Long-term accumulation of amyloid-beta, beta-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma

Plaques composed of amyloid beta (Abeta) have been found within days following brain trauma in humans, similar to the hallmark plaque pathology of Alzheimer's disease (AD). Here, we evaluated the potential source of this Abeta and long-term mechanisms that could lead to its production. Inertial brain injury was induced in pigs via head rotational acceleration of 110 degrees over 20 ms in the coronal plane. Animals were euthanized at 3 hours, 3 days, 7 days, and 6 months post-injury. Immunohistochemistry and Western blot analyses of the brains were performed using antibodies specific for amyloid precursor protein (APP), Abeta peptides, beta-site APP-cleaving enzyme (BACE), presenilin-1 (PS-1), caspase-3, and caspase-mediated cleavage of APP (CCA). Substantial co-accumulation for all of these factors was found in swollen axons at all time points up to 6 months following injury. Western blot analysis of injured brains confirmed a substantial increase in the protein levels of these factors, particularly in the white matter. These data suggest that impaired axonal transport due to trauma induces long-term pathological co-accumulation of APP with BACE, PS-1, and activated caspase. The abnormal concentration of these factors may lead to APP proteolysis and Abeta formation within the axonal membrane compartment.     

Diffuse axonal injury in infants with nonaccidental craniocerebral trauma: enhanced detection by beta-amyloid precursor protein immunohistochemical staining

OBJECTIVE: Accurate identification of diffuse axonal injury is important in the forensic investigation of infants who have died from traumatic brain injury. beta-Amyloid precursor protein (beta-APP) immunohistochemical staining is highly sensitive in identifying diffuse axonal injury. However, the effectiveness of this method in brain-injured infants has not been well established. The present study was undertaken to assess the utility of beta-APP immunohistochemistry in detecting diffuse axonal injury in infants with either shaken baby syndrome or blunt head trauma. MATERIALS AND METHODS: Archival formalin-fixed, paraffin-embedded blocks from infants (<1 year old) with shaken baby syndrome (7 cases) and blunt head trauma (3) and blocks from 7 control cases that included nontraumatic cerebral edema (1), acute hypoxic-ischemic encephalopathy (1), and normal brain (5) were immunostained for beta-APP. A semiquantitative assessment of the severity of axonal staining was made. Corresponding hematoxylin-eosin-stained sections were examined for the presence of axonal swellings. RESULTS: Immunostaining for beta-APP identified diffuse axonal injury in 5 of 7 infants with shaken baby syndrome and 2 of 3 infants with blunt head trauma. Immunoreactive axons were easily identified and were present in the majority of the sections examined. By contrast, hematoxylineosin staining revealed axonal swellings in only 3 of 7 infants with shaken baby syndrome and 1 of 3 infants with blunt head trauma. Most of these sections had few if any visible axonal swellings, which were often overlooked on initial review of the slides. No beta-APP immunoreactivity was observed in any of the 7 control cases. CONCLUSIONS: Immunostaining for beta-APP can easily and reliably identify diffuse axonal injury in infants younger than 1 year and is considerably more sensitive than routine hematoxylin-eosin staining. We recommend its use in the forensic evaluation of infants with fatal craniocerebral trauma.     

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.     

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.     

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 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.     

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.     

AMPA/kainate receptors mediate axonal morphological disruption in hypoxic white matter

We used acute brain slices to investigate the hypothesis that oxygen-glucose deprivation (OGD) induced loss of axon function and neurofilament labeling are correlated to axonal morphological disruption in the corpus callosum of adult brain. Coronal brain slices including corpus callosum were prepared from adult mice. White matter immunohistochemical properties and conduction along axons remained stable over 12 h after preparation. White matter injury was assessed by recording compound action potentials (CAPs) across corpus callosum, combined with immunofluorescence for axonal neurofilaments and by bright field microscopy of myelin profiles in semi-thin sections. OGD for 30 min resulted in irreversible loss of the CAPs, formation of axon heads and bulbs, and swelling of myelin profiles in slices examined 1h after OGD. In slices followed for 9 h after OGD, there was complete loss of neurofilament labeling and myelin profiles. Because overactivation of AMPA/kainate receptors mediates axon structural and functional disruption in hypoxic corpus callosum slices, we tested whether blockade of AMPA/kainate receptors reduced OGD-induced axonal morphological disruption. NBQX (30 microM), an AMPA/kainate receptor antagonist, prevented OGD-induced formation of axon heads and bulbs, swelling of myelin profiles, loss of neurofilament staining and preserved axonal morphology. These results expand our previous findings that the AMPA/kainate receptor activation contributes to axonal morphological disruption, as well as loss of electrical function.     

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.     

Intraneuronal APP/A beta trafficking and plaque formation in beta-amyloid precursor protein and presenilin-1 transgenic mice

Neuropil deposition of beta-amyloid peptides A beta40 and A beta42 is believed to be the key event in the neurodegenerative processes of Alzheimer's disease (AD). Since A beta seems to carry a transport signal that is required for axonal sorting of its precursor beta-amyloid precursor protein (APP), we studied the intraneuronal staining profile of A beta peptides in a transgenic mouse model expressing human mutant APP751 (KM670/671NL and V7171) and human mutant presenilin-1 (PS-1 M146L) in neurons. Using surface plasmon resonance we analyzed the A beta antibodies and defined their binding profile to APP, A beta40 and A beta42. Immunohistochemical staining revealed that intraneuronal A beta40 and A beta42 staining preceded plaque deposition, which started at 3 months of age. A beta was observed in the somatodendritic and axonal compartments of many neurons. Interestingly, the striatum, which lacks transgenic APP expression harbored many plaques at 10 months of age. This is most likely due to an APP/A beta transport problem and may be a model region to study APP/A beta trafficking as an early pathological event.     

What's new in the diagnosis of head injury?

The diagnosis of DAI is not always easy, and should be based on adequate sampling of appropriate anatomical areas from a sliced, fixed brain. It is now recognised that there is a continuum of traumatic white matter damage, and that DAI represents only the severe end of the scale. Such damage may be detected from very shortly after a head injury-a fact that may give rise to some challenging diagnostic problems. Early axonal injury detected by means of beta APP immunostaining should be interpreted with caution. The most useful tools currently available for detecting axonal damage are antisera to beta APP, PG-M1, and GFAP, used in conjunction with a routine haematoxylin and eosin stain, but even with immunocytochemistry precise dating of histological changes may not be possible.     

β-Amyloid precursor protein (βAPP) as a marker for axonal injury after head injury

It has been demonstrated recently that β-amyloid protein (βAP), generally associated with the plaques of Alzheimer's disease, can also be found in the brains of survivors of head injury. In this study the distribution of the βAP precursor protein (βAPP) was examined immunohistochemically to determine if it is colocalized with βAP in such cases. βAPP immunoreactivity was observed in neuronal perikarya in the neocortex and in dystrophic neurites surrounding βAP immunoreactive plaques i.e. in a distribution similar to that seen in Alzheimer's disease. In addition, βAPP immunoreactivity was noted within white matter tracts where it marked damaged axons. However, no colocalisation of βAPP with βAP was observed in any white matter region. These results indicate that processing of βAPP to produce βAP occurs in the synaptic terminal field of axons and illustrate the utility of βAPP immunoreactivity as a general marker for axonal injury.     

Hypoxia increases Abeta generation by altering beta- and gamma-cleavage of APP

Environmental factors are significant contributors for the development of Alzheimer's disease (AD). The greatly increased incidence of AD following stroke and cerebral ischemia suggests that hypoxia is a risk factor which may accelerate AD pathogenesis by altering amyloid precursor protein (APP) processing. However, the molecular mechanisms underlying the hypoxia mediated AD pathogenesis have not been fully elucidated. In the present study we demonstrated that repeated hypoxia increased beta-amyloid (Abeta) generation and neuritic plaques formation by elevating beta-cleavage of APP in APP(swe)+PS1(A246E) transgenic mice. We also found that hypoxia enhanced the expression of APH-1a, a component of gamma-secretase complex, which in turn may lead to increase in gamma-cleavage activity. Furthermore, we demonstrated that repeated hypoxia treatment can activate macroautophagy, which may contribute to the increases in Abeta production since pretreatment with macroautophagy inhibitor 3-methyladenine significantly blocked chemical hypoxic condition-induced increase in Abeta production in SH-SY5Y cells. Taken together, our results suggest an important role of hypoxia in modulating the APP processing by facilitating both beta- and gamma-cleavage which may result in a significant increase of Abeta generation.     

Traumatic injury in the developing brain – effects of hypothermia

Little is known about the underlying mechanisms of head trauma in the developing brains, despite considerable social and economic impact following such injuries. Age has been shown to substantially influence morbidity and mortality. Children younger than 4 years of age had worse cognitive, motor, and brain atrophy outcomes than children 6 years of age and older. Younger children tend to more frequently suffer from diffuse cerebral swelling compared to adults. Typical autoptic findings also include axonal injury and ischemic neurodegeneration. These differences impact not only the primary response of the brain to injury but the secondary response as well. The complexity of damaging mechanisms in traumatic brain injury contributes to the problem of determining effective therapy. As an alternative/ adjunct to pharmacological approaches, hypothermia has been shown to be cerebroprotective in traumatized adult brains. Although a large number of animal studies have shown protective effects of hypothermia in a variety of damaging mechanisms after TBI, little data exist for young, developing brains. The injury mechanisms of TBI in the immature, effects of hypothermia following resuscitation on adult and immature traumatized brains, and some possible mechanisms of action of hypothermia in the immature traumatized brain are discussed in this review.     

Novel aspects of accumulation dynamics and A beta composition in transgenic models of AD

A homogeneous time-resolved fluorescence immunoassay for detection of beta-amyloid (A beta) peptides has been adapted for quantification of A beta(40) and A beta(42) accumulation in brains of APP695SWE transgenic mice. These over-express human beta APP(swe), beta-amyloid precursor protein (beta-APP) containing the K670N/M671L 'Swedish' familial Alzheimer's disease (FAD) mutation. Both peptides start to accumulate in this line from about 260 to 280 days of age. Co-expression of a human presenilin-1 (PS1) transgene containing the A246E FAD mutation accelerates deposition and also favors-at least initially-accumulation of A beta(42) so that the A beta(2):A beta(40) ratio of peptides from 7- to 12-month-old APP695SWE x PS1A246E animals is significantly elevated above that observed throughout the lifetime of APP695SWE mice. These findings, supported by parallel immunohistochemical staining and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry data, offer important longitudinal characterization of two mouse models of cerebral amyloidosis. Application of the same extraction and quantitation procedures to samples of temporal cortex from AD sufferers indicates however that A beta(40) is only a minor component of beta-amyloid in humans.     

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.     

Protective effect of hypoxic preconditioning on hypoxic-ischemic injured newborn rats

Brief episodes of cerebral hypoxia-ischemia cause transient ischemic tolerance to subsequent ischemic events that are otherwise lethal. This study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic-ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning. The rats were preconditioned with hypoxia (8% oxygen, 92% nitrogen) for three hours, subjected to ischemia using ligation of the right common carotid artery, and then exposed to another three hours of hypoxia. Using proton magnetic resonance spectroscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining, and morphologic scores, this study shows that hypoxic preconditioning 6-hr to 1-day before hypoxic-ischemic injury increases survival rates and has neuroprotective effects against subsequent hypoxic-ischemic injury. The mechanism of the protective effects of hypoxic preconditioning in the newborn rat brain may involve downregulation of apoptotic cell death.     

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.     

Minocycline suppresses hypoxic activation of rodent microglia in culture

Hypoxia is one of the important physiological stimuli that are often associated with a variety of pathological states such as ischemia, respiratory diseases, and tumorigenesis. In the central nervous system, hypoxia that is accompanied by cerebral ischemia not only causes neuronal cell injury, but may also induce pathological microglial activation. We have previously shown that hypoxia induces inflammatory activation of cultured microglia, and the hypoxic induction of nitric oxide production in microglia is mediated through p38 mitogen-activated protein kinase pathway. Now, we present evidence that minocycline, a tetracycline derivative, suppresses the hypoxic activation of cultured microglia by inhibiting p38 mitogen-activated protein kinase pathway. The drug markedly inhibited hypoxia-induced production of inflammatory mediators such as nitric oxide, TNFalpha, and IL-1beta as well as iNOS protein expression. The signal transduction pathway that leads to the activation of p38 mitogen-activated protein kinase was the molecular target of minocycline. Thus, the known neuroprotective effects of minocycline in animal models of cerebral ischemia may be partly due to its direct actions on brain microglia.