [(backcrossing approach to colony maintenance. Importantly, however, all mouse models of HD studied so far have significantly )] TJ ET BT 26.250 115.145 Td /F1 9.8 Tf [(less striatal neuronal loss than the 90% reported in advanced human HD )] TJ ET 0.267 0.267 0.267 rg BT 342.189 116.652 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 347.008 115.145 Td /F1 9.8 Tf [( . While striatal neuronal loss in R6/2 mice and other )] TJ ET BT 26.250 103.240 Td /F1 9.8 Tf [(mouse HD models is modest, the presence of dramatic loss in human HD brain suggests that estimation of striatum neuronal )] TJ ET BT 26.250 91.335 Td /F1 9.8 Tf [(number should be an important outcome in HD mice which can be used to evaluate therapies in pre-clinical studies. Finally, we )] TJ ET BT 26.250 79.431 Td /F1 9.8 Tf [(undertook a sample size analysis to determine the number of mice required to detect rescue of neuronal loss in R6/2 mice in a )] TJ ET BT 26.250 67.526 Td /F1 9.8 Tf [(hypothetical treatment study. Parameters used were: 80% power, a significance level of 0.05, standard deviations proportional )] TJ ET BT 26.250 55.621 Td /F1 9.8 Tf [(to the mean and a one-tailed t-test )] TJ ET 0.267 0.267 0.267 rg BT 177.463 57.128 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 187.100 55.621 Td /F1 9.8 Tf [( . Based on these parameters we calculated that for 50% and 100% rescue of striatal )] TJ ET BT 26.250 43.716 Td /F1 9.8 Tf [(neuronal loss it would be necessary to have group sizes of 10 and 5, respectively. While stereologic approaches to estimate )] TJ ET Q q 15.000 29.431 577.500 747.569 re W n 0.965 0.965 0.965 rg 26.250 699.136 555.000 77.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l f q 35.250 710.386 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Striatal neuronal loss occurs in R6/2 HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Striatal neuronal loss occurs in R6/2 HD mice. )] TJ ET BT 236.812 748.106 Td /F4 9.8 Tf [(A.)] TJ ET BT 246.562 748.106 Td /F1 9.8 Tf [( R6/2 HD mice have 12.7% fewer neurons than wild-type litter-mate mice )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(at 12 weeks of age. p=0.035, )] TJ ET BT 163.443 734.370 Td /F4 9.8 Tf [(B.)] TJ ET BT 173.193 734.370 Td /F1 9.8 Tf [( R6/2 HD mice do no differ from wild-type litter-mate mice in striatal neuronal number )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(estimates at 5 weeks of age. P-value: *=p<0.05 )] TJ ET Q 0.965 0.965 0.965 rg 26.250 307.773 555.000 383.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 691.636 m 581.250 691.636 l 581.250 690.886 l 26.250 690.886 l f 26.250 307.773 m 581.250 307.773 l 581.250 308.523 l 26.250 308.523 l f q 450.000 0 0 290.250 35.250 391.636 cm /I4 Do Q q 35.250 319.023 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 376.113 Td /F4 9.8 Tf [(Fig. 2: Striatal neuronal atrophy and loss in R6/2 HD mice.)] TJ ET BT 35.250 356.743 Td /F1 9.8 Tf [(Striatal neuronal atrophy and loss in R6/2 HD mice. )] TJ ET BT 259.588 356.743 Td /F4 9.8 Tf [(A.)] TJ ET BT 269.338 356.743 Td /F1 9.8 Tf [( R6/2 HD mice have fewer neurons in the cell body size range 550-)] TJ ET BT 35.250 343.006 Td /F1 9.8 Tf [(1050 m)] TJ ET BT 73.187 348.726 Td /F1 8.7 Tf [(3 )] TJ ET BT 80.415 343.006 Td /F1 9.8 Tf [(at 12 weeks of age. )] TJ ET BT 167.678 343.006 Td /F4 9.8 Tf [(B. )] TJ ET BT 180.138 343.006 Td /F1 9.8 Tf [(There are no significant differences in neuronal number estimates per cell body size )] TJ ET BT 35.250 329.270 Td /F1 9.8 Tf [(category in 5-week-old mice. Round circles = R6/2 HD; closed circles=wild-type. P-values: *=p<0.05, **=p<0.01)] TJ ET Q BT 26.250 273.551 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 253.597 Td /F1 9.8 Tf [(Numerous studies have documented a reduction in striatal neuronal cell body size in R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 455.962 255.104 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 465.599 257.485 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 468.008 255.104 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 477.646 253.597 Td /F1 9.8 Tf [( . Here we took a new )] TJ ET BT 26.250 241.692 Td /F1 9.8 Tf [(approach to analysis by determining estimates of neuronal numbers in specific size categories. Our analysis cannot clearly )] TJ ET BT 26.250 229.788 Td /F1 9.8 Tf [(reveal in what population neuronal loss occurs in R6/2 striatum as decreased numbers of neurons in the size range 550-1050 )] TJ ET BT 26.250 217.883 Td /F1 9.8 Tf [(m)] TJ ET BT 39.793 221.771 Td /F1 8.7 Tf [(3)] TJ ET BT 44.611 217.883 Td /F1 9.8 Tf [( \()] TJ ET BT 50.569 217.883 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 83.075 217.883 Td /F1 9.8 Tf [(\) could result from atrophy, loss or a combination. However, the results do suggest that larger medium spiny )] TJ ET BT 26.250 205.978 Td /F1 9.8 Tf [(neurons are more vulnerable to degeneration in R6/2 HD.)] TJ ET BT 26.250 186.573 Td /F1 9.8 Tf [(Taken together, our findings corroborate and extend those reported previously )] TJ ET 0.267 0.267 0.267 rg BT 366.047 188.081 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 375.685 186.573 Td /F1 9.8 Tf [( . Our analysis of striatal neuron number within )] TJ ET BT 26.250 174.669 Td /F1 9.8 Tf [(whole striatum reveals a 12% loss of neurons in R6/2 mice which is less than the 26% loss reported in an analysis based on )] TJ ET BT 26.250 162.764 Td /F1 9.8 Tf [(examination of the anterior striatum to the level of the anterior commissure, which is about 2/3 of the striatum )] TJ ET 0.267 0.267 0.267 rg BT 497.702 164.271 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 507.339 162.764 Td /F1 9.8 Tf [( . This )] TJ ET BT 26.250 150.859 Td /F1 9.8 Tf [(discrepancy could be for a number of reasons that includes differences in neuronal loss in whole striatum versus anterior )] TJ ET BT 26.250 138.954 Td /F1 9.8 Tf [(striatum and differences in the respective R6/2 mouse models including CAG size and background genetic effects due to the )] TJ ET BT 26.250 127.050 Td /F1 9.8 Tf [(backcrossing approach to colony maintenance. Importantly, however, all mouse models of HD studied so far have significantly )] TJ ET BT 26.250 115.145 Td /F1 9.8 Tf [(less striatal neuronal loss than the 90% reported in advanced human HD )] TJ ET 0.267 0.267 0.267 rg BT 342.189 116.652 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 347.008 115.145 Td /F1 9.8 Tf [( . While striatal neuronal loss in R6/2 mice and other )] TJ ET BT 26.250 103.240 Td /F1 9.8 Tf [(mouse HD models is modest, the presence of dramatic loss in human HD brain suggests that estimation of striatum neuronal )] TJ ET BT 26.250 91.335 Td /F1 9.8 Tf [(number should be an important outcome in HD mice which can be used to evaluate therapies in pre-clinical studies. Finally, we )] TJ ET BT 26.250 79.431 Td /F1 9.8 Tf [(undertook a sample size analysis to determine the number of mice required to detect rescue of neuronal loss in R6/2 mice in a )] TJ ET BT 26.250 67.526 Td /F1 9.8 Tf [(hypothetical treatment study. Parameters used were: 80% power, a significance level of 0.05, standard deviations proportional )] TJ ET BT 26.250 55.621 Td /F1 9.8 Tf [(to the mean and a one-tailed t-test )] TJ ET 0.267 0.267 0.267 rg BT 177.463 57.128 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 187.100 55.621 Td /F1 9.8 Tf [( . Based on these parameters we calculated that for 50% and 100% rescue of striatal )] TJ ET BT 26.250 43.716 Td /F1 9.8 Tf [(neuronal loss it would be necessary to have group sizes of 10 and 5, respectively. While stereologic approaches to estimate )] TJ ET Q q 15.000 29.431 577.500 747.569 re W n 0.965 0.965 0.965 rg 26.250 699.136 555.000 77.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l f q 35.250 710.386 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Striatal neuronal loss occurs in R6/2 HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Striatal neuronal loss occurs in R6/2 HD mice. )] TJ ET BT 236.812 748.106 Td /F4 9.8 Tf [(A.)] TJ ET BT 246.562 748.106 Td /F1 9.8 Tf [( R6/2 HD mice have 12.7% fewer neurons than wild-type litter-mate mice )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(at 12 weeks of age. p=0.035, )] TJ ET BT 163.443 734.370 Td /F4 9.8 Tf [(B.)] TJ ET BT 173.193 734.370 Td /F1 9.8 Tf [( R6/2 HD mice do no differ from wild-type litter-mate mice in striatal neuronal number )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(estimates at 5 weeks of age. P-value: *=p<0.05 )] TJ ET Q 0.965 0.965 0.965 rg 26.250 307.773 555.000 383.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 691.636 m 581.250 691.636 l 581.250 690.886 l 26.250 690.886 l f 26.250 307.773 m 581.250 307.773 l 581.250 308.523 l 26.250 308.523 l f q 450.000 0 0 290.250 35.250 391.636 cm /I4 Do Q q 35.250 319.023 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 376.113 Td /F4 9.8 Tf [(Fig. 2: Striatal neuronal atrophy and loss in R6/2 HD mice.)] TJ ET BT 35.250 356.743 Td /F1 9.8 Tf [(Striatal neuronal atrophy and loss in R6/2 HD mice. )] TJ ET BT 259.588 356.743 Td /F4 9.8 Tf [(A.)] TJ ET BT 269.338 356.743 Td /F1 9.8 Tf [( R6/2 HD mice have fewer neurons in the cell body size range 550-)] TJ ET BT 35.250 343.006 Td /F1 9.8 Tf [(1050 m)] TJ ET BT 73.187 348.726 Td /F1 8.7 Tf [(3 )] TJ ET BT 80.415 343.006 Td /F1 9.8 Tf [(at 12 weeks of age. )] TJ ET BT 167.678 343.006 Td /F4 9.8 Tf [(B. )] TJ ET BT 180.138 343.006 Td /F1 9.8 Tf [(There are no significant differences in neuronal number estimates per cell body size )] TJ ET BT 35.250 329.270 Td /F1 9.8 Tf [(category in 5-week-old mice. Round circles = R6/2 HD; closed circles=wild-type. P-values: *=p<0.05, **=p<0.01)] TJ ET Q BT 26.250 273.551 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 253.597 Td /F1 9.8 Tf [(Numerous studies have documented a reduction in striatal neuronal cell body size in R6/2 HD mice )] TJ ET 0.267 0.267 0.267 rg BT 455.962 255.104 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 465.599 257.485 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 468.008 255.104 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 477.646 253.597 Td /F1 9.8 Tf [( . Here we took a new )] TJ ET BT 26.250 241.692 Td /F1 9.8 Tf [(approach to analysis by determining estimates of neuronal numbers in specific size categories. Our analysis cannot clearly )] TJ ET BT 26.250 229.788 Td /F1 9.8 Tf [(reveal in what population neuronal loss occurs in R6/2 striatum as decreased numbers of neurons in the size range 550-1050 )] TJ ET BT 26.250 217.883 Td /F1 9.8 Tf [(m)] TJ ET BT 39.793 221.771 Td /F1 8.7 Tf [(3)] TJ ET BT 44.611 217.883 Td /F1 9.8 Tf [( \()] TJ ET BT 50.569 217.883 Td /F4 9.8 Tf [(Fig. 2A)] TJ ET BT 83.075 217.883 Td /F1 9.8 Tf [(\) could result from atrophy, loss or a combination. However, the results do suggest that larger medium spiny )] TJ ET BT 26.250 205.978 Td /F1 9.8 Tf [(neurons are more vulnerable to degeneration in R6/2 HD.)] TJ ET BT 26.250 186.573 Td /F1 9.8 Tf [(Taken together, our findings corroborate and extend those reported previously )] TJ ET 0.267 0.267 0.267 rg BT 366.047 188.081 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 375.685 186.573 Td /F1 9.8 Tf [( . Our analysis of striatal neuron number within )] TJ ET BT 26.250 174.669 Td /F1 9.8 Tf [(whole striatum reveals a 12% loss of neurons in R6/2 mice which is less than the 26% loss reported in an analysis based on )] TJ ET BT 26.250 162.764 Td /F1 9.8 Tf [(examination of the anterior striatum to the level of the anterior commissure, which is about 2/3 of the striatum )] TJ ET 0.267 0.267 0.267 rg BT 497.702 164.271 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 507.339 162.764 Td /F1 9.8 Tf [( . This )] TJ ET BT 26.250 150.859 Td /F1 9.8 Tf [(discrepancy could be for a number of reasons that includes differences in neuronal loss in whole striatum versus anterior )] TJ ET BT 26.250 138.954 Td /F1 9.8 Tf [(striatum and differences in the respective R6/2 mouse models including CAG size and background genetic effects due to the )] TJ ET BT 26.250 127.050 Td /F1 9.8 Tf [(backcrossing approach to colony maintenance. Importantly, however, all mouse models of HD studied so far have significantly )] TJ ET BT 26.250 115.145 Td /F1 9.8 Tf [(less striatal neuronal loss than the 90% reported in advanced human HD )] TJ ET 0.267 0.267 0.267 rg BT 342.189 116.652 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 347.008 115.145 Td /F1 9.8 Tf [( . While striatal neuronal loss in R6/2 mice and other )] TJ ET BT 26.250 103.240 Td /F1 9.8 Tf [(mouse HD models is modest, the presence of dramatic loss in human HD brain suggests that estimation of striatum neuronal )] TJ ET BT 26.250 91.335 Td /F1 9.8 Tf [(number should be an important outcome in HD mice which can be used to evaluate therapies in pre-clinical studies. Finally, we )] TJ ET BT 26.250 79.431 Td /F1 9.8 Tf [(undertook a sample size analysis to determine the number of mice required to detect rescue of neuronal loss in R6/2 mice in a )] TJ ET BT 26.250 67.526 Td /F1 9.8 Tf [(hypothetical treatment study. Parameters used were: 80% power, a significance level of 0.05, standard deviations proportional )] TJ ET BT 26.250 55.621 Td /F1 9.8 Tf [(to the mean and a one-tailed t-test )] TJ ET 0.267 0.267 0.267 rg BT 177.463 57.128 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 187.100 55.621 Td /F1 9.8 Tf [( . Based on these parameters we calculated that for 50% and 100% rescue of striatal )] TJ ET BT 26.250 43.716 Td /F1 9.8 Tf [(neuronal loss it would be necessary to have group sizes of 10 and 5, respectively. While stereologic approaches to estimate )] TJ ET Q q 450.000 0 0 290.250 35.250 391.636 cm /I4 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 223 0 obj << /Type /Annot /Subtype /Link /A 224 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 391.6365 485.2500 681.8865 ] >> endobj 224 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2013/11/Fig2.jpg) >> endobj 225 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 387 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 27809>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?J(?_V* -8)QGz(EPEPE(((@((/|WYȗSy+1\ka;-"T9}A~J]ZSֳG$*mى8*m;K HlicRŝ;1$$fӦ/Fdcq+j^z0+7qIn18 Ȫ)9.n,\ZQujD04G#cVDf hֹ ŭ[Nn4_woJ~bc~9ʀ}"#">651[+i-.|̢H-\)o_eq-OҪ>_'|%-'c=L$)+$@Q@Q@(PEPE ((((@Q@J(QEvv(((1QEQҊ(1@j(EQEA}WͿ?soE&RaP5U95yA\ж$(;hQKI3JzQA-PvZ:QK(4RPA>gs}+M-YjcIK`'BnQ e!L^asIԠO=6i:ƞ4T` q}^ԴIuGIQ[&.`aʺHAA/<|eccx-HX$Zs=[cUHpϛdPFX1@C9|Wm2$nHbX@cC<*ƅ]+Fq}c<3rrz$qN95rMNfII!K"Q 1|QX~q43A͎U=+G#=Eq?e+p[ҊZO-'ZZJ3KFi:N3K~)m4+VkFvӚlr (q@-PffK@ ֌K(4is@ KIK@%Q@fv ZJS@%-'z8GZ^QMR)8(RfQE y\6TQ}WͿHKyA\T- (E0QEQGj())h(((QE(7Z4:5WVH~oY4!OrBH◉i!RdFYv~|-Լ{ j-tS9gGfV`3P| җD 23֝H*1K@(1EP (Q@(PGz(QE(E(Q@w ( ( Q((ъ(omCl?UU?Ub!ފA֊`(E'z-RPNbGjJZ) њJ_@hE ;Z%[?Q,Bm}7 ZNŧϣBt36Fg2FѬD#/% R&N}e`a!YYa\ 6J?.O$p?fCJ _g^eb(pXB T֩⦻c;M2EyKcw;l,QWp 6Fj^6Ψ5;]R Tsmha]*ReT# W@m.Nlڅ;-&2kGic-] (撀)(Iz 3_ f~xKbZ'j,G. ~&iw d?)ea1<_|dVƝ|Vmv?LHVYxv H x'w~ EVxzy$$FHq""rT7$fn|[oƙ4mgXgֵ==>kh2'FrSXzQšO_xGOs o0e O-a3t;>"ִ-2*bu*T/pTC^UHถ?Hd̎Uao_t),| }W._qV$?{IsiG]GP71T@ o?OZ_GK,w; |_,|֨uŬ)7 ԓ< ώ~"JH-¼y>|3;XMkI34h@^_`95+8揦kK{ ݘi 08cw.@Hq@{/ឣrm ,_Mu j$9*[wm*sԑ8R| .|ey|Wq:5QKl!f ]\@M =Q-Q@((1F( QE(P1EbQEbEb1F( 1EP1F( Q(QA/soE-y\6TRe!s_V;{qXBؐE `QޓQI@ FhE3EJ-E-PIEGt-&AOqןJ7n',7_SM*&ӯnu S]CinʗCB,~g ĪX9eTbcÐF z@xOw>1^kڍ\$MPG ᑧ`Vݣެ6c|iY3XjQjSE{MܶI`Y RPc >OkVZޡ-nȊBAo<(PO*v>?'>|!}*? 趚NtGM]zy'5 JK(7|) sǶ(no`!e?D^g%Ys$6 _!n㟊Ѽ![ēW5 'ĕx͜jm0vRBui,-e# ڼ|qoC5O =q[%rBH> B ísiJ{:徱u=ֲQY:&0HLlB9?++3话f_S|ǍhWxt} "r+`u8ɭ"J(E%-JZ(QJZ(@ F)hJZ((Z(J)hPQKE!R@_ǔͿmAaPʧU?ʬP$(EPFh (Q@J((QEQGZ3EPGJ((ols|. 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/@<;'#eh-TIWM3`ĜHh88ː ]_#׆oo~5|=uWѵ'× f𥙷F(D0c[Kd$dgѯ%jfZ]i0Cre =?8_7 ? xbm?^4fM2X-5|=_7\xgU|Gon|eU[994|t0A;,it{4 33e4~4|='?xk]s'x;>ׯcD72"dX{5Sӟ׍& y!#-l<12i_YФXD$dD[ݠ*0pxgօ$M#K5kaq (Si #~vsGvw]V7gl=иq""T qfOj5Im!cvFuk|7x[Iu]we ʡWɀy98 ? <=sKⷀ5 ݽwvZ$"X䶝F,c3 7J'Ekz͆7Ea2 L:_ ~lrgsPEPEf ( (EQEQFh3@Q@Q@_ǕsoE ) aPʬaP5U`ж$;wE0Q@Av QEQE(@ E(QE#(n EQ֎h )h+~q6ypFcMpS2`cMwZ/5AW:kӠtejUF pN4xkguk>л`b]ŘF1f9>t>Dkmg&uAIA>q2 {~=^ځm42&#f8a43lWIÕhԂ=JGq'_-- I?~?7oϑ0΢|H<$Ə¶Eݧ&/ ^e3F 뺴R{^M&ZK$ѽ@?*e݀0R:FS#>(k>6Y_kZZkVq1M,29 d` 0I*Yi5oX5[!iuTg Hv!Tzt4m%56B^Q~m`(dͻWl< i`ȒȮq&Ƒ"($OĽ1/C3\hI>&>ZVYp?'`L+ro!x9+i`k7K5BcnyLy{ɮC_ 4_k0:# n𬘎T+"g4Fw yUdҴ/, 1s]Env=H+$Q4tO#jZ7z\;BڂM pZA*$wTE'\f=oLMi|Wٳ;<Ӄ,( *drx_į '/Yes>Wӯn^?7d>`@Uw2su1kgLmWO^gԺ}M'W'v@hv![<7#`U? ȵ5iZv9(2%~}9qWl^2{cªiR^O&i4'(\FAPy~58, o ǯj$pGYpWc(`pl|1p/i7,c̖]1'=+Ȯ>"x_?R>EFȎQ@FV~8Xmo<[ddd7rIES٠Tmug|olŨI1>bц5ំ-mt}F{,+vy8n?կ~odڌqIxm#3*/PQ@ s>Zlͦ&KHځ>L`}t4Ѯ o8r[#)sy;E7H𦉠4{ ;VT%E6mi]*/r&WԱ{(`yςEjz%} F"|,.w1{ch2k6 vLnQ@'3nҒ H%-s(ۇ䟽|okakiiV6ЬQ-aIva(6zAEz> \-u-O/-ʘn.RI#*IRIS -K>rKO,2qr`~(ѵ+ۭ&?dKezQ|9E;AܥBE@ې|Px֞-t[ ="$Im_* z zơN}S:K_"5b6OF>(ѿҲmGϐW)iE>l/$rٻ;]O@֊(Sb(iJ(((((((((((( mL endstream endobj 226 0 obj << /Type /Annot /Subtype /Link /A 227 0 R /Border [0 0 0] /H /I /Rect [ 455.9617 254.3027 465.5991 263.1210 ] >> endobj 227 0 obj << /Type /Action >> endobj 228 0 obj << /Type /Annot /Subtype /Link /A 229 0 R /Border [0 0 0] /H /I /Rect [ 468.0084 254.3027 477.6458 263.1210 ] >> endobj 229 0 obj << /Type /Action >> endobj 230 0 obj << /Type /Annot /Subtype /Link /A 231 0 R /Border [0 0 0] /H /I /Rect [ 366.0473 187.2789 375.6846 196.0972 ] >> endobj 231 0 obj << /Type /Action >> endobj 232 0 obj << /Type /Annot /Subtype /Link /A 233 0 R /Border [0 0 0] /H /I /Rect [ 497.7015 163.4694 507.3388 172.2877 ] >> endobj 233 0 obj << /Type /Action >> endobj 234 0 obj << /Type /Annot /Subtype /Link /A 235 0 R /Border [0 0 0] /H /I /Rect [ 342.1890 115.8504 347.0077 124.6687 ] >> endobj 235 0 obj << /Type /Action >> endobj 236 0 obj << /Type /Annot /Subtype /Link /A 237 0 R /Border [0 0 0] /H /I /Rect [ 177.4627 56.3267 187.1001 65.1450 ] >> endobj 237 0 obj << /Type /Action >> endobj 238 0 obj << /Type /Annot /Subtype /Link /A 239 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 391.6365 485.2500 681.8865 ] >> endobj 239 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2013/11/Fig2.jpg) >> endobj 240 0 obj << /Type /Annot /Subtype /Link /A 241 0 R /Border [0 0 0] /H /I /Rect [ 455.9617 254.3027 465.5991 263.1210 ] >> endobj 241 0 obj << /Type /Action >> endobj 242 0 obj << /Type /Annot /Subtype /Link /A 243 0 R /Border [0 0 0] /H /I /Rect [ 468.0084 254.3027 477.6458 263.1210 ] >> endobj 243 0 obj << /Type /Action >> endobj 244 0 obj << /Type /Annot /Subtype /Link /A 245 0 R /Border [0 0 0] /H /I /Rect [ 366.0473 187.2789 375.6846 196.0972 ] >> endobj 245 0 obj << /Type /Action >> endobj 246 0 obj << /Type /Annot /Subtype /Link /A 247 0 R /Border [0 0 0] /H /I /Rect [ 497.7015 163.4694 507.3388 172.2877 ] >> endobj 247 0 obj << /Type /Action >> endobj 248 0 obj << /Type /Annot /Subtype /Link /A 249 0 R /Border [0 0 0] /H /I /Rect [ 342.1890 115.8504 347.0077 124.6687 ] >> endobj 249 0 obj << /Type /Action >> endobj 250 0 obj << /Type /Annot /Subtype /Link /A 251 0 R /Border [0 0 0] /H /I /Rect [ 177.4627 56.3267 187.1001 65.1450 ] >> endobj 251 0 obj << /Type /Action >> endobj 252 0 obj << /Type /Annot /Subtype /Link /A 253 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 391.6365 485.2500 681.8865 ] >> endobj 253 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2013/11/Fig2.jpg) >> endobj 254 0 obj << /Type /Annot /Subtype /Link /A 255 0 R /Border [0 0 0] /H /I /Rect [ 455.9617 254.3027 465.5991 263.1210 ] >> endobj 255 0 obj << /Type /Action >> endobj 256 0 obj << /Type /Annot /Subtype /Link /A 257 0 R /Border [0 0 0] /H /I /Rect [ 468.0084 254.3027 477.6458 263.1210 ] >> endobj 257 0 obj << /Type /Action >> endobj 258 0 obj << /Type /Annot /Subtype /Link /A 259 0 R /Border [0 0 0] /H /I /Rect [ 366.0473 187.2789 375.6846 196.0972 ] >> endobj 259 0 obj << /Type /Action >> endobj 260 0 obj << /Type /Annot /Subtype /Link /A 261 0 R /Border [0 0 0] /H /I /Rect [ 497.7015 163.4694 507.3388 172.2877 ] >> endobj 261 0 obj << /Type /Action >> endobj 262 0 obj << /Type /Annot /Subtype /Link /A 263 0 R /Border [0 0 0] /H /I /Rect [ 342.1890 115.8504 347.0077 124.6687 ] >> endobj 263 0 obj << /Type /Action >> endobj 264 0 obj << /Type /Annot /Subtype /Link /A 265 0 R /Border [0 0 0] /H /I /Rect [ 177.4627 56.3267 187.1001 65.1450 ] >> endobj 265 0 obj << /Type /Action >> endobj 266 0 obj << /Type /Page /Parent 3 0 R /Contents 267 0 R >> endobj 267 0 obj << /Length 21448 >> stream 0.271 0.267 0.267 rg q 15.000 40.792 577.500 736.208 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(neuronal loss can be labor intensive, our calculations indicate that only modest group sizes are required. Analysis of neuronal )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(numbers may be especially valuable for interventions that are protective in mice and when further characterization of protective )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effects is needed prior to considering human testing.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(Lindsay Dodds and Jianfang Chen contributed equally to this work. We thank Dr. Kenneth Gerow, Department of Statistics, )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(University of Wyoming, for assistance with the sample size analysis.)] TJ ET BT 26.250 646.103 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 618.648 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 618.648 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, et al. \(1985\) Neuropathological classification of Huntington's )] TJ ET BT 26.250 606.744 Td /F1 9.8 Tf [(disease. J Neuropathol Exp Neurol 44: 559-577.)] TJ ET BT 26.250 587.339 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 587.339 Td /F1 9.8 Tf [(Tabrizi SJ, Scahill RI, Durr A, Roos RA, Leavitt BR, et al. \(2011\) Biological and clinical changes in premanifest and early )] TJ ET BT 26.250 575.434 Td /F1 9.8 Tf [(stage Huntington's disease in the TRACK-HD study: the 12-month longitudinal analysis. Lancet Neurol 10: 31-42.)] TJ ET BT 26.250 556.029 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 556.029 Td /F1 9.8 Tf [(Ferrante RJ \(2009\) Mouse models of Huntington's disease and methodological considerations for therapeutic trials. Biochim )] TJ ET BT 26.250 544.125 Td /F1 9.8 Tf [(Biophys Acta 1792: 506-520.)] TJ ET BT 26.250 524.720 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 524.720 Td /F1 9.8 Tf [(Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, et al. \(1996\) Exon 1 of the HD gene with an expanded CAG )] TJ ET BT 26.250 512.815 Td /F1 9.8 Tf [(repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493-506)] TJ ET BT 26.250 493.410 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 493.410 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, et al. \(2013 \) Iron Accumulates in Huntingtons Disease Neurons: Protection by )] TJ ET BT 26.250 481.506 Td /F1 9.8 Tf [(Deferoxamine. PLoS ONE 8)] TJ ET BT 26.250 462.101 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 462.101 Td /F1 9.8 Tf [(Chopra V, Quinti L, Kim J, Vollor L, Narayanan KL, et al. \(2012\) The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's )] TJ ET BT 26.250 450.196 Td /F1 9.8 Tf [(disease mouse models. Cell Rep 2: 1492-1497.)] TJ ET BT 26.250 430.791 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 430.791 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 418.887 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 399.482 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 399.482 Td /F1 9.8 Tf [(Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, et al. \(2003\) Selective striatal neuronal loss in a YAC128 )] TJ ET BT 26.250 387.577 Td /F1 9.8 Tf [(mouse model of Huntington disease. Hum Mol Genet 12: 1555-1567.)] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 368.172 Td /F1 9.8 Tf [( Hickey MA, Kosmalska A, Enayati J, Cohen R, Zeitlin S, et al. \(2008\) Extensive early motor and non-motor behavioral )] TJ ET BT 26.250 356.268 Td /F1 9.8 Tf [(deficits are followed by striatal neuronal loss in knock-in Huntington's disease mice. Neuroscience 157: 280-295.)] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 336.863 Td /F1 9.8 Tf [(Zhang Y, Ona VO, Li M, Drozda M, Dubois-Dauphin M, et al. \(2003\) Sequential activation of individual caspases, and of )] TJ ET BT 26.250 324.958 Td /F1 9.8 Tf [(alterations in Bcl-2 proapoptotic signals in a mouse model of Huntington's disease. J Neurochem 87: 1184-1192.)] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 305.553 Td /F1 9.8 Tf [(Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, et al. \(2003\) Mutant huntingtin causes context-dependent neurodegeneration in )] TJ ET BT 26.250 293.649 Td /F1 9.8 Tf [(mice with Huntington's disease. J Neurosci 23: 2193-2202.)] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 274.244 Td /F1 9.8 Tf [(Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, et al. \(2002\) Tauroursodeoxycholic acid, a bile acid, is )] TJ ET BT 26.250 262.339 Td /F1 9.8 Tf [(neuroprotective in a transgenic animal model of Huntington's disease. Proc Natl Acad Sci U S A 99: 10671-10676.)] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 242.934 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, et al. \(2007\) A small-molecule therapeutic lead for Huntington's )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proc Natl Acad Sci U S A 104: 16685-)] TJ ET BT 26.250 219.125 Td /F1 9.8 Tf [(16689.)] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 199.720 Td /F1 9.8 Tf [(Stack EC, Kubilus JK, Smith K, Cormier K, Del Signore SJ, et al. \(2005\) Chronology of behavioral symptoms and )] TJ ET BT 26.250 187.815 Td /F1 9.8 Tf [(neuropathological sequela in R6/2 Huntington's disease transgenic mice. J Comp Neurol 490: 354-370.)] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 168.411 Td /F1 9.8 Tf [(Fox JH, Connor T, Chopra V, Dorsey K, Kama JA, et al. \(2010\) The mTOR kinase inhibitor Everolimus decreases S6 kinase )] TJ ET BT 26.250 156.506 Td /F1 9.8 Tf [(phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of )] TJ ET BT 26.250 144.601 Td /F1 9.8 Tf [(Huntington's disease. Mol Neurodegener 5: 26.)] TJ ET BT 26.250 125.196 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 125.196 Td /F1 9.8 Tf [(West MJ, Slomianka L, Gundersen HJ \(1991\) Unbiased stereological estimation of the total number of neurons in )] TJ ET BT 26.250 113.292 Td /F1 9.8 Tf [(thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231: 482-497.)] TJ ET BT 26.250 93.887 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 93.887 Td /F1 9.8 Tf [(Turmaine M, Raza A, Mahal A, Mangiarini L, Bates GP, et al. \(2000\) Nonapoptotic neurodegeneration in a transgenic )] TJ ET BT 26.250 81.982 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Proc Natl Acad Sci U S A 97: 8093-8097.)] TJ ET BT 26.250 62.577 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 62.577 Td /F1 9.8 Tf [(Wang CZ, Johnson KM \(2007\) The role of caspase-3 activation in phencyclidine-induced neuronal death in postnatal rats. )] TJ ET BT 26.250 50.673 Td /F1 9.8 Tf [(Neuropsychopharmacology 32: 1178-1194.)] TJ ET Q q 15.000 40.792 577.500 736.208 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(neuronal loss can be labor intensive, our calculations indicate that only modest group sizes are required. Analysis of neuronal )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(numbers may be especially valuable for interventions that are protective in mice and when further characterization of protective )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effects is needed prior to considering human testing.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(Lindsay Dodds and Jianfang Chen contributed equally to this work. We thank Dr. Kenneth Gerow, Department of Statistics, )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(University of Wyoming, for assistance with the sample size analysis.)] TJ ET BT 26.250 646.103 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 618.648 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 618.648 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, et al. \(1985\) Neuropathological classification of Huntington's )] TJ ET BT 26.250 606.744 Td /F1 9.8 Tf [(disease. J Neuropathol Exp Neurol 44: 559-577.)] TJ ET BT 26.250 587.339 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 587.339 Td /F1 9.8 Tf [(Tabrizi SJ, Scahill RI, Durr A, Roos RA, Leavitt BR, et al. \(2011\) Biological and clinical changes in premanifest and early )] TJ ET BT 26.250 575.434 Td /F1 9.8 Tf [(stage Huntington's disease in the TRACK-HD study: the 12-month longitudinal analysis. Lancet Neurol 10: 31-42.)] TJ ET BT 26.250 556.029 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 556.029 Td /F1 9.8 Tf [(Ferrante RJ \(2009\) Mouse models of Huntington's disease and methodological considerations for therapeutic trials. Biochim )] TJ ET BT 26.250 544.125 Td /F1 9.8 Tf [(Biophys Acta 1792: 506-520.)] TJ ET BT 26.250 524.720 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 524.720 Td /F1 9.8 Tf [(Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, et al. \(1996\) Exon 1 of the HD gene with an expanded CAG )] TJ ET BT 26.250 512.815 Td /F1 9.8 Tf [(repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493-506)] TJ ET BT 26.250 493.410 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 493.410 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, et al. \(2013 \) Iron Accumulates in Huntingtons Disease Neurons: Protection by )] TJ ET BT 26.250 481.506 Td /F1 9.8 Tf [(Deferoxamine. PLoS ONE 8)] TJ ET BT 26.250 462.101 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 462.101 Td /F1 9.8 Tf [(Chopra V, Quinti L, Kim J, Vollor L, Narayanan KL, et al. \(2012\) The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's )] TJ ET BT 26.250 450.196 Td /F1 9.8 Tf [(disease mouse models. Cell Rep 2: 1492-1497.)] TJ ET BT 26.250 430.791 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 430.791 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 418.887 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 399.482 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 399.482 Td /F1 9.8 Tf [(Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, et al. \(2003\) Selective striatal neuronal loss in a YAC128 )] TJ ET BT 26.250 387.577 Td /F1 9.8 Tf [(mouse model of Huntington disease. Hum Mol Genet 12: 1555-1567.)] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 368.172 Td /F1 9.8 Tf [( Hickey MA, Kosmalska A, Enayati J, Cohen R, Zeitlin S, et al. \(2008\) Extensive early motor and non-motor behavioral )] TJ ET BT 26.250 356.268 Td /F1 9.8 Tf [(deficits are followed by striatal neuronal loss in knock-in Huntington's disease mice. Neuroscience 157: 280-295.)] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 336.863 Td /F1 9.8 Tf [(Zhang Y, Ona VO, Li M, Drozda M, Dubois-Dauphin M, et al. \(2003\) Sequential activation of individual caspases, and of )] TJ ET BT 26.250 324.958 Td /F1 9.8 Tf [(alterations in Bcl-2 proapoptotic signals in a mouse model of Huntington's disease. J Neurochem 87: 1184-1192.)] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 305.553 Td /F1 9.8 Tf [(Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, et al. \(2003\) Mutant huntingtin causes context-dependent neurodegeneration in )] TJ ET BT 26.250 293.649 Td /F1 9.8 Tf [(mice with Huntington's disease. J Neurosci 23: 2193-2202.)] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 274.244 Td /F1 9.8 Tf [(Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, et al. \(2002\) Tauroursodeoxycholic acid, a bile acid, is )] TJ ET BT 26.250 262.339 Td /F1 9.8 Tf [(neuroprotective in a transgenic animal model of Huntington's disease. Proc Natl Acad Sci U S A 99: 10671-10676.)] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 242.934 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, et al. \(2007\) A small-molecule therapeutic lead for Huntington's )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proc Natl Acad Sci U S A 104: 16685-)] TJ ET BT 26.250 219.125 Td /F1 9.8 Tf [(16689.)] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 199.720 Td /F1 9.8 Tf [(Stack EC, Kubilus JK, Smith K, Cormier K, Del Signore SJ, et al. \(2005\) Chronology of behavioral symptoms and )] TJ ET BT 26.250 187.815 Td /F1 9.8 Tf [(neuropathological sequela in R6/2 Huntington's disease transgenic mice. J Comp Neurol 490: 354-370.)] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 168.411 Td /F1 9.8 Tf [(Fox JH, Connor T, Chopra V, Dorsey K, Kama JA, et al. \(2010\) The mTOR kinase inhibitor Everolimus decreases S6 kinase )] TJ ET BT 26.250 156.506 Td /F1 9.8 Tf [(phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of )] TJ ET BT 26.250 144.601 Td /F1 9.8 Tf [(Huntington's disease. Mol Neurodegener 5: 26.)] TJ ET BT 26.250 125.196 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 125.196 Td /F1 9.8 Tf [(West MJ, Slomianka L, Gundersen HJ \(1991\) Unbiased stereological estimation of the total number of neurons in )] TJ ET BT 26.250 113.292 Td /F1 9.8 Tf [(thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231: 482-497.)] TJ ET BT 26.250 93.887 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 93.887 Td /F1 9.8 Tf [(Turmaine M, Raza A, Mahal A, Mangiarini L, Bates GP, et al. \(2000\) Nonapoptotic neurodegeneration in a transgenic )] TJ ET BT 26.250 81.982 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Proc Natl Acad Sci U S A 97: 8093-8097.)] TJ ET BT 26.250 62.577 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 62.577 Td /F1 9.8 Tf [(Wang CZ, Johnson KM \(2007\) The role of caspase-3 activation in phencyclidine-induced neuronal death in postnatal rats. )] TJ ET BT 26.250 50.673 Td /F1 9.8 Tf [(Neuropsychopharmacology 32: 1178-1194.)] TJ ET Q q 15.000 40.792 577.500 736.208 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(neuronal loss can be labor intensive, our calculations indicate that only modest group sizes are required. Analysis of neuronal )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(numbers may be especially valuable for interventions that are protective in mice and when further characterization of protective )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effects is needed prior to considering human testing.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(Lindsay Dodds and Jianfang Chen contributed equally to this work. We thank Dr. Kenneth Gerow, Department of Statistics, )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(University of Wyoming, for assistance with the sample size analysis.)] TJ ET BT 26.250 646.103 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 618.648 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 618.648 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, et al. \(1985\) Neuropathological classification of Huntington's )] TJ ET BT 26.250 606.744 Td /F1 9.8 Tf [(disease. J Neuropathol Exp Neurol 44: 559-577.)] TJ ET BT 26.250 587.339 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 587.339 Td /F1 9.8 Tf [(Tabrizi SJ, Scahill RI, Durr A, Roos RA, Leavitt BR, et al. \(2011\) Biological and clinical changes in premanifest and early )] TJ ET BT 26.250 575.434 Td /F1 9.8 Tf [(stage Huntington's disease in the TRACK-HD study: the 12-month longitudinal analysis. Lancet Neurol 10: 31-42.)] TJ ET BT 26.250 556.029 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 556.029 Td /F1 9.8 Tf [(Ferrante RJ \(2009\) Mouse models of Huntington's disease and methodological considerations for therapeutic trials. Biochim )] TJ ET BT 26.250 544.125 Td /F1 9.8 Tf [(Biophys Acta 1792: 506-520.)] TJ ET BT 26.250 524.720 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 524.720 Td /F1 9.8 Tf [(Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, et al. \(1996\) Exon 1 of the HD gene with an expanded CAG )] TJ ET BT 26.250 512.815 Td /F1 9.8 Tf [(repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493-506)] TJ ET BT 26.250 493.410 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 493.410 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, et al. \(2013 \) Iron Accumulates in Huntingtons Disease Neurons: Protection by )] TJ ET BT 26.250 481.506 Td /F1 9.8 Tf [(Deferoxamine. PLoS ONE 8)] TJ ET BT 26.250 462.101 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 462.101 Td /F1 9.8 Tf [(Chopra V, Quinti L, Kim J, Vollor L, Narayanan KL, et al. \(2012\) The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's )] TJ ET BT 26.250 450.196 Td /F1 9.8 Tf [(disease mouse models. Cell Rep 2: 1492-1497.)] TJ ET BT 26.250 430.791 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 430.791 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 418.887 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 399.482 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 399.482 Td /F1 9.8 Tf [(Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, et al. \(2003\) Selective striatal neuronal loss in a YAC128 )] TJ ET BT 26.250 387.577 Td /F1 9.8 Tf [(mouse model of Huntington disease. Hum Mol Genet 12: 1555-1567.)] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 368.172 Td /F1 9.8 Tf [( Hickey MA, Kosmalska A, Enayati J, Cohen R, Zeitlin S, et al. \(2008\) Extensive early motor and non-motor behavioral )] TJ ET BT 26.250 356.268 Td /F1 9.8 Tf [(deficits are followed by striatal neuronal loss in knock-in Huntington's disease mice. Neuroscience 157: 280-295.)] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 336.863 Td /F1 9.8 Tf [(Zhang Y, Ona VO, Li M, Drozda M, Dubois-Dauphin M, et al. \(2003\) Sequential activation of individual caspases, and of )] TJ ET BT 26.250 324.958 Td /F1 9.8 Tf [(alterations in Bcl-2 proapoptotic signals in a mouse model of Huntington's disease. J Neurochem 87: 1184-1192.)] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 305.553 Td /F1 9.8 Tf [(Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, et al. \(2003\) Mutant huntingtin causes context-dependent neurodegeneration in )] TJ ET BT 26.250 293.649 Td /F1 9.8 Tf [(mice with Huntington's disease. J Neurosci 23: 2193-2202.)] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 274.244 Td /F1 9.8 Tf [(Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, et al. \(2002\) Tauroursodeoxycholic acid, a bile acid, is )] TJ ET BT 26.250 262.339 Td /F1 9.8 Tf [(neuroprotective in a transgenic animal model of Huntington's disease. Proc Natl Acad Sci U S A 99: 10671-10676.)] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 242.934 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, et al. \(2007\) A small-molecule therapeutic lead for Huntington's )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proc Natl Acad Sci U S A 104: 16685-)] TJ ET BT 26.250 219.125 Td /F1 9.8 Tf [(16689.)] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 199.720 Td /F1 9.8 Tf [(Stack EC, Kubilus JK, Smith K, Cormier K, Del Signore SJ, et al. \(2005\) Chronology of behavioral symptoms and )] TJ ET BT 26.250 187.815 Td /F1 9.8 Tf [(neuropathological sequela in R6/2 Huntington's disease transgenic mice. J Comp Neurol 490: 354-370.)] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 168.411 Td /F1 9.8 Tf [(Fox JH, Connor T, Chopra V, Dorsey K, Kama JA, et al. \(2010\) The mTOR kinase inhibitor Everolimus decreases S6 kinase )] TJ ET BT 26.250 156.506 Td /F1 9.8 Tf [(phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of )] TJ ET BT 26.250 144.601 Td /F1 9.8 Tf [(Huntington's disease. Mol Neurodegener 5: 26.)] TJ ET BT 26.250 125.196 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 125.196 Td /F1 9.8 Tf [(West MJ, Slomianka L, Gundersen HJ \(1991\) Unbiased stereological estimation of the total number of neurons in )] TJ ET BT 26.250 113.292 Td /F1 9.8 Tf [(thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231: 482-497.)] TJ ET BT 26.250 93.887 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 93.887 Td /F1 9.8 Tf [(Turmaine M, Raza A, Mahal A, Mangiarini L, Bates GP, et al. \(2000\) Nonapoptotic neurodegeneration in a transgenic )] TJ ET BT 26.250 81.982 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Proc Natl Acad Sci U S A 97: 8093-8097.)] TJ ET BT 26.250 62.577 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 62.577 Td /F1 9.8 Tf [(Wang CZ, Johnson KM \(2007\) The role of caspase-3 activation in phencyclidine-induced neuronal death in postnatal rats. )] TJ ET BT 26.250 50.673 Td /F1 9.8 Tf [(Neuropsychopharmacology 32: 1178-1194.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 268 0 obj << /Type /Page /Parent 3 0 R /Contents 269 0 R >> endobj 269 0 obj << /Length 3337 >> stream 0.271 0.267 0.267 rg q 15.000 668.072 577.500 108.929 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Witting A, Muller P, Herrmann A, Kettenmann H, Nolte C \(2000\) Phagocytic clearance of apoptotic neurons by )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Neurochem 75: 1060-1070.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Ferrante RJ, Kubilus JK, Lee J, Ryu H, Beesen A, et al. \(2003\) Histone deacetylase inhibition by sodium butyrate )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci 23: 9418-9427.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Owen DB \(1965\) The Power of Student's t-test. Journal of the American Statistical Association 60: 320-333.)] TJ ET Q q 15.000 668.072 577.500 108.929 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Witting A, Muller P, Herrmann A, Kettenmann H, Nolte C \(2000\) Phagocytic clearance of apoptotic neurons by )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Neurochem 75: 1060-1070.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Ferrante RJ, Kubilus JK, Lee J, Ryu H, Beesen A, et al. \(2003\) Histone deacetylase inhibition by sodium butyrate )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci 23: 9418-9427.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Owen DB \(1965\) The Power of Student's t-test. Journal of the American Statistical Association 60: 320-333.)] TJ ET Q q 15.000 668.072 577.500 108.929 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Witting A, Muller P, Herrmann A, Kettenmann H, Nolte C \(2000\) Phagocytic clearance of apoptotic neurons by )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Neurochem 75: 1060-1070.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Ferrante RJ, Kubilus JK, Lee J, Ryu H, Beesen A, et al. \(2003\) Histone deacetylase inhibition by sodium butyrate )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci 23: 9418-9427.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Owen DB \(1965\) The Power of Student's t-test. Journal of the American Statistical Association 60: 320-333.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj xref 0 270 0000000000 65535 f 0000000008 00000 n 0000000073 00000 n 0000000119 00000 n 0000000399 00000 n 0000000436 00000 n 0000000699 00000 n 0000001307 00000 n 0000030732 00000 n 0000030839 00000 n 0000030947 00000 n 0000031058 00000 n 0000031171 00000 n 0000031559 00000 n 0000036040 00000 n 0000036167 00000 n 0000036356 00000 n 0000036483 00000 n 0000036672 00000 n 0000036799 00000 n 0000036906 00000 n 0000037032 00000 n 0000037134 00000 n 0000037261 00000 n 0000037372 00000 n 0000037500 00000 n 0000037596 00000 n 0000037724 00000 n 0000037825 00000 n 0000037951 00000 n 0000037987 00000 n 0000038115 00000 n 0000038151 00000 n 0000038279 00000 n 0000038315 00000 n 0000038441 00000 n 0000038477 00000 n 0000038605 00000 n 0000038641 00000 n 0000038769 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