| BackgroundPeripheral nerve injury(PNI) is a common complication of severe trauma, the most common types including crush injury and strech injury,et al. Severity of the injury is an important factor for clinicians to determine what kind of therapy to apply. Conservative approach is sufficient for mild nerve injury, but in severe injury, surgical repair is often needed to ensure the fuctional recovery of the nerve. To judge the severity of PNI, observation of nerve regeneration for several weeks to months is needed, and many patients lost the best opportunity of therapy and result in irreversible disability. Therefore, an auxiliary method that can accurately detect the severity and regeration of the injuried nerve is necessary to help clinicians to apply proper therapy for PNI.Electrophysiological method is mainly used to detect the degree of PNI in addition to physical examination in clinical practice nowadays, which is known as gold standard to evaluate the function of peripheral nerve. However, Electrophysiological method is largely dependent on the experience of the operator, lack of sensitivity and specificity and can not obtain the anatomical information which is very important for preoperative evaluation and intraoperative localization of PNI.Although some research showed that conventional MRI is useful to reveal abnormality of signal and time of relaxation of PNI, but it lacks of specificity in detection of nerve regeneration and can not display the running course of nerves with complex anatomy. It is generally accepted that diffusion tensor MR imaging (DTI) can display nerve fibers and diagnose diseases in central nervers system, some researches also showed it is helpful in PNI. Nevertheless, systemic research of in vivo DTI evaluating PNI using conventional MR scanner has never been reported. It is reported that manganese enhanced magnetic resonance imaging(MEMRI) can accurately reveal pathways in the brain, but research of MEMRI evaluating PNI is very few.The purpose of this study lies in:①To explore the feasibility of in vivo diffusion tensor fiber tractography of rabbit sciatic nerve at 1.5 T MR. (2)To establish animal model of peripheral nerve crush injury and investigate the value of DTI in evaluation of peripheral nerve crush injury and regeneration by comparing with histopathology and nerve function assessment.③To investigate the value of MEMRI in evaluation of peripheral nerve crush injury and regeneration by comparing with histopathology and nerve function assessment. This study aims at providing a reliable imaging method to accurately evaluate PNI and direct therapy.Part One:Study on Optimal Parameter and Feasibility of Diffusion Tensor Tractography of Rabbit Sciatic NerveObjectiveTo determine the effect of b value on the image quality of DTI tractography by comparing the image quality under different b values and optimal b value of diffusion tensor tractography of rabbit sciatic nerve at 1.5 T MR. To determine the effect of b value on measurement of the derived DTI paramertes; To investigate the feasibility of diffusion tensor tractography of rabbit sciatic nerve at 1.5 T MR by comparing reconstructed sciatic nerve fiber with gross anatomy and conventional MR images of sciatic nerve.1 Materials and methods1.1 Subjects10 healthy New Zealand white rabbits weighting 2.0-3.OKg provided by Guangzhou traditional Chinese Medicine University1.2 Instruments, equipment and drugsPhilips Achieva 1.5T Nova Dual MR scannerDedicated rabbit coil manufactured by Chenguang medical technology companyLtd. Of Shanghai.Philips EWS (Extended Workspace) v2.6 post processing workstation withFiberTrak software package for fiber tracking.3% sodium pentobarbital and Su-Mian-XinⅡ1.3 MR scan1.3.1 Animal preparation:New Zealand rabbits underwent MR scan after anesthetized by 3% sodium pentobarbital (30mg/kg) intravenously and 0.2-0.3ml Su-Mian-XinⅡintramuscularly1.3.2 MR scan method and parametersMR sequence including conventional T2WI,T1WI and DTI.TSE sequence were used for T2WI scan, following parameters were used:TR, 2500ms; TE,120ms; TSE factor,25; slice thickness,2mm; gap, 1mm; FOV, 120mm; matrix:344×344, in plane pixel size,0.35×0.35mm; NSA,4.TSE sequence were used for T1WI scan:TR,572ms; TE,17ms; TSE factor,3; SPIR were used for fat suppression, other parameters were the same as T2WI.Single shot SE-EPI sequence and SENSE parallel scanning technique were used for DTI scan. Six different b values were used:400,600,800,1000,1200和1400 s/mm2, scan parameters were as follow:TR,1550-3650ms; TE,55,59,63,66,68 and 71ms respectively; Direction, range and other parameters were same under 6 b values:slice thickness,1.6 mm; gap,0mm; FOV,130mm; matrix,80×80; in plane pixel size,1.6×1.6; NSA,2; MPG:32.1.4 Data processingPhilips EWS (Extended Workspace) v2.6 post processing workstation with FiberTrak software package were used. Multiple ROI method was used:2 ROIs of 5mm apart were drawn in freehand mode at the level of greater trochanter of femur, including the outer diameter of sciatic nerve. Fibers running through the 2 ROIs were computed automatically by the software. The lower limit of FA value was defined as 0.4, maximum angle change was 27 degree, minimum fiber length was 10mm, then the fibers was fused with the T1 WI or T2WI images.1.5 Data analysis:1.5.1 Number, mean length, FA, ADC and three Eigen value (λ1,λ2,λ3) of the fibers were computed by the software automatically1.5.2 Measurement and computation of signal to noise ratio (SNR) DWI images were produce by the software, a ROI (5 pixels) was drawn on the sciatic nerve to measure the signal intensity of the nerve (SIn); ROI of 20 pixels was drawn on the air region that lcm outside the body margin of rabbit limb to measure the standard deviation of background noise (SDbg); SNR was computed according to the following formula:SNR=SIn/SDbg。1.6 subjective assessment of image qualityAssessment of the quality of fiber tractography under 6 different b values was conducted by 2 radioglogists with more than10 years experience of MR diagnosis. Blind method was adopted. The principle to consider including:fiber lenth, diameter, homogeneity and smoothnesss of fiber margin. Sorting of the image quality was made afterwards(l is defined as the best quality and 6 the worst)Four rabbits were sacrificed; the sciatic nerve was dissect and compared with the reconstructed fibers of the best quality to evaluate the consistency between the tractography and the gross anatomy. Contralateral legs were frozen and transversely desected at the level of great trochanter of femur. Reconstructed fibers fused with the T2WI and T1WI images were compared with sectional anaotomy to test whether the position of fibers was in accordance.1.7 Statistical analysisSPSS 13.0 software was used. All data was presented as the mean±standard deviation, Test of normality was done in advance, One-way Anova was used to compare means of number, mean length, FA, ADC and three Eigen value (λ1,λ2,λ3) of the fibers under different b values if the data is of normal distribution. Fisher testwas used if variance was homogeneous, otherwise Welch test was used; If anova was significant, multiple comparison was conducted, Bonferroni test was used when the variance was homogeneous, Dunnett's T3 test was used otherwise. Multiple related sample nonparametic test was used in the comparison of scores of subjective quality assessment, and kappa consistency test was used to check interobserver agreement on tractographic image. Correlation between SNR, FA, ADC,λ1,λ2,λ3 and b value was conducted using the Spearman correlation test. P<0.05 was defined as statistical significant.2 Result2.1 Relation between SNR of DWI image and b valueSNR decreased with the rising of b value, SNR was negtively correlated with the b value (P=0.000,r=-0.589)2.2 Number and mean length of fibers under different b value Number of fibers of b=1000 s/mm2 was more than that of other b values, anova was significant (F=21.641, P=0.000); Multiple comparison showed significant difference between number of fibers of b= 1000 s/mm2 group and that of b=400,600, 1200,1400 s/mm2 group (P<0.001), but no significant difference between b=800 s/mm2 group.Mean length of fibers of b=1000 s/mm2 was longer than that of other b values, anova is significant (F=19.736, P=0.000). Multiple comparison showed significant difference between mean length of b=1000 s/mm2 group and that of b=400,600,1200,1400 s/mm2 group (P=0.000,0.008,0.013,0.002, respectively), but no significant difference between b=800 s/mm2 group.2.3 Relationship between FA, ADC,λ1,λ2,λ3 and b valueMeans of FA in all of the groups was between 0.51±0.02 and 0.53±0.03, there was no significant difference between FAs of different groups, and there was no correlation between FA and b value.ADC,λ1,λ2.λ3 decreased with the rising of b value. Negative correlation between ADC,λ1,λ2,λ3 and b value were detected by Spearman correlation test (P=0.000, r=-0.787,-0.898,-0.829,-0.559, respectively).2.4 Subjective assessment of image qualityImage quality ranking of b=1000 s/mm2 was always in the first 3 position with the lowest score(best quality), and score of b=400 s/mm2 group was the highest(worst quality)(X2=33.714,P=0.000). Interobserver agreement on tractographic image quality ranking was good (k=0.76, P=0.000)Reconstructed fiber of b=1000 s/mm group was compared with the gross anatomy and transactional anatomy, comparison showed that the running course and location of the tractographic fiber is consistant with that of sciatic nerve anatomy, the two main branch within the sciatic nerve-tibial nerve and peroneal nerve was clearly revealed. Conclusion1. Different b value had significant effect on the SNR, number and length of fibers of peripheral nerve, thereby effect the image quality of DTI tractography..2. Different b values had significant effect on ADC and eigenvalues measurement, but had no effect on FA measurement.3. The optimal b value for DTI and fiber tractography of rabbit sciatic nerve at 1.5 T was 1000 s/mm2.4. In vivo diffusion tensor fiber tractography of rabbit sciatic nerve at 1.5 T MR was feasible with parameters we used.Part Two:Experimental Study on MR Diffusion Tensor Tractography in Evaluating Sciatic Nerve Crush Injury of RabbitObjectiveTo investigate the FA, ADC,λ//andλ(?) of distal and proximal to the crush injury changes with the different time course after injury. Comparing with histopathology and functional assessment, to determine the relationship between FA, ADC,λ//,λ(?) of distal part crushed sciatic nerve and histopathology and nerve function.1 Materials and methods1.1 Subjects36 New Zealand white rabbits weighting 2.0-3.0Kg provided by Guangzhou traditional Chinese Medicine University were used.36 rabbits were randomly divided into normal control group(4 rabbits) and crush injury group(32 rabbits), the crush injury group(32 rabbits) were randomly divided into 8 groups(24hours,4 days, 8 days,2 weeks,4 weeks,6 weeks,8weeks and 10weeks) according to the time course after crush injury. 1.2 Instruments, equipment and drugsMR scanner, coil, and anesthetic agent were the same as part I.Sterile operating instrument setA 16cm needle holder1.3 Establishment of rabbit sciatic nerve crush injury model.Rabbit was anesthetized with 3% sodium pentobarbital (30rng/kg) intravenously and 0.2-0.3ml Su-Mian-Xin II intramuscularly, sciatic nerve of right leg was exposed under sterile operation, sciatic nerve was crushed using a 16cm needle forceps at the level 1cm distal to the greater trochanter of femur, the forceps was locked to the first point of its ratchets and retained for 5 minutes before releasing. An epineurial suture was placed right above the crush site to exactly localize the position of the crush.Sham operation was performed on the contralateral leg.1.4 MR scanMR scan were performed 24 hours,4 days,8 days,2 weeks,4 weeks,6 weeks, 8weeks and 10weeks after injury,as well as the normal control group.Animal preparation were the same as part I.MR scan sequence and parameters were the same as part I except for some parameters of DTI scan:b=1000 s/mm2; TR/TE:8184/66 ms; slices:35; scan time:9 minute and 46 seconds.1.5 Post processing of Data.Philips EWS (Extended Workspace) v2.6 post processing workstation with FiberTrak software package were used. Multiple ROI method was used:2 ROIs of 10mm apart were drawn in freehand mode at the level of greater trochanter of femur(proximal to the injury site), including the outer diameter of sciatic nerve. Fibers running through the 2 ROIs were computed automatically by the software. The lower limit of FA value was defined as 0.35, maximum angle change was 27 degree, minimum fiber length was 30mm, then the fibers was fused with the T1WI or T2WI images.1.6 Data measurementThree ROIs were drawn in freehand mode on the color coded FA map generated by the software, two of the ROIs was drawn on the sciatic nerve of the injuried side,one of which is lcm distal and the other lcm proximal to the injury site; the third ROI was placed on the sham operated sciatic nerve on the site corresponding to the distal ROI of the injuried side.FA, ADC,and the three eigenvalues(λ1,λ2,λ3) were computed automatically by the software,λ(?) andλ//were computed as following formula:λ(?)=(λ2+λ3)/2,λ//=λ11.7 Histopathological examinationAnimals were sacrificed after MR scan, and sciatic nerves of the injuried side that 1cm distal to the injured site were harvested. One part of the nerve were fixed in 4% paraformaldehyde, dehydrated, osmificated, and embedded in plastic resin. Semithin sections were then made and stained with uranyl acetate and lead citrate for transmission electron microscopic examination (Hitachi H7500). Another part of the sample were fixed in 10% paraformaldehyde and sectioned longitudinally at a 2-mm thickness. The sections were stained with standard hematoxylineosin staining for light microscopic examination.1.8 Functional assessmentFunctional assessment was performed befor every MR scan.Toe-spreading reflex:grade 1,barely visible spreading of toes; grade 2, readily discernible though slight spreading of the toes; grade 3, unequivocal spreading of toes (though less forceful than normal); and grade 4, full spreading of the toes equal to normal.Modified Tarlov score:grade 0, complete paraplegia of hind limbs; grade 1, hind limbs barely respond to hind limb pinch (barely detectable movement); grade 2, spontaneous movement at all hind limb joints but no resistance when flexing the foot; grade 3, obvious resistance when flexing the foot with abnormal gait; and grade 4, walks with normal gait.1-4 marks were recorded for one rabbit of corresponding toe spreading reflex grade;0-4 marks were recorded for one rabbit of corresponding Tarlov score grade. Full marks for a single rabbit is 8, and for a group is 32 marks.1.9 Statistical analysisSPSS13.0 software was used. All data was presented as the mean±standard deviation, One-way Anova was used to compare FA, ADC,λ//andλ(?) of sciatic nerve on the eight time point after injury as well as the normal control group. Fisher test was used if variance was homogeneous, otherwise Welch test is used; If anova was significant, multiple comparison was conducted, LSD test is used when the variance is homogeneous, Dunnett's T3 test was used otherwise. P<0.05 is defined as statistical significant.2 Results2.1 FA, ADC,λ//andλ(?) changes of sciatic nerve after crush injury2.1.1 FA, ADC,λ,//andλ(?) changes of distal portion of injuried sciatic nerve.One-way Anova showed that FA value of the distal portion injuried sciatic nerve at different time point is different significantly. (F=47.283, P=0.000), LSD multiple comparison test showed:FA of normal control group is significantly different from that of all the other group (P<0.01);Other significant differences of FA between consecutive groups were:24 hours group and 4 days group(P=0.000),4 weeks group and 6 weeks group(P=0.000).There was no significant difference between ADC of every time point of distal portion injuried sciatic nerve.There was no significant difference betweenλ//of every time point of distal portion injuried sciatic nerve. There was significant difference betweenλ(?) of time points of distal portion injuried sciatic nerve (F=11.847, P=0.000). LSD multiple comparison test showed:λ(?) of normal control group is significantly different from that of 4 days,8 days,2 weeks,4 weeks,6 weeks group (P<0.05), but not significantly different from that of 24 hours,8weeks and 10 weeks group, Other significant differences ofλ(?) between consecutive groups were:24 hours group and 4 days group (P=0.000),4 weeks group and 6 weeks group(P=0.007)2.1.2 FA, ADC,λ//andλ(?) changes of proximal portion of the injuried injuried sciatic nerve.One-way Anova showed that There was no significant difference between FA,ADCλ//andλ(?) of every time point of proximal portion injuried sciatic nerve.2.1.3 FA, ADC,λ//andλ(?) changes of sham operated sciatic nerve.One-way Anova showed that There was no significant difference between FA,ADC,λ//andλ(?) of every time point of sham operated sciatic nerve.2.2 Histopathological changes of distal portion of the injuried sciatic nerve.Normal sciatic nerve showed that nerve fibers lines up in order, myelin sheath and axon was clearly seen.Swelling of the axon an myelin sheath was seen 24 hours after injury; axon began degeneration 4 days after injury, and fragment of axons can be seen.Proliferation of Schwann cells was not obvious; large amounts of axons disappeared and myelin sheath generally disintegrate 2-4 weeks after injury,and Schwann cells began to proliferate generally, inflammatory cell infiltration is obvious. Small amount of regenerated axons were detected until 6 weeks after injury, which wass very thin and amyelinated. Large amount of regenerated axons were revealed 8 weeks after injury.10 weeks after injury, most regenerated axons-were myelinated.2.3 Functional recovery of sciatic nerve after injuryToe-spreading reflex disappered 24 hours after injury, but the injuried leg respond to pinch but have no tension (Tarlov 2); Slight tension of the injured leg can be felt in individual animals 2 weeks after injury (Tarlov 3),but Toe-spreading reflex was still undetectable.Indivadule animals can be seen slight spreading of injuried side toes, strong force of the injuried leg can be detected in most animals; 6 weeks after injury, most animals revealed slight spreading and strong force of the injured leg, but the gait was still abnormal; 10 weeks after injury, some animals showed normal toe-spreading reflex, almost all the animals had normal gait.Sham-operated legs was strong and moved freely after injury, and the fur of the legs was smooth and intacted.The toe spreading reflex was normal.Conclusion:1. FA andλ(?) of distal portion of injuried sciatic nerve changed significantly after injury, and the change were in good correlation with the histopathology and fuctional recovery. FA andλ(?) distal portion of injuried sciatic nerve can reflect Wallerian degeneration and regeneration after crush injury of sciatic nerve.2. ADC andλ,//of distal portion of injuried sciatic nerve had no significant change after injury.3. FA, ADC,λ,//andλ(?). of proximal portion of the injuried injuried sciatic nerve had no significant change after injury.Part Three:Preliminary Study of Manganese Enhanced MRI in Evaluating Sciatic Nerve Crush Injury of RabbitObjectiveTo investigate the value of MEMRI in evaluation of nerve regenertation after sciatic nerve crush infury of rabbit by comparing with histopathology and functional assessment, crush injury; To explore the transporting pathway of Mn2+ in MEMRI of peripheral nerve.1 Materials and methods1.1 Subjects24 New Zealand white rabbits weighting 2.0-3.OKg provided by Guangzhou Traditional Chinese Medicine University were used.24 rabbits were randomly divided into normal group(4 rabbits) and crush injury group(20 rabbits), the crush injury group were randomly divided into 5 groups(24hours,2 weeks,4 weeks,6 weeks and 10weeks) according to the recovery time after injury. There were 4 rabbits in each group.1.2 Instruments, equipment and drugsMR scanner, coil, and anesthetic agent were the same as part I. Sterile operating instrument set for crush injury modle was the same as part II. AR class MnCl2·4H2O crystal (molecular weight 197.9g/mol; purity>99%) polyethylene glycol(PEG) 15401.3 Establishment of rabbit sciatic nerve crush injury modelThe procedure was the same as part II.1.4 Preparation of MnCl2 solution50% PEG 1540 solution was prepared by PEG 1540 solid melted and dissolved in 0.9% NaCl solution. MnCl2·4H2O crystal was dissolved in 50% PEG 1540 to form 400mM MnCl2 solution.1.5 MnCl2 injectionAfter corresponding recovery time, rabbits were anesthetized again in the same manner as the initial operation. The sciatic nerve of the right leg was exposed, then 30 and 20ul of 400 mM MnCl2 were injected into the two major branches of the sciatic nerve, the tibial nerve and peroneal nerves, respectively. The injection site was 2cm distal to the injury site. Sham operation were performed on the contralateral legs. 1.6 MR scanT1WI and T2WI sequence were scanned after 24 hours after MnCl2 injection.The technique and parameters used were the same as part II 1.7 Data measurement ROIs were placed on three different portion of injuried sciatic nerve proximal to injury site:Greater trochanter of femur, 1cm proximal to Greater trochanter of femur and ischial tuberosity, which were defined as distal, middle and proximal segment respectively in this study. Signal intensity of the three ROIs on the sciatic nerve were recorded as S and the signal intensity of the contralateral sciatic nerve was recorded as Sc, the signal intensity of muscle medial to the proximal femur (Sm) was recorded as a reference. Relative signal intensity of injuried sciatic nerve was computed as AS, which was calculated according to the formula:ΔS=(S-Sc)/Sm.1.8 Histopathologic examinationThe same as partⅡ.1.9 Functional assessmentThe same as partⅡ.1.10 Statistical analysisSPSS 13.0 software was used. All data was presented as the mean±standard deviation, One-way Anova and Two-way Avova were used respectively to compare AS of different recovery time after injury as well as the normal control group andAS between three different portion of injuried sciatic nerve. Fisher test was used if variance was homogeneous, otherwise Welch test was used; If anova was significant, multiple comparison was conducted, LSD test was used when the variance is homogeneous, Dunnett's T3 test was used otherwise. If anova was not significant, multiple comparison would not be conducted. P<0.05was defined as statistical significant.2 Results2.1 MEMRI manifestation of normal group and injuried groups of sciatic nerveRemarkable enhancement of the sciatic nerve on the T1WI images was revealed in the normal group after MnCl2 injection, the extent of enhancement was generally beyond the vertical portion of the sciatic nerve, and the enhancement decreased gradually from distal to proximal portion. There was no enhancement of the nerve in most animals of the 24 hours group after injury.2-4 weeks after injury, the sciatic nerve proximal to injury site revealed slight and short enhancement that decreased gradually from distl to proximal part.6 and 10 weeks after injury, the proximal part of the injuried sciatic nerve showed remarkable enhancement,but not as strong as normal group, the enhancement was also decreased gradually from distal to proximal portion.2.2 Relative signal intensity (ΔS) changes of sciatic nerve after injuryOne-way Anova showed that there were significant difference betweenAS of injuried sciatic at different time point after injury at proximal, middle and distal portion (F=5.819, P=0.002; F=26.987, P=0.000; F=45.406, P=0.000)In the proximal portion, multiple comparison showed thatΔS of normal control group was significantly different from that of 24 hours,2 weeks,4 weeks and 6 weeks group (P<0.05), there was no significant difference betweenAS of normal group and 10 weeks group. There was no significant differences ofΔS between other consecutive groups.In the middle portion, multiple comparison showed that AS of normal control group was significantly different from that of all the other groups (P<0.001),there was no significant difference betweenAS of 24 hours,2 weeks and 4 weeks groups.ΔS of 6 weeks and 10 weeks group was significantly different from that of 24 hours, 2 weeks and 4 weeks group(P<0.001,P=0.000). There was no significant differences ofΔS between 6 weeks and 10 weeks group.In the distal portion, multiple comparison showed thatΔS of normal control group is significantly different from that of all the other groups (P=0.000), there was no significant difference betweenAS of 24 hours,2 weeks and 4 weeks groups. AS of 6 weeks and 10 weeks group was significantly different from that of 24 hours,2 weeks and 4 weeks group (P≤0.011,P=0.000). There was no significant differences ofΔS between 6 weeks and 10 weeks group. 2.3 Two-way Anova showed that there was significant difference between the three portion of injuried sciatic nerve at different recovery time point (F=90.928, P=0.000); LSD multiple comparision showed:There were significant differences betweenAS of proxilmal and distal portion (P=0.000), proxilmal and middle portion (P=0.000) as well as middle portion and distal portion (P=0.001)2.4 Histopathological changes of distal portion of the injuried sciatic nerve. The same as partⅡ2.5 Functional recovery of sciatic nerve after injury The same as partⅡ2.6 Correlation between MEMRI relative signal intensity of sciatic nerve proximal to injury site and functional assessment.Speraman correlation test showed that the proximal,middle and distal portion of scitic nerve promximal to injuried site was significantly correlated with functional scores(r=0.670,P=0.000;r=0.888,P=0.000,r=0.899(P=0.000).Conclusion1. The MEMRI signal intensity of sciatic nerve crush injury was in good correlation with histopathology and nerve function recovery, MEMRI was able to reflect the regeneration of crushed sciatic nerve.2. Enhancement of sciatic nerve decreased gradually from distal to proximal portion, infered that there was passive diffusion factor in the retrograde Mn2+ transportation.
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