The diagnosis of diffuse axonal injury: implications for forensic practice

The diagnosis of diffuse axonal injury (DAI), which may be of considerable importance in forensic medicine, necessitates widespread sampling of the brain for histology. Because a limited sampling method for screening brains for axonal damage would be of value for medico‐legal work, the authors have tested the findings of an earlier study which suggested that a standard set of three blocks from above and below the tentorium could reliably be used in routine practice as a basis for the diagnosis of DAI. A series of 22 previously diagnosed cases of DAI, with a range of survival times, was studied using immunohistochemistry with antibodies to β‐amyloid precursor protein (βAPP), the microglial‐associated antigen CD68 (PG‐M1) and for GFAP. Strict histological criteria were used to assess traumatic damage, and the evolution of the histological changes with increasing survival is described. In four cases, the sampling scheme employed yielded evidence of axonal damage in only one block, and a diagnosis of DAI could have been made in only 13/22 cases. In six of the shortest surviving cases, βAPP positivity in the corpus callosum and brainstem outlined areas of early ischaemia, as well as of traumatic damage, so that interpretation of immunolabelling was not always clear‐cut. The findings suggest that DAI cannot be reliably diagnosed on a restricted number of blocks from vulnerable areas, and that the use of βAPP and PG‐M1 immunocytochemistry may bring interpretative problems that can only be resolved by taking a larger series of tissue samples for histology.     

弥漫性轴突损伤早期超微结构改变

目的通过观察弥漫性轴突损伤(DAI)患者伤后早期轴突的超微结构变化以探索DAI的发生机理.方法对12例DAI患者的14份活体脑组织标本进行透射电镜检查.结果 DAI患者在伤后早期可发生多方面的轴突改变,包括(1)轴突的细胞骨架破坏;(2)轴膜改变;(3)膜性细胞器的变化;(4)髓鞘的改变;(5)轴突出现肿胀和离断,轴突近侧断端呈现球状.结论在DAI的发生中,可能有多种机理参与.推测,在受到足够强的外力作用时,一些管径较细的轴突可能会立即断裂;其它受损轴突则会出现进行性的延迟性轴突断裂.在此演化过程中,细胞骨架破坏和轴膜受损继而通透性改变可能是造成轴突局灶性轴浆转运障碍最终离断的最重要的因素.     

Graded model of diffuse axonal injury for studying head injury‐induced cognitive dysfunction in rats

Diffuse axonal injury (DAI) plays a major role in the development of cognitive dysfunction, emotional difficulties and behavioral disturbances in patients following closed head injury, even when they have no definite abnormalities on conventional MRI. This study aimed to develop a highly controlled and reproducible model for DAI that simulates post‐traumatic cognitive dysfunction in humans. Sprague‐Dawley (SD) rats were subjected to impact acceleration head injury, using a pneumatic impact targeted to a steel disc centered onto their skull. The severity of injury was graded as three levels by adjusting the driving pressure at 60, 70 or 80 pounds per square inch. In vivo MRI was obtained 2 days post‐injury. Cognitive function was evaluated using the Morris water maze at 1 and 2 weeks post‐injury. HE staining and immunohistochemistry were performed to assess neuronal and axonal damages after 2 weeks. MRI demonstrated that this model induced no gross structural modification in the brain. The degree and duration of cognitive dysfunction were dependent on the force of impact. Histological analysis revealed the force‐dependent damage of the neurons and microtubule‐associated protein 2‐positive axons in the neocortex. Hippocampal damage was much less pronounced and was not linked to cognitive dysfunction. This is the first report that precisely evaluates the threshold of impact energy to lead to neocortical damage and cognitive dysfunction in rodents. This model would be suitable for clarifying the complex mechanisms of post‐traumatic brain damage and testing novel therapeutic approaches against post‐traumatic cognitive dysfunction due to diffuse axonal damage.     

Traumatic axonal damage in the brain can be detected using beta-APP immunohistochemistry within 35 min after head injury to human adults

Immunohistochemistry staining for beta-amyloid precursor protein (beta-APP) is a sensitive method to detect early axonal damage in traumatic brain injury, which was previously estimated to be of minimum 60-90 min after head injury. We present seven cases of well-documented posttraumatic survival of 35-60 min where beta-APP detects early axonal damage. Cases were selected from routine work where documentation about survival is judged to be accurate. These are divided into three groups: group 1: severe head injury (n = 7) with documented survival between 35 and 60 min. Group 2: severe head injury (n = 4) with documented survival of less than 30 min. Group 3: cases (n = 4) where death was not due to head injury but survival is documented between 45 and 109 min. The brains were fixed in formalin for 4 weeks and six regions (frontal lobe with anterior corpus callosum, parietal lobe with deep white matter, basal ganglia with posterior limb of internal capsule, cerebellum with white matter and middle cerebellar peduncle and pons with basis pontis and superior cerebellar peduncle) were sampled. All blocks were stained for haematoxylin and eosin and beta-APP and selected ones for CD68, using antigen retrieval method. In group 1 sections revealed beta-APP immunoreactivity in forms of small globules and granules and occasionally as thin and short filaments. These were detected in the pons, corpus callosum, internal capsule and cerebral white matter, with some variation in localization and intensity. In groups 2 and 3 all the sections were negative for beta-APP staining. None of the cases showed evidence of severe brain swelling, increased intracranial pressure, ischaemia or infection. Using the antigen retrieval method, beta-APP immunohistochemistry can detect axonal damage within 35 min after severe head injury. These results may have an implication in the consideration of minimal survival time after traumatic head injury in medico-legal practice.     

Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury

Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as "varicosities." Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury.     

Neuropathological investigation of cerebral white matter lesions caused by closed head injury

In order to ascertain whether there is widespread axonal disruption of cerebral white matter in the so‐called ‘diffuse axonal injury’ (DAI), a type of closed head injury, proposed by Adams et al. the author investigated his own cases clinicopathologically. Twenty‐six male autopsied cases of head injury, aged between 19 and 84, 15 of which had sustained road traffic accidents, were examined; the others were due to falling from heights and so on. The study group all belonged to non‐missile head injuries and included 12 cases of diffuse brain injury, as well as 14 cases of focal brain injury, according to the classification of Gennarelli et al. The survival time ranged from 2 h to 21 years. Formalin‐fixed brains were cut coronally so as to make paraffin‐embedded hemispheric sections. Then these sections were stained conventionally (HE, Bodian, Kluver‐Barrera and Holzer) and immunohistochemically (GFAP) to assess axonal decrease, myelin pallor and gliosis by the use of light microscopy. In the 13 chronic cases that died more than 1 month after the accidents, the intensities of gliosis, myelin pallor and axonal decrease tended to correlate with each other. In the 13 acute cases who died less than 1 month after their accident, the degree of axonal decrease in white matter seemed to correlate with the severity of myelin pallor. Regardless of types of trauma, however, axonal retraction balls, the so‐called hallmark of DAI, were found only with myelin pallor suggesting the presence of brain swelling after the injury. Therefore these findings indicate that it may be difficult to accept the notion of DAI, that is, the presence of axonal retraction balls without brain swelling. In addition, diffuse vascular injury (2 cases) as well as rarefaction of subcortical white matter (6 cases) were presented and their pathogenesis individually discussed based on a literature review.     

直线加速度所致的猫弥漫性轴突损伤

目的：为了解直线加速度在颅脑损伤特别是在弥漫性轴突损伤（ＤＡＩ）中的作用。方法：用一套撞击装置对２６只猫进行实验。结果：直线加速度可以导致几乎所有类型的颅脑损伤，包括颅骨骨折、蛛网膜下腔出血、硬膜下血肿、硬膜外血肿、脑挫伤、脑干损伤，特别是ＤＡＩ。ＤＡＩ的特征性改变－轴突回缩球在２４小时内死亡的动物脑标本中看不到，２４小时后可见到，７２小时则多而典型，７天时仍然存在。结论：凡能使脑的神经纤维受到广泛剪力和（或）张力的任何形式的外部作用均可产生ＤＡＩ。     

弥漫性轴突损伤胆碱能纤维改变的实验研究

目的通过大鼠弥漫性脑损伤模型观察海马及乳头体内的轴突损伤,了解轴突损伤后上述结构中胆碱酯酶纤维的变化,探讨轴突损伤与伤后记忆功能障碍的相关性.方法用Marmarou介绍的落体打击装置致伤动物,对海马和乳头体区脑组织进行胆碱酯酶(AChE)纤维染色.结果该模型较好地模拟了轴突损伤的表现,简便实用.在这个模型中轴突损伤的最常见部位为桥脑基底部和小脑上脚,其次为大脑半球白质、海马和乳头体.海马结构内含有大量胆碱酯酶阳性染色纤维.与对照组相比中,损伤10天海马CA1区,CA3区,齿状回分子层和乳头体内纤维密度明显低于对照组大鼠(P＜0.01).结论大鼠损伤后海马区和乳头体内胆碱酯酶阳性纤维明显减少,这可能是弥漫性轴突损伤病人记忆功能损害的原因.     

Diffusion Tensor and Functional Magnetic Resonance Imaging of Diffuse Axonal Injury and Resulting Language Impairment

Diffuse axonal injury (DAI) is a common aftermath of brain trauma. The diagnosis of DAI is often difficult using conventional magnetic resonance imaging (MRI). We report a diffusion tensor imaging (DTI) study of a patient who sustained DAI presenting with language impairment. Fractional anisotropy (FA) and DTI tractography revealed a reduction of white matter integrity in the left frontal and medial temporal areas. White matter damage identified by DTI was correlated with the patient's language impairment as assessed by functional MRI (fMRI) and a neuropsychological exam. The findings demonstrate the utility of DTI for identifying white matter changes secondary to traumatic brain injury (TBI).     

Correlation of survival time with size of axonal swellings in diffuse axonal injury

Widespread damage to axons in the white matter of the brain is a well-recognised consequence of non-missile head injury. This diffuse axonal injury is characterised by a gradual swelling of the axon associated with an accumulation of cellular organelles and proteins. We have investigated the relationship between the size of the swellings of the damaged axon with survival time in post-mortem brain tissue. Sixty-six cases of head injury with known length of post-traumatic survival were selected for study, and immunohistochemistry for β-amyloid precursor protein (βAPP) was carried out. The minimum diameter of the βAPP-immunolabelled damaged axons was measured in micrometers using the IBAS image analysis system. There was a strong, positive and significant relationship between the mean size of axonal swelling and survival time which plateaued at around 85 h post injury. With longer survival times the situation becomes more complex. βAPP immunolabelling of damaged axons can contribute evidence about trauma and post-injury survival time in the forensic setting but should always be assessed with other evidence. Received: 13 June 1996 / Revised: 18 July 1997, 20 October 1997, 4 November 1998 / Accepted: 5 January 1999     

Neuropathology of the vegetative state after head injury

A detailed neuropathological study of patients identified clinically after head injury as either severely disabled (SD, n = 30) or vegetative (VS, n = 35) has been carried out to determine the nature and frequency of the various pathologies that form the basis of these clinical states. Patients who were SD were older (SD median 49.5 yrs vs. VS median 38 yrs, p = .04), more likely to have a lucid interval (SD 31% vs. VS 9%, p = .03), and to have had an acute intracranial haematoma (SD 70% vs. VS 26%, p < .001). SD patients less often had severe, Grades (2 or 3) of traumatic diffuse axonal injury (SD 30% vs. VS 71%, p = .001) and less often had thalamic damage (SD 37% vs. VS 80%, p < .001). Similar features of both focal and diffuse damage were present in some SD and VS cases with both groups having considerable damage to white matter and to the thalamus. It is concluded that the principal structural basis of both SD and VS is diffuse traumatic axonal injury (DAI) with widespread damage to white matter and changes in the thalami. However, both ischaemic brain damage and the vascular complications of raised intracranial pressure contributed to the clinical signs and symptoms.     

Clinicoradiological and therapeutic considerations in severe diffuse traumatic brain injury in children

Forty-one children with severe head injuries and diffuse brain lesions were selected from a consecutive series of 62 children in traumatic coma (21 focal mass lesions) and studied. According to the CT pattern, two main types of intracranial lesions were considered: diffuse axonal injury (DAI) and diffuse brain swelling (DBS). High mortality, due to secondary increases of intracranial pressure (ICP), correlated well with the patterns of severe DBS, absence of perimesencephalic cisterns, and obliteration of the ventricles. However, children with normal CTs, and/or obvious shearing injuries indicative of DAI, had favorable outcomes; there was no mortality if increased ICP was not present. We conclude that although there does not seem to be any routine indications for ICP monitoring in children with pure DAI, early ICP monitoring and aggressive management of increasing ICP should be considered in comatose children with DBS, especially when associated with subarachnoid hemorrage and respiratory or circulatory failure.Presented at the 11th Meeting of the European Society for Paediatric Neurosurgery, Naples 1988     

弥漫性轴突损伤形态学改变的实验研究

目的　观察弥漫性轴突损伤的常见部位, 病理改变过程, 以及其发生原因和机制。探讨其与临床的相关性。方法　用 Ｍａｒｍａｒｏｕ 的落体打击装置致伤动物, 大鼠脑组织标本在光镜和电镜下观察。结果　轴突回缩球密度在桥脑基底部和小脑上脚最高。伤后轴突内的微丝、微管结构紊乱, 轴突肿胀。伤后３ 天, 多数肿胀的轴突断裂, 形成轴突回缩球。１ 小时组轴突内钙颗粒数量是对照组的１７ 倍, 提示钙的内流。结论　本实验中弥漫性轴突损伤的最常见部位是桥脑基底部和小脑上脚。轴突损伤的过程为： 轴突内结构的紊乱, 轴突肿胀及断裂。轴突损伤的主要发生原因和机制可能是细胞外钙的内流。在伤后１２ 小时以内, 损伤轴突尚未断裂, 可能仍存在可逆性, 这可能是临床上有效治疗的最佳时机。     

Non-missile head injury: report of a patient surviving for 6 years

A female patient with non-missile head injury is described. She showed slight improvement in her level of consciousness, and survived for 6 1/2 years after injury. At autopsy, the white matter lesions were localized rather than diffuse in distribution. In addition to lesions in the corpus callosum, anterior commissure and dorsolateral quadrant of the rostral brain stem, those in the parasagittal cerebral white matter, and in the hilus of the dentate nucleus and superior cerebellar peduncle were considered to be due to primary axonal injury. A cavity in the frontal white matter was remarkable in that there was no evidence to indicate expansion of the lesion due to haemorrhage. These features suggested that the injurious physical forces had acted parallel to the direction of the axons.     

Diffuse axonal injury and traumatic coma in the primate

Traumatic coma was produced in 45 monkeys by accelerating the head without impact in one of three directions. The duration of coma, degree of neurological impariment, and amount of diffuse axonal injury (DAI) in the brain were directly related to the amount of coronal head motion used. Coma of less than 15 minutes (concussion) occurred in 11 of 13 animals subjected to sagittal head motion, in 2 of 6 animals with oblique head motion, and in 2 of 26 animals with full lateral head motion. All 15 concussed animals had good recovery, and none had DAI. Conversely, coma lasting more than 6 hours occurred in none of the sagittal or oblique injury groups but was present in 20 of the laterally injured animals, all of which were severely disabled afterward. All laterally injured animals had a degree of DAI similar to that found in severe human head injury. Coma lasting 16 minutes to 6 hours occurred in 2 of 13 in the sagittal group, 4 of 6 in the oblique group, and 4 of 26 in the lateral group; these animals had less neurological disability and less DAI than when coma lasted longer than 6 hours. These experimental findings duplicate the spectrum of traumatic coma seen in human beings and include axonal damage identical to that seen in severe head injury in humans. Since the amount of DAI was directly proportional to the severity of injury (duration of coma and quality of outcome), we conclude that axonal damage produced by coronal head acceleration is a major cause of prolonged traumatic coma and its sequelae.     

Ultrastructural evidence of axonal shearing as a result of lateral acceleration of the head in non-human primates

Summary The concept of shearing of axons at the time of non-impact injury to the head was first suggested in the middle of this century. However, no experimental model of diffuse axonal injury (DAI) has provided morphological confirmation of this concept. Evidence from experiments on invertebrate axons suggests that membrane resealing after axonal transection occurs between 5 and 30 min after injury. Thus, ultrastructural evidence in support of axonal shearing will probably only be obtained by examination of very short-term survival animal models. We have examined serial thin sections from the corpus callosum of non-human primates exposed to lateral acceleration of the head under conditions which induce DAI. Tearing or shearing of axons was obtained 20 and 35 min after injury, but not at 60 min. Axonal fragmentation occurred more frequently at the node/paranode but also in the internodal regions of axons. Fragmentation occurred most frequently in small axons. Axonal shearing was associated with dissolution of the cytoskeleton and the occurrence of individual, morphologically abnormal membranous organelles. There was no aggregation of membranous organelles at 20 and 35 min but small groups did occur in some axons at 60 minutes. We suggest that two different mechanisms of injury may be occurring in non-impact injury to the head. The first is shearing of axons and sealing of fragmented axonal membranes within 60 min. A second mechanism occurs in other fibres where pertubation of the axon results in axonal swelling and disconnection at a minimum of 2 h after injury.Supported by NIH grant number NS-08803-21, the Wellcome Trust, the Royal Society, London and the Institute of Neurological Sciences, University of Glasgow. Part of this work was presented at the Ist International Neurotrauma Symposium, Fukishima, Japan and the IVth European Meeting of Neuropathology, Berlin Dedication: The authors would like this work to be a tribute to Professor J. H. Adams upon his retiral. His research into diffuse axonal injury inspired the authors to undertake this study.     

Neuropathology of the Head Injuries

Structural abnormalities develop progressively after trauma to the central nervous system suggesting that injury is a process of events rather than a singular event. Thus, numerous types of neuropathologies can occur, depending on the exact nature of the processes of cellular damage that are set into motion after injury. Four general interrelated processes of delayed cellular damage occur in different amounts and in different locations to result in the numerous types of traumatic brain damage. These include direct damage caused by calcium influx into cells, free radical-mediated damage, receptor-mediated damage, and inflammation. By these mechanisms, the general response of the brain to mechanical energy causes damage to vascular or neural components of the brain, and results in principally focal or diffuse brain damage, respectively. These form the phenotypic types of damage to the brain and the resulting clinically associated traumatic syndromes. Focal brain damage consists principally of vascular injury that results in confusions and hemorrhages in various locations. Diffuse brain damage mainly involves scattered damage to axons in the white matter (diffuse axonal injury) or secondary damage attributable to raised intracranial pressure, hypoxia, or ischemia.     

Perisomatic thalamic axotomy after diffuse traumatic brain injury is associated with atrophy rather than cell death

Morbidity and mortality associated with traumatic brain injury (TBI) stem from diffuse axonal injury (DAI) throughout subcortical and brainstem white matter and subcortical nuclei. After midline fluid percussion brain injury, DAI in the thalamus includes perisomatic axotomy and resembles human post-traumatic pathology where the degree of morbidity correlates with thalamic damage. After axotomy, acute somatic perturbations resolve and appear compatible with cell survival; however, the long-term fate of neurons in an area with perisomatic axotomy is unknown. From brain-injured and uninjured rats at 1, 7 and 28 days after injury (injury, n = 5/group; sham, n = 4), alternate sections were immunostained for amyloid precursor protein (APP) to detect perisomatic axotomy or Giemsa stained for quantification of neuronal number, neuronal density, regional volume, and neuronal nuclear volume using design-based stereology. One day postinjury, APP-immunoreactive axons were identified consistently within the perisomatic domains of thalamic neurons of the ventral basal complex. Bilateral systematic-random quantification of the ventral basal complex indicated a significant reduction in neuronal density (number per mm, but not number alone) at 1 week after injury, compared with sham and 1 day postinjury. Furthermore, by 1 day and persisting through 1 week after injury, the mean neuronal nuclear volume was atrophied significantly compared with sham. Therefore, diffuse TBI results in early perisomatic axonal injury followed by neuronal atrophy in the ventral basal complex, without gross degeneration. Enduring atrophy in thalamic relays could underlie circuit disruption responsible for post-traumatic morbidity.     

弥漫性轴索损伤的影像与临床分析

目的探讨弥漫性轴索损伤的影像学及临床特点,为诊断和治疗提供参考。方法对47例弥漫性轴索损伤患者的临床资料及影像学进行回顾性分析。结果随访3～12个月,根据格拉斯哥预后评分（GOS）：恢复良好12例,中残14例,重残8例,植物生存3例,死亡10例。不同影像学分级组的预后无显著差异。结论弥漫性轴索损伤是临床上常见的颅脑损伤类型,其诊断主要根据临床和影像学表现;目前CT和常规MRI尚不能作为诊断的必要条件,也不能作为准确评估患者预后的独立依据。