Experimental Study On Multimodal Imaging Of Mild Craniocerebral Blast Injury In Rats

Background and purpose:Due to the frequent use of improvised explosive devices and high-speed and high-energy weapons,both military and police may suffer from traumatic brain injury（TBI）caused by high-speed impact in operations such as riot control,anti-terrorism,and war.Craniocerebral penetrating injuries caused by high-speed explosive fragments and projectiles have serious damage,high casualty rate,and are easy to identify;however,blast-induced mild Traumatic brain injury（bmTBI）islatent and difficult to identify.According to the U.S.Defense and Veterans Brain Injury Center,350,000 soldiers were diagnosed with TBI,and82.3%were mild traumatic brain injury（m TBI）.m TBI has become a characteristic trauma of modern warfare.The mechanism of craniocerebral injury under the action of high-speed impact is rather special.Mild craniocerebral impact injury does not necessarily show neurological symptoms immediately after injury or the symptoms are mild,and it is very likely that the original mild injury will migrate and cause chronic neuropsychiatric disorders in the later stage.The specific diagnosis and identification method is still unclear.Clinically,routine imaging examinations of m TBI patients often showunobvious positive manifestations,and rely on scale scoring.Early diagnosis is difficult.Faced with this practical problem,it is extremely important to explore and apply new imaging techniques to establish an objective,accurate and quantitative method for bmTBI diagnosis.In this study,the bmTBI rat model was first established by using BST-I shock tube.Then,we quantitatively evaluated the rat model of bmTBI by combining¹⁸F-FDG PET and various magnetic resonance imaging（MRI）functional imaging sequences.Changes in brain metabolism,vascular perfusion,structure and function characteristics provide objective scientific basis for early and accurate clinical diagnosis and treatment plan.Materials and Methods:Part I:Establishment and evaluation of an animal model of blast-induced mild Traumatic brain injury（1）Twenty adult male Sprague-Dawley rats,weighing 200-225 g,were randomly divided into two groups,b TBI group（n=10）and sham operation group（n=10）.BST-I type biological shock tube was used for shock injury,and the sham operation group only received intraperitoneal anesthesia.（2）The ten-point Modified Neurological Severity Score（m NSS）method was used to evaluate the neurological damage in rats.（3）Magnetic resonance T1-weighted imaging（T1WI）,T2-weighted imaging（T2WI）and paramagnetic weighted imaging（SWI）were used to observe whether there are signs of intracranial hemorrhage and skull fracture.（4）The water content of rat brain tissue samples was measured,and HE staining was performed after sectioning.（5）One-way ANOVA was used to evaluate the difference between m NSS and brain water content of two groups of rats.Part II:¹⁸F-FDG PET combined with MRS to assess early metabolic changes after bmTBI in rats（1）Forty adult male Sprague-Dawley rats,weighing 200-225 g,were randomly divided into two groups,bmTBI group（n=20）and sham operation group（n=20）.BST-I type biological shock tube was used for shock injury,and the sham operation group only received intraperitoneal anesthesia.（2）PET/CT imaging experiments were performed before blasting and 1-3 hours,1 day,and 7 days after blasting in m TBI group and sham operation group.¹⁸F-FDG was injected into the tail vein of rats after fasting for 4-6 hours.40 min after ingestion,static PET scans were performed using a small animal PET/CT scanner,and¹⁸F-FDG PET data were processed and analyzed using PMOD software version 3.6.（3）The m TBI group and the sham-operated group were subjected to MRI imaging experiments before blasting and 1-3 hours,1 day,and 7 days after blasting.Scanning sequences include:T1WI,T2WI,Magnetic resonance spectroscopy（MRS）,and MRS voxels are located in the somatosensory and motor cortex and hippocampus regions of the rat brain,using the advanced method of accurate,robust and efficient spectral fitting in j MRUI software（AMARES）to analyze the parameters.（4）Two rats in each group were euthanized at each time point,and the brain tissue was sectioned for HE and immunohistochemical staining to detect the expressions of GFAP,Iba1,and Neu N.（5）IOD/area value data for¹⁸F-FDG uptake changes in PET data,various neurometabolite changes in MR,and immunohistochemistry were analyzed using two-way repeated measures ANOVA.Part III:Dynamic contrast-enhanced magnetic resonance imaging for dynamic monitoring of early cerebral perfusion and blood-brain barrier changes in rats after bmTBI（1）Forty adult male Sprague-Dawley rats,weighing 200-225 g,were randomly divided into two groups,bmTBI group（n=20）and sham operation group（n=20）.BST-I type biological shock tube was used for shock injury,and the sham operation group only received intraperitoneal anesthesia.（2）Dynamic contrast-enhanced（DCE）magnetic resonance imaging experiments were performed before blasting and at 1-3 hours,1 day,and 7 days after blasting in m TBI group and sham operation group.Omni Kinetics software was used to analyze the data,and two vascular perfusion parameters,K^transand Vp,were obtained based on Patlak model fitting.（3）2%Evans blue solution was injected into the tail vein of three rats in each group at each time point,sacrificed 1 hour later,and the brains were harvested by perfusion with normal saline,and the permeability of Evans blue in the brain was calculated.（4）Two-way ANOVA with repeated measures was used to analyze the changes in K^transand Vp values in the DCE data,and the changes in the permeability of Evans blue in the brain.Part IIII:Resting-state functional magnetic resonance imaging combined with diffusion tensor imaging to assess early brain function and microstructural changes after bmTBI in rats（1）Ten adult male Sprague-Dawley rats,weighing 200-225g,were all included in the bmTBI group.After intraperitoneal anesthesia,the rats in the bmTBI group were placed in a BST-I biological shock tube for shock injury.（2）Rats in m TBI group were subjected to resting-state functional magnetic resonance imaging（rs-fMRI）and diffusion tensor imaging（DTI）before blasting,day1 and day7 after blasting.The ALFF and ReHo values of the functional images were calculated by the ALFF-ReHo toolkit,and the obtained ALFF and ReHo values were normalized with the mean of the whole brain.DTI parametric images were calculated by FMRIB’s Diffusion Toolbox.（3）One-way ANOVA was performed on the ALFF,ReHo and FA images of the three groups using the statistical module in SPM12,and the statistical F map was obtained.（4）Changes in ALFF,ReHo,and FA values in functional and structural data were analyzed using two-way repeated measures ANOVA.ResultsPart I（1）The BST-I device generates two-stage shock waves of lower intensity.The incident peak overpressure is 140.86k Pa,the reflection peak overpressure is 392.34k Pa,the duration is 52.22ms,the incident-reflection overpressure interval is 1.41ms,the sensitivity coefficient is 7.104mv/Kpa,and the first peak forward impulse is 4753.09 k Pa.ms.All rats survived the explosion.（2）The m NSS score of the rats in the b TBI group was 2（1.75,3）at 6 hours after injury,which was significantly higher than that in the control group of 0（0,0）（P<0.01）.It’s classified as mild craniocerebral injury according to the injury degree.（3）There were no obvious contusion and intracranial hemorrhage in conventional T1and T2 scans 6 hours after blasting in the b TBI group and the sham-operated group,and no microbleeding was found in the SWI scan.（4）In the gross morphological observation,no skull fracture was found in the brain,no subdural hemorrhage,and no contusion changes in the brain tissue after blasting.No obvious abnormality was found in HE staining,and no obvious edema and necrotic changes were found in the cells.（5）The whole brain water content of rat brain tissue was measured 6 hours after blasting.The control group was 77.67±0.17,and the b TBI group was 78.09±0.14.There was no significant difference between the two groups（P=0.081）.Part II（1）Rats in both the sham and m TBI groups did not show any visible sign of injury in the brain on T1 and T2-weighted images at any time points after blast.（2）Among 58 brain regions,5 VOIs were found significantly changed between sham and m TBI rats in¹⁸F-FDG uptake from baseline to day 7,including amygdala(F_1,5=4.137,p=0.0253),somatosensory cortex(F_1,5=13.43,p=0.0145),motor cortex(F_1,5=7.479,p=0.0410),colliculus inferior(F_1,5=38.77,p=0.0016),and colliculus superior(F_1,5=16.82,p=0.0093).At 1-3h post-injury,amygdala（+5.97%,p=0.0103）and somatosensory cortex（+6.82%,p=0.0245）presented increased¹⁸F-FDG uptake in m TBI group,compared with sham group.Subsequently,¹⁸F-FDG uptake in amygdala and somatosensory cortex gradually returned to the baselinefrom 1 day to 7 days post-injury in m TBI group,with significant difference in somatosensory cortex by 1 day（+6.12%,p=0.0429）compared to the sham group（Fig.4b-c）.¹⁸F-FDG uptake in motor cortex increased in m TBI group from 1 day（+3.60%,p=0.0326）to 7days post-injury（+3.41%,p=0.0446）（Fig.5d）.However,at 1-3 h post-injury,decreased¹⁸F-FDG uptake was observed in two midbrain structures,i.e.the inferior colliculus（-17.21%,p<0.0001）and superior colliculus（-6.75%,p=0.0094）.Then¹⁸F-FDG uptake in superior colliculus,and inferior colliculus gradually returned to the baseline from 1 day to 7 days post-injury in m TBI group,with significant difference in inferior colliculus by 1 day（-12.43%,p<0.0001）and 7 days（-7.68%,p=0.0049）,superior colliculus by 1 day（-5.95%,p=0.0230）compared to the sham group.（3）MRS revealed relative levels of glia marker Ins(F_1,5=54.49,p=0.0007),oxidative stress and gliosis marker Glx(F_1,5=31.38,p=0.0025),and hypoxic indicator Lac were markedly elevated as early as 1-3 h and day 1 post-injury,together with day 7.The major osmolyte,relative level of Tau(F_1,5=21.90,p=0.0054)immediately increased at 1-3 h（+160.37%,p<0.0001）,were continuously elevated by 1 day（+276.48%,p<0.0001）,but were deceased to sham level on 7 days post-injury（+11.92%,p=0.9444）.The m TBI group showed significant increased relative level of Cho on 7 days post-injury,compared with the sham（+24.04%,p=0.0366）.No significant reduction in relative level of NAA was found in m TBI group,compared with sham group at each timepoint(F_1,5=0.9721,p=0.3694).（4）HE staining showed no signficant alteration in tissue composition of the frontal cortex and hippocampus after blast at each time point.Furthermore,immunohistochemicalstaining for Iba 1,the microglia marker,revealed more Iba 1-positive cells in the frontal cortex of m TBI rats at each timepoint and were elevated as the time went on（p<0.0001）,which was consistent with the findings of¹⁸F-FDG PET and Ins alterations in MRS.Glial changes in the frontal cortex were also among the most marked after blast-induced m TBI.GFAP immunostaining for astrocytes demonstrated the sametrend as Iba1 in the m TBI group（p<0.0001）.However,no marked difference was found on Neu N expression of the neuron markerin the frontal cortex between the m TBI groupand the sham group at each time point after injury（p=0.1025）.On the contrary,no significant difference was shown between the m TBI group and the sham group on theexpressions of Iba 1（p=0.8591）,GFAP（p=0.5935）,and Neu N（p=0.7671）in the hippocampus.Part III（1）K^transvalues in the frontal cortex of bmTBI rats were significantly increased at 1-3hours,day 1,andday 7 after injury compared with the sham-operated group（p<0.01）.Compared with the sham-operated group,the K^transvalue at 1 hour and 1 day was significantly increased（p<0.01）,and returned to the baseline level 7 days after the injury,and the K^transvalue was not statistically significant compared with the sham-operated group（p>0.05）.Compared with the sham-operated group,the K^transvalues of the hippocampus of bmTBI rats were significantly increased at 1-3 hours and 1 day and 7 days after injury（p<0.01）.The overall trend of the K^transvalue of bmTBI rats were increased first and were then decreased,and the peak appeared 1 day after the blast shock.（2）The Vp values of the frontal cortex,hippocampus and corpus callosum of bmTBI rats had no significant changes compared with the sham operation group at 1-3 hours and 1 day after injury（p<0.01）,and the Vp value at 7 days after injury was compared with the sham operation group.All three brain regions in the group were significantly increased（p<0.01）.（3）Evans blue（EB）penetration test showed that the content of EB in the brain tissue of bmTBI rats was significantly increased at 1-3 hours and 1 day after injury compared with the sham-operated group（p<0.01）,and the content of EB at 7 days after injury It was still elevated compared to the sham group,but not statistically significant（p>0.05）.Evans blue（EB）penetration test showed that the trend of EB content in brain tissue first increased and then decreased,and the peak appeared 1 day after blast injury,which was consistent with the trend of K^transvalue.Part IV（1）There are two brain regions with differences in the dynamic changes of ReHo values in bmTBI rats,which are located in the left primary somatosensory cortex and motor cortex and the right primary motor cortex respectively.The change trend of ReHo value is shown in Figure 5-1,which shows that the ReHo value of the right primary motor cortex was decreased significantly in the acute phase after the explosion（p<0.01）,but did not change significantly in the subacute phase（P=0.95）,but was similar to that in the subacute phase（P=0.95）.Baseline ReHo values were still significantly lower（p<0.01）.ReHo values in the left primary somatosensory cortex and motor cortex were significantly decreased in the acute phase after the explosion（p<0.01）,recovered in the subacute phase（p<0.01）,and were significantly higher than baseline levels（p<0.01）.（2）There were 3 brain regions with differences in the dynamic changes of ALFF values in bmTBI rats,which were located in the left thalamus,the left scapular granular cortex and the right corpus callosum.The change trend of ALFF value is shown in Figure 5-2,which shows that the ALFF value in three brain regions decreased to varying degrees in the acute phase after the explosion,among which the granular cortex of the left slum and the corpus callosum of the right were more significant（p<0.01）,and there was no statistical difference in the descending trend of the lateral thalamus（p>0.05）.In the subacute stage,the ALFF values of the left squat granular cortex and the right corpus callosum began to recover,the left squat granular cortex returned to the baseline level,and the right corpus callosum was comparable to the baseline ALFF.The value comparison was still significantly lower（p<0.01）,and the ALFF value in the subacute phase of the left thalamus did not change significantly compared with the acute phase（p>0.05）.（3）There were 4 brain regions with different dynamic changes of FA value in bmTBI rats,which were located in the left thalamus,right thalamus,bilateral brainstem and bilateral corpus callosum.The change trend of FA value is shown in Figure 5-3,which showed that the FA value in the acute subacute phase were decreased continuously after bilateral brainstem explosion（p<0.01）,and the FA value in the acute subacute phase continued after bilateral corpus callosum explosion（p<0.01）,although the FA values of the left thalamus and the right thalamus were decreased significantly in the subacute phase of the acute phase（p<0.01）,and the values in the subacute phase were recovered somewhat compared with the acute phase.Conclusion（1）Based on imaging,behavioral,and pathological studies,it has been proved that the establishment of rat bmTBI model by BST-I shock tube is safe and reproducible,and provides a reliable experimental animal model for human bmTBI research.（2）In this study,the combined application of¹⁸F-FDG PET,MRS,DCE-MRI,rs-fMRI,and DTI advanced imaging techniques was used to evaluate bmTBI rats from multiple perspectives,revealing the early brain metabolism,blood flow,perfusion and BBB permeability,function and microstructural changes after blast shock.It hasgreat significance for the early diagnosis and prognosis evaluation of clinical bmTBI patients in the future.