Optic nerve damage in shaken baby syndrome: detection by beta-amyloid precursor protein immunohistochemistry

BACKGROUND: Rapid acceleration-deceleration of an infant's head during intentional shaking should in theory exert stretch or shear forces upon the optic nerves sufficient to cause axonal injury. beta-Amyloid precursor protein (beta-APP) immunohistochemistry recently has been shown to be a highly effective method for identifying diffuse axonal injury in the brains of infants with shaken baby syndrome. In this study, we investigated the utility of beta-APP in identifying optic nerve damage in infants who have sustained fatal whiplash shaking. MATERIALS AND METHODS: beta-Amyloid precursor protein immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of eyes (including optic disc and distal optic nerve) from infants less than 1 year of age with shaken baby syndrome (5 cases), combined shaken baby syndrome/blunt head trauma (3 cases), and "pure" blunt head trauma (1 case). Nontraumatic control cases included infants who died of suffocation (1 case), sudden infant death syndrome (1 case), and positional asphyxia (1 case) and an enucleation from a child with a retinoblastoma (1 case). Matched hematoxylin-eosin-and neurofilament-stained sections were used for comparison. RESULTS: Three of the 5 shaken baby cases and all 3 combined shaken baby/blunt head trauma cases had optic nerve axonal injury identified by the presence of strongly beta-APP-immunoreactive beaded or swollen axonal segments. Axonal injury could not be detected in the corresponding hematoxylin-eosin-or neurofilament-stained sections. Optic nerve axonal injury was not seen in the case involving pure blunt head trauma or in the nontraumatic control cases. CONCLUSIONS: Optic nerve axonal injury is a prominent feature of intentional fatal whiplash head trauma in infants less than 1 year of age. beta-Amyloid protein precursor immunohistochemistry appears to be the most effective method for demonstrating axonal damage in the optic nerve.     

The possible role of hypoxia in the formation of axonal bulbs.

AIMS: To assess the possible role of hypoxia in the formation of axonal bulbs. METHODS: Study material comprised sections from 28 brains showing evidence of cerebral hypoxia with no history of head injury, four with a history of head trauma but no evidence of hypoxic change, eight with a history of head trauma and hypoxic change, and four from control brains originally described as "diffuse axonal injury." These were subjected to microwave antigen retrieval and immunohistochemistry using monoclonal antibodies to beta amyloid precursor protein (beta APP), glial fibrillary acid protein (GFAP), and CD68-PGM1. RESULTS: Positive staining for beta APP was seen in all four controls, all four cases of head injury only, seven of eight cases of head injury and hypoxic changes, and 12 of 28 cases of hypoxia without history of head injury; 22 of 25 cases who had been ventilated showed positive staining. The majority of cases showed evidence of cerebral swelling. CONCLUSIONS: Axonal bulbs staining positively for beta APP may occur in the presence of hypoxia and in the absence of head injury. The role of hypoxia, raised intracranial pressure, oedema, shift effects, and ventilatory support in the formation of axonal bulbs is discussed. The presence of axonal bulbs cannot necessarily be attributed to shearing forces alone.     

Anatomy of the shaken baby syndrome

Shaken baby syndrome refers to the constellation of nonaccidental injuries occurring in infants and young children as a consequence of violent shaking. The typical victim of shaken baby syndrome is a male infant younger than six months of age who is alone with the perpetrator at the time of injury. Occurrence of the syndrome is unrelated to race, gender, socioeconomic status, or education. The characteristic injuries observed in shaken baby syndrome include subdural hemorrhages, retinal hemorrhages, and fractures of the ribs or long bones. Although each of these injuries may result from violent shaking of the victim, the most severe brain injuries result from the addition of a forceful impact of the infant's or child's head against a firm surface. The unique anatomic features of the infant's head and skeletal system, which account for the type and pattern of injuries observed in shaken baby syndrome, are emphasized in this article. Anat. Rec. (New Anat.) 253:13–18, 1998. © 1998 Wiley-Liss, Inc.     

Diffuse axonal injury in early infancy.

Diffuse axonal injury typified by retraction balls and axonal swellings was identified in the brains of a series of infants, 5 months old and younger, who had suffered closed head injuries. These axonal discontinuities were shown by using Nauomenko and Feigin's silver method, which is particularly useful for showing fine axons such as those found in the developing brain. Diffuse axonal injury in early infancy may occur in the same way as that described in adults. The low incidence of intracerebral haematomata suggests that recurrent trauma to the head from a combination of direct contact and shaking results in axonal damage to the poorly myelinated axons and that blood vessels are rarely damaged.     

Intracranial diffuse axonal injury at autopsy

An illustrative case of diffuse axonal injury (DAI) emphasizes features that help to separate focal outer head trauma owing to blows and/or falls from angular acceleration head injuries associated with diffuse inner brain lesions. In the past, explaining significant neurological deficits and death as the result of diffuse closed head trauma received from high-speed automobile accidents has been difficult as well as confusing. The long-term consequences from such diffuse inner cerebral trauma are still poorly defined. Head injuries sustained in automobile accidents have been associated with diffuse brain damage characterized by axonal injury at the moment of impact. The reported victim of a motor vehicle accident showed post-mortem findings for both inner cerebral trauma and focal outer cerebral damage. The diffuse degeneration of cerebral white matter is associated with sagittal and lateral acceleration with centroaxial trauma and has a different pathogenesis from outer focal head trauma, typified by subdural hematomas and coup injuries. Unlike outer cerebral injury, over 50 percent of victims with diffuse axonal injury die within two weeks. These individuals characteristically have no lucid interval and remain unconscious, vegetative, or severely disabled until death. Compared to head trauma victims without diffuse axonal injury, there is a lower incidence of skull fractures, subdural hemorrhages, or other intracranial mass effect as well as outer brain contusions. Primary brainstem injuries often demonstrated at autopsy are seen in the reported victim. Diffuse axonal injury is produced by various angles of acceleration with prolonged acceleration/deceleration usually accompanying traffic accidents. Less severe diffuse axonal injury causes concussion.     

CLARITY reveals a more protracted temporal course of axon swelling and disconnection than previously described following traumatic brain injury

Diffuse axonal injury (DAI) is an important consequence of traumatic brain injury (TBI). At the moment of trauma, axons rarely disconnect, but undergo cytoskeletal disruption and transport interruption leading to protein accumulation within swellings. The amyloid precursor protein (APP) accumulates rapidly and the standard histological evaluation of axonal pathology relies upon its detection. APP+ swellings first appear as varicosities along intact axons, which can ultimately undergo secondary disconnection to leave a terminal “axon bulb” at the disconnected, proximal end. However, sites of disconnection are difficult to determine with certainty using standard, thin tissue sections, thus limiting the comprehensive evaluation of axon degeneration. The tissue‐clearing technique, CLARITY, permits three‐dimensional visualization of axons that would otherwise be out of plane in standard tissue sections. Here, we examined the morphology and connection status of APP+ swellings using CLARITY at 6 h, 24 h, 1 week and 1 month following the controlled cortical impact (CCI) model of TBI in mice. Remarkably, many APP+ swellings that appeared as terminal bulbs when viewed in standard 8‐µm‐thick regions of tissue were instead revealed to be varicose swellings along intact axons when three dimensions were fully visible. Moreover, the percentage of these potentially viable axon swellings differed with survival from injury and may represent the delayed onset of distinct mechanisms of degeneration. Even at 1‐month post‐CCI, ~10% of apparently terminal bulbs were revealed as connected by CLARITY and are thus potentially salvageable. Intriguingly, the diameter of swellings decreased with survival, including varicosities along intact axons, and may reflect reversal of, or reduced, axonal transport interruption in the chronic setting. These data indicate that APP immunohistochemistry on standard thickness tissue sections overestimates axon disconnection, particularly acutely post‐injury. Evaluating cleared tissue demonstrates a surprisingly delayed process of axon disconnection and thus longer window of therapeutic opportunity than previously appreciated. Intriguingly, a subset of axon swellings may also be capable of recovery.     

Axonal injury in cerebral malaria

Impairment of consciousness and other signs of cerebral dysfunction are common complications of severe Plasmodium falciparum malaria. Although the majority of patients make a complete recovery a significant minority, particularly children, have sequelae. The pathological process by which P. falciparum malaria induces severe but usually reversible neurological complications has not been elucidated. Impairment of transport within nerve fibers could induce neurological dysfunction and may have the potential either to resolve or to progress to irreversible damage. Beta-amyloid precursor protein (beta-APP) immunocytochemistry, quantified using digital image analysis, was used to detect defects in axonal transport in brain sections from 54 Vietnamese cases with P. falciparum malaria. The frequency and extent of beta-APP staining were more severe in patients with cerebral malaria than in those with no clinical cerebral involvement. Beta-APP staining was often associated with hemorrhages and areas of demyelination, suggesting that multiple processes may be involved in neuronal injury. The age of focal axonal damage, as determined by the extent of the associated microglial response, varied considerably within tissue sections from individual patients. These findings suggest that axons are vulnerable to a broad range of cerebral insults that occur during P. falciparum malaria infection. Disruption in axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria.     

Fate of reactive axonal swellings induced by head injury

The fate of those reactive axonal swellings seen following head injury was assessed in cats subjected to mild to moderate fluid-percussion head injury. To allow for the ready visualization of any traumatically induced reactive axonal change at both the light and electron microscopic level, the anterograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase was employed over a 21-day posttraumatic period in selected cerebral and cerebellar efferents coursing through the brain stem. At the designated posttraumatic survival time, the animals were perfused with aldehydes, processed for the light and electron microscopic visualization of the peroxidase reaction product, and examined for any evidence of reactive axonal change. At the 3rd and 4th posttraumatic days, peroxidase-laden swellings could be identified. Some reactive swellings were packed with organelles and were either encompassed by a distended myelin sheath or lacked myelin investment. Other reactive swellings demonstrated either lobulation or increased electron density with macrophage accumulation, all of which indicated degeneration. Wallerian change occurred distal to the reactive swellings; however, with the exception of these changes the related brain parenchyma and vasculature demonstrated no significant abnormality. With continued survival, reactive swellings comparable to those just described were consistently observed; however, now regenerative responses were also seen. At the 5th and 7th days, reactive sprouts were observed originating from reactive swellings which displayed a reduction both in size and in organelle content. By the 9th and 14th posttraumatic days, some sprout-containing swellings demonstrated several robust extensions. These regenerative changes were seen in both myelin- and nonmyelin-invested swellings and persisted through the 21st day, occurring in concert with lobulated, electron dense, and unchanged, swollen reactive axons. This study suggests that head injury elicits axonal swelling that may persist unchanged, degenerate, or undergo a regenerative response. The sustained regenerative responses are considered intriguing and may have relevance both for head-injured humans and for future studies of central nervous system regeneration.     

Axonal cytoskeletal changes after non-disruptive axonal injury

In animal models of human diffuse axonal injury, axonal swellings leading to secondary axotomy occur between 2 and 6 h after injury. But, analysis of cytoskeletal changes associated with secondary axotomy has not been undertaken. We have carried out a quantitative analysis of cytoskeletal changes in a model of diffuse axonal injury 4 h after stretch-injury to adult guinea-pig optic nerves. The major site of axonal damage was the middle portion of the nerve. There was a statistically significant increase in the proportion of small axons with a diameter of 0.5 m and smaller in which there was compaction of neurofilaments. Axons with a diameter greater than 2.0 m demonstrated an increased spacing between cytoskeletal elements throughout the length of the nerve. However, in the middle segment of the nerve these larger axons demonstrated two different types of response. Either, where periaxonal spaces occurred, there was a reduction in axonal calibre, compaction of neurofilaments but no change in their number, and a loss of microtubules. Or, where intramyelinic spaces occurred there was an increased spacing between neurofilaments and microtubules with a significant loss in the number of both. Longitudinal sections showed foci of compaction of neurofilaments interspersed between regions where axonal structure was apparently normal. Neurofilament compaction was correlated with disruption of the axolemma at these foci present some hours after injury. We suggest that the time course of these axonal cytoskeletal changes after stretch-injury to central axons is shorter than those changes documented to occur during Wallerian degeneration.     

Primary brain trauma in non-accidental injury

The brains from 12 babies up to 21/2 years of age, who died after repeated non-accidental injury to the head, were subjected to detailed neuropathological examination. The nine brains from infants under 5 months showed contusional tears--slit like lesions in the white matter surrounded by astrocytes and associated with evidence of old and recent haemorrhage. The three brains from infants over 5 months showed white matter lesions similar to those seen in adults after closed head injury, including damage in the dorsolateral quadrant of the brain stem without axonal hemispheric damage, which may have been a result of whiplash injury after shaking. In addition, all the brains examined showed diffuse gliosis. This paper draws attention to contusional tears and other white matter lesions, which the authors believe are manifestations of mechanical damage produced by trauma. The long term neurological and intellectual defects observed in patients suffering non-accidental injury early in life are increasingly being recognised, although it is difficult to identify the extent to which these are due to social or neuropathological factors. We suggest that the white matter damage we describe has an important role.     

Dimethylsulfoxide enhances CNS neuronal plasma membrane resealing after injury in low temperature or low calcium

The inability to repair the damaged membrane may be one of the key mechanisms underlying the severe neuronal degeneration and overall functional loss seen in in vivo spinal cord injury and traumatic axonal injury in blunt head trauma. Promoting membrane resealing following damage may therefore constitute a potential effective therapeutic intervention in treating head trauma and spinal cord injuries. In our previous studies, we have shown that the axolemma failed to reseal following transection in clinically related situations, such as low extracellular calcium and low temperature. Our current studies indicate that DMSO is capable of rendering significant improvement in guinea pig axonal membrane resealing following transection in both 0.5 mM [Ca(2+)](0) and 25 degrees C situations. This was demonstrated physiologically by monitoring membrane potential recovery and anatomically by conducting HRP-exclusion assays 60 minutes after injury. Further, we have shown that the addition of DMSO in normal Krebs' solution (2 mM [Ca(2+)](0) and 37 degrees C) resulted in a decrease in membrane repair following injury. This indicates that DMSO-mediated membrane repair is sensitive to temperature and calcium. This study suggests the role of DMSO in axonal membrane resealing in clinically relevant conditions and raises the possibility of using DMSO in combination with other more established therapies in spinal cord injury treatment.     

Diffusion tensor imaging of diffuse axonal injury in a rat brain trauma model

Diffusion tensor imaging (DTI) was used to study traumatic brain injury. The impact-acceleration trauma model was used in rats. Here, in addition to diffusivities (mean, axial and radial), fractional anisotropy (FA) was used, in particular, as a parameter to characterize the cerebral tissue early after trauma. DTI was implemented at 7 T using fast spiral k-space sampling and the twice-refocused spin echo radiofrequency sequence for eddy current minimization. The method was carefully validated on different phantom measurements. DTI of a trauma group (n = 5), as well as a sham group (n = 5), was performed at different time points during 6 h following traumatic brain injury. Two cerebral regions, the cortex and corpus callosum, were analyzed carefully. A significant decrease in diffusivity in the trauma group versus the sham group was observed, suggesting the predominance of cellular edema in both cerebral regions. No significant FA change was detected in the cortex. In the corpus callosum of the trauma group, the FA indices were significantly lower. A net discontinuity in fiber reconstructions in the corpus callosum was observed by fiber tracking using DTI. Histological analysis using Hoechst, myelin basic protein and Bielschowsky staining showed fiber disorganization in the corpus callosum in the brains of the trauma group. On the basis of our histology results and the characteristics of the impact-acceleration model responsible for the presence of diffuse axonal injury, the detection of low FA caused by a drastic reduction in axial diffusivity and the presence of fiber disconnections of the DTI track in the corpus callosum were considered to be related to the presence of diffuse axonal injury.     

Diffuse axonal swellings in a case of acquired immunodeficiency syndrome

We report a case of a 25-year-old woman affected by the acquired immunodeficiency syndrome in whom the neuropathologic examination disclosed the presence of diffuse axonal swellings in the brain stem and in selected areas of cerebral hemispheres. These lesions, although microscopically similar to those previously described as focal pontine leukoencephalopathy, differ in that their distribution was wider, there was an absence of associated necrosis of the adjacent nervous tissue, and there was a lack of calcification. It is suggested that axonal swellings represent a nonspecific reaction of the central nervous system to infection by opportunistic agents and/or human immunodeficiency virus in patients with the acquired immunodeficiency syndrome.     

Delayed white matter injury in a murine model of shaken baby syndrome

Shaken baby syndrome, a rotational acceleration injury, is most common between 3 and 6 months of age and causes death in about 10 to 40% of cases and permanent neurological abnormalities in survivors. We developed a mouse model of shaken baby syndrome to investigate the pathophysiological mechanisms underlying the brain damage. Eight-day-old mouse pups were shaken for 15 seconds on a rotating shaker. Animals were sacrificed at different ages after shaking and brains were processed for histology. In 31-day-old pups, mortality was 27%, and 75% of survivors had focal brain lesions consisting of hemorrhagic or cystic lesions of the periventricular white matter, corpus callosum, and brainstem and cerebellar white matter. Hemorrhagic lesions were evident from postnatal day 13, and cysts developed gradually between days 15 and 31. All shaken animals, with or without focal lesions, had thinning of the hemispheric white matter, which was significant on day 31 but not earlier. Fragmented DNA labeling revealed a significant increase in cell death in the periventricular white matter, on days 9 and 13. White matter damage was reduced by pre-treatment with the NMDA receptor antagonist MK-801. This study showed that shaking immature mice produced white matter injury mimicking several aspects of human shaken baby syndrome and provided evidence that excess release of glutamate plays a role in the pathophysiology of the lesions.     

Congenital giant axonal neuropathy

Giant axonal neuropathy (GAN) is a distal sensorimotor neuropathy, characterized by neurofilamentous axonal swellings, with usual onset at 2 to 3 years of age. We report a case of congenital GAN with hypotonia at birth. At 7 months of age, nerve conduction studies showed almost complete lack of sensory and motor responses in the lower extremities. A sural nerve biopsy specimen disclosed absence of myelinated axons. Autopsy, following death at 15 months of age, revealed axonal swellings in peripheral nerves and distal degeneration of long spinal cord tracts. The neurofilamentous content of the axonal swellings was confirmed by Glees-Marsland staining and immunoperoxidase reaction with antibodies to neurofilaments. Axonal swellings did not stain with periodic acid-Schiff and were not seen in the cerebral cortex or brain stem, distinguishing this process from infantile neuroaxonal dystrophy. This patient illustrates congenital GAN with subsequent rapid progression.     

Neuroprotective effects of resveratrol against traumatic brain injury in immature rats

Childhood trauma resulting in traumatic brain injury (TBI) due to accidents and abuse is the major cause of death and dysfunction in the young. Since there are no approved specific pharmacological agents that block the progression of the secondary injury, the current management of TBI is mainly supportive. We aimed to determine the effect of resveratrol on hippocampal damage and behavioral deficits in 7-day-old rat pups subjected to contusion injury. Resveratrol was injected intraperitoneally at the doses of 100 mg/kg of body weight immediately after induction of traumatic injury. Hippocampal damage was examined by cresyl violet staining and behavioral alterations were evaluated using open field and novel object recognition tests 2 weeks after trauma. Histopathological evaluation showed that treatment with a single dose of 100 mg/kg resveratrol (i.p.) after the trauma significantly ameliorated the trauma induced hippocampal neuron loss at ipsilateral and contralateral hippocampal brain regions of rats. Additionally, treatment with resveratrol decreased anxiety and increased cortex/hippocampus dependent memory of animals subjected to blunt head trauma. These results show that acute treatment of resveratrol has a neuroprotective role against trauma induced hippocampal neuron loss and associated cognitive impairment in rats.     

川芎嗪干预钝性肺挫伤急性期大鼠肺组织细胞的凋亡

背景：急性胸部撞击后所致的肺挫伤(钝性肺挫伤)常引起呼吸功能异常和继发性炎性反应,并参与全身炎性反应综合征和多器官功能障碍综合征,其发病原因及致病机制亟待明确。 目的：观察胸部撞击所致钝性肺挫伤急性期细胞凋亡的变化及其川芎嗪对其的影响。 方法：健康雄性SD大鼠随机分为正常对照组、模型组、川芎嗪治疗组,后两组制备胸部撞击伤模型,川芎嗪治疗组建模后立即腹腔注射川芎嗪80 mg/kg 1次。在创伤发生后1,2,3 h观察肺组织病理形态学及细胞凋亡的改变、检测肺水肿程度和肺血管通透性改变,免疫组织化学检测肺组织Bcl-2、Bax和Caspase-3的表达及血液中肿瘤坏死因子α水平变化。 结果与结论：模型组肿瘤坏死因子α水平在创伤后1 h即显著增加,创伤后2 h及3 h间急剧增加(P < 0.05);创伤后2 h及3 h肺组织细胞凋亡指数及肺组织损伤程度显著增高(均P < 0.05);肺血管通透性及肺水肿程度增加(P < 0.05);Caspase-3表达显著增高(P < 0.05),Bcl-2/Bax比值显著降低(P < 0.05)。川芎嗪治疗组在相应时间点相对于模型组肿瘤坏死因子α水平显著降低(P < 0.05),肺组织内细胞凋亡指数及肺组织损伤程度降低(P < 0.05),肺血管通透性及肺水肿程度减轻(P < 0.05);Caspase-3表达下降(P < 0.05),Bcl-2/Bax比值增加(P < 0.01)。结果提示,川芎嗪可通过抑制肿瘤坏死因子α表达,下调Caspase-3的表达并提高Bcl-2/Bax的比值,以降低胸部撞击所致肺组织急性期的异常凋亡并减轻胸部撞击所致急性期肺挫伤。     

Cerebral endothelial damage after severe head injury.

We demonstrate that in head injuries the degree of cerebral endothelial activation or injury depends on the type of brain injury and the patients age, and that in severe head injuries measuring the serum levels of thrombomodulin (TM) and von Willebrand factor (vWF) is useful in evaluating cerebral endothelial injury and activation. The values of vWF in the cases of focal brain injury were significantly higher than in the cases of diffuse axonal injury. The serum levels of TM in focal brain injuries were higher than in diffuse axonal injuries, but the differences were not statistically significant. In patients with delayed traumatic intracerebral hematoma (DTICH), vWF levels were much higher than in patients without DTICH. The values of TM and vWF in elderly patients were significantly higher than in younger patients. These findings indicate that: 1) the degree of endothelial activation in focal brain injury is significantly higher than in diffuse brain injury; 2) the degree of cerebral endothelial injury in patients with DTICH is much higher than in those without DTICH; and 3) the degree of cerebral endothelial activation and injury in elderly head injury patients is significantly higher than in younger patients.     

Brain damage in fatal non-missile head injury without high intracranial pressure.

As part of a comprehensive study of brain damage in 635 fatal non-missile head injuries, the type and prevalence of brain damage occurring in the absence of high intracranial pressure were analysed. Of 71 such cases, 53 sustained their injury as a result of a road traffic accident; only 25 experienced a lucid interval. Thirty eight had a fractured skull, a mean total contusion index of 12.9 and diffuse axonal injury in 29: severe to moderate ischaemic damage was present in the cerebral cortex in 25, brain swelling in 13, and acute bacterial meningitis in nine. The prevalence and range of brain damage that may occur in the absence of high intracranial pressure are important to forensic pathologists in the medicolegal interpretation of cases of fatal head injury.     

Race and the shaken baby syndrome: experience at one hospital

At Children's Hospital of Michigan there seemed to be a disproportionate number of white infants with shaken baby syndrome (SBS) relative to the proportion of white infants in our physically abused population. All reports of suspected child abuse on children less than or equal to 18 months from Children's Hospital from 1980-1985 were reviewed. The total number of abused children 18 months or younger was 545 (447 black, 87 white, 1 unknown). There were 20 children in the SBS group. Eight of 87 (9%) of white abused infants had SBS compared with 12 of 447 (2.7%) of black abused infants. The occurrence of SBS in white abused infants was disproportionately higher than in blacks. This finding has not been previously reported. Before generalizations can be made, additional data must be obtained.