The Study Of The Correlations Between DTI And CD4 Cell Counts 、CD4/CD8 Ratio And CSF Viral Load Of SIVmac239 Infected Rhesus Monkeys

ObjectiveWe aimed to longitudinally study the T1WI、T2WI and White matter abnormalities of SIVmac239 infected macaques on baseline(which is 2weeks prior to inoculation)、the first time point(12weeks post virus inoculation)and the third time point(24 weeks post virus inoculation)with MRI,we also had the CD4 cell counts、 CD4/CD8、CSF viral load performed before each MRI scan.By longitudinally calculating the correlations among DTI parameters (FA、MD、AD、RD) and CD4 cell counts、CSF viral load and CD4/CD8,we might early obtain precise diagnosis of HAND from the perspective of imaging, so as to realize the expectation that HAND could be treated timely.Only in this way shall we bring people infected with HIV to enjoy better life.Materials and Methods1. SubjectsEleven male rhesus monkeys(ages3-5 years,weight 5.0-7.Okg) were utilized in our longitudinal study.Eight of the eleven monkeys were inoculated intravenously with SIVmac239,three others were for contral;②All the animals underwent health screening and were confirmed to be healthy before being enrolled to this project.Indirect immune influscent assay (IFA) was also used to exclude the possible infection of simian immunodeficiency(SIV), simian type-D retrovirus (SRV), or simian T cell lymphotropic virus-I (STLV-I).③All animals were provided by and housed at the Institute of Chinese Academy of Medical Sciences & Peking Union Medical College,meanwhile mainly evaluated by the specialist on syndromes caused by brain damage,the stands are as follows:persistant anorexia,lost weight,invalid diarrhea,respiratory disease,abonormal behavior and hematology abnormality,and so on.@The housing atmosphere were maintained at 16～26℃, humidity 40-70%,and 12-h/12-h light/dark recycle.And Water was available adlibitum. Monkeys formula feeds were provided twice daily without dietary restrictions.⑤MRI scans were obtained prior to virus inoculation (as the baseline) and in the 12thand 24th week.96th week post virus inoculation. All rhesus monkeys had laboratory evaluations (such as peripheral blood CD4+T cell counts, CD8+T cell counts and CSF viral load) performed before every MRI scan. In the preparation for imaging peripheral blood T cells counts and CSF viral load, all rhesus monkeys were anesthetized by the intramuscular injection of ketamine hydrochloride (5-10 mg/kg). Temperature and vital signs of each animal were monitored during each MRI scan, and a water blanket was also used to prevent hypothermia.. During the experiment,four monkeys died of non AIDS, they were 130007、130008、130004、 130011.2. T cell markers monitoringThe blood samples(for quantitation of peripheral blood CD4+ cells and CD8+ T cells) and CSF viral load were collected before each DTI scan. CD4+/CD8+ ratio was finally calculated using the obtained data above. DTI data analysis.3.Behavioral testCertain electronic equipment for neuropsychological test was employed to tract alternative behavior of rhesus monkeys,including delayed non-matching-to-sample, self-ordered spatial search task,progressive-ratio,reaction time,bimanual motor skill, which is special for attention and memory of animal models(completed by specialist at the Institute of Chinese Academy of Medical Sciences & Peking Union Medical College).4.DTI scan protocolThe DTI scans were acquired 8 times on each animal for the longitudinal assessment of SIV infection. All DTI scans were performed using the 3T Siemens Tim TRIO whole-body magnetic resonance scanner (Siemens, Germany) employing the 32 channel head coil at Beijing You An Hospital, Capital Medical University. For every animal, structural and diffusion weighted MRI scans were acquired after the animal was anesthetized and placed in the fixed position.1)T1 weighted images were acquired with a turbo flash sequence. The parameters were repetition time/echo time (TR/TE)=1800/4.5 ms, FOV=160 mm×160 mm, data matrix=320×320, flip angle=9°, slice thickness=1 mm (voxel size=0.5×0.5×1.0 mm3). Scan time was 7 min 50 s.2) T2 weighted images parameters were repetition time/echo time(TR/TE)=3000/300ms,FOV= 160×160mm, data matrix= 130x130mm, slice thickness=1mm,The T2 weighted images scan was repeated 2 times and were averaged to improve the signal-to-noise ratio Scan time was 5min14s;3)the turbo spin echo(TSE) sequence in the axial orientation was used in the DTI scans. Diffusion tensor images (DTI) were obtained along 30 non collinear and noncoplanar directions with b=800 s/mm2 and a b=0 s/mm2 image. The parameters were TR/TE=4200/96 ms, FOV=160 mm×160 mm, data matrix=130×130, flip angle=90°,28 slices with 2mm slice thickness, and no gap (voxel size=1.2×1.2×2.0 mm3). DTI scan was repeated four times and were averaged to improve the signal/noise ratio. The scan time was 8 min 53 s. Shimming before scanning so as to reduce image distortion caused by uneven static magnetic field strength. 5.Preprocessing and data analysis1) Preprocessing:The FMRIB Software Library[1] was used for data preprocessing. Firstly, all raw DTI volumes were aligned to the b0 image by Eddy Correct Tool to be corrected,for the head motion and eddy current distortions. Then, the skull and non brain tissues were removed using Brain Extraction Tool [4]. Finally, the diffusion tensor, or ellipsoid, was model at each voxel by Dtifit Tool[5]. Based on the eigenvalues of the tensor imaging, FA, MD, AD, and RD values were obtained on a voxel by voxel basis.2) Tract-based spatial statistic:To identify all the major white matter tracts in brain, voxel-wise analysis on FA images was conducted using the TBSS toolbox of FSL [3]. TBSS modified for processing nonhuman primate (NHP) brains was employed in our study,following the previous published approaches [2]. Firstly, each FA was non linearly registered to the common space. For our search, T1 template from Wisconsin 112RM-SL rhesus atlas[6]was used as the target image. After registration, all the images were in the coordinate space of Saleem-Logothetis rhesus brain stereotaxic atlas[7] and in the resolution of 0.5×0.5×0.5 mm3. Secondly, the registered images were then averaged to create the mean FA image.The mean FA image was skeletonized by suppressing non max FA values perpendicular to the local tract structure,and then thresholded by 0.2 to create the mean FA skeleton. The threshold was employed to exclude small peripheral tracts and suppress the high inter subject variability. After being thresholded, the mean FA skeleton represented the major white matter tract common to all the subjects. Finally, all the registered FA images were projected to the mean FA skeleton by feeding the skeleton with FA values from the nearest relevant tract center. The skeletonized FA images were subsequently filled to statistical analysis. Statistical analysis To uncover the white impairments owing to SIV infection,repeated ANOVA was performed on the skeletonized FA images from the three DTI scans. ANOVA analysis was conducted with the Randomise tool in FSL[8].ANOVA results were thresholded at P<0.05(uncorrected).Anato micregions which were survived from the threshold identified and labeled according to structures defined in Saleem-Logothetis rhesus brain stereotaxic atlas [7]. Results were displayed in the T1 template from Wisconsin 112RM-SL macaque atlas and considered to be regions of interest (ROIs). The mean and max FA values,AD values,RD values, and MD value were acquired in each ROI for each subject.3) To access the longitudinal changes of the mean and max FA, AD, RD, and MD values in each ROI across the three time points, three groups were conducted with paired.t test analysis (baseline vs.12th week,12th week vs.24th week and baseline vs.24th week). CSF viral load、peripheral blood CD4+ cell counts and CD4+/CD8+ ratio were analyzed in the same manner. Furthermore,we applied Pearson correlation between the diffusion tensor imaging parameters (FA values,AD values, RD values, and MD values) and CSF viral load、peripheral blood CD4+ cell counts and CD4+/CD8+ ratio) to calculate whether the white matter impairments were correlated with the progression of HIV infection or Immune dysfunction.The investigation was limited to the data acquired on the base line and two other time points post virus inoculation. Paired t test and Pearson correlations of SPSS 17 were used in the our study. threshold was set at P<0.05.The relationship of the diffusion tensor images parameters with the peripheral blood plasma SIV RNA viral load and the peripheral blood white cell counts was analyzed in the same manner.Results1、White matter alterations:We found significant DTI parameters alterations in temporal region by repeated ANOVA.(TE, P<0.05, uncorrected,Fig.1).Result was displayed on the T1 template from Wisconsin 112RM-SL rhesus atlas, which was in the coordinate space of Saleem-Logothetis rhesus brain stereotaxic atlas. Significant longitudinal changes of the mean and max FA, AD values in TE were illustrated in Fig.1.2、Longitudinal alterations of CSF viral load、peripheral blood CD4+ cell counts and CD4+/CD8+ ratio were illustrated in Fig.3. CSF viral load increased significantly in the 24th week in comparison with the baseline values (Fig.3a). CD4+ T cell counts declined significantly in the 12th week in comparison with the baseline values (t=5.77,P=0.01,Fig.3b).CD4+/CD8+ ratio increased significantly in the 24th week in comparison with the values of 12th week (t=-6.761, P=0.021,Fig.3c).3n Longitudinal alterations of DTI parameters:Compared with the baseline values, the mean and max FA values in TE significantly decreased in the 12th weeks post inoculation (t=4.353, P=0.022 for the mean FA values; t=5.301, P=0.013 for the max FA values). In comparison with the values of the baseline, Max AD values in TE significantly increased in the 24th week (t=-0.547, P=0.038),mean AD values in TE significantly increased in the 24th week (Z=-2.201, P=0.028).No significant changes were observed in max and mean MD and RD values.4、Correlations between diffusion tensor imaging parameters and CSF viral load peripheral blood indices:For the data obtained post virus inoculation, both the max AD values in TE were significantly positively correlated with peripheral blood CD4+/CD8+ ratio (r=0.824π,P=0.023, R2=0.679, scatter diagrams were showed in Fig.4a). Both the max and mean AD values were positively correlated with the CSF viral load (r=0.867π,P=0.012, R2=0.706 for max and mean AD values,scatter diagrams were dispiayed in Fig.4b、4c).The research above indicated that the peripheral blood CD4+/CD8+ratio significantly increased, while the max AD values in TE increased significantly with the advantage of disease. So, the increased AD values in TE were associated with the elevated peripheral blood CD4+/CD8+ ratio. Meanwhile,the analysis above also indicate that AD values increased significantly with the elevated CSF viral load.No significant correlations were found between other diffusion tensor imaging parameters and peripheral blood indices.The peripheral blood plasma SIV RNA viral load and the peripheral blood white cell counts showed no significant association with the diffusion parameters.ConclusionOur study above indicated that SIV contributed to synapse impairment,further induced the neuropathological alterations,with decreased FA and increased AD,the latter was positively correlated with increased CD4/CD8 ratio and increased CSF Viral load,and decreased CD4+ cell counts.Meanwhile,while there is no significant changes with max and mean MD and RD.we also found that white matter alterations existed with the alleviated immune injury in brain,and that AD changes significantly correlated with CD4/CD8 ratio,while there is no significant correlations among other DTI parameters and CD4+ cell counts、CD4/CD8 ratio and CSF Viral load,Therefore we prove that AD is a relative sensitive imaging biomarker during the HIV infection process.By the way,a longitudinal study with great sample and short intervals is to be developed.