Interpreting scan data acquired from multiple scanners : A study with Alzheimer 's disease Large , multi-site studies utilizing MRI-derived measures from multiple scanners present an opportunity to advance research by pooling data . On the other hand , it remains unclear whether or not the potential confound introduced by different scanners and upgrades will devalue the integrity of any results . Although there are studies of scanner differences for the purpose of calibration and quality control , the current literature is devoid of studies that describe the analysis of multi-scanner data with regard to the interaction of scanner -LRB- s -RRB- with effects of interest . We investigated a data-set of 136 subjects , 62 patients with mild to moderate Alzheimer 's disease and 74 cognitively normal elderly controls , with MRI scans from one center that were acquired over 10 years with 6 different scanners and multiple upgrades over time . We used a whole-brain voxel-wise analysis to evaluate the effect of scanner , effect of disease , and the interaction of scanner and disease for the 6 different scanners . The effect of disease in patients showed the expected significant reduction of grey matter in the medial temporal lobe . Scanner differences were substantially less than the group differences and only significant in the thalamus . There was no significant interaction of scanner with disease group . We describe the rationale for concluding that our results were not confounded by scanner differences . Similar analyses in other multi-scanner data-sets could be used to justify the pooling of data when needed , such as in studies of rare disorders or in multi-center designs . Introduction Techniques utilizing in vivo MRI-derived measures of brain tissue morphometrics show increasing promise for aiding clinicians in assessment and diagnosis of disease . Studies that use longitudinal and/or multi-site data-sets , such as the Alzheimer 's Disease Neuroimaging Initiative -LRB- Mueller et al. , 2005 -RRB- , have the potential to provide a wealth of information . The large numbers of subjects resulting from pooling multi-scanner data-sets has numerous advantages . It increases sensitivity thus allowing detection of subtle effects and enables the analysis of subgroups within a cohort . Additionally , pooling offers increased reliability and confidence about the size of effect by averaging out unforeseen confounds and hence a method for carrying out meta-analyses or analyses of rare subjects with orphan-diseases scanned at home rather than in distant centers . Any one study may have unforeseen bias which is lessened by pooling . However , one important confound of combining images gathered from different scanners is the potential for scanner effects to introduce systematic error , thus making the interpretation of results difficult . Partial volume effects and image intensity inhomogeneity can introduce error into automatic segmentation with any given scanner -LRB- Li et al. , 2005 -RRB- . Noise of the electronics of the MRI system , subject-specific physiological noise and imaging gradient non-linearities also contribute to image intensity variability -LRB- Jovicich et al. , 2006 ; Littmann et al. , 2006 -RRB- . Furthermore , differences in subject positioning between sites add to the variability in distortion fields -LRB- Jovicich et al. , 2006 -RRB- and images can vary as a function of protocol differences between a baseline and a later scan or with drifts in instrument signal to noise over time -LRB- Preboske et al. , 2006 -RRB- . The interaction of scanner differences with segmentation remains a particular concern and potential cause of varied measures of regional tissue volume in the brain -LRB- Ashburner and Friston , 2000 -RRB- . It is therefore vital to confirm that there is no important interaction between scanner and effect of interest and/or account for the effects of different scanners in a principled manner before pooling from different scanners can be recommended as a routine . The use of phantoms to calibrate different scanners and account for scanner drift or upgrades is well established -LRB- Tofts , 1998 ; Van Haren et al. , 2003 ; Schnack et al. , 2004 ; Jovicich et al. , 2006 -RRB- and a useful way to guarantee quality . Studies using a small number of healthy subjects repeatedly scanned on different scanners or after upgrades are also recommended to test reliability -LRB- Tofts , 1998 ; Han et al. , 2006 ; Jovicich et al. , 2006 -RRB- . However , it is not clear that the extra calibration efforts can actually correct for the majority of these sources of variability -LRB- Jovicich et al. , 2006 -RRB- , or are even necessary . Also , it has yet to be established how confidently the results from multi-scanner data-sets can be interpreted irrespective of up-front calibration . Multi-scanner studies , though not common , are increasing . A longitudinal aging study by Raz and colleagues , one of the few which discussed the analysis of images derived from multiple scanners , concluded that using different scanners in 4 subjects did not affect measured intracranial volume with a manual tracing method -LRB- Raz et al. , 2005 -RRB- . Likewise , manual hippocampal measurements performed on both 1.5 T and 3.0 T scanners in 8 healthy controls were not affected by field strength -LRB- Breillmann et al. , 2001 -RRB- . We are not aware of any study that has described an automated analysis of a large , multi-scanner data-set with the aim of assessing whether scanner-associated biases are significant . We set out to investigate scanner effects in an automatically preprocessed data-set of subjects with mild to moderate AD and cognitively normal elderly people whose MRI scans had been collected over a period of 10 years with multiple scanners and upgrades on a consistent platform and repeated calibrations . Even though the concern has been raised that segmentation errors are a particular problem in atrophic brains -LRB- Good et al. , 2002 -RRB- , we did not find this to be an issue in our sample , possibly because the disease was early . We hypothesized that the changes due to AD pathology are large compared to scanner induced distortions and that there would be no interaction of scanner with case and/or control groups . In this paper , we report the results in our data-set and outline a method by which multi-scanner data-sets might be pooled with confidence . Methods and results Our data-set included a total of 6 scanners and 136 subjects scanned over a 10-year period . None of the subjects were scanned more than once . All scans were done on the same platform , General Electric Signa 1.5 T scanners -LRB- slice thickness 1.6 , matrix dimensions 256 × 192 -RRB- . There were minor variations in the TR , TE , and flip angle -LRB- see Table 1 -RRB- . Scanners also underwent upgrades over time . Importantly , all scanners were monitored with daily phantom quality checks which calibrated the gradients to within ± 1 mm over a 200-mm volume centered at iso-center , monitored signal to noise and radio frequency -LRB- RF -RRB- transmit gain . The major hardware elements -LRB- body resonance module gradient coil and birdcage head transmit -- receive volume coil -RRB- were unchanged throughout time and across scanners , except that for the oldest scanner -LRB- scanner 2 -RRB- , the wiring in the resistive shim set was not cooled to super conducting temperatures , whereas for the other 5 scanners , the shim coils were inside the dewar and were cooled to the superconducting range . We used voxel-based morphometry -LRB- VBM -RRB- to evaluate the interaction of scanner and grey matter segmented modulated images for the 6 different scanners . VBM has the advantage of assessing the whole brain and not being biased to one particular region or structure . It entails a voxel-wise comparison of local volume of grey matter between groups after the images are spatially normalized into the same space , segmented , modulated , and smoothed . Voxel-wise statistical parametric maps result from statistically thresholded contrasts after corrections for multiple comparisons -LRB- Ashburner and Friston , 2000 -RRB- using false discovery rate -LRB- FDR -RRB- -LRB- Benjamini and Hochberg , 1995 -RRB- . Images were visually inspected for artifacts or structural abnormalities unrelated to AD . They were firstly segmented into white -LRB- WM -RRB- and grey matter -LRB- GM -RRB- using SPM5 -LRB- Wellcome Trust Centre for Neuroimaging , Institute of Neurology , UCL , London UK -- http://www.fil.ion.ucl.ac.uk/spm -RRB- . Then , WM and GM segments were further normalized to a population template generated from the complete image set using a diffeomorphic registration algorithm . This non-linear warping technique minimizes structural variation between subjects -LRB- Ashburner , 2007 -RRB- . For comparison , we also repeated the analysis using the more widely used standard SPM5 segmentation code -LRB- Ashburner and Friston , 2005 -RRB- instead of the diffeomorphic registration algorithm . Resolution before normalization was − .9 , .9 , 1.6 and after normalization was − 1.5 , 1.5 , 1.5 for the diffeomorphic registration algorithm and − 2.0 , 2.0 , 2.0 with the standard SPM5 procedure . A separate ` modulation ' step -LRB- Ashburner and Friston , 2000 -RRB- was used to ensure that the overall amount of each tissue class was not altered by the spatial normalization procedure . Modulation was performed by multiplying the warped tissue probability maps by the Jacobian determinant of the warp on a voxel-by-voxel basis , which represents the relative volume ratio before and after warping , thus allowing voxel intensities in the segmented grey matter map , together with the size of the voxels , to reflect regional volume and preserve total grey matter volume from before the warp . Modulated grey matter scans were smoothed using a 6-mm full-width at half-maximum Gaussian kernel . The smoothed grey matter images were analyzed in a factorial design , with the 6 different scanners as one factor -LRB- SCANNER -RRB- with 6 levels and the presence of AD -LRB- GROUP -RRB- as the second factor with two levels -LRB- present and absent -RRB- . Age , gender , and total intracranial volume were entered as covariates -LRB- Fig. 1 -RRB- . We performed F-tests correcting for multiple comparisons across the brain -LRB- FDR correction -RRB- . The degrees of freedom was 121 for all comparisons . The effect of group revealed strong effects -LRB- p < 0.001 -RRB- in the left medial temporal lobe -LRB- − 29 , − 24 , − 9 -LSB- x , y , z -RSB- ; F = 100.34 -RRB- and right medial temporal lobe -LRB- 30 , − 27 , − 9 -LSB- x , y , z -RSB- ; F = 91.44 -RRB- -LRB- Fig. 2 -RRB- . The effect of scanner showed significant differences -LRB- p < 0.05 -RRB- in the right -LRB- 9 , − 30 , 0 -LSB- x , y , z -RSB- ; F = 11.93 -RRB- and left -LRB- − 9 , − 11 , 8 -LSB- x , y , z -RSB- ; F = 9.69 -RRB- thalami -LRB- Fig. 3 -RRB- , but this effect was less than the effect of group . T-tests contrasting each scanner against the others revealed the scanner effect in the thalamus was mostly due to the oldest scanner -LRB- scanner 2 -RRB- . One newer scanner -LRB- scanner 6 -RRB- revealed an effect that did not survive FDR correction in the right thalamus -LRB- 8 , − 5 , − 3 -LSB- x , y , z -RSB- ; T = 4.03 -RRB- , whereas the oldest scanner contrasted with the others revealed a significant effect -LRB- p < 0.05 -RRB- in the left thalamus -LRB- − 9 , − 11 , 6 -LSB- x , y , z -RSB- ; T = 5.94 -RRB- . Despite the effect of scanner , there was no significant interaction of scanner with group , the highest Z-score being 3.82 with a corrected p value of 0.942 . We performed F-tests for each possible combination of scanners and found no significant interaction with any of these groupings . When using the standard VBM procedure implemented in SPM5 for normalization and segmentation , the results of the various contrasts followed the same patterns as when using the diffeomorphic normalization/segmentation procedure . We failed to find an interaction of scanner with group ; the highest Z-score was 3.78 with an FDR corrected p value of 0.954 . The effect of scanner showed significant differences -LRB- p < 0.05 -RRB- in the left thalamus -LRB- − 10 , − 10 , 6 -LSB- x , y , z -RSB- ; F = 9.49 -RRB- and the right thalamus -LRB- 8 , − 28 , − 4 -LSB- x , y , z -RSB- ; F = 9.41 -RRB- . The effect of group also showed strong effects -LRB- p < 0.001 -RRB- in the left medial temporal lobe -LRB- − 26 , − 14 , − 20 -LSB- x , y , z -RSB- ; F = 101.46 -RRB- and right medial temporal lobe -LRB- 26 , − 8 , − 18 -LSB- x , y , z -RSB- ; F = 77.98 -RRB- . Importantly , we found no significant scanner effects in the medial temporal lobe cluster -LRB- − 29 , − 24 , − 9 -LSB- x , y , z -RSB- ; F = 0.01 -RRB- and the disease effect size in the thalamus was minimal compared to the effect size in the medial temporal lobe -LRB- 9 , − 30 , 0 -LSB- x , y , z -RSB- ; F = 1.06 -RRB- , suggesting minimal scanner effects in the areas that are most affected by AD and minimal disease effects in the areas showing scanner differences . Confidence intervals , which are reflective of the standard deviations , for the contrast estimates are shown in Fig. 4 . At the voxel of greatest effect of group , the confidence interval is small relative to the effect size for the main effect of group . The opposite is true for the effect of scanner at the area of greatest disease , which is further evidence of lack of effect . For the main effect of scanner , confidence intervals are similar between the different scanner contrasts -LRB- see Fig. 4A -RRB- , indirectly suggestive of relatively little variance across scanners . Because there were 4 software upgrades , we also analyzed the interaction of software version and disease . Using the same basic design matrix as described for the interaction with scanners , this time the contrasts included cases and controls from each software version , covaried with age , gender and intracranial volume . As with the effect of scanner , there was no significant interaction of software version with group , the highest Z-score being 3.65 with a corrected p value of 0.880 . Discussion In our data-set , we found the effect of disease to be substantially larger than the effect of scanner and failed to find a significant interaction of disease with scanner or software upgrades . In general , the effect of disease in AD is liable to be larger than the effect of scanner , which is supported by our result of no important interaction between scanner and effect of interest . Ideally , further studies with an even larger data set to better calculate the effect sizes and quantify the distance between the scanner effect cluster and group effect cluster could be done to validate our findings . However , comparison of the magnitude of the scanner effect versus disease effect in the medial temporal lobe cluster in our data-set demonstrates that scanner differences had minimal effects in the areas that are important in the study of AD . Furthermore , even though there are likely to be some differences among data from different scanners , our experiments were explicitly designed to detect scanner-related differences . By modeling appropriate confounds in the design matrix , it appears possible to remove these small effects . We were able to model this interaction easily because relatively homogeneous cases and controls were scanned in each machine . We are unable to say whether lesser or more subtle and distributed changes would be as resistant to scanner effects . Though we did not detect a significant interaction of scanner with disease , we can not be absolutely certain it is indeed due to the absence of such effect . The lack of significance may be a reflection of the lack of statistical power , e.g. , insufficient number of scans . Other causes such as a high average residual variance or residual variance inhomogeneities could also under-power the detection of the effects . However , the variance inhomogeneity was considered in our analysis by assuming unequal variance for the different levels of each of the two factors in our full factorial design -LRB- the two factors are the scanner and group -RRB- . In a post-hoc manner , we explored the residual variance across scanners to assess whether that explained the lack of sensitivity in the results . In the area of greatest disease effect , the variance was low and did not reflect significant inhomogeneity , but in the area of greatest scanner effect there was lower average residual variance and more variability . Though our tests for variance inhomogeneity across the 6 scanners was not voxel-by-voxel over the whole brain volume , our findings seem to support that the variance inhomogeneity is location-dependent and should be accounted for when analyzing data acquired from different scanners as we attempted to do in our analysis . The greatest effect of scanner was in the thalamus . The effect of the thalamus was largely driven by the scanner with the resistive shim set that was not cooled to superconducting temperatures , which suggests an impact of such hardware differences on thalamic segmentation . The composition of the thalamus is an issue of debate as it is not completely grey matter receiving numerous white matter tracts from other parts of the brain . Additionally , the grey matter intensity value of the thalamus is different from that of cortical grey matter . The intrinsically poor intensity contrast in the thalamus renders it susceptible to small differences in image contrast due to scanner differences . There is also less variability in this part of the brain , so tests will be more sensitive to such differences . These factors may contribute to the difficulty of accurate segmentation of the thalamus in addition to scanner effects . The relatively small effect of scanner is potentially attributable to quality control measures during data acquisition and/or the robustness of the segmentation method . The SPM5 algorithm , by using spatial information together with intensity information , should be more robust to such differences than a segmentation algorithm that is purely intensity-based . However , this study did not directly investigate which forms of preprocessing are most affected by scanner parameters . It is important to note , though , that the effect was not due to the diffeomorphic normalization procedure , since we obtained similar patterns to those with the standard SPM5 normalization . Presumably , the use of daily scanner calibrations allows for a relatively constant intensity contrast between grey and white matter voxels between scans . Routine procedures designed to minimize image intensity differences over time are useful , particularly since it has been demonstrated that several common scan functions can potentially introduce measurement errors as high as 100 % -LRB- i.e. , much greater than the disease effect of AD -RRB- , without appropriate quality control measures in place -LRB- Preboske et al. , 2006 -RRB- . Even though it is common experience that current generation scanners are remarkably stable over time , they do still drift in a way that is correctable with judicious calibration . Though we collected no data to determine the frequency and level of sophistication necessary , basic calibration akin to the current standard in most major centers is likely sufficient . Furthermore , while it can be useful to collect data on inter-subject variability by scanning the same subjects in different scanners , such efforts would not directly answer the question of whether data from different subjects on different scanners can be pooled . The unified segmentation method of SPM5 -LRB- Ashburner and Friston , 2005 -RRB- produces an estimate of the tissue class intensities from a fitting of spatial priors to the image , which allows for differences in image intensities between scans . Additionally , any image intensity differences due to subject to subject variation in coupling of the RF coil to the head that may be introduced by different head shapes and sizes are likely to be well-accounted for by bias corrections in the unified segmentation algorithm . As long as provision for different scanners and/or upgrades is made within an analysis , the effect of scanner regardless of magnitude is not likely to devalue the integrity of results . However , if there is a true physical interaction of the biological effect of interest with the method of measurement , perfect calibration or even using a single scanner would not prevent bias . On the other hand , any unusually large effect from one scanner would be attenuated by the totality of scans from different scanners that make up the template , which averages the different scanner effects for normalization . To date , methodological differences between individual studies have prevented comprehensive pooling of data in meta-analyses of AD MRI studies -LRB- Zakzanis et al. , 2003 ; Whitwell and Jack , 2005 ; Wahlund et al. , 2005 -RRB- . To the extent that data pooling was possible , the meta-analysis compared regional volume between disease groups -LRB- Zakzanis et al. , 2003 -RRB- . Although there may be concern about error introduced by the variability inherent in preprocessing many individuals across studies , automated VBM methods successfully overcome the problems to combine information from multiple scans and studies . VBM meta-analyses of pooled scans permit the usual range of analyses beyond simple categorical comparisons -LRB- e.g. , regression , age interaction , subgroup , nonlinear , etc. -RRB- . Our results suggest that for Alzheimer 's disease , particularly if the imaging platform remains constant , variations attributable to individual scanners and upgrades may have negligible effects on segmented grey matter images . We expect , though do not yet have the data , that the approach is robust enough to accommodate using both 1.5 T and 3 T scans in the same meta-analysis . A principled approach to test the validity of a meta-analysis is to carry out an a priori interaction analysis of scanner by biological effect to be studied to exclude scanner-specific compromising effects . Additional comfort can be gained by finding that the biological effect is of greater magnitude than any effect of scanner . An alternative approach is to model each scanner 's effects separately , thereby permitting a principled meta-analysis on pooled data . That data can be pooled from different scanners without corroding the integrity of results is reassuring for large multi-site studies .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

multi-scanner
magnetic resonance imaging (mri)
alzheimer's disease
voxel based morphometry


Human Coenzyme Q10 Deficiency Ubiquinone -LRB- coenzyme Q10 or CoQ10 -RRB- is a lipid-soluble component of virtually all cell membranes and has multiple metabolic functions . Deficiency of CoQ10 -LRB- MIM 607426 -RRB- has been associated with five different clinical presentations that suggest genetic heterogeneity , which may be related to the multiple steps in CoQ10 biosynthesis . Patients with all forms of CoQ10 deficiency have shown clinical improvements after initiating oral CoQ10 supplementation . Thus , early diagnosis is of critical importance in the management of these patients . This year , the first molecular defect causing the infantile form of primary human CoQ10 deficiency has been reported . The availability of genetic testing will allow for a better understanding of the pathogenesis of this disease and early initiation of therapy -LRB- even presymptomatically in siblings of patients -RRB- in this otherwise life-threatening infantile encephalomyopathy . Introduction Coenzyme Q10 -LRB- CoQ10 -RRB- is the predominant human form of endogenous ubiquinone . Synthesized in the mitochondrial inner membrane , CoQ10 is comprised of a ubiquinone head group attached to a tail of 10 five-carbon isoprenoid units , that anchors the molecule to the membranes -LSB- 1 -RSB- . In additions to its central role in the mitochondrial respiratory chain as electrons carrier from complex I and II to complex III -LSB- 2 -RSB- , CoQ10 is now thought to be involved in a number of cellular functions -LRB- Table 1 -RRB- -LSB- 1 -RSB- . Intracellular synthesis is the major source of CoQ10 , although a small proportion is acquired through diet . The complexity of the biosynthesis -LRB- Fig. 1 -RRB- suggests that defects in different biosynthetic enzymes or regulatory proteins may cause different clinical syndromes . Table 1Functions of coenzyme Q -LRB- modified from -LSB- 1 -RSB- -RRB- Electron carrier in mitochondrial respiratory chain -LSB- 3 -RSB- Extra-mitochondrial electron transport -LSB- 4 -RSB- Antioxidant -LSB- 5 -RSB- Regulation of mitochondrial permeability transition pore -LSB- 6 -RSB- Regulation of physiochemical properties of membranes -LSB- 7 -RSB- Modulation of the amount of β-integrins on the surface of blood monocytes -LSB- 8 -RSB- Modulator of endothelial function -LSB- 9 -RSB- Oxidation of sulfide in yeast -LSB- 10 -RSB- Introduction of disulfide bonds in bacteria -LSB- 11 -RSB- Fig. 1CoQ10 biosynthetic pathway with eight known biosynthetic enzymes denoted as COQ1-8 . CoQ10 is composed of a benzoquinone and a decaprenyl side chain . While the quinone ring is derived from amino acids tyrosine or phenylalanine , the isoprenoid side chain is produced by addition of isopentenyl pyrophosphate molecules to geranylgeranyl pyrophosphate -LRB- derived from mevalonate pathway -RRB- by decaprenyl diphosphate synthase . After para-hydroxybenzoate and decaprenyl pyrophosphate are produced , at least seven enzymes -LRB- encoded by COQ2-8 -RRB- catalyze condensation , methylation , decarboxylation , and hydroxylation reactions to synthesize CoQ10 Primary CoQ10 deficiency is an autosomal recessive condition with a clinical spectrum that encompasses at least five major phenotypes : -LRB- 1 -RRB- encephalomyopathy characterized by the triad of recurrent myoglobinuria , brain involvement and ragged red fibers ; -LRB- 2 -RRB- severe infantile multisystemic disease ; -LRB- 3 -RRB- cerebellar ataxia ; -LRB- 4 -RRB- Leigh syndrome with growth retardation , ataxia and deafness ; and -LRB- 5 -RRB- isolated myopathy -LSB- 12 -- 26 -RSB- . Generalized weakness , exercise intolerance , and recurrent myoglobinuria are the distinctive features in the first four patients reported with the encephalomyopathic form , -LSB- 12 -- 15 -RSB- ; however , myoglobinuria was absent in another patient -LSB- 16 -RSB- . All the patients described so far have proximal muscle weakness -LSB- 12 -- 16 -RSB- The brain involvement is variable ; seizures are a common finding -LSB- 12 , 13 -RSB- , in association with cognitive impairment -LSB- 12 -RSB- and cerebellar symptoms -LSB- 12 , 15 , 16 -RSB- . The second variant , described in three families , manifests as an infantile encephalopathy with renal involvement . Rötig and colleagues initially described two siblings with defects of multiple quinone-dependent enzymes ascribed to a deficiency of CoQ10 in various tissues -LSB- 17 -RSB- . The patients presented with retinitis pigmentosa , optic nerve atrophy , bilateral sensorineuronal deafness , nephrotic syndrome , progressive ataxia , and cardiomyopathy . In Rahman 's patient , hypothermia , lactic acidosis , cerebral and cerebellar atrophy , and developmental delay were associated with renal tubulopathy and ventricular hypertrophy -LSB- 18 -RSB- . Last year , we described two siblings who presented with nephrotic syndrome due to glomerulosclerosis , and the older sibling developed hypotonia , psychomotor delay , seizures , stroke-like episodes , and cerebellar and cerebral atrophy -LSB- 19 -RSB- . Interestingly , in these infantile-onset patients , deficiency of CoQ10 is present not only in skeletal muscle , but also in other tissues and cells including skin fibroblasts . In addition , two adult sisters with encephalopathy , growth retardation , infantilism , ataxia , deafness and lactic acidosis have been described by Van Maldergam -LSB- 23 -RSB- . Cerebellar ataxia is the most common phenotype associated with CoQ10 deficiency with 21 reported patients -LSB- 20 -- 22 , 26 -RSB- . Cerebellar ataxia and cerebellar atrophy are present in all the patients . Epilepsy is the most common associated feature , but pyramidal signs , mental retardation , myopathic weakness , and delayed motor milestones are other variably associated symptoms and signs . Except for two adult brothers with cerebellar ataxia and hypogonadism -LSB- 22 -RSB- , the condition begins in childhood or adolescence . Muscle morphology does not show ragged-red fibers and lipid storage myopathy and CoQ10 is moderately reduced in skin fibroblasts . The clinical presentation of the variant isolated myopathy , recently described in four patients , is subacute onset of exercise intolerance and proximal limb weakness at variable ages . All patients have lipid storage and ragged-red fibers in muscle , as well as increased serum lactate and creatine kinase -LRB- CK -RRB- -LSB- 24 , 25 -RSB- . In all the patients with different phenotypes , respiratory chain enzymes analyses show reduced activities of complex I + III and II + III with normal activities of isolated complex I and III . In most of the reported patients , the exact site and nature of the defects in the biosynthesis of CoQ10 have not yet been identified . Because ubiquinone biosynthesis is complex and not fully defined -LRB- Fig. 1 -RRB- , identification of the molecular genetic defect is not straightforward . Undetectable CoQ10 and low levels of decaprenylpyrophosphate , an intermediate compound in the synthesis of the lateral chain of CoQ10 were found in the fibroblasts of the patient reported by Rötig and colleagues suggesting a defect in the synthesis of the decaprenyl side-chain , but they did not find any mutation in the cDNA of COQ1 , the gene encoding transprenyltransferase , the enzyme that synthesizes decaprenylpyrophosphate -LSB- 17 -RSB- . This year , in two siblings with the infantile form of CoQ10 deficiency , we reported a homozygous missense mutation -LRB- Y297C -RRB- in the COQ2 gene , which encodes para-hydroxybenzoate -LRB- PHB -RRB- polyprenyl transferase . PHB-polyprenyl transferase mediates the conjugation of the benzoquinone ring with the decaprenyl side chain . Biochemical assays measuring incorporation of radiolabeled PHB or decaprenyl-PP into CoQ10 in skin fibroblasts from the proband showed 23 -- 25 % of control fibroblasts activity -LSB- 27 -RSB- confirming the defect of CoQ10 biosynthesis . The finding of a homozygous stop codon mutation in the APTX gene which is known to cause ataxia-oculomotor-aprataxia 1 -LRB- AOA1 -RRB- , in three siblings with cerebellar ataxia and CoQ10 deficiency , supports the hypothesis that the ataxic form is a genetically heterogeneous disease in which CoQ10 deficiency can be secondary -LSB- 28 -RSB- . Aprataxin is a member of the histidine triad superfamily and may be involved in nuclear DNA single strand break repair -LSB- 29 -- 31 -RSB- . Despite the lack of an obvious link between aprataxin and regulation of CoQ10 synthesis or catabolism , our findings , coupled with the clinical improvement of patients after CoQ10 supplementation , suggest that CoQ10 deficiency plays a pathogenic role in AOA1 . Intriguingly , both CoQ10 and cholesterol share a common biosynthetic pathway , therefore , in AOA1 , altered levels of these molecules could be due to aberrant biosynthesis . Patients with all forms of CoQ10 deficiency have shown clinical improvement with oral CoQ10 supplementation . The beneficial effects of exogenous CoQ10 require high doses and long-term administration -LSB- 32 , 33 -RSB- ; its oral bioavailability is poor due to its extreme hydrophobicity ; therefore , only a small fraction of orally administered CoQ10 reaches the circulatory system , and augmentation of mitochondrial CoQ10 content is even less effective . Beneficial effects of CoQ10 supplementation are supported by in vitro correction of biochemical and histological abnormalities -LSB- 12 -- 14 , 17 -RSB- . However , while muscle abnormalities improve clinically and biochemically , in general , cerebral symptoms are only partially ameliorated -LSB- 16 -RSB- . This difference could be explained by the presence of irreversible structural brain damage before treatment or the poor penetration of CoQ10 across the blood-brain barrier . Increases in plasma concentrations of CoQ10 after oral supplementation in both humans and animals has been well-documented , but early work questioned whether CoQ10 accumulates in tissues -LSB- 34 -RSB- and studies of 45-day-old rodents showed little or no change in rodent brain CoQ10 concentrations after oral administration perhaps because levels of CoQ10 are tightly regulated in young animals or may be saturated in membranes -LSB- 34 , 35 -RSB- . In contrast , administration of very high doses of CoQ10 -LRB- 200 mg/kg daily -RRB- to 12 -- 24 month-old rats produced significant increases in brain CoQ10 levels -LSB- 36 -RSB- , but a more recent study of the regional distribution of CoQ in 16 week-old rat brain before and after 4 weeks of moderately high exogenous dietary CoQ10 -LRB- 20 mg/kg daily -RRB- supplementation showed that the concentration of CoQ was essentially unchanged in the cortex and in the striatum -LSB- 37 -RSB- . That study also demonstrated that the cerebellum , of the 7 brain regions dissected , contains the lowest levels of CoQ . If human brain has the same regional distribution of CoQ10 as rat brain , the cerebellum may have the narrowest safety margin and therefore would be the first tissue to suffer from a pathological shortage of CoQ10 . Moreover , to function as an antioxidant , CoQ10 must be in the reduced form but normally only 20 % of the lipid is reduced in the brain . The high proportion of oxidized CoQ10 in the brain could be a reflection of the high oxygen consumption in this tissue , causing an increased demand of anti-oxidants -LSB- 38 -RSB- . Therefore , CoQ10 deficiency could be another form of inherited ataxia due to oxidative damage , as vitamin E deficiency . Oxidative damage and mitochondrial dysfunction have also been implicated in neurodegenerative diseases such Parkinson disease -LRB- PD -RRB- , Alzheimer disease -LRB- AD -RRB- , and Friedreich 's ataxia -LRB- FRDA -RRB- -LSB- 39 -- 41 -RSB- . Preclinical studies have indicated that CoQ10 can protect the nigrostriatal dopaminergic system and high doses -LRB- 1200 mg daily -RRB- of CoQ10 for 16 months appeared to slow progression PD -LSB- 40 , 42 -RSB- . In 10 FRDA patients treated for 47 months high doses of CoQ10 -LRB- 400 mg daily -RRB- with vitamin E -LRB- 2100 IU daily -RRB- , improved bioenergetics in heart and skeletal muscle , improved cardiac function by echocardiography , and , possibly less clinical decline compared to historical controls -LSB- 43 -RSB- . Larger studies are necessary to better define the short - and long-term effects of CoQ10 as primary or adjunctive therapy in neurodegenerative diseases . Deficiency of CoQ10 has been associated with a variety of neurological disorders that respond to oral supplementation . We have demonstrated that CoQ10 deficiency can be primary due to a defect of biosynthesis or secondary as in patients with APTX mutations . The response of both groups of patients to oral supplementation suggests that exogenous CoQ10 is taken up by affected tissues and can correct biochemical derangements . Further studies to define CoQ10 deficiency syndrome will reveal new causes , enhance our understanding of its pathogenesis , and may provide novel insights that may be relevant to other neurodegenerative diseases .

O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O

coq10
encephalopathy
myopathy
cerebellar ataxia


Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model . NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined , particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary , no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice . There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus . Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice . Further , anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease . These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels . Introduction Influenza associated encephalopathy -LRB- IE -RRB- is one of the most severe neurological diseases amongst children , mostly under the age of 5 . It is marked by rapid progression and the mortality rate is estimated to range from 27 to 44 % -LSB- 1 -RSB- . Recently , we reported that production of the biological free radical gas , nitric oxide -LRB- NO -RRB- , in the serum and cerebrospinal fluid -LRB- CSF -RRB- of patients with IE was increased in the initial stages and correlated with disease severity -LSB- 2 , 3 -RSB- . Yokota et al. hypothesized that the influenza virus activates the limbic system followed by glial cell activation which in turn produces NO and inflammatory cytokines which flood the brain and cause a cytokine storm -LSB- 4 -RSB- . Recent in vitro studies by Imanishi et al. showed that a mouse lung-adapted strain of influenza virus could induce inducible NO synthase -LRB- NOS-2 -RRB- mRNA expression and NOS-2-mediated NO production in murine macrophage cell -LSB- 5 -RSB- . Moreover , Ino et al. studied five autopsies cases of IE patients . They reported that four out of the five patients showed degenerated astrocytic spines in the cortex , by glial fibrillary acidic protein -LRB- GFAP -RRB- staining . They suspected that the IE was largely due to these altered glial cells -LRB- Ino et al. unpublished -RRB- . Taken together , it is likely that IE is related to both the influence of NO and glial cells changes . Nevertheless , there have been no reports on a relationship between NOS-2 and glial cells within the brain after infection with the influenza virus . Thus , the aim of these studies is to better understand the clinical state of IE by focusing on NO and astrocytes in the central nervous system -LRB- CNS -RRB- after influenza virus infection . By using the A/NWS/33 influenza virus infected BALB/c model mouse , we were able to demonstrate that influenza virus infection leads to an up regulation of NOS-2 and astrocytes , mostly around capillary blood vessels of the hippocampus and olfactory bulb , starting at an early stage of the disease . Although IE has been reported for more than a decade , a therapy for IE has not yet been defined . We expect that our results will provide evidence which will aid in the development of a novel IE therapy . Experimental procedures Virus Influenza A/NWS virus , which is a mouse brain adapted type of human influenza A/NWS/33 virus was used . -LRB- A kind gift from Dr. K. Hayashi , Toyama University , Japan . -RRB- Experimental animals In total , 82 male BALB/c mice -LRB- 5-week-old -RRB- were purchased from Japan Charles River Co. and given free access to food and water -LRB- Oriental Yeast , Co. , Tokyo -RRB- and acclimatized for at least a week before the experiment . Anesthetics Each mouse was anesthetized by inhalation of ether before being inoculated intranasally with 50 μl NWS , 5.0 × 107 plaque-forming units -LRB- PFU -RRB- / ml -LRB- 5.0 × 105 PFU/mouse -RRB- . For negative control mice , the same amounts of 0.1 M phosphate-buffered saline -LRB- PBS -RRB- were intranasally inoculated . The mice were examined at 1 , 3 and 6 days postinfection -LRB- p.i. -RRB- . Body weight Body weight changes were monitored daily . Euthanasia All mice were decapitated whilst under ether inhalational anesthesia . Each brain was divided into eight regions -LRB- frontal cortex , occipital cortex , cerebellum , medullar oblongata , hippocampus , corpus striatum , thalamus/hypothalamus and olfactory bulb -RRB- . Then each section was immediately frozen with liquid nitrogen . NO2 − and NO3 − measurements by HPLC-UV method Both NO2 and NO3 -LRB- NOx -RRB- levels in each brain region were measured by HPLC-UV system -LRB- ENO-10 , EICOM -RRB- which was reported by us -LSB- 3 -RSB- . Briefly , thawed brain regions were deproteinized with a ten times volume of methanol and then centrifuged -LRB- 12,000 r.p.m. for 20 min at 4 °C -RRB- to avoid an occlusion in the polystyrene polymer column used in this system , by protein or other macromolecules . Detection was measured at 540 nm -LRB- absorption -RRB- and the concentrations were calculated from the area under the curve -LRB- AUC -RRB- of NO2 and NO3 -LRB- Power Chrome , EICOM , Kyoto -RRB- by Griess reaction . RNA extraction RNA was extracted from each part of the brain by using a QIA amp RNA extraction kit -LRB- QIAGEN K.K. , Tokyo -RRB- , according to the protocol suggested by the manufacturer . cDNA synthesis cDNA was synthesized from purified RNA by using the Transcriptor First Strand cDNA Synthesis Kit -LRB- Roche , Switzerland -RRB- . Random hexamer primer was used and the protocol was as follows ; after annealing for 10 min at 25 °C , and incubating 30 min at 55 °C , the reaction was heated to 85 °C for 5 min and chilled on ice . Real time reverse transcriptase-polymerase chain reaction -LRB- RT-PCR -RRB- Messenger RNA levels of NOS-1 , NOS-2 and NOS-3 were quantified by using a Light Cycler Fast Start DNA Master HybProbe -LRB- Roche -RRB- . This was done according to the following protocol ; Pre-incubation was 95 °C for 30 s . This was followed by 40 cycles of denaturation at 95 °C for 10 s , annealing at 62 °C for 15 s , amplification at 72 °C for 8 s for 45 cycles . Cycles were followed by 30 s 40 °C cooling program . The primers and probes were designed by the Light Cycler primer and probe set -LRB- Roche Diagnostics Applied Science , Tokyo -RRB- . NOS-2 , NOS-3 and NOS-1 primer sequences were designed on GenBank database NM_0100927 , NM_008713 , and NM_008712 respectively . All data were normalized by dividing with the corresponding GAPDH mRNA from the same sample . Immunohistochemistry Anesthetized animals were perfused intracardially with 20 ml of 0.1 M PBS , followed by 30 ml of 4 % paraformaldehyde in 0.1 M phosphate buffer . The brains were removed , postfixed overnight and cryoprotected in 30 % sucrose , processed through graded alcohols and embedded in paraffin . Then , they were cut as 6 μm thick coronal sections . They were incubated with 0.3 % H2O2 for 20 min . The sections were then rinsed with tris-buffered saline , pH 7.6 -LRB- TBS -RRB- . To identify reactive astrocytes , we stained with Anti-GFAP -LRB- rabbit polyclonal , DAKO , Denmark -RRB- . Sections were incubated over night at a 1:2,000 dilution . The NOS-2 stains were incubated for 4 h at a 1:1,500 dilution -LRB- Anti-iNOS/NOSII , rabbit polyclonal IgG ; Upstate Biotechnology , Lake Placid , NY -RRB- . After the first staining , each section was incubated with the secondary antibody for 30 min -LRB- Nichirei Histofine Simple Stain , anti-rabbit IgG -RRB- at room temperature after TBS rinsing . Labeling was visualized by incubating with a solution containing 2 % , 3,3 ′ - diaminobenzidine , 0.1 M imidazole and 30 % H2O2 . All sections were counterstained with Mayer 's hematoxylin -LRB- SIGMA , MI -RRB- . Statistical analysis Fisher 's exact test was used when comparing body weights . All results obtained from the real time RT-PCR were subjected to one - or two-way analysis of variance -LRB- ANOVA -RRB- and differences among the means were analyzed by two-way ANOVA followed by a Newman -- Keuls range test at the 0.05 significance level . Results are expressed as mean ± standard error . Results Body weight Figure 1 shows the change in body weight of the non-infected -LRB- PBS -RRB- and infected -LRB- Flu -RRB- mice over day . The mice infected with virus showed a decrease in weight and ruffled hair from 3 days p.i. and by 6 days p.i. and there was a significant decrease in the body weight of the infected mice as compared to the non-infected mice -LRB- P < 0.05 -RRB- . Prominent neurological signs in the infected mice did not appear during the experimental period and none of the mice died during the experiment , although spontaneous locomotor activity was reduced in the infected mice . Fig. 1Changes in body weight of A/NWS/33 influenza virus infected BALB/c model mice after virus infection . Each result shows the mean ± SE value of eight experiments . -LRB- -RRB- non-infected -LRB- PBS treatment -RRB- mice group , -LRB- -RRB- infected -LRB- Flu -RRB- mice group Viral titers At one day p.i. , 120 PFU/50 mg virus was detected in the infected mouse brain and 136 × 104 PFU/100 mg virus was detected in the infected mouse lung . Real-time RT-PCR for NOS-2 , NOS-3 and NOS-1 Figure 2 shows the NOS-2 / GAPDH and NOS-3 / GAPDH ratios as measured in the olfactory bulb and hippocampus of infected mouse brains as compared to the non-infected mice on days 1 , 3 and 6 p.i. Due to the scattered results obtained in both the infected and non-infected mice , data on the NOS-1 / GAPDH ratio is not reported . The increases in the NOS-2 / GAPDH ratio as seen in the olfactory bulb -LRB- OB -RRB- and hippocampus -LRB- HIP -RRB- at day 1 and 3 p.i. , were obvious as shown in Fig. 2a and b. However , no changes were seen in the levels of NOS-3 / GAPDH in the same regions -LRB- Fig. 2a ' , b ' -RRB- . Fig. 2NOS-2 / GAPDH and NOS-3 / GAPDH ratios in the olfactory bulb and hippocampus of non-infected and infected mice on days 1 , 3 and 6 p.i. . The open column shows the mean and S.E. value obtained from nine non-infected mice and the closed column shows the mean and S.E. value obtained from eight infected mice . * Significant difference between non-infected mice and infected mice . * P < 0.05 . -LRB- a and a ' -RRB- : olfactory bulb , -LRB- b and b ' -RRB- : hippocampus Changes in brain NO levels between non-infected and infected mice Figure 3 shows that NO levels in the eight brain regions examined , either with or without NWS virus infection , changes in a day-by-day manner after virus infection . The levels of NO2 and NO3 were measured separately , as shown in the Materials and methods . In the isolated brain regions , NO3 levels were more than 40-fold higher than NO2 levels , and the final data are shown as the total value -LRB- NO -RRB- of NO2 plus NO3 . At day 1 p.i. , the NO levels detected in olfactory bulb -LRB- OB -RRB- , hippocampus -LRB- HIP -RRB- and frontal cortex -LRB- FC -RRB- of the infected mice -LRB- Flu -RRB- were significantly increased as compared to the levels in the same brain regions of the non-infected -LRB- PBS -RRB- mice -LRB- P < 0.05 -RRB- . Further , at day 6 p.i. , the increased levels of NO in the olfactory bulb -LRB- OB -RRB- and the frontal cortex -LRB- FC -RRB- returned to the levels seen in non-infected mice . However , the levels of NO in the hippocampus -LRB- HIP -RRB- were significantly increased -LRB- P < 0.05 -RRB- at all the time points examined . Fig. 3Day to day changes in NO levels in the eight brain regions either with or without NWS virus infection . The open column shows the mean and S.E. value obtained from eight non-infected mice and the closed column shows the mean and S.E. value obtained from seven infected mice . * Significant difference between non-infected mice and infected mice . * P < 0.05 . OB : olfactory bulb , HIP : hippocampus , St : striatum , Hypo/Thal : hypothalamus and thalamus , MO : medulla oblongata , CE : cerebellum , FC : frontal cortex , and OC : occipital cortex Immunohistochemistry Figure 4 shows the results of anti-NOS-2 staining and GFAP staining in the hippocampus of non-infected -LRB- PBS -RRB- and infected mice -LRB- Flu -RRB- at 1 -LRB- Fig. 4a -RRB- and 6 -LRB- Fig. 4b -RRB- days p.i. in the same magnification each . The brown-yellow staining was recognized as positive staining for anti-NOS-2 . We found positive staining in the hippocampus , especially in the area surrounding capillary blood vessels of the infected mice at 1 day p.i. -LRB- Fig. 4a , Day 1 , Infected -RRB- . By 6 days p.i. , positive staining had spread to the striatum , hippocampus and cortex of the infected mice and positive staining were seen in the areas surrounding capillary vessels -LRB- Fig. 4b , Day 6 , Infected -RRB- . Add that infected mice showed an increase in GFAP staining on days 1 much more so than that seen in non-infected mice . Positive staining was most notable around hippocampal blood vessels in infected mice . Additionally , most of the blood vessels in hippocampi of infected mice were markedly expanded as compared to those seen in the non-infected mice . Fig. 4Anti-NOS-2 immunohistochemistry and GFAP immunohistochemistry in the hippocampus at 1 -LRB- Fig. 4a -RRB- and 6 -LRB- Fig. 4b -RRB- days p.i Discussion Since our aim was to see the effect to the brain after influenza infection , this experiment was undertaken by using the A/NWS/33 virus because it is proven by Li et al that the NWS/33 strain can replicate in the brain of adult mice after intracerebral injection , add that the virus usually fails to spread beyond the respiratory epithelium after internasal instillation -LSB- 6 -RSB- . During the experiment , we observed that the NWS virus infected mice lose significantly more body weight at 6 days p.i. -LRB- Fig. 1 -RRB- in agreement with previous studies -LSB- 5 , 7 -RSB- . Since bodyweight changes are an indicator of the overall condition of the mice , this bear out that the mice was infected to the virus . More over , we have checked that RT-PCR for A/NWS/33 virus were positive in all brain regions expect medullar oblongata on day 1 p.i. -LRB- data not shown -RRB- . The reason why we determined the amounts of influenza virus in this experiment is as followed . During the past experiment , we had experienced that the mice die within 3 days p.i. when inoculating any more than the virus we used this time and when we use fewer viruses , there were hardly any difference in the non-infected and infected behavioral change . Our results show that an enhancement NO biosynthesis was induced in the NWS virus infected mouse brain . Over-production of NO was substantiated by determining an increase in the expression of NOS-2 mRNA as shown in Fig. 2 , and increased immuno-histochemical staining for anti-NOS-2 as shown in Fig. 4 . At 1 day p.i. , the amount of NOS-2 mRNA within the infected mouse brain was greater than that seen in control mice in all brain regions studied . Above all , the olfactory bulb showed the greatest increase . On the basis of these results , we compared NOS-2 mRNA expression in the olfactory bulb on days 1 , 3 , and 6 p.i. , respectively . This showed that the NOS-2 mRNA ratio was greater in the infected mouse brain at all experimental times studied -LRB- Fig. 2 -RRB- . On the contrary , there was no difference in NOS-3 or NOS-1 mRNA levels between infected and control mice -LRB- data not shown -RRB- . These results are important since these three isoforms of NOS , NOS-2 the inducible form of NOS , is capable of producing a continuous flux of NO and a large amount of NO generated by NOS-2 over a sustained period is implicated in the pathogenesis of various disorders and with the response to infectious organisms -LSB- 8 -RSB- . In regards to the immuno-histochemical analysis , positive staining for anti-NOS-2 was mostly detected in the hippocampus of infected mouse brains , whereas hardly any staining was seen in control brains -LRB- Fig. 4 -RRB- . Furthermore , positive staining had spread to the hippocampus and striatum by 6 days p.i. , which correlated with the transition of NO levels . NOS-2 has been shown to play a role in the defense against bacteria , parasites and viruses . However , it seems that NO induced by the influenza virus has a dual role . Rimmelzwaan et al. reported an inhibitory effect of NO on virus growth -LSB- 9 -RSB- , whereas Akaike et al. reported that NO played a pathological role in the influenza virus-induced pneumonia in mice -LSB- 10 -RSB- . The fact that the influenza virus can induce NO has been reported previously -LSB- 2 , 3 -RSB- . Yao et al. reported that the mRNA 's of both NOS-2 and NOS increased in the brain after the intranasal infection with influenza A virus of newborn mice -LSB- 11 -RSB- . Further , Imanishi et al. pointed out that a mouse lung-adapted strain of influenza virus could induce NOS-2 mRNA expression and NOS-2-mediated NO production in murine macrophage cells -LSB- 5 -RSB- . Our study has demonstrated that the NWS virus can induce NOS-2 expression in the infected mouse brain , especially in the olfactory bulb and hippocampus . These results may relate to the fact that intranasally administered NWS virus can invade the brain through the olfactory pathway as has been reported previously -LSB- 12 -- 16 -RSB- . Recently , Yossi et al. reported that astrocytes are capable of rapid and massive NOS-2-dependent NO production in brains of normal mice in situ -LSB- 17 -RSB- . According to the report by Ino et al. on the brain autopsy of patients who had died of IE , four out of five IE patients showed degenerated astrocytic spines in the cortex by GFAP staining . They suspected that the IE was largely due to altered glial cells -LRB- Ino et al. unpublished -RRB- . Takahashi et al. also made a statement on the autopsied case of a 2-year-old girl with encephalopathy associated with influenza A virus infection and according to their report ; reactive astrocytes were seen in the vicinity of blood vessel in the brain but astrocytosis was not seen anywhere , including the areas where virus antigen-positive neurons were abundant -LSB- 18 -RSB- . Additionally , Yokota et al. hypothesized that the influenza virus activates the limbic system followed by the activation of glial cells which produce NO and other inflammatory cytokines which together flood the brain and cause a cytokine storm -LSB- 4 -RSB- . Astrocytes are the major glial cell in the CNS and have important physiological properties in terms of CNS homeostasis . Astrocytes also influence the formation and maintenance of the blood -- brain barrier -LRB- BBB -RRB- . The importance of astrocytes is demonstrated by a study from Bush et al. . According to their report , an astrocyte ablation led to failure of BBB repair and vasogenic edema -LSB- 19 -RSB- . There have been several reports related to glial cells and NO -LSB- 17 , 20 , 21 -RSB- . However , there has not been a report considering the relationship between influenza virus infection , NOS-2 and astrocytes together . By determining an increase in GFAP staining as shown in Fig. 4 , our results show that astrocytes are activated in the infected mouse brain and especially in hippocampus . Interestingly , we noticed that both the increased NOS-2 and GFAP staining were mostly found around capillary blood vessels of the hippocampus starting at an early stage . Our results suggest that since NOS-2 and astrocytes became activated around capillary blood vessels after viral infection , this may lead to brain capillary blood vessels break down . However , we were unable to determine whether the NOS-2 measured is derived from vascular endothelial cells or macrophages which will be an issue for future experiments . Although IE has been mostly reported in Asian countries , influenza infection occurs in epidemics in both the northern and southern hemispheres and is a pandemic disease throughout the year . Recently , influenza virus which is resistant to anti-influenza drugs has been shown to emerge and there is real concern in regards to a new type of influenza pandemic -LSB- 22 -RSB- . However , no standardized therapy for the treatment of IE has yet been defined . In this paper , we suggest that the influenza virus causes the activation of astrocytes and NOS-2 in the brain . Thus it is likely that our results will add supportive evidence for the development of a novel IE therapy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

no
astrocyte
olfactory bulb
hippocampus
brain capillary blood vessels

influenza a/nws/33 virus
balb/c mouse
nos-2