In this study we examined the extent to which AD biomarkers measured in CSF predict brain microstructure indexed by diffusion tensor imaging (DTI) and volume indexed by T1-weighted imaging. Forty-three middle-aged adults with parental family history of AD received baseline lumbar puncture and MRI approximately 3.5 years later. Voxel-wise image analysis methods were used to test whether baseline CSF Aβ42, total tau (T-Tau), phosphorylated tau (P-Tau) and neurofilament light protein predicted brain microstructure as indexed by DTI and gray matter volume indexed by T1-weighted imaging. T-Tau and T-Tau/Aβ42 were widely correlated with indices of brain microstructure , notably in white matter regions adjacent to gray matter structures affected in the earliest stages of AD. None of the CSF biomarkers were related to gray matter volume. Elevated P-Tau and P-Tau/Aβ42 levels were associated with lower recognition performance on the Rey Auditory Verbal Learning Test. Overall, the results suggest that CSF biomarkers are related to brain microstructure in healthy adults with elevated risk of developing AD. Furthermore, the results clearly suggest that early pathological changes in AD can be detected with DTI and occur not only in cortex, but also in white matter.Cerebrospinal fluid (CSF) biomarkers T-Tau and Aβ Several cerebrospinal fluid (CSF) markers that are presumably related to the core pathology in AD show promise for early detection of the disease. Lower levels of CSF AβWhile studies resulting from volumetric imaging have been very useful for revealing gross structural changes, it is possible that some of the earliest brain changes involved in AD are subtle and below the detection threshold for volumetric imaging. Sensitive to water molecule motion, maps derived from diffusion tensor imaging (DTI) 181, and Aβ42. In addition to these markers of AD pathology, we analyzed CSF for a structural protein of neurons that is predominantly localized in large-caliber axons : neurofilament light chain protein (NFL) 42 in CSF would be related to lower FA, higher MD, and altered radial and axial diffusivity in AD-sensitive brain regions. Additionally, we expected that NFL, being sensitive to axonal alteration, would be especially related to axial diffusivity–reflecting altered axonal health. Although the brain regions affected in AD are well known, the relationship between CSF biomarkers and brain health is a relatively new area of study, accordingly we used voxel-wise analyses (corrected for multiple comparisons) In the present study, we examined the relationship between brain microstructure indexed by DTI and proteins associated with brain health in CSF collected in healthy middle-aged and older adults from the Wisconsin Registry for Alzheimer’s Prevention Study procedures were approved by the University of Wisconsin Health Sciences Institutional Review Board and were in accordance with U.S. federal regulations. All participants provided written informed consent.We enrolled 47 participants, four of whom were excluded due to unexpected abnormalities found on their MRI scan by the reviewing radiologist (HAR). The remaining 43 middle- to older- aged participants were 37 years of age to 66 years of age at time of lumbar puncture and CSF collection ; and 42 to 71 years of age at time of follow-up scan . There were 12 men and 31 women. As a group, participants were well educated . All participants were from the Wisconsin Registry for Alzheimer’s Prevention (WRAP) BVMT  total raw score, and delayed recall, which index visuospatial learning and memory COWAT  total over the five learning trials, recognition, and 20 minute delayed recall raw scores As part of their participation in WRAP, participants received at least one comprehensive neuropsychological assessment. Neuropsychological testing occurred an average of 1.80 years (SD = 1.28 years) out from the MR scan and 2.06 years (SD = 1.20 years) out from their lumbar puncture. In order to screen for changes in cognitive status, participants who were >3 months out from a neuropsychological testing visit also received the Mini Mental State Examination (MMSE) 181, and Aβ42 analysis was accomplished via xMAP technology and utilizing the INNO-BIA AkzBio3 kit (Innogenetics) as previously described The CSF collection was performed via lumbar puncture at a baseline visit in a simvastatin trial Participants were invited for brain imaging on the condition that baseline CSF samples were available for analysis. Participants were imaged on a General Electric 3.0 Tesla Discovery MR750  MRI system with an 8-channel head coil and parallel imaging (ASSET). DTI was acquired using a diffusion-weighted, spin-echo, single-shot, echo planar imaging pulse sequence in 40 encoding directions, B0 = 1300, with eight non-diffusion weighted reference images. The cerebrum was covered using contiguous 2.5 mm thick axial slices, FOV = 24 cm, TR = 8000, E = 67.8, matrix = 96×96, resulting in isotropic 2.5 mm voxels. High order shimming was performed prior to the DTI acquisition to optimize the homogeneity of the magnetic field across the brain and to minimize EPI distortions. A T1-weighted volume was acquired in the axial plane with a 3D fast spoiled gradient-echo (3D EFGRE) sequence using the following parameters: TI = 450 ms; TR = 8.1 ms; TE = 3.2 ms; flip angle = 12°; acquisition matrix = 256×256×156, FOV = 260 mm; slice thickness = 1.0 mm.http://afni.nimh.nih.gov/). FA, MD, and lambda maps  were generated via the FMRIB Software Library (FSL) (http://www.fmrib.ox.ac.uk/fsl/fdt/index.html using the following procedures: (1) image distortions in the DTI data caused by eddy currents were corrected; (2) estimation of diffusion tensors was achieved using DTIFIT; (3) three-dimensional maps of FA, MD, and the 3 eigenvalues  were computed from the tensors from step (2). Each participant’s FA map was aligned to an FA template in Montreal Neurological Institute (MNI) space, comprised of an average of 121 FA maps acquired from healthy participants with similar demographics as the study cohort. Transformations were achieved via 12-parameter affine transformation and nonlinear deformation using Statistical Parametric Mapping software (SPM8 available at http://www.fil.ion.ucl.ac.uk/spm). Estimated transforms from each participant’s FA map warping were applied to the participant’s remaining DTI maps  resulting in a transformation of all the original images into MNI space. The normalized FA maps were used to visually inspect for accurate normalization using the “check registration” function in SPM and selecting specific fiber tracts for comparison . λ2 & λ3 maps were averaged to create radial diffusivity maps, and λ1 was the axial diffusivity map.Diffusion-weighted DICOM images were converted into NIFTI format using AFNI , phosphorylated tau (P-Tau181), T-Tau/Aβ42, and P-Tau181/Aβ42, while the dependent variables consisted of participants’ DTI maps and gray matter probability maps. Due to relatively low levels of NFL in the majority of participants , NFL was treated as a categorical variable. The detection limit of the assay is NFL<125 ng/L, thus we used this as a cut-point to determine the groups. Differences in brain health based on NFL was tested using voxel-wise ANCOVA, where DTI maps or gray matter probability maps were the dependent variable and the groups were NFL>125 ng/L, and NFL<125 ng/L. Age (at time of MR scan) and gender were included as covariates in all models due to known effects on brain microstructure In order to test the relationship between CSF proteins, age, and cognitive function, we performed linear correlation analysis using SPSS . CSF biomarkers were log transformed to normalize distribution prior to inclusion in analysis. Differences in CSF protein levels based on APOE status and gender were tested using independent t-tests in SPSS. In order to test the extent to which CSF biomarkers were associated with brain health as indexed by the DTI maps and T1-weighted imaging, voxel-wise linear regression models were implemented via statistical modules available in SPM8. The independent variables were Aβ42, and P-Tau181/Aβ42  compared to carriers (m = 3.90), t(41) = 2.038, p<.05, and Aβ42 was lower in the carriers (m = 5.57) compared to non-carriers (m = 5.83), t(41) = 3.062, p<.05. APOE4 carriers and non-carriers did not differ on age (p = .78).There was a significant positive correlation (P<.05) between age at time of lumbar puncture and T-Tau, T-Tau/Aβ42 . However, a scatter-plot revealed that the participant with the highest tau value (>2 SD higher than the mean) performed well on working memory–skewing the results. When this participant was removed from the analysis, the relationships were no longer significant. RAVLT recognition score was negatively correlated with both P-Tau181  and P-Tau181/Aβ42  but was not related to T-Tau, Aβ42, or their ratio. No other RAVLT sub-scores or other neuropsychological tests were significantly correlated with the CSF markers.All participants were cognitively normal as determined by comprehensive neuropsychological testing and as assessed by MMSE (MMSE ≥27). Controlling for age and education, linear correlation analysis indicated that baseline CSF biomarker levels were related to a subset of the neuropsychological test scores. WAIS Working Memory Index  was positively correlated with T-Tau  and with T-Tau/Aβ42 showed robust and widespread positive relationships with several of the DTI measures, specifically MD, axial and radial diffusion. These relationships were extensive in white matter and were prevalent in temporal, parietal and frontal lobes. The locations of peak T-value for MD and FA clusters obtained in the voxel-wise analysis are tabulated in Table S1). The brain regions where CSF T-Tau/Aβ42 predicted MD values in the voxel-wise regression analysis are shown in 42 and MD is shown in 42 SPM result maps  was extensive, and is summarized in terms of percent overlap in Voxel-wise regression analysis indicated that both T-Tau and T-Tau/Aβ42 and FA. Furthermore, neither P-Tau181 nor P-Tau/Aβ42 were related to any of the DTI measures . Higher Aβ42 in CSF was related to higher FA in medial frontal gyrus, but not related to MD, axial or radial diffusivity. Participants with NFL>125 ng/L showed a single region of higher FA in middle temporal gyrus white matter compared to control, with no differences in MD, axial or radial diffusivity. Complete results  for all CSF measures and FA and MD maps are in Table S1). None of the CSF biomarkers predicted gray matter volume as indexed by the modulated gray matter probability maps (FDR p<.05).At a statistical threshold of p<.05 (FDR corrected for multiple comparisons) there was no relationship between T-Tau or T-Tau/AβAD-related CSF biomarkers are linked with global and regional brain volumes in healthy elderly adjacent to critical gray matter structures that are known to be affected in AD. This was especially true for T-Tau and T-Tau/Aβ42, where higher CSF levels were related to higher axial, radial, and mean diffusivity in a large swath of temporal lobe white matter adjacent to hippocampus. Several studies now point toward early involvement of white matter in AD development, including human studies on AD risk While gray matter volume was not related to any of the CSF biomarkers in this study, CSF biomarkers were related to white matter health as indexed by DTI; interestingly, the regions of significant association were mostly White matter degeneration in diagnosed AD is confirmed by several human post mortem studies 42 predicted axial and radial diffusivity, suggesting these CSF biomarkers are potentially related to both neuronal axon integrity and health of oligodendrocyte synthesized myelin. Furthermore, the analyses of axial, radial, and mean diffusivity produced statistical maps that were highly overlapping, suggesting that alterations in one cell component  could very well be associated with related alterations in another cell component (myelin).In our study we measured the relationship between CSF biomarkers and DTI measures that are presumably related to axonal health  and myelin health . We found that both T-Tau and T-Tau/AβAlthough CSF measures of myelin were not available to us in this study, we assessed axonal degeneration via CSF measured neurofilament light protein. Neurofilament proteins are major constituents of the axonal cytoskeleton, consisting of three polypeptides; light (NFL), medium and heavy subunits. NFL proteins are most related to large-caliber axons 42 . Although the localized source of tau protein measured in CSF is unknown, higher levels in our study were related to higher diffusion across temporal, parietal, and frontal lobes–particularly in white matter. We speculate CSF tau protein may have originated from axons in these brain regions, but we can not rule out that tau may have also originated from oligodendroglia FDR threshold. Although some studies suggest that CSF P-Tau181 does not correlate with neuropathological burden in AD 181 and tissue microstructure was too subtle to survive our statistical threshold. Indeed, when we performed exploratory analyses at uncorrected (for multiple comparisons) thresholds, higher P-Tau181 was associated with altered tissue microstructure. Furthermore, P-Tau181 and P-Tau181/Aβ42 were the only biomarkers related to memory function in this study. Nonetheless, considering alternative sites of tau phosphorylation will likely be important for understanding early AD and in AD biomarker development.In AD, abnormal phosphorylation of tau protein and deposition of amyloid are considered primary processes underlying neuronal degeneration. Hyperphosphorylation of tau and its subsequent release from the cell means markers of tau measured in CSF increase compared to controls 42 showed widespread relationships with the DTI measures, CSF Aβ42 was associated with lower anisotropy of water (FA) only in medial frontal cortex and underlying white matter. Deposition of brain amyloid in AD likely involves frontal involvement at the earliest stages While T-Tau and T-Tau/Aβ42, and P-Tau181/Aβ42. Alone, Aβ42 was not related to age. We also found that both T-Tau and Aβ42 differed between APOE4 carriers and non-carriers. Similar to previous studies, Aβ42 was lower in the carriers compared to non-carriers–conceivably suggesting greater amyloid deposition in the group at greater risk for AD. Interesting, we found that the APOE4 carriers in this sample showed lower T-Tau compared to non-carriers. These T-Tau findings are consistent with our previous report on CSF biomarkers in middle-aged at-risk adults In healthy older adults, levels of AD biomarkers measured in CSF are related to age, cognition, structural brain volume, and AD risk factors including APOE4 status 181 and P-Tau181/Aβ42 levels had lower recognition on a list learning test (RAVLT). Glodzik et al. have also found a relationship between P-Tau (P-Tau231) and memory function 181 and Aβ42 has also been shown to predict longitudinal subjective memory impairment in healthy elderly All of the participants in our study tested cognitively normal. Among the cognitive tests, only recognition memory was related to any of the CSF measures. Participants with higher P-Tau42 levels have significantly higher whole brain loss over time, ventricular expansion, and greater rates of hippocampal atrophy 42 correlated with 1-year change in several regions including putamen, thalamus, superior temporal cortex, cingulate, and frontal brain regions, whereas higher T-Tau/Aβ42 was related to atrophy in amygdala, paracentral cortex, and ventricular regions. Higher P-Tau was related to 1-year change in amygdala, paracentral cortex, posterior cingulate, and pallidum. Fjell et al.’s study is also one of the few to show a relationship between P-Tau and cerebral white matter, with higher P-Tau predicting lower white matter volume. P-Tau reported in these studies was assayed for tau phosphorylated at threonine 181, although a similarly aged healthy cohort evaluated at baseline and 2 years later by Glodzik et al. 231 is also associated with longitudinal medial temporal lobe volume decline and memory function.Previous brain imaging studies in healthy adults have found that CSF biomarkers are related to volumetric measures. Healthy elderly individuals from ADNI with low CSF AβIn our study of adults with parental family history of AD and a high frequency of APOE4, we found relationships between CSF measures and DTI in several brain regions shown in previous volumetric studies of healthy participants, including temporal, cingulate, and frontal cortices. We did not find a relationship between the CSF biomarkers and gray matter volume (indexed by T1-weighted imaging) in any brain regions, possibly due to the younger age of our cohort in whom tissue changes are likely to be more subtle and less easily detected with volumetric imaging. Using DTI, we had the additional advantage of being able to evaluate potentially subtle effects in white matter, and these relationships were robust and widespread.While the results of this study are promising, potential limitations should be noted. We adopted a voxel-wise approach in order to assess the extent to which CSF biomarkers were related to brain microstructure and volume across gray and white matter tissue. While we took great care to evaluate for potential errors during registration of the DTI maps to a common template, methods that employ registration to a template may be susceptible to subtle error and statistical effects may be influenced by the size of smoothing filters used in voxel-wise analyses. As a further comment on the DTI results, we would like to note that this study used a tensor model which resolves a single fiber direction within each voxel. We found very robust results when analyzing the MD, axial, and radial diffusivity maps, compared to the FA maps. Due to the complex architecture of white matter, employing FA derived from the tensor model may not have captured the full information available with alternative methods of DTI acquisition and higher order modeling CSF biomarkers previously associated with AD are related to brain tissue microstructure. This study supports an emerging theory that brain white matter alterations are an early event in AD pathogenesis. The mechanisms that underlie white matter alterations and the nature of these alterations deserve further study.Table S1Regions where CSF biomarkers were significantly correlated with axial and radial diffusivity in the voxel-wise analyses.(DOC)Click here for additional data file.


To investigate the efficacy of a new method (TT-Upslope) for transit time (Δt) estimation from phase contrast CMR flow curves, and to compare it with three previously described methods based on the commonly used foot-to-foot approaches, as well as the point-to-point and wave-to-wave approaches.CMR is increasingly used for measuring aortic arch PWV (arch-PWV) by using accurate aortic length and transit time between flow waves. If robust aortic length measurement is an obvious strength of CMR thanks to many 3D imaging approaches available, Δt measurement remains a major challenge. Consequently, different methods have been previously described to estimate the Δt using CMR, but there is to date no fully standardized method for its determination.Fifty healthy subjects underwent carotid-femoral pulse wave velocity (cf-PWV), and carotid pulse pressure (CPP) measurements by applanation tonometry, as well as CMR exams with steady-state free-precession (SSFP) and Phase Contrast (PC) acquisitions at the level of the aortic arch. These data were used for the automated estimation of the arch-PWV, and the aortic areas of the ascending aorta, which were combined with CPP to estimate the local distensibility (aa-Dist). The 3D length of the aortic arch was calculated from axial and coronal SSFP acquisitions. Δt was defined as the time shift between the flow curves of the ascending (CA) and descending (CD) aorta and calculated with: 1) TT-Upslope by minimizing the area delimited by two sigmoid curves fitted to the systolic up-slope of CA and CD, 2) TT-Point using the half maximum of CA and CD, 3) TT-foot using CA and CD feet, 4) TT-Wave by minimizing the area between the whole CA and the CD curves using the cross correlation technique.The arch-PWV estimated with both estimators TT-Upslope and TT-Wave resulted in a better correlation with aging, cf-PWV, as well as aa-Dist Table . FurtherTT-Upslope and TT-Wave appear to be less sensitive to the low temporal resolution, signal-to-noise ratio, and varying profile of velocity curves. Indeed, they avoid the restriction of the analysis to a few points of the velocity curve, and provide better correlations with the physiological and stiffness parameters.


Prevalence of neurocognitive impairment in HIV-1 infected patients is reported to be high. Whether this is a result of active HIV-related neurodegeneration is unclear. We examined axonal injury in HIV-1 patients by measuring the light subunit of neurofilament protein (NFL) in CSF with a novel, sensitive method.With a cross-sectional design, CSF concentrations of neurofilament protein light (NFL) , neopterin  and CSF/Plasma albumin ratio (blood-brain barrier integrity) were analyzed on CSF from 252 HIV-infected patients, subdivided into untreated neuroasymptomatics (n = 200), HIV-associated dementia (HAD) (n = 14) and on combinations antiretroviral treatment (cART) (n = 85), and healthy controls (n = 204). 46 HIV-infected patients were included in both treated and untreated groups, but sampled at different timepoints. Furthermore, 78 neuroasymptomatic patients were analyzed before and after treatment initiation.While HAD patients had the highest NFL concentrations, elevated CSF NFL was also found in 33% of untreated neuroasymptomatic patients, mainly in those with blood CD4+ cell counts below 250 cells/μL. CSF NFL concentrations in the untreated neuroasymptomatics and treated groups were equivalent to controls 18.5 and 3.9 years older, respectively. Neopterin correlated with NFL levels in untreated groups while the albumin ratio correlated with NFL in both untreated and treated groups.Increased CSF NFL indicates ongoing axonal injury in many neuroasymptomatic patients. Treatment decreases NFL, but treated patients retain higher levels than controls, indicating either continued virus-related injury or an aging-like effect of HIV infection. NFL correlates with neopterin and albumin ratio, suggesting an association between axonal injury, neuroinflammation and blood-brain barrier permeability. NFL appears to be a sensitive biomarker of subclinical and clinical brain injury in HIV and warrants further assessment for broader clinical use. HIV invades the central nervous system (CNS) shortly after transmission where it can then be detected in cerebrospinal fluid (CSF) throughout the course of untreated systemic infection in most patients including the majority of patients without neurological symptoms in the earlier phase of infection Currently, the formal diagnosis of HAND, including particularly its milder forms, relies on the results of neuropsychological testing compared to normative controls, and hence confounded by other conditions affecting performance and may be overestimated using currently recommended methods Recently, a new and more sensitive immunoassay for measuring CSF NFL has been introduced. This assay is more sensitive, and in particular, has a broader dynamic range at the lower level of biological concentrations, CSF NFL can now be measured in most normal subjects allowing the detection of smaller increases in pathological states. In this study we applied this new assay to examine the frequency of neuronal injury at different stages of untreated systemic HIV infection and the effects of antiretroviral treatment. Because of the natural increase of CSF NFL concentration with age in the uninfected population, we also examined the relation of HIV-associated NFL changes to subject age. We further examined whether CSF NFL was associated with the level of systemic and CNS viral replication as measured by plasma and CSF HIV RNA concentration, with systemic immunosuppression as reflected in blood CD4+ T cell counts, with intrathecal immune activation measured by CSF neopterin concentrationsThe report describes two related studies, one cross-sectional and the other a longitudinal cohort design. Both used archived specimens and data from volunteer subjects from two academic centers: Sahlgrenska University Hospital in Gothenburg, Sweden and San Francisco General Hospital, California, USA. All subjects were studied under research protocols approved by institutional review boards of the Sahlgrenska Academy and University of California Committee on Human Research and followed guidelines of the Helsinki declaration. Informed verbal consent was obtained from all patients and documented in their respective patient file, a procedure accepted by the ethic committees. Written consent was not required by the ethic committees. All patients received written and verbal study information.NA) subdivided according to levels of blood CD4+ T-cells into <50 CD4+ T-cells per μL (n = 42), 50–199 cells per μL (n = 49), 200–349 cells per μL (n = 52) and >350 (n = 57) cells per μL; a group of HAD patients (n = 14); one group on suppressive cART with plasma HIV-RNA <50 copies/mL for at least one year (n = 85). Forty-six subjects were included in both untreated and treated groups sampled at different time points, usually several years apart . We also included healthy HIV-negative controls (n = 204), neuroasymptomatic if they were without symptoms or signs of neurological or cognitive disability on clinical examination and follow-up. Neuropsychological examinations were performed only on subjects with symptoms or complaints consistent with neurocognitive disorder. The diagnosis of HAD was based on CDC and American Academy of Neurology Task Force Criteria using standard laboratory and clinical evaluations In the cross-sectional study, 252 HIV-infected subjects were segregated into 6 subgroups : Four grThe longitudinal cohort study included 78 neuroasymptomatic patients who were treatment-naive or had been off treatment for >6 months. CSF samples were analyzed before and after treatment initiation, with a median time of 15 weeks . Subject characteristics are also shown in CSF samples were submitted to low-speed centrifugation to remove cells, aliquoted, and frozen to −70°C within 1 hour of performing the lumbar puncture for storage until analysis. CSF NFL protein was measured in the Laboratory of Neurochemistry at the University of Gothenburg using a novel, sensitive sandwich ELISA method (NF-light® ELISA kit) as described by the manufacturer . The lower limit of quantification was 50 ng/L and intra- and inter-assay coefficients of variation were 4·1–13%. The upper normal refence limits of CSF NFL were <380 ng/L (18–30 years), <560 (30–39 years), <890 (40–59 years), <1850 (>59 years); these limits were established using 108 CSF samples from neurologically healthy control individuals aged 18–76 years.me HIV-1 assay . The lowest limit of detection was defined as 20 copies per mL. Neopterin was analyzed in CSF using a commercially available immunoassay  with an upper normal reference value of 5.8 nmol/L HIV RNA in CSF and plasma was measured using the Roche Amplicor Monitor version 1.5, Roche Taqman assay version 1 or 2  or Abbott RealTi10 CSF NFL levels and CD4+ cell counts were analyzed with non-linear regression. The relationship between log10 CSF NFL levels and age in controls and the partly overlapping treated and untreated groups were analyzed with a linear mixed effects model.The Mann-Whitney test was used to compare variables between independent groups. Wilcoxon matched-pairs signed rank test was used to compare CSF NFL levels before and after treatment. The relationship between log10 CSF NFL were analyzed by multiple linear regression analysis with backward elimination in treated and untreated groups separately. In these analyses WBC and HIV RNA were log transformed and CD4+ T-cell count was inverse transformed (1/(CD4 + 45.3)) based on the previous non-linear regression. General descriptive statistics are presented as median and interquartile range (IQR). All statistical analyses were performed using IBM SPSS Statistics© version 20 or Prism© version 5 .Predictors of log252 HIV-positive patiens subdivided into three groups were studied: Neuroasymptomatics, further stratified into four groups according to levels of blood CD4 cells, HAD and patients on suppressive antiretroviral treatment. 204 HIV-negative subjects served as controls. For further subject characteristics, see Using the laboratory age-related cutoffs for CSF NFL described earlier, nearly all  of the HAD patients had elevated CSF NFL concentrations. Additionally, elevated CSF NFL concentrations were found in a substantial number of the untreated neuroasymptomatic subjects, particularly those with lower blood CD4+ T cell counts. Thus, 29/42 (69%) of those with CD4+ T-cell count below 50 cells/mL and 15/49 (31%) with 50–199 CD4+ cells/mL had elevated concentrations, and even 11/52 (21)% of subjects with 200–349 CD4 cells and 11/57 (19%) of those with CD4 cell counts above 350 cells/mL were also elevated—indicating a high level of clinically unappreciated ongoing CNS injury that varied with degree of systemic disease progression. By comparison, in the treated group and HIV-negative subjects the frequency was much lower, 7/85 (8%) and 4/204 (2%), respectively.The influence of disease progression was more directly shown as a strong correlation of CSF NFL with the blood CD4+ T-cell counts in neuroasymptomatic untreated patients, p = <0.001. A loess regression suggested an inverse transformation of CD4+ T-cell counts . Visual No significant correlation was found between CD4+ T-cell counts and CSF NFL within the treated, virologically suppressed group.Since CSF NFL increases with age in uninfected populations, we analyzed the differences between treated and untreated subjects and HIV negatives with age as covariate. This resulted in a model of three parallel regression lines . The groA multiple non-linear regression analysis that included CSF NFL versus age, CD4+ T-cell counts, CSF WBC, CSF:blood albumin ratio, plasma and CSF HIV RNA and CSF neopterin was performed on both untreated and treated patients. Univariate and multivariate analyses are shown in Overall, CSF NFL levels decreased in 63% of the patients (n = 49) from a pre-treatment CSF NFL median level of 520 ng/L (IQR 310–1070) to 475 ng/L (IQR 315–782.5) after initiation of cART (p<0.01) . Of the Although CSF NFL decreased in some patients with normal baseline CSF NFL (n = 52), overall there was no significant reduction for the group as a whole. All patients with normal CSF NFL concentrations at baseline remained normal except for three subjects who exhibited increases to abnormal levels from 470 to 1500 ng/L, from 290 to 2460 ng/L and from 420 to 990 ng/L, respectively (reference range for their ages is <890 ng/L). These changes were not accompanied by notable neurological symptoms and all three showed decrease in plasma and CSF HIV RNA and exhibited increases in their CD4 counts after treatment. The patient with the highest CSF NFL increase developed a cutaneous vasculitis, along with a proximal venous thrombosis requiring anticoagulants and high doses of corticosteroids. No symptoms and signs associated with immune reconstitution or other disorders to explain the elevations were noted. One patient was lost to follow-up.Our results provide compelling evidence supporting the value of CSF NFL as a biomarker of axonal disruption in untreated patients with HIV infection. They show that CSF NFL is not only a sensitive biomarker of symptomatic HAD, but also demonstrates that a substantial number of untreated asymptomatic patients suffer subclinical CNS injury. The prevalence of this injury increases as systemic disease progresses and blood CD4+ T cells fall. Thus, CSF NFL was strongly inversely correlated with CD4+ T-cell counts. Indeed, a clear majority of patients with counts below 50 cells/μL exhibited elevation of this neuronal marker in CSF and nearly half of those with counts below 200 had abnormal levels showing that subclinical injury in more advanced disease is common.This common, presumably chronic, brain injury at reduced cell counts provides one explanation for the reported high prevalence of neurological symptoms and reduced cognitive performance in treated patients with lower CD4+ T cell nadirs CSF NFL increases with age in the absence of HIV infection. Though the mechanisms underlying this increase are not established, it may confound analysis of the consequences of the HIV infection itself and potential treatment effects on CSF NFL over time. However, when comparing neuroasymptomatic untreated patients as a whole to HIV-negative controls and the HIV-positive group on cART, CSF NFL was higher in untreated subjects regardless of age, equivalent to 18.5 years older HIV negative controls. Fortunately, this elevation is largely reversible. Yet, and perhaps of greater interest, levels of CSF NFL in the treated and virologically suppressed group remained significantly higher than those of the HIV-negative population, a difference equivalent to the addition of 3.9 years of life in this group. This finding might reflect a discrete, continuous neuroinflammation and axonal degradation due to ongoing, yet undetected, infection or continued local infection with mild, but important neuropathogenic effect. Or it might indicate an effect of the prior injury that is similar to the effect of aging on the metabolism of NFL and their neuronal substrate. Apart from neuroinflammatory and neurodegenerative diseases, such as MS Future studies of both aging and treated HIV infection need to address the sources of this elevated CSF NFL, whether due to increased release or turnover of NFL or, alternatively, reduced clearance from CSF. Additionally, it will be important to assess correlations of elevated CSF NFL in treated patients with detailed neuropsychological test performance in addition to pretreatment neurological status to dissect the effects of earlier injury from evidence of ongoing damage in determining post-treatment neurological status and CSF NFL.Earlier studies have shown that microglia/macrophage activation, reflected by increased levels of CSF neopterin is an important factor in HIV neuropathogenesis CSF neopterin was not found to be a predictive factor for CSF NFL in treated patients. However, CSF albumin ratio was an independent predictor for CSF NFL in both treated and untreated HIV infected subjects. Increased CSF albumin ratio reflects a compromised BBB with increased permeability of serum proteins and other substances into the brain. Perivascular inflammation and BBB dysfunction are frequent findings in patients with HAD Several reports indicate that milder forms of HAND are fairly common also in HIV infected patients on effective treatment There are a number of limitations of this study. It involved a convenience sample studied through archived samples. Importantly, extensive neuropsychological testing required for Frascati criteria diagnosis The findings of this study support the value of CSF NFL as an independent marker of CNS damage at different stages of HIV infection. We present data indicating subclinical brain injury reflected by levels of CSF NFL in a substantial proportion of untreated HIV infected patients. The most important influencing factors in addition to subject age were CD4+ T-cell counts, CSF neopterin levels, CSF WBC counts and CSF/plasma albumin ratio, reflecting the importance of systemic disease progression, immune activation and blood brain barrier dysfunction in HIV neuropathogenesis. Antiretroviral treatment has a substantial effect on intrathecal immunoactivation and neurodegeneration, but even virologically suppressed patients exhibited higher levels of CSF NFL than HIV negative controls, suggesting ongoing low-grade injury or an aging effect of HIV infection that is not fully reversed by treatment. To better understand the prognostic meaning of fluctuations of CSF NFL in HIV disease and to evaluate this marker as a diagnostic tool for HIV-associated neurocognitive disorders, future prospective studies with concurrent neuropsychological testing will be important.


To investigate whether uncharacterized infectious agents were associated with neurologic disease, we analyzed cerebrospinal fluid specimens from 12 children with acute central nervous system infection. A high-throughput pyrosequencing screen detected human parvovirus 4 DNA in cerebrospinal fluid of 2 children with encephalitis of unknown etiology. Encephalitis is a major cause of death and disability globally. In Asia, Japanese encephalitis virus is the most commonly recognized cause, with 30,000–50,000 cases and ≈10,000 deaths annually  is a single-stranded DNA virus first detected in 2005  hospitalized with a suspected acute central nervous system (CNS) infection at the Vijayanagar Institute of Medical Sciences, Bellary, India, October 2005–October 2007, as previously described (>5 cells/mm3 or protein >45 mg/dL). These patients had negative diagnostic test results for pathogens known to cause CNS infection in this region of India at the time of investigation ; in addition, CSF culture was performed and no bacterial organisms were found  hit. For 2 of the 12 patients, patients VES085 and VES065, viral sequences were detected in the CSF.The raw sequence reads were deconvoluted on the basis of the barcode and then processed through a standardized bioinformatic pipeline (We identified 17 (92.6%–98.2% sequence identity) distinct sequence reads in the CSF of patient VES085 and 6  distinct reads from patient VES065 with pairwise identities based on BLASTn alignment of each read to the reference genome (GenBank accession no. EU175855.1). To verify the results, we used PARV4-specific PCR primers to screen all 12 original CSF samples as described , and endpoint titers of 50% positivity were calculated by using the Reed-Muench formula ,.To genetically characterize PARV4 variants infecting the patients, we generated overlapping sets of amplicons spanning the complete genome of PARV4 from the 3 samples. No variation was seen between the CSF and serum-derived sequences from VES085. The fully assembled coding regions of the variants generated from both patients were compared to previously described PARV4 variants  . PhylogeThe serum sample was screened for immunoglobulin (Ig) G and IgM PARV4-specific antibodies by using a previously developed serologic assay for PARV4 antibodies .Through serologic screening of age-matched controls, we were able to largely rule out the possibility that detection of PARV4 DNA in samples from 2 of the 12 patients was simply incidental to a high background incidence of PARV4 infection in this age group. Although published data on the duration of viremia in patients with acute PARV4 infections has not been directly determined, viral loads as found in the study patients are of relatively short duration in other parvovirus infections  . It is more common to have CSF pleocytosis than not, though of course there are well characterized cases of CNS infection  with no pleocytosis  is cutting edge; this technique’s utility also extends to a recent identification of astrovirus in a case of encephalitis (


Conscious assessment for organ dysfunction in infected patients is not uniformly performed since the prognostic performance of organ dysfunction has not been validated. We hypothesize that the number of organ dysfunctions is a prognostic marker in emergency department (ED) patients with suspected infection and that an increasing number of organ dysfunctions correlates with in-hospital mortality.A prospective observational study of adult (18+ years) ED patients with suspected infection presenting to one of two urban, academic medical center EDs. The inclusion criterion was clinically suspected infection at ED presentation. At Beth Israel Deaconess Medical Center (BIDMC), Boston, USA, consecutive patients were enrolled over a 1-year period  and at Aarhus University Hospital (AUH), Aarhus, Denmark, a case-control study was performed . Laboratory and clinical data were collected at enrollment to assess organ dysfunction. Primary outcome was in-hospital mortality. Logistic regression was performed to determine the independent mortality odds.Four thousand, nine hundred and fifty-two patients were enrolled at BIDMC and 483 patients at AUH. Overall mortality rates were 4% and 11% with mean ages of 58 ± 21 and 69 ± 16 years, respectively. The mortality rate increased with increasing number of organ dysfunctions: BIDMC: 0 organ dysfunctions, 0.6% mortality; 1 dysfunction, 3.3%; 2 dysfunctions, 7.8%; 3 dysfunctions, 15.9%; and ≥4 dysfunctions, 34.3%; and AUH: 2.2%, 6.7%, 17%, 41%, and 57% mortality (Figure Using readily available criteria in the ED to assess the number of organ dysfunctions is a reliable tool in predicting in-hospital mortality in both validation sets and could assist in risk prognostication and aid with earlier, targeted therapy.


Mannan-binding lectin (MBL), a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF) can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb), i.e., with CSF flow rate.MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction.MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram) 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3) The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66%) compared to the MBL concentrations in serum (CV = 146%) indicate an independent source of MBL in CSF. 4) The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear) increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb.MBL in CSF is predominantly brain-derived and all results pointed to the leptomeningeal cells as the source of the protein. The evaluation of this protein requires the interpretation of its absolute concentrations in CSF as a function of the albumin quotient, QAlb. This recognition of MBL in brain cells opens a new field of discussion about the function of the innate immune response in CNS in cases of acute and chronic neurological diseases. Increased protein concentrations in the cerebrospinal fluid (CSF) of patients with neurological diseases, frequently ascribed to a blood-CSF barrier dysfunction, are due to pathologically-reduced CSF flow rates . This viAlbumin in CSF is derived exclusively from blood even in all kinds of pathological processes of neurological disease ,2. ThereThree sources for CSF proteins can be identified. These are: firstly, blood-derived proteins in CSF  evaluatInappropriate referencing of brain-derived proteins to their serum concentration using the CSF/serum quotient instead of the absolute CSF concentration leads to a loss of sensitivity for discriminations between groups, i.e., eventually to false interpretations of CSF data and disease pathologies. These different dynamics in CSF have been shown empirically and were derived quantitatively from the molecular diffusion/CSF flow model of Reiber ,4,6. ThiThe increasing awareness for the innate immune system and its possible role in the CNS is documented in recent publications : MannIn previous studies MBL has been detected in CSF ,14. In tLumbar CSF and serum samples originated from patients of the Department of the Neurological University Hospital, Goettingen. All samples were taken for routine analysis ,3, indicPatients with non-inflammatory diseases but with blood–CSF barrier dysfunction had increased CSF/serum albumin quotients (QAlb) as well as all other blood-derived CSF/serum protein quotients  but without any intrathecal contribution to the CSF values, as shown in the quotient diagrams  is shown in Table From the statistics in Table For MBL with a molecular weight of 6 × 96 kDa = 576 kDa, the mean transfer gradient was similar to IgM. i.e., 3000:1 [The biological variability, expressed as the coefficient of variation (CV), for MBL in CSF is much smaller CV = 66%) than that of the serum MBL value CV = 146%). In the case of a blood-derived protein in CSF, an increased coefficient of variation would be expected, due to the additional biological variation originating from the individual barrier function. These consequences of the data in Table 6%. In th6% than tThe correlation of the absolute CSF concentration of MBL with the albumin CSF/serum quotient, QAlb, is shown in Figure The sufficiently sensitive analysis of MBL in CSF represents an analytical challenge . For theIn fact, we do not know the effective molecular size of MBL, relevant for its passage from blood to CSF. The blood-derived MBL fraction in CSF may be much smaller than calculated on the base of the molecular size of free MBL (results). It is possible that MBL with its tendency to bind other proteins does not pass the blood CSF barriers as a free MBL molecule but as a larger MBL-MASP complex, i.e., with bound serine proteases ,13. If tThe intrathecal MBL fractions are shown directly in the IgM quotient diagram in Figure It may be a surprising application of the coefficient of variation (CV) to characterize the connection of a parameter in two different compartments of a biological system.-3). Of course there are different options in a biological system to get such a result, but if the inverse combination with a smaller or still equal variation in CSF compared to serum samples of the same group is obtained, it can be concluded that there is no diffusion-dependent association of the parameter in the different compartments. But this is exactly what was obtained for MBL  additionally influencing the protein concentration in CSF. Due to such variation propagation there is a larger variation in the albumin concentration in CSF samples CV = 26%) compared to the corresponding variation in blood samples (CV = 15%) in a control group  accumulate in CSF with decreasing turnover rate, i.e. slower CSF flow rate. Correspondingly their concentration correlates with QAlb. As shown for beta trace protein the correlation with QAlb is linear, as expected from the theory . Figure As a consequence of empirical data and theory  the rostAn important confirmation of our conclusions that MBL is a leptomeningeal protein comes from histochemical investigations in animals ,18 whichHence, the following arguments are found for MBL in CSF as a predominantly leptomeningeal protein:1. The CSF concentration of MBL is larger than expected from a corresponding molecular size-dependent passage from blood.2. An intrathecal MBL fraction in CSF is observed in more than 80% of the controls.3. The smaller variability in CSF compared to blood concentrations of MBL, i.e. the absence of variation propagation, clearly excludes blood as the primary source for the MBL concentration in CSF.4. The dynamics of the absolute MBL concentration in CSF as a (linear) function of QAlb point to a leptomeningeal protein.5. Morphological data from animals show that MBL can be present in the leptomeninges.Using established methods for evaluation of CSF data ,3,5,15, This study has shown that mannan-binding lectin in CSF is predominantly brain-derived, most likely from the leptomeningeal cells. There is a negligible physiological connection between the MBL fractions in CSF and serum. The evaluation of the clinical role of this protein requires the measurement of absolute concentration in CSF as a function of the CSF/serum albumin quotient. The recognition that MBL originates in brain cells opens a new field of discussion about the function of the innate immune response in CNS in acute and chronic neurological diseases.MASP: MBL-associated serine protease; MBL: Mannan-binding lectin; QAlb, QIgG, QIgA, QIgM, QMBL: CSF/serum concentration quotient of albumin, IgG, IgA, IgM, MBL correspondingly; Qlim: Upper limit of the reference range for blood-derived proteins in CSF.There are no competing interests for any of the authors.H R: Sample acquisition, theoretical interpretation of data. B P-D: Supervisor for clinical interpretation of MBL, coordination of the patient data acquisition. J C J: Laboratory analysis of MBL in CSF and serum. A J D-C: Organizer of the study, immunological interpretation of MBL data. All authors have read and approved the final version of the manuscript.


Herpes simplex encephalitis (HSE) often leads to severe disability or death. Factors usually associated with outcome include Simplified Acute Physiology Score, age and delay of initiation of acyclovir treatment.Herpes simplex virus (HSV) load in cerebrospinal fluid (CSF) upon HSE outcome.Our aim was to determine the impact of We retrospectively determined HSV load in the CSF of 43 patients with confirmed HSE, hospitalized in northern France from 1998 to 2005, using CSF samples collected the day of hospital admission and stored at −20°C. We analyzed the association between HSV load and mortality/morbidity by the Glasgow Outcome Scale. Fisher’s exact test and Wilcoxon’s test were used for statistical analysis.p=0.03). Longer delay in acyclovir initiation tended to be associated with higher mortality but did not reach statistical significance (p=0.08). Severe disability and death due to HSV were associated with a higher Knaus score (p=0.004), later acyclovir initiation (p=0.006), older age (p=0.04) and presence of red blood cells in CSF (p=0.05). HSV load in CSF was neither associated with mortality (p=1.00) nor with morbidity (p=0.90).The M/F sex ratio was 1.7 and median patient age was 61 years. Median HSV load in CSF was 2.0 log copies/μL (IQR 25-75=1.2-2.6). The mortality rate was 32.6% six months after HSE diagnosis. Higher age was associated with mortality (In this study, HSV load in CSF was not found to be associated with poor outcome in patients with HSE. These data do not support measurement of HSV load at admission in patients with HSE. Herpes simplex virus (HSV) is the primary cause of viral necrotizing encephalitis in developed countries; when untreated, the disease has a 70% mortality rate[ity rate,2. Despiity rate,4: 15 toFactors commonly reported to be associated with HSE outcome are age, level of consciousness at initial management of the patient and timeThe aim of this study was to assess the association between HSV load in CSF (before initiation of acyclovir treatment) and HSE outcome.We conducted a retrospective study in patients diagnosed with HSE in northern France from 1998 to 2005. We focused on patients for whom a CSF sample was available prior to acyclovir treatment initiation in order to assess the association between CSF HSV load and morbidity and mortality six months after HSE onset.Data were collected retrospectively from patient medical records using a standardized questionnaire. Morbidity was assessed using the Glasgow Outcome Scale (GOS) divided into 5 categories ,13. The HSV load was measured in CSF of patients with HSE using a frozen sample taken before acyclovir treatment was initiated. Samples were prospectively collected and stored at −20°C. When frozen CSF was available (n=30), we extracted HSV DNA using the QIAamp® DNA Blood Qiagen kit according to the manufacturer’s instructions. We performed the extraction and quantified HSV load on 200 μL of the sample for 6 patients and less than 200 μL for 24 patients. When no CSF was available to extract DNA (n=13), we quantified the viral load directly on DNA that had been extracted on the date of sampling and stored frozen at −20°C. Then, quantification of the viral load was performed using the artus® HSV-1/2 LC PCR kit manufactured by Qiagen. This PCR kit uses real time PCR for quantification and contains a second system of heterologous amplification to monitor for the possible presence of inhibitors. It allows the distinction between HSV1 and 2.First, we used descriptive statistics to evaluate patient characteristics according to CSF sample availability. Secondly, we assessed univariate associations between HSV load (or other variables collected) and HSE outcome defined as mortality at six months and morbidity at six months .Non-parametric tests were used for all comparisons. Fisher’s exact test and Wilcoxon’s test were used to compare qualitative and quantitative variables, respectively. A significance threshold of 0.05 was retained. Statistical analysis was performed using SAS software .All patients included in the study were followed in different hospitals from northern France. The care practices included CSF sampling for diagnosis and management of these patients with suspected HSE. HSV qualitative PCR were performed routinely for this diagnosis. We performed quantitative PCR retrospectively on the residual frozen CSF. No additional sampling was necessary. This study was approved by the “Comité de Protection des Personnes Nord Ouest IV”, the ethic committee of Lille Universitary Hospital.Between 1998 and 2005, 78 patients sought care for HSE in northern France. Those patients belonged to a previously published cohort. Frozen We first studied factors associated with mortality six months after HSE onset Table. Median We next studied factors associated with morbidity six months after HSE onset Table. ResultsWe assessed factors associated with HSE outcome in 43 patients with symptomatic HSV encephalitis who sought care in northern France between 1998 and 2005. To our knowledge, this cohort is the largest ever used for evaluating the impact of HSV load in CSF upon HSV encephalitis outcome. Moreover, we used a technique of quantitative real time PCR, limiting the risks of false positive samples. We found no association between HSV load and HSE outcome, nor in mortality or morbidity six months after HSE onset.Our results are consistent with other studies concerning factors usually associated with poor outcome in HSV encephalitis: older age, delayed initiation of acyclovir treatment and a high Knaus score,6,17,18.et al.[. However, in that study, HSV load was studied as a dichotomous variable with a cut-off of 100 copies/μL separating high and low viral load levels. In our study, we analyzed the relationship between viral load and outcome, introducing viral load as a continuous variable. Our results are in agreement with Wildeman et al.[et al.[et al.[et al., who evaluated serial changes of intrathecal viral loads by chemiluminescence assay and nested PCR, did not find any evident association between viral load and outcome either[et al., comparing nested PCR to quantitative real-time PCR[Regarding the absence of association between CSF HSV load and HSE outcome, our results are in contrast to results reported by Dominges et al.. Howeverl.[et al. and Hjal.[et al.. Kamei Se either. Our restime PCR. HoweverThe absence of an association between HSE outcome and CSF HSV load could suggest that the pathogenicity of HSE may not be directly linked to extent of viral replication, but rather depends on the host response. This hypothesis is consistent with the fact that IgG levels were described to be significantly higher in patients with good prognostic than in patients with poor outcome.Surprisingly, in our study, we found a significant association between a high red cell count in CSF and severe morbidity. To our knowledge, this association had not been described previously. It can be hypothesized that a higher red cell count in CSF could reflect more severe brain cell necrosis, which may be due to an intense and inappropriate immunological response of the host to viral aggression. Some experimental data suggested that an intense and inappropriate inflammatory response could be harmful, and might explain the higher risk of morbidity,23. Howeet al. showed the absence of genetic matter loss in samples frozen at −20°C[et al., measured HSV loads in the same samples directly after sampling and after storage at 4°C for 16 months. HSV loads in freshly analyzed samples did not differ from those that had been stored at 4°C[et al.: accurate quantitation of viral DNA is not possible in samples with low levels of virus. In addition, we sometimes had to work with low quantities of CSF due to limited availability of samples. This occurred more frequently in patients whose samples gave negative results by quantitative PCR. In one of these 5 patients, enough CSF was available to re-perform a real time qualitative PCR, which was positive at the 39th cycle, corresponding to a low viral load. Thus, we can consider that these false negative results by quantitative PCR do not bias our results, because they effectively correspond to low HSV loads.Our study had several limitations. First, although our sample size was greater than in other studies,10,11, tat −20°C. Jerome d at 4°C. It is tIn the present study, we found no relationship between poor outcome in patients suffering from HSV encephalitis and HSV load in CSF prior to acyclovir treatment initiation. But we found a relation between poor outcome and the presence of red blood cells in CSF. Our results suggest that, in herpetic meningo-encephalitis, severity is probably not due to the intensity of viral replication. Whether it is related to other physiopathological causes, such as intense and inappropriate inflammatory responses, remains to be elucidated. According to our results and that of similar studies, quantitative real-time PCR is not useful to diagnose HSE. Qualitative real-time PCR assay is perfectly suited.Herpes simplex encephalitis; HSV: Herpes simplex virus; CSF: Cerebrospinal fluid.HSE: The authors declare no competing interests concerning this article.JP collected the data, performed the quantitative PCR, the statistical analysis and wrote the manuscript; KC performed the statistical analysis, helped to the interpretation of the results and to the redaction of the manuscript; AD helped to identify the CSF samples, coordinated the realization of the quantitative PCR and helped to the redaction of the manuscript; HM gave a critical analysis of the work; HG gave a critical analysis of the work; ES gave a critical analysis of this work; YY had the idea of the study, coordinated its realization, verified the statistical analysis and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/356/prepub


Previous studies indicate that successful resolution of Lyme neuroborreliosis (NB) is associated with a strong T helper (Th) 1-type cytokine response in the cerebrospinal fluid (CSF) followed by a down-regulating Th2 response, whereas the role of the recently discovered Th17 cytokine response is unknown.To investigate the relative contribution of different Th associated cytokine/chemokine responses, we used a multiple bead array to measure the levels of CXCL10 (Th1 marker), CCL22 (Th2 marker), IL-17 (Th17 marker) and CXCL8 , in serum and in CSF from untreated patients with confirmed NB (n = 133), and non-NB patients (n = 96), and related the findings to clinical data. Samples from patients with possible early NB (n = 15) and possible late NB (n = 19) were also analysed, as well as samples from an additional control group with orthopaedic patients (n = 17), where CSF was obtained at spinal anaesthesia.Borrelia-antibodies.The most prominent differences across groups were found in the CSF. IL-17 was elevated in CSF in 49% of the patients with confirmed NB, but was not detectable in the other groups. Patients with confirmed NB and possible early NB had significantly higher CSF levels of CXCL10, CCL22 and CXCL8 compared to both the non-NB group and the control group . Patients in the early NB group, showing a short duration of symptoms, had lower CCL22 levels in CSF than did the confirmed NB group (p < 0.0001). Furthermore, patients within the confirmed NB group showing a duration of symptoms <2 weeks, tended to have lower CCL22 levels in CSF than did those with longer symptom duration (p = 0.023). Cytokine/chemokine levels were not correlated with clinical parameters or to levels of anti-Our results support the notion that early NB is dominated by a Th1-type response, eventually accompanied by a Th2 response. Interestingly, IL-17 was increased exclusively in CSF from patients with confirmed NB, suggesting a hitherto unknown role for Th17 in NB. However, for conclusive evidence, future prospective studies are needed. Neuroborreliosis (NB) is the most common manifestation of disseminated borreliosis in Europe ,2. Most The Th1/Th2 concept has recently been extended to include a population called Th17, based on their secretion of interleukin (IL)-17 . Th17 cee.g. monocytes, endothelial cells and fibroblasts [Chemokines are small chemotactic cytokines that are induced during an immune response to promote migration of immune cells to the site of infection . Chemokiroblasts  in resporoblasts . Previouroblasts  as well roblasts . The Th2roblasts , and is roblasts . CXCL8 , respectively. CXCL8 was analyzed as a general marker of inflammation. In addition, we also wanted to relate the cytokine/chemokine levels to age, sex, clinical presentation and course of the disease.Borrelia antibody index (AI) or increased levels of Borrelia-specific IgG or IgM antibodies in CSF were found in 152 patients, whereas 111 patients had no detectable Borrelia-specific antibodies in CSF. In the entire study group (n = 263), there were 155 men (59%) and 108 women (41%). The median age in the whole group was 41 years, range 2-87 years. There were 95 (36%) patients aged 15 years or less, and 168 (64%) patients were over 15 years of age. The patients were divided into four groups based on the CSF findings  where CSF was not obtained due to suspicion of NB; 17 patients undergoing elective orthopaedic surgery, where CSF was collected prior to spinal anaesthesia. The reference material was collected at the University Hospital of Linköping from patients that had not experienced NB and they were negative for anti-Borrelia antibodies in serum and in CSF. Further characteristics of the patient groups are presented in Table Serum and CSF samples of 263 patients were investigated for suspected NB during 2003 through 2005 at the hospitals of Kalmar n = 165) and Jönköping (n = 98). The domiciles of the patients were distributed all over the Counties of Kalmar and Jönköping, respectively. None of the patients had, in conjunction with the current study, received antibiotic treatment for NB prior to the lumbar puncture (LP). Elevated anti-65 and JöBorrelia-specific antibodies, i.e. AI, . Results were interpreted as negative or positive according to the manufacturer's instructions. Serum samples from patients in Kalmar County were investigated with Immunetics Quick ELISA Borrelia C6 Assay kit, Immunetics, Cambridge, MA, USA.The Clinical Laboratory of Microbiology (CLM) in Kalmar used an ELISA kit measuring intrathecal production of nd Generation  was used for measuring Borrelia-specific antibodies in serum and in CSF. Results were interpreted as positive or negative according to a cut-off adjusted to a local sample collection used when validating the method.At the CLM in Jönköping the Lyme Borreliosis ELISA kit 2Borrelia-specific antibodies by ELISA kit measuring serum and intrathecal production of antibodies .The control samples (group 5) were analysed for All samples had been stored at -20°C and thawed once before the current analyses. The cytokine/chemokine and albumin analyses were performed in 2009 using Luminex technology (see below) and rate nephelometry (Beckman Coulter Immage 800), respectively.Concentrations were measured by Luminex multiple bead technology  according to the instructions provided by the manufacturer. The lower detection limits were as follows; CXCL8: 1.6 pg/mL, IL-17: 1.6 pg/mL, CXCL10: 16 pg/mL, CCL22: 80 pg/mL in serum, 20 pg/mL in CSF. Values under the detection limit were given half the value of the lowest point of the standard curve. The inter-assay coefficient of variation (CV) was 9.3-11.6%, and the intra-assay CV was 4.5-7.1% according to the manufacturer. In the following, CXCL8, CXCL10 and CCL22 are collectively referred to as chemokines; IL-17 is referred to as cytokine, whereas all  are referred to as cytokine/chemokines.2 test was used. Correlations between parameters were calculated using Spearman correlation analysis . P-values < 0.01 were considered to be significant, this p-value was chosen to avoid mass-significance problems. By analogy to the IgG-index used for calculations of intrathecal IgG production [Statistical analyses were performed using SPSS for Windows, version 15.0. Since most of the variables had skewed distributions, non-parametric tests were used. Kruskal-Wallis ANOVA was performed to compare multiple study groups, and Mann-Whitney U test was applied as a post-hoc test. For ordered categorical variables, the Chioduction , we calcThe study was approved by the Regional Ethical Review Board in Linköping, Sweden.There were no differences in cytokine/chemokine levels in serum or in CSF between men and women. When comparing the patients under 15 years of age to patients over 15 in the entire study population (groups 1-5), the children had significantly higher CSF levels of CXCL8 (p < 0.0001), CXCL10 (p < 0.0001), CCL22 (p = 0.0001) and IL-17 (p = 0.001). In serum, there were no differences in cytokine/chemokine levels between younger or older patients, except for CCL22 that was higher in the patients under 15 years of age (p < 0.0001).The cytokine/chemokine data from the aspect of the different study groups are presented in Figure i.e. confirmed NB and possible early NB, showed increased levels of CXCL8 . Furthermore, cytokine/chemokine levels in CSF did not correlate with the corresponding serum levels. The chemokine intrathecal production indices for CXCL8, CXCL10 and CCL22 were increased in groups 1 and 3  age, ii) duration of symptoms prior to diagnosis, iii) degree of pleocytosis in CSF, iv) intrathecal Borrelia-specific antibodies, or v) duration of symptoms after treatment (data not shown). Group 1 also showed increased CSF levels of IL-17, being detected (i.e. >0.80 pg/mL) in 65 patients (49%), whereas IL-17 was not detected in CSF of the other groups. However, there were no significant differences between patients with elevated IL-17 levels in CSF and the remaining group 1 patients regarding age, sex, duration of symptoms prior to diagnosis or duration of symptoms after treatment.Group 1, the confirmed NB group, showed significantly higher CSF levels of all cytokine/chemokines compared with groups 4, the non-NB group, and 5, the control group  compared to the patients in group 1 with undetectable levels of IL-17 in CSF. There was no correlation within group 1 between serum and CSF levels of IL-17 , or between the CSF-albumin/S-albumin ratio and IL-17 in CSF; . Further, there was no significant difference in CSF-albumin/S-albumin ratio when comparing patients with elevated IL-17 in CSF to those with low levels (p = 0.172) . The same differences between the groups were obtained as when analysing the entire study population  and in group 3 (possible early NB). CCL22 in CSF was particularly elevated in group 1, and moderately elevated in group 3. Here, the time aspect is probably of importance for the chemokine profile. Early in the inflammation process, a Th1-type immune response dominates , but is then counter-balanced by a Th2 response . This pattern is also found within group 1, e damage -19.Borrelia-antibodies in CSF (group 3) is then puzzling. However, it could be speculated that children show defective or delayed Th17 responses. The presence of IL-17 in CSF of NB patients has only recently been reported in another cohort of NB patients on the Islands of Åland [Remarkably, half of the patients in group 1 had elevated levels of IL-17 in CSF, whereas IL-17 was not detectable in any of the other patient groups. The role of the increase in IL-17 is still puzzling since there were no significant differences between those with high levels of IL-17 in CSF and those with low levels regarding sex, age, duration of symptoms prior to diagnosis, or duration of symptoms after treatment. The significant findings distinguishing patients with high levels of CSF-IL-17 from those with low levels, were a more pronounced pleocytosis, slightly more elevated CXCL10 in CSF and a higher frequency of fatigue. These findings are hard to interpret and need to be confirmed. The association with pleocytosis suggests IL-17 to be associated with ongoing inflammation, although the absence of IL-17 in children with pleocytosis but no anti-of Åland . Since Iof Åland , furtherCXCL8 is one important effector mechanism associated with IL-17, which is in line with the high CXCL8 levels in group 1. However, also the patients in group 3 (possible early NB) showed high levels of CXCL8 in CSF, but no detectable IL-17, which suggests that CXCL8 might be induced by other pathways independent of IL-17.We found several evidence supporting an intrathecal origin of the induced cytokine/chemokines; no correlations between serum and CSF levels, the levels being of the same magnitude (or higher) in CSF compared to serum for CXCL8, CXCL10 and IL-17, and increased chemokine intrathecal production indices for CXCL8, CXCL10 and CCL22 in groups 1 and 3. For IL-17, an intrathecal production index could not be reliably calculated due to the absence of IL-17 in CSF of several groups. Yet, it seems that IL-17 is produced intrathecally since neither the serum levels of IL-17 nor the CSF-albumin/S-albumin ratios correlated with the CSF levels of IL-17.As to findings in serum, the patients with confirmed NB showed significantly higher levels of CXCL10 as compared to groups 2 and 4, possibly reflecting that the strong Th1 response in the CNS also is associated with a systemic response. However, the orthopaedic control patients also displayed elevated CXCL10 levels in serum. It could be speculated that this finding might be related to age . SurprisWe also attempted to find correlations between cytokine/chemokine levels and clinical parameters including the course of disease. However, no such correlations were found, which implies that cytokine/chemokine levels do not seem to be useful clinical predictors of the disease course. However, we can not exclude that such correlations would have been revealed if a standardized follow-up protocol had been applied in a prospective manner.Borrelia infection, nor any signs of CNS inflammation, and it is our opinion that the inflammation in NB is mainly restricted to the CNS compartment. Furthermore, there were no significant differences found between the non-NB group and the control group (group 5), except for higher serum levels of CXCL10 in the control group as discussed above. Another possible limitation to this study is the difference in age between patients in group 3 (children with possible early NB) and patients in the other groups. However, when comparing group 3 to the children in groups 1 and 4, we obtained the same results as when including the adults in the analysis.One limitation to this study is the difficulty to find an appropriate control group. The use of completely healthy control patients with no symptoms and no other disorders would of course be desirable, but it is generally very difficult to obtain CSF samples from healthy volunteers. Importantly, we included a control group (group 5) where CSF was not obtained due to suspicion of NB. We also believe that our non-NB group (group 4) is quite useful, since the patients have neither signs of ongoing The samples had been stored at -20°C and thawed once before. However, chemokines are fairly robust, and concentrations were measurable using the methods described here. Furthermore, all samples had been handled and stored under the same conditions. It is also worth noting that we here measured the concentrations of circulating cytokine/chemokines, and the results could be different at a cellular level. However, CXCL10 and CCL22 seem to roughly reflect previous data on Th1 and Th2 -15.Our results support the notion that a Th1-type immune response dominates in the CNS in early NB, and is then followed by a Th2-type immune response. We suggest that chemokines are suitable and feasible markers for measuring of Th1/Th2 responses. We also show that IL-17 levels are increased in CSF in a substantial proportion of NB cases, suggesting a role for Th17 in NB. However, the precise role of Th17 in NB pathogenesis and clinical course remains to be evaluated.The authors declare that they have no competing interests.The study was approved by the Regional Ethical Review Board in Linköping, Sweden. A copy of the written approval is available for review by the Editor-in-Chief of this journal. The patients had given a general consent to their samples being stored in a biobank and used for research purposes.AJH participated in the design of the study, performed the statistical analysis and drafted the manuscript. IT, BEM and PF participated in the analysis and interpretation of data and helped to draft the manuscript. JE conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors have read and approved of the final manuscript.


Background: The radial forearm free flap (RFFF) is the most commonly used free flap in head and neck reconstructive surgery. However, despite excellent results with respect to the site of reconstruction, donor site morbidity cannot be neglected. This review summarizes the current state of knowledge and analyzes the level of evidence with regard to perioperative management of the reduction of RFFF donor site morbidity. Methods: The medical Internet source PubMed was screened for relevant articles. All relevant articles were tabulated according to the levels of scientific evidence, and the available methods for reduction of donor site morbidity are discussed. Results: Classification into levels of evidence reveals 3 publications (1.5%) with level I , 29 (14.0%) with level II , 3 (1.5%) with level III , 121 (58.7%) with level IV , and 15 (7.3%) with level V . Thirty-five (17.0%) articles could not be classified, because they focused on a topic other than donor site morbidity of the RFFF. Conclusions: Although great interest has been expressed with regard to reducing the donor site morbidity of the workhorse flap in microvascular reconstruction procedures, most publications fail to provide the hard facts and solid evidence characteristic of high-quality research. Microvascular free flaps have the advantage of providing healthy vascularized nonirradiated tissue for recipient sites that may have been compromised by surgery, radiation, chemotherapy, or a combination of all 3. Since its introduction by Yang et al2This article reviews current evidential knowledge of the earlier named topics and perioperative aspects with regard to the RFFF donor site.We used the PubMed (www.pubmed.gov) electronic information sources and database. The last electronic update was carried out on October 5, 2011, with the key words: “donor site morbidity” and “radial forearm flap” within the “all fields” search builder, including title and abstract, if available. The scientific quality of each identified contribution was evaluated by the first author (D.J.L.) and the last author (M.R.K.) by using a modified version of the Antes classification for the levels of evidence in therapyWe classified the publications that included meta-analyses or individual randomized controlled trials (RCTs) as level I, individual cohort studies, experimental studies with no randomization, and quality-of-life research as level II, case-control studies as level III, nonexperimental studies and case reports as level IV, and expert opinion as level V. To differentiate between publications classified within evidence level IV, we categorized them according to the number of participants . Level VI, which is not a level of evidence, recorded articles that were not classifiable.The computer-assisted search of the Internet PubMed database yielded 188 hits. In addition, 18 relevant articles were found, which were not identified by the database, during preparation of the manuscript. All articles (n = 206) were tabulated according to the levels of scientific evidence. After evaluation of the manuscripts, or if not available, the abstracts, 35 contributions were classified as irrelevant to the discussed topic and were recorded as level VI.No systematic review of RCTs was published during the period covered by our search (evidence level Ia).Three contributions (1.5%) were articles about RCTs (evidence level Ib). Two of these papers focused directly and solely on the improvement of donor-site morbidity: Moazzam and Gordon,Although the radius bone is significantly weakened by the harvest of a graft, a level IIb study of Edmonds et al12The third level Ib study by Chau et alTwenty-nine contributions (14.0%) were identified having evidence level II: none in level IIa, 8 in level IIb, and 21 in level IIc. Three papers (1.5%) were recognized as having evidence level III; all of them were in level IIIb. One hundred twenty-one contributions (58.7%) were identified as having evidence level IV and 15 (7.3%) with level V. Thirty-five (17.0%) articles could not be classified, because they focused on a topic other than that of donor site morbidity of the RFFF and were therefore classified as level VI  the flap raising techniques of the RFFF, (2) donor site closure methods, and (3) the comparison of donor site morbidity to other free flaps.-20,Research in suprafascial preparation or prelamination techniques of the RFFF, together with donor site morbidity, is mainly based on retrospective studies. Only 2 prospective studies investigated and compared different dissection techniques with different reconstructive methods: first, the earlier discussed, prospective, randomized controlled level Ib study by Chau et al16One further option during flap raising to decrease postoperative complications, namely, the exposure of the flexor carpi radialis tendon and possible consecutive skin graft failure, is to mobilize the belly of the flexor digitorum sublimis muscle to lie over to that of the flexor pollicis longus muscle, so that the flexor carpi radialis tendon is covered. This technique, described in 1985, eliminates tendon exposure but can provoke a compression of the median nerveAnother idea to reduce donor site morbidity and to avoid a second donor site is the prelamination of the RFFF with autologousThe authors recommend that the flap is raised in the classic secure subfascial way on the nondominant arm. The use of a tourniquet is not mandatory in our opinion and even prolongs the ischemia time. When transecting the fibrous attachments between the undersurface of the forearm fascia and the paratenon, the paratenon that envelops the muscle tendons must crucially be left untouched to achieve an accurate wound bed. Extensive mobilizing and oversewing of the surrounding muscle bellies are not necessary and not recommended. The flap should not be extended to the dorsal aspect of the arm for aesthetic reasons. Opinions differ, if an integration of the cephalic vein increases donor site morbidityDirect closure of the radial forearm flap donor site is commonly considered as the method of choice if possible; it avoids the complication of delayed wound healing, but its application is restricted to narrow wounds. If the donor site defect exceeds a range of 2 to 3 cm (depending on the elasticity of the tissue), direct closure is not possible without special approaches. An evidence level Ib study by Moazzam and Gordon9From other methods for the reduction and closure of the skin defect all over the body, only the “purse-string” suture has been researched, on an evidential basis, in cases of the RFFF donor site defect. This technique has been evaluated by Winslow et al28Local skin flaps are adjacent to the defect margin and can be considered for RFFF donor site closure, when defect size is limited, and when the elasticity of the surrounding tissue is sufficient.The Z-plasty technique has been described for the RFFF donor site closure by Hui et al,32,Elliot, Bardsley, and colleagues302. The bilobed flap consists of a large lobe and a small lobe. After elevation, the flap is rotated, and the large lobe is used to cover the radial forearm donor defect, whereas the small lobe is used to repair the resultant defect from the large lobe , Akyürek and SafakTissue expansion is another alternative that can be used to diminish the donor site defect so that direct closure can be performed, but only if time permits. Tissue expanders are silicone envelopes with self-sealing injection ports. These devices are subcutaneously implanted near to the donor region, either pre- or posttransfer. Unfortunately, at least 2 operative stages are required for each of these options. The expander is implanted in a subcutaneous pocket that is located in the epifascial plane. Modern expanders are self-inflating absorbing body fluid from the surrounding tissue through osmotic action. The major advantage of this technique is the possibility of direct wound closure after flap harvesting without the need for a skin graft.With regard to using an autologous or artificial skin graft instead of performing primary closure or a local flap plasty to close the RFFF donor site defect, various ideas have been proposed as to the best method to cover the full-thickness donor site defect. Most investigations into this topic are retrospective studies that examined aspects such as the rate of complete skin graft take, wound healing distributions, and aesthetical and functional impairments, especially with regard to hand function and health-related quality of life.With regard to a high level of evidence, only 1 prospective RCT, namely, that by Sidebottom et al-Other available literature  reports better aesthetic results after closure of RFFF donor sites with FTSG, although these differences are marginal compared with STSG grafts.42To avoid creating a secondary donor site defect , Kawashima et alAllogenic grafts, instead of autologous skin grafts for coverage of the donor site defect of RFFF, did not become popular for a long time, until Rowe et al,51,When artificial dermal templates are used in conjunction with STSG, for example Terudermis or Integra, the donor site is primarily covered with this artificial skin directly after flap raising. The collagen fibers of the artificial dermis resemble those of dermal tissue; host fibroblasts and capillaries become incorporated into the collagen sponge soon after its application to the wound. Dermal tissue is thereby regenerated as a result of the conversion of the collagen sponge into a pseudodermis, a neodermal tissue is formed within about 2 weeks, and this new tissue is then covered by an STSG. Accordingly, the artificial dermis has been designed with the intention of achieving the positive effects of a full-thickness skin graft, both mechanically and aesthetically, by combining the organized collagen matrix with an ultrathin STSG19Considering different flap raising options when using Integra, the suprafascial method seems to be superior to the subfascial method. Andreas et al,With regard to the topic of vacuum-assisted closure (VAC), marketed by Kinetic Concepts, Inc, San Antonio, Tex, Vidrine et al56,15,30,34,56,59-In summary, with regard to the results of the closure of the donor site, a nonnegligible functional forearm and wrist range-of-motion morbidity is widely accepted to occur in the early postoperative period30-However, only a moderate and acceptable incidence of long-term functional morbidity is associated with the radial artery removal63Nevertheless, Emerick and Deschler have demonstrated that only 1.9% of patients have severe complications requiring surgical interventionProgress in microsurgical procedures has led to the development of other free flaps for head and neck reconstruction that are comparable in their usage to the RFFF but with fewer donor site drawbacks. Predominately, the (thinned) anterolateral thigh cutaneous flap has become famous because of its comparable texture and versatility. It gives optimal results, either at the donor site or at the accepting site, and is easy to harvest. Furthermore, it can provide a long and constant pedicle of large caliber and can even be raised without the sacrifice of a main vessel. Studies that have compared both these flap types for head and neck reconstruction accentuate the lower donor site morbidity of the anterolateral thigh (ALT) flap compared with the RFFF.Morrissey et alAn evaluation of donor-site morbidity in an evidence level IIb study by Huang et alAnother evidence level IIb study by de Vicente et al72Two other evidence level IIc studies have also reported an improvement in donor site morbidity in the ALT group compared with the RFFF group.74Recently, our department evaluated the peroneal perforator flap for intraoral reconstruction of an ablative tumor defect of the floor of the mouth and tongue and cheek defects and found comparable results with either ALT or RFFF, with the limitation that no long-term follow-up was available at this early stage. We think that this flap is a good alternative in small- and medium-sized intraoral defects, particularly if direct closure at an inconspicuous donor site is desiredNo further studies were found, which compared the RFFF donor site with other free flap donor sites directly.An obvious discrepancy between quality and quantity exists in the literature with regard to RFFF donor site morbidity. Practicing evidence-based medicine (EBM) means integrating individual clinical experience with the best available external clinical evidence. The difficulty is that EBM demands stronger evidence  than traditional medicine has used, and these studies are rare in a field like plastic surgery.The finding that 81.3% (n = 139) of all relevant contributions  have an evidence level of III, IV, or V Table  should sIf the best available evidence (levels I and II) is represented by only one-sixth of the available literature, as was the case in this analysis, the results are apt to be misinterpreted, and there is a latent danger of following non–evidence-based treatment strategies. This review is, however, limited because we have searched only the electronic databases, possibly leading to incomplete results. The results suggest that some treatment strategies are promoted with great self-confidence by certain authors with regard to the purported additional benefits for the donor-site morbidity of the RFFF, but that this is not based on solid scientific grounds.Short-term donor site morbidity cannot be avoided in reconstructive surgery when using free flaps. Whenever switching to a free flap with less donor site morbidity than the RFFF is not possible, we conclude, on the basis of the reviewed literature, that small RFFF donor defects should be closed directly or with straightforward local advancement flaps if possible. The authors themselves mostly trust in a safe and reliable way of covering the RFFF donor site with autologous STSG after a classic subfascial flap raising technique. We consider it worth repeating that the paratenon of the exposed muscles should be left untouched to achieve an accurate wound bed and thus to avoid mobilizing and oversewing the surrounding muscle bellies.Experimentation in a prospective and controlled way seems to be promising, as some articles have shown promising high-evidence–based results with closure by allogenic grafts, which offer equal or even better results than the use of autografts and, moreover, prevent the need for a secondary donor site. Further available methods, such as VAC therapy or tissue expanders, can be considered in extremely special situations.This article should encourage researchers to carry out high-evidence clinical and experimental trials. When investigating new operative techniques for the closure of the RFFF donor site defect, a multicenter study with high volumes of RFFFs would be desirable to provide systematic research in decreasing donor site morbidity.


Developing controlled hypercarbia is a known scheme of lowering the suprasellar part of the adenoma in order to assist the surgeon, which acts through raising the ICP and therefore the CSF pressure.The purpose of this study is to compare the effect of introducing a lumbar drain with that of controlled hypercapnia on the quality of transsphenoidal pituitary tumor resection and CSF leak.Fifty two patients with pituitary adenoma who underwent transsphenoidal hypophysectomy by the same surgeon were included. They were randomly divided into two groups. A lumbar drain catheter introduced into the L3-L4 subarachnoid space under local anesthesia in all patients. The same anesthesia was performed in both groups. In the study group, we used a saline injection into the subarachnoid space versus hypoventilation in the control group in order to increase the ICP according to the surgeon's request. The surgeon's satisfaction during the tumor resection and the resection time were assessed during the surgery. The CSF catheter was closed and sent with the patient for CSF drainage. If there was no CSF leak, the catheter removed 24 hours later. With evidence of a CSF leak, we used the catheter as a lumbar drain. The time taken for the leakage control was assessed.The satisfaction came from 21 (87.5%) and 2 (9.1%) for surgeon in the first and the second group respectively (P = 0.0001). CSF leakage time in the first and the second group was 1.6 ± 0.24 and 5 ± 0.50 respectively. It revealed a significant difference between the two groups (P = 0.001). The mean resection time was 13.54 ± 0.66 minutes in the study group; and 30.91 ± 0.98 minutes in the control group.In summary, the method described here for ICP manipulation is an effective procedure for a better visualization of the pituitary tumor during transphenoidal resection by surgeon and beneficial in managing the CSF leak following surgery. Transsphenoidal pituitary surgery has evolved over the past decades. The evolution is due to the technological advancements that have served as a catalyst for minimally invasive surgeries . AdenomaThe aim of this study is to compare the effect of introducing a lumbar drain with that of controlled hypercapnia on the quality of pituitary tumor resection via transsphenoidal approach.Fifty two patients with pituitary adenoma who underwent transsphenoidal hypophysectomy by the same surgeon were included. Imaging revealed suprasellar extension in all subjects. They were divided into two groups by a simple randomization. Inclusion criteria were an age range between 30-60 and the presence of a suprasellar macroadenoma. We excluded all patients with rising ICP, cardiovascular disease or COPD. In the operating room, the monitoring included an electrocardiogram, central vein pressure, and intracranial pressure, invasive and noninvasive blood pressure. There was no monitoring of CCP however it was calculated by (MAP-ICP = CPP) while saline injection and rising ICP therefore it was not monitored continuously. All of the patients were turned over to on to their sides. An 18 gauge catheter was introduced into the l3-l4 subarachnoid space under local anesthesia. A 50 cc syringe was attached to the CSF catheter via a three-way port for the injection of saline and monitoring of CSF pressure. The induction of anesthesia was conducted with Fentanyl 2μg/kg, Midazolam/01 mg/kg, Thiopentone 4-5 mg/kg, Cisatracuriom. /015 mg/kg; It was maintained with 50% N2O in 50% O2 and Isoflurane 1%-1.5% with an intermittent positive pressure ventilation to achieve an end-tidal CO2 around 30 mmHg. Further relaxation was maintained by repeating cisatracuriom. Following securing the airway with the endotracheal tube, the throat was packed to protect blood leakage into the stomach and causing postoperative nausea and vomiting. The patient was positioned with a 10° head-up tilt, and the surgery commenced. The baseline arterial blood gas was measured. By means of the technique assigned to the patient, the cerebrospinal fluid pressure was raised at the surgeon's request, usually when the floor of the sella was removed. The surgeon was unaware of the technique used. A screen was placed between the surgeon on one side and the anesthetists on the other side. In the first group, increments of 2.5 m of normal saline were purged via the spinal catheter up to 30 cc, and the descent of the tumor was assessed by the surgeon. At the same time ICP as well as CPP, was monitored and calculated. Then saline volume and the time to reach satisfactory exposure were recorded. In the second group, at first the circle absorber was switched off and the tidal volume and respiratory rate were decreased until end- Tidal (ET) CO2 reached to maximum level of CO2, 50mm Hg and again time to reach satisfactory was recorded. Complete visualization, descent of tumor, visualization of arachnoid or suprasellar capsule was considered satisfactory and was recorded. If the surgeon was satisfied prior achieving a CSF pressure of 20 mmHg or ETCO2 of 50 mm Hg, no further attempts were made, however if the pressure did not rise with our hypoventilation, we again used a saline infusion to raise the pressure. During the dissection, if an excessive descent of the central part of the tumor made the periphery not accessible, CSF pressure by CSF aspiration in the control group has been decreased or hyperventilated in the study group. The cerebral perfusion pressure was continuously monitored and kept above 60 mm Hg, and the mean blood pressure was maintained below 90 mmHg. Then the second arterial blood gas was measured. Muscle relaxation was reversed after the surgeon closed the sellar floor with subcutaneous fat. The patient was extubated and the CSF catheters were closed and sent with the patient for CSF drainage. If there was no CSF leak, the catheters were removed 24 hours later.The obtained data were expressed as Means standard deviations, frequencies and percentages. The data analyzed by an SPSS version 17. To compare the quantitative data, an independent sample T-test was used and for the qualitative data, a Chi-square test or Fisher's exact test was used. In all the investigated cases, the results were considered statistically significant in case of P ≤ 0.05.Statistical analysis was used for 46 out of 52 patients . There wTranssphenoidal microsurgery is a preferred approach for more than 95% of pituitary tumors and for a large proportion of other sellar abnormalities. Decompression of the intrasellar portion of the tumor frequently permits a suprasellar extension to prolapse into view within the sella. Following resection, the diaphragm prolapses and generally signifies that the resection is complete. When a spontaneous prolapse of the tumor capsule or the diaphragm does not occur, instillation of 15 to 20 cc of air or lactated Ringer’s solution into a lumbar subarchnoid catheter may facilitate the descent . Alterna