| Introduction:The diagnosis of diffuse axonal injury remains a quite important and difficult project in (DAI) in the research and practice of neuroscience and forensic pathology due to the short of objective and sensitive detection method, especially for the cases that die instantly after DAI. The used delinations of axonal injury were mainly achieved by HE staing, silver staining, and immunohistochemical staining, which were primarily concentrated on sub-cellular level to acquire the morphological changes and the expression of biomarkers following axonal injury. Importantly, the disadvantages involved in these methods should not be disgarded. For instance, it is quite difficult to expertise axonal injuries by silver staining in the acute phase, as well as the differentiation between the primary and secondary axotomy when verified by these biomarkers. Moreover, the feasibility of these biomarkers depends seriously on the the morphological characteristics of injured axons, in other word, they are typically feasible in the time widows of the appearances of morphological changes after axonal injury.In the current study, on the basis of the changes of myelin, axolemma, ulstructure in axons and intercellular space following axonal injury, diffusion characteristics of water in the injured axons, including diffusion directions and values, as well as the time imformation were acquired from DTI imaging. As a result, the onefold research on morphological changes of injured axons were abandoned, and the diagnosing approaches of DAI are introduced to the molecular level to prevent from the limitations of classical methods that depend primarily on the morphological changes. The combined using of diffusion parameters in DTI and immunohistochemical biomarkers is benefit for screening the most optimal biomarker or a diagnosing program using multiple biomarkers, making up the defects of the determination towards DAI using only single biomarker.Furthermore, on the basis of the investigation of DAI and DTI in animal, a comparative study of DTI and immnohistochemistry on prefixed traumatic brain injury (TBI) human brains were also performed to establish the feasibility of DTI in the diagnosing of axonal injuries in forensic pathological expertise. In brief, the study deserves to be a crossover investigation on forensic pathology, neuroscience and medical imaging.Objective:1. To observe the changing characteristics and temporal profiles of diffusion parameters of DTI in rats following DAI in the acute phase between3-72h.2. To view the features of β-APP immunostaining in rats following DAI. and the numbers of APP stained axons per square millimeter and the percentage of the positive area of axons in the ROIs by quantitative analysis.3. To observe the characteristics of NF-L immunostaining, and the mean density and sum density of NF-L immunostaining in the ROIs by quantitatively analysis.4. To establish the correlations between diffusion parameters in DTI and the indicators of β-APP or NF-L immunostaining. and to evaluate the feasibility of DTI in the diagnosing of DAI.5. To explore the characteristics and applicability of diffusion parameters of DTI in unfixed human brain following TBI, as well as the relationships between diffusion parameters and β-APP immunostaining. Material and Method:Meterial and machines60male Sprague-Dawley rats (the Animal Department of Tongji Medical College, Huazhong University of Science and Technology and the Experimental Animal Center of Wuhan University Medicine Division); Small-animal MR Imaging System (7T/20cm Bruker Biospec scanner, Germany); ImagePro Plus (Paravision5.0, Bruker Biospec, Germany); Signa EXCITE1.5T HDECHO MR Imaging System (General Electric Co., USA); Constant temperature freezing microtome (CM3050S. Leica, Germany); Nikon Eclipse90i microscope (Japan); Nikon image analysis software3.1(NIS Element BR3.1, Japan); Ultrapure Water Polishing System (AJY-660-U, Shanghai Aquapro co., China); Electronic analytical balance (Shanghai Minqiao precise Science Instrument co., Ltd, China); Constant temperature Magnetism Heating Mixer (Jintan instrument plant, China); High speed refrigerated centrifuge5702R (Eppendorf Corporate, Germany); Acidometer (PHS-3D, Shanghai REX Instrument Factory, China); Slide Drier (KPJ-1A, Tianjin Tianli Aviation Electro-Mechanical Co., Ltd., China); Monoclonal rabbit anti-beta-APP antibody (Y188, IgG, Abcam, UK); Monoclonal mouse anti-68-kD neurofilament (NF-L) antibody (NR4, IgM, Sigma, USA); Monoclonal mouse anti-beta-APP antibody (M066-3, MBL, IgG, Japan) Biotinylated secondary goat anti-rabbit IgG for β-APP, goat anti-mouse IgM for NF-L and goat anti-mouse IgG for β-APP (Boster Biological Technology, Ltd, China); VECTASTAIN ABC kit (Vector Loboratories, USA); Trihydroxymethyl aminomethane (Tris)(Dow chemical, USA); Paraformaldehyde (PFA)(Sigma, USA); Disodium hydrogen phosphate (NaaHPO4.12H2O), sodium dihydrogen phosphate (NaH2PO4.2H2O), chloral hydrate, glycerol,30%H2O2, TritonX-100(Sinopharm Chemical Reagent Co. Ltd., China); Ethanol. dimethylbenzene, ammonia, hydrochloric acid and neutral balsam (Reagent No.l Factory of Shanghai Chemical Reagent Co., Ltd., China).Preparation of the reagents1.10×TBS:60.5g Tris+90.0g NaCl+800ml double distilled water (DDW), followed by the regulation of pH to7.6by HC1, then the solution fixed to1000ml by DDW.2.4%paraformaldehyde (PFA):20g PFA+5.75g Na2HPO4.12H2O+500ml DDW, dissolved fully by stir and heat in a Magnetism Heating Mixer, followed by the add of1.31g NaH2PO4.H?O. After cooling, the solution reserved at4C in a refrigerator.3.0.9%saline:2.7g NaCl+300ml DDW, reserved at4℃in a refrigerator after the solution.4.10%chloral hydrate:10g chloral hydrate dissolved by DDW, then fixed to1000ml.5.30%sucrose TBS:60g sucrose dissolved by1×TBS, and fixed to200ml, thereafter2.0ml5%NaNj is added and reserved at4C in refrigerator.6. Antibody diluent (Supermix):0.25g gelatin (?)500μl TritoX100fixed to100ml by1x TBS. then dissolved after stirring and heating and reserved at4C in a refrigerator.7. Avidin-biotin incubation system:2.0μl avidin+2.0μl biotin+796.0μl Supermix (1:400).8. DAB developing agent:DAB50mg+1×TBS10ml, after the solution, the agent subpackaged to1.0ml in each eppendorf tube.9. DAB developing system:6.5ml1×TBS+0.5ml DAB+2.5μl30%H2O2.Methods1. The animals were randomly devided into a sham group and four injured groups (n=5for each). For injured groups, four time points were designated at3,12,24and72h after TBI. The Marmarou model was established in the rats according to the following procedures:General anesthesia was induced by intraperitoneal (i.p.) injection with10% chloral hydrate at0.3ml/100g body weight. Following a mid-line incision between coronal and lambdoid sutures, a steel disk10mm in diameter and3mm thick was adhered to the skull between the bregma and lambda suture. Then, the animal was placed on a foam bed with a prone position and fastened by a belt around the trunk, with the disk centered right under the lower end of a1-m plexiglass tube. A cylindrical steel weight of450g was allowed to fall through the tube at a designated height of1m to impact the disk. Immediately, the rat was removed and the disk was gently removed and the scalp sutured to prevent from the second impact from rebounce. In the sham group, the rats underwent the surgical procedure without impact.2. The animals were imaged by the small-animal MR Imaging System (7T/20cm Bruker Biospec scanner, Germany), reaching the end of post-injury time, either3,12,24, or72h。 And, the scanning area was set ranging from the genu of corpus callosum to the end of medulla oblongata.3. Using a region-of-interest-based approach, DTI parameters (λ1, λ2, λ3,FA, ADC) in the CC, LIC, RIC, LEC, REC, LPY and RPY were obtained double-blinded by outlining the FA images through twenty coronal slices for the image sets with the same anatomical landmark-based rules according to the Rat Brain in Stereotaxic Coordinates. And, the image software (Paravision5.0) binding feature allowed for the replication of the traced ROI to other images. The boundaries of CC and EC in rats were defined as followings:in the most rostral regions (Bregma+1.60mm to-0.92mm), the boundary was the lateral edge of the cingulum; in the regions (Bregma-0.92mm to-2.80mm), the boundary of CC was defined by a horizontal line extending laterally from the bottom of the fimbria until it intersected with external capsule; moving more caudally (Bregma-2.80mm to-5.30mm), a horizontal line extending laterally from the lateral-inferior edge of the hippocampus (HP) was employed. Bilateral IC (Bregma-0.26mm to-4.52mm) and PY (Bregma-8.3mm to-14.08mm) were also well outlined. The program then returned the mean signal intensity for each traced ROI on each slice. Overall, for each diffusion parameter in each animal,5 values of CC and EC,4values of IC and7values of PY were obtained, includingλ1, λ2and λ3, FA and ADC. Moreover, according to the value of λ1, λ2and λ3. RD and RA were calculated.4. Immediately after imaging, the rats were perfused transcardiacally with4%paraformaldehyde. Then, the brains were removed and immersed in the same fixative overnight (16-18h). The brainstem and cerebrum were harvested using two transverse cuts: the first at the level of the inferior colliculus within the midbrain region, and the second2mm caudal to the pyramidal decussation within the medulla oblongata. The brainstem was then divided equally by a sagittal cut to include the basal interpeduncular regions of the brain, pons, and pyramids inclusive of the left and right pyramidal tract. The cerebral blocks were trimmed to contain complete corpus callosum. The blocks were then immersed in30%sucrose for further tissue processing. To strictly distinguish the side of slices, serial coronal sections of the cerebrum, and serial sagittal sections from the midline to lateral of bilateral brain stem blocks were cut on a freezing microtome with the thickness of30μm, and mounted on gelatin covered microscope slides. Every sixth section was used for histology. For each block of unilateral brain stem,36sections were obtained as the width of unilateral PY is about1.2mm.5.(3-APP and NF-L immunostaining were performed to observe the characteristics of p-APP and NF-L in ea.-h ROI. For each section of each animal, the ROI was outlined at a low magnification (40x) and followed by systematic counts of individual injured axons at a high magnification (200x), which were randomly chosen by the NIS-Elements BR software over the entire sections of ROIs. Then, the numbers of APP stained axons per square mm and percentage of the positive area of axons in the ROIs were obtained by dividing the counts or the area of injured axons by the area of each sampled region generated by the software. Also, the mean density and sum density of NF-L immunostaining were employed to evaluate the axonal damage.6. Normalized AD, FA and RA were correlated with the numbers of APP stained axons per square millimeter or mean density of NF-L immunostaining in the ROIs to establish the relationships between between diffusion paramaters and immunostaining results, as well as to screen the most optimal biomarkers for diagnosing DAI.7. Unfixed human brains following TBI were collected in the Tongji Center for Medicolegal Expertise in Hubei (TCMEH), and scanned with DTI to acquire the ADC and FA images and values in the ROIs of CC, IC and CSTs. Moreover, a comparative study between DTI and β-APP immunostaining was also performed in the brains to explore the feasibility of DTI in diagnosing TAI in human.Stastatical analysisAll data were analyzed using IBM SPSS Statistics17.0. There were no prespecified hypotheses about the histological or DTI parameters between the left and right side of IC, EC or PY at each time point or that between invitro control, injured group and vivo control, so a two-tailed/test was used. The threshold for statistical significance was set to P<0.05without correction for multiple comparisons. For the comparisons of immunostaining profiles or DTI diffusion parameters between different groups, a one-way ANOVA was used followed by a Fisher post-hoc test to correct for multiple comparisons.Results:1. The CC and PY in the control images had high FA, as expected for a highly organized tissue. Acutely after injury, there were dr matic reductions of signals in FA in CC, LPY and RPY in the post-TBI images. The gradient of reduced signal of FA was also visible in these images. For AD and ADC images, no apparent grayscale changes were seen in PY, and decreased signal intensities were only seen in CC at3h, while the remaining imaging modalities showed no visible grayscale changes. In addition, no visible grayscale changes were seen in the three parameters in LEC. REC. LIC and RIC. As to the images ofλ2and λ3, no visible changes were noted in the ROIs.2. A quantitative analysis of DTI parameters within the ROIs was performed. The AD, FA and RA in each ROI were significantly reduced after injury compared with control. Multiple comparisons showed that they also decreased consistently over time in each ROI (Oneway ANOVA, P<0.05). Namely, AD, FA and RA were consistently lower as the increasing survival time, and the injured groups could be reliably separated from each other using the two parameters. There was consistently decreased ADC over time in bilateral IC and EC in injured groups, whereas in CC and bilateral PY, ADC decreased congruously before24h, but there was no difference between24and72h. For RD, it did not change consistently over time. In CC, a slight increased RD was found since24h, but it was only slightly elevated at72h in bilateral IC, EC and PY. Of the diffusion parameters evaluated, only AD, FA and RA permitted a complete separation between injured and uninjured rats. In CC,λ2increased slightly since24h, while for λ3, no significant differences were found between different groups. In LEC,λ2rised slightly at72h, whileλ3increased since24h. In REC, bilateral IC and PY, both of λ2and λ3were elevated slightly at72h. No significant differences were found between the left and right side of IC, EC, or PY within each time point (two-tailed t test, P>0.05).3. No APP stained axons were found in each ROI of the control animals, as well as in bilateral IC and EC in the injured animals. Spatial differences and temporal gradient of axonal injury were observed in the injured groups. APP stainings were substantial obvious in PY, the posterior body and splenium of CC in the injured animals. Oblong and round APP stained varicosities were noticed under high magnification. At3h after injury, APP antibody labeled axonal deposits mainly presented as dot-like profiles. At12-72h post-injury, the immunoreactive profiles including axonal swellings and axonal retraction balls (ARBs) markedly increased in size and number in the same regions. The numbers of APP stained axons per square millimeter and the percentage of the positive area of axons in these ROIs increased since3h, increased congruously postinjury, reaching peak at72h (one way ANOVA, P<0.05). No differences were found in the numbers of APP stained axons per square millimeter and the percentage of the positive area of axons between LPY and RPY at each time point (two-tailed t test P>0.05). 4. In controls, there was no NF-L staining in CC, EC and IC with the exception of PY, which presented weak positive staining. In the injured groups, both diffuse staining and focal staining were present in the ROIs, with increasing immunostaining density since3h and increased density between12and72h in each ROI. Moreover, several axonal swellings were also seen in the ROIs since12h post-TBI, including axonal varicosities and ARBs. The mean density and sum density of the NF-L staining increased congruously after injury in each ROI by quantitative analysis, with a sharp increase from3to24h and a slight increase at72h (ANOVA, P<0.05). There were no differences observed in the mean density and sum density of NF-L immunostaining between the left and right EC, IC or PY (two-tailed(?)test, P>0.05).5. The changes in AD, FA and RA were correlated with the numbers of APP immunostained axons in CC and bilateral PY, and the mean density of NF-L staining in the ROIs. In CC, the coefficient of determination (R2) between AD, FA or RA and the numbers of APP immunostained axons was0.735,0.762and0.742, respectively. In LPY, the R" between AD, FA or RA and the numbers of APP immunostained axons was0.815.0.833and0.828, respectively. In RPY, the corresponding R2was0.857,0.877and0.873, respectively. Moreover, significant correlations were found between the changes in AD, FA or RA and the mean density of NF-L in each ROI. In CC, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.838,0.847and0.837, respectively. In LIC, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.787,0.707and0.653, respectively. In RIC, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.687,0.627and0.654, respectively. In LEC, the R" of between AD, FA or RA and the mean density of NF-L immunostaining was0.838,0.802and0.846, respectively. In REC, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.902,0.849and0.885, respectively. In LPY, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.743,0.738and0.738, respectively. In RPY, the R2of between AD, FA or RA and the mean density of NF-L immunostaining was0.844,0.849and0.851, respectively.6. No significant differences were found in FA values of the regions of interest between vivo and invitro control group, while decreased ADC was found between invitro control or injured group and vivo control. Three were no differences presented ADC between invitro control and injured group, moreover, with the exception of the genus of corpus callosum, no differences were observed in FA in other areas. No significant correlation was observed with the comparative study of DTI and β-APP immunostaining. However, positive β-APP immunostaining was presented in the side of white matter with lower FA in the symmetrical white matter.Conclusions:1. Reliablely, DAI could be established in rats in the Marmarou model. The defects of biomechanics involved in this model could be prevented with well controlled experimental conditions.2. NF-L and β-APP immunostaining are feasible for early diagnosis of axonal injury, and the immunostaining characteristics may well indicate the time imformation of axonal injuries in the acute phase.3. The changes of diffusion parameters in DTI correspond well with β-APP and NF-L immunostaining. It is suggested that diffusion parameters could be served as biomarkers for demonstrating DAI, as well as provide the temporal profiles of axonal injuries.4. DTI is conductive to demonstrate axonal injury in invitro human brain following TBI. Keywords:Diffuse axonal injury; Diffusion tensor imaging; Traumatic brain injury; Diffusion parameter;β-amyloid precursor protein; Neurofilament light chain; Biomarkers...
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