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Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability
Abstract
It has recently been demonstrated [Pettus et al., J. Neurotrauma, 11 (1994) 507-522] that moderate traumatic brain injury evokes alterations in axolemmal permeability associated with rapid local compaction of axonal neurofilaments (NF). The current communication fully characterized these local NF changes, while also exploring the possibility of other related cytoskeletal abnormalities. A tracer normally excluded by the intact axolemma (horseradish peroxidase) was administered intrathecally in cats, which were then subjected to moderate/severe fluid percussion brain injury (FPI). After survival times ranging from 5 min to 6 h post-traumatic brain injury (TBI), the animals were perfused and processed for light microscopic (LM) and electron microscopic (EM) visualization of horseradish peroxidase (HRP). HRP-containing axons identified by LM, were investigated by EM in both the sagittal and coronal planes. Electron micrographs were videographically captured, digitized, and analyzed for cytoskeletal distribution. Local alterations in axolemmal permeability to HRP were detected, and consistently linked with distinct cytoskeletal changes. Within 5 min of injury, the injured HRP-containing axons displayed a significant decrease in inter-NF spacing associated with a lack of NF side arm projections. Density analysis proved a significant increase in NF packing in the HRP-containing axons, and further revealed an associated significant decrease in microtubule (MT) density. All ultrastructural changes were seen within 5 min of injury, and persisted unchanged for up to 6 h post-TBI. Collectively, these abnormalities suggest that altered axolemmal permeability triggers a rapid, yet persisting general cytoskeletal change most likely linked to local ionic disregulation. We posit that this local cytoskeletal collapse/alteration marks a site of impaired axonal transport, associated with upstream axoplasmic swelling and eventual axonal detachment.
... Specifically, axon membrane infolding and occasional disconnection are noted [17], which is likely representative of the degradation of the sub-axolemmal protein network that anchors the membrane to the cytoskeleton. Furthermore, the mechanical deformation associated with primary injury can create membrane pores, with an immediate increase in axolemma permeability [18][19][20][21][22][23][24], a process known as mechanoporation. Mechanoporation is principally assessed via the application of cell-impermeable dyes, with the intra-axonal presence of dye indicative of increased axolemma permeability [18][19][20][21][22][23][24], demonstrated in a severity-dependent manner. ...
... Furthermore, the mechanical deformation associated with primary injury can create membrane pores, with an immediate increase in axolemma permeability [18][19][20][21][22][23][24], a process known as mechanoporation. Mechanoporation is principally assessed via the application of cell-impermeable dyes, with the intra-axonal presence of dye indicative of increased axolemma permeability [18][19][20][21][22][23][24], demonstrated in a severity-dependent manner. For example, in primary cortical neurons, severe stretch (10 s −1 , 0.3 magnitude) caused immediate uptake of all tested dyes (380-150,000 Da) in up to 60% of neurons, whereas a milder stretch (1 s −1 , 0.1 magnitude) was associated with minimal dye uptake, regardless of dye size [25]. ...
... In vivo models show a similar injury severity effect with moderate-severe [18][19][20][21][22][23], but not mild injury [20,29] associated with acute axonal dye uptake, evident within 5 min of injury. Like in vitro models, variable uptake was seen within bundles of fibers, with some axons exhibiting dye uptake, but not others, supporting the differential responses of axons in the same anatomical location to injury [18][19][20][21][22][23]. ...
Diffuse axonal injury (DAI) is a significant feature of traumatic brain injury (TBI) across all injury severities and is driven by the primary mechanical insult and secondary biochemical injury phases. Axons comprise an outer cell membrane, the axolemma which is anchored to the cytoskeletal network with spectrin tetramers and actin rings. Neurofilaments act as space-filling structural polymers that surround the central core of microtubules, which facilitate axonal transport. TBI has differential effects on these cytoskeletal components, with axons in the same white matter tract showing a range of different cytoskeletal and axolemma alterations with different patterns of temporal evolution. These require different antibodies for detection in post-mortem tissue. Here, a comprehensive discussion of the evolution of axonal injury within different cytoskeletal elements is provided, alongside the most appropriate methods of detection and their temporal profiles. Accumulation of amyloid precursor protein (APP) as a result of disruption of axonal transport due to microtubule failure remains the most sensitive marker of axonal injury, both acutely and chronically. However, a subset of injured axons demonstrate different pathology, which cannot be detected via APP immunoreactivity, including degradation of spectrin and alterations in neurofilaments. Furthermore, recent work has highlighted the node of Ranvier and the axon initial segment as particularly vulnerable sites to axonal injury, with loss of sodium channels persisting beyond the acute phase post-injury in axons without APP pathology. Given the heterogenous response of axons to TBI, further characterization is required in the chronic phase to understand how axonal injury evolves temporally, which may help inform pharmacological interventions.
... 1 Traumatic brain injury (TBI) is defined as "an alteration in brain function, or other evidence 2 of brain pathology, caused by an external force". 1 In 2013 approximately 2.8 million TBI-3 related emergency department visits, hospitalization, and deaths occurred in the United 4 States. 2 In a recent study reporting the epidemiology of TBI in Europe, Peeters and coworkers 5 analyzed data from 28 studies on 16 European countries and reported an average mortality 6 rate of ≈ 11 per 100,000 population over an incidence rate of 262 per 100,000 population per 7 year. 3 Diffuse axonal injury (DAI), a multifocal damage to white matter axons, is the most 8 common consequence of TBIs of all severities including mild TBIs or concussions. ...
... 4 Invisible 9 to conventional brain imaging, DAI can only be histologically diagnosed and its hallmark is 10 the presence of axonal swellings or retraction balls observable under microscopic 11 examination. 5 ...
... Another peculiarity of the axonal membrane is that both in unmyelinated and myelinated 4 axons, protein channels span the lipid bilayer, making possible the propagation of the action 5 potential. In particular, voltage-gated sodium channels are the main responsible for the 6 continuous and saltatory conduction in unmyelinated and myelinated axons respectively. ...
Traumatic brain injuries are a leading cause of morbidity and mortality worldwide. With almost 50% of traumatic brain injuries being related to axonal damage, understanding the nature of cellular level impairment is crucial. Experimental observations have so far led to the formulation of conflicting theories regarding the cellular primary injury mechanism. Disruption of the axolemma, or alternatively cytoskeletal damage has been suggested mainly as injury trigger. However, mechanoporation thresholds of generic membranes seem not to overlap with the axonal injury deformation range and microtubules appear too stiff and too weakly connected to undergo mechanical breaking. Here, we aim to shed a light on the mechanism of primary axonal injury, bridging finite element and molecular dynamics simulations. Despite the necessary level of approximation, our models can accurately describe the mechanical behavior of the unmyelinated axon and its membrane. More importantly, they give access to quantities that would be inaccessible with an experimental approach. We show that in a typical injury scenario, the axonal cortex sustains deformations large enough to entail pore formation in the adjoining lipid bilayer. The observed axonal deformation of 10-12% agree well with the thresholds proposed in the literature for axonal injury and, above all, allow us to provide quantitative evidences that do not exclude pore formation in the membrane as a result of trauma. Our findings bring to an increased knowledge of axonal injury mechanism that will have positive implications for the prevention and treatment of brain injuries.
... Reduced number of microtubules [19] Yes [14] Increased density of neurofilaments [19] Yes [15] Neurofilament side arms less evident or non-existent [19] Yes [15] Axon contours uneven with alternating swollen and shrunken sections [18,20] Yes [16,17] ...
... Reduced number of microtubules [19] Yes [14] Increased density of neurofilaments [19] Yes [15] Neurofilament side arms less evident or non-existent [19] Yes [15] Axon contours uneven with alternating swollen and shrunken sections [18,20] Yes [16,17] ...
... Reduced number of microtubules [19] Yes [14] Increased density of neurofilaments [19] Yes [15] Neurofilament side arms less evident or non-existent [19] Yes [15] Axon contours uneven with alternating swollen and shrunken sections [18,20] Yes [16,17] ...
This etiologic model proposes that Alzheimers Disease (AD) arises when an unusually rapid increase in ventricle volume triggers axon stretch that culminates in the physical separation of trans-synaptic proteins. As a result, these proteins, such as neurexin, neuroligin, N-Cadherin and Amyloid Precursor Protein (APP), experience a change in the configuration of their cytoplasmic tail, so that instead of transmitting signals to create and maintain synaptic structure they activate enzymes, and generate molecules, that stimulate neurite growth; for example, the transformation of the N-Cadherin tail dissolves its complex with presenilin and β-catenin triggering activation of glycogen synthase kinase 3 beta (GSK3β) and cytoskeletal disruption. This disruption leads to an increase in pro nerve growth factor (proNGF), a molecule that stimulates neurite growth via the p75 neurotrophin receptor (p75). GSK3β contributes to this growth by increasing microtubule instability through the phosphorylation of tau. Separation of trans-synaptic APPs leads to their cis dimerization and this stimulates production of amyloid beta (Aβ), an autocrine growth factor that interacts with both the p75 and alpha 7 nicotinic acetylcholine receptors. Cis dimerization of APPs may also allow the autophosphorylation of Y682 and T668 in the APP cytoplasmic tail, triggering activation of c-Jun N terminal kinase, Abelson kinase and cyclin dependent kinase 5, all of which play a role in neurite growth. ProNGF, Aβ and the kinase cascades work together to transform synapses into growth cones and stimulate sprouting of neuropil threads in an attempt to reconnect axons and dendrites. Neurofibrillary tangles, located in neural cell soma, consist of neurofilaments and microtubules needed to fuel this renewal of neurite growth. The model suggests that the best way to treat AD is to prevent synaptic separation by identifying individuals experiencing unusually high rates of ventricle growth and reducing this to more normal levels by shunting or some other technique.
... High-power views (Fig. 3D-F) revealed that the aggregate was composed of unmyelinated axons and astroglial processes (arrowheads, Fig. 3D). The former were rich in neurofilaments (arrows, Fig. 3F) but had few microtubules (Fig. 3F), as seen in axons receiving a traumatic injury (Pettus et al., 1994;Pettus and Povlishock 1996;Okonkwo et al., 1998) axons (Fig. 3F). Therefore, a scalpel-transection of the white matter would immediately produce aggregates of neurofilament-rich axons and astroglial processes, "axon-glial complex (AGC)" at the lesion site, which would be a physicochemical barrier for regenerative pioneering axons. ...
... These events were followed by a secondary axotomy near the transection site, which finally formed abnormal zipper-like axon segments (fragments) at the transection site. Povlishock and co-workers have reported a similar axonal pathology immediately after a traumatic brain injury as a focal axonal injury, which includes a focal perturbation of axolemmal permeability, a rapid compaction of axonal neurofilament, an enhanced expression of NF-IR and a microtubular loss (Pettus et al., 1994;Pettus and Povlishock 1996;Okonkwo et al., 1998). The present study further confirmed the abundance of neurofilaments and poverty of microtubules in the abnormal axon segments by an electron microscopy. ...
In general, axonal regeneration is very limited after transection of adult rat spinal cord. We previously demonstrated that regenerative axons reached the lesion site within 6hours of sharp transection with a thin scalpel. However, they failed to grow across the lesion site, where injured axon fragments (axon-glial complex, AGC) were accumulated. Considering a possible role of these axon fragments as physicochemical barriers, we examined the effects of prompt elimination of the barriers on axonal growth beyond the lesion site. In this study, we made additional oblique section immediately after the primary transection and surgically eliminated the AGC (debridement). Under this treatment, regenerative axons successfully traversed the lesion site within 4hours of surgery. To exclude axonal sparing, we further inserted a pored sheet into the debrided lesion and observed the presence of fascicles of unmyelinated axons traversing the sheet through the pores by electron microscopy, indicating bona fide regeneration. These results suggest that the sequential trial of reduction and early elimination of the physicochemical barriers is one of effective approaches to induce spontaneous and rapid regeneration beyond the lesion site.
... TBIs result from a primary injury induced by an external force to the brain, such as direct impact, acceleration/deceleration or blast. The primary injury initiates a secondary pathophysiological cascade, which is characterized by excitotoxicity, the generation of free radicals and lipid peroxidation [22], mitochondrial dysfunction [23], swelling and loss of astrocytes [13], axonal swelling [24] and neuronal injury [25]. Secondary injury is associated with inflammatory response and alterations in both metabolism and cerebral blood flow [26], axonal lysis and breakdown with parenchymal accumulation of tau and amyloid beta (Aβ) protein [27], demyelination [28] and subsequent axonal degeneration [29] and programmed neuronal death through a caspase-3 activation mechanism [25]. ...
... As pointed out earlier, DAI is the signature injury in TBI [13,24,29]. Likewise, DAI was recently identified by the presence of APP-positive axonal swellings typical after blast exposure [93]. ...
Traumatic brain injury (TBI) is one of the leading causes of death and disability around the world. The lack of validated biomarkers for TBI is a major impediment to developing effective therapies and improving clinical practice, as well as stimulating much work in this area. In this review, we focus on different settings of TBI management where blood or cerebrospinal fluid (CSF) biomarkers could be utilized for predicting clinically-relevant consequences and guiding management decisions. Requirements that the biomarker must fulfill differ based on the intended context of use (CoU). Specifically, we focus on fluid biomarkers in order to: (1) identify patients who may require acute neuroimaging (cranial computerized tomography (CT) or magnetic resonance imaging (MRI); (2) select patients at risk for secondary brain injury processes; (3) aid in counseling patients about their symptoms at discharge; (4) identify patients at risk for developing postconcussive syndrome (PCS), posttraumatic epilepsy (PTE) or chronic traumatic encephalopathy (CTE); (5) predict outcomes with respect to poor or good recovery; (6) inform counseling as to return to work (RTW) or to play. Despite significant advances already made from biomarker-based studies of TBI, there is an immediate need for further large-scale studies focused on identifying and innovating sensitive and reliable TBI biomarkers. These studies should be designed with the intended CoU in mind.
... Immediately after LFPI, injured animals exhibit cortical contusion, changes in blood pressure, increased intracranial pressure, reduced perfusion pressure and cerebral blood flow, and increased blood-brain barrier permeability (McIntosh et al., 1989;Pfenninger, Reith, Breitig, Grunert, & Ahnefeld, 1989;Schmidt & Grady, 1995;Tanno, Nockels, Pitts, & Noble, 1992). In addition, histopathological analyses have verified the presence of gradual gray matter damage, brain edema, and intracerebral hemorrhage in these animals (Dietrich, Alonso, & Halley, 1994;Graham, Mcintosh, Maxwell, & Nicoll, 2000;Pettus & Povlishock, 1996). Moreover, LFPI induces cognitive, behavioral, and sensorimotor function alterations similar to those experienced by TBI patients (Faden et al., 2003;Floyd et al., 2002;Hamm, Lyeth, Jenkins, O'Dell, & Pike, 1993;Hogg, Moser, & Sanger, 1998;Johnstone et al., 2018;Smith, Okiyama, Thomas, Claussen, & McIntosh, 1991). ...
... Interestingly, we found that white matter tract permeability was relatively rare with only the highest angular velocity head rotation in the sagittal plane inducing appreciable primary axonal permeability. This finding is partially supported by seminal studies done in the 1990's using a feline model of fluid percussion injury that showed that following mild TBI, axons did not exhibit flooding with HRP yet displayed characteristic neurofilament compaction and axonal swelling consistent with classic DAI neuropathology (Pettus et al., 1994;Pettus and Povlishock, 1996;Okonkwo et al., 1998). However, these studies did show focal axolemmal permeability to HRP following moderate-to-severe TBI that was also associated with local mitochondrial abnormalities in addition to increased neurofilament packing and microtubule loss. ...
Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY ⁺ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY ⁺ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY ⁺ and LY – cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.
... Multiple factors are involved in this process, but the key role is played by the neurofilament protein subunits, with the misalignment of the filaments, the disruption of axoplasmic flow, and swelling [34]. Ultrastructural studies [35] indicated that the neurofilament response to injury depends on the severity of the event. Changes occurring in the axon trigger the regulation of cellular proteins in the cell body (as, for instance, actin and tubulin) towards successful axonal regeneration and synaptogenesis [36]. ...
Wound healing is characterized by the formation of a granulation tissue consisting of inflammatory cells, newly formed blood vessels, and fibroblasts embedded in a loose collagenous extracellular matrix. Tumors behave as wounds that fail to heal. Neuronal loss in neurodegenerative disease is associated with the synthesis and release of new components of the extracellular matrix by activated fibroblasts and astrocytes. This condition is responsible for a perpetuation of the wound healing state and constitutes a condition very similar to that which occurs during tumor progression. The aim of this article is to emphasize and compare the role of wound healing in two different pathological conditions, namely tumor growth and central nervous system neurodegenerative diseases. Both are conditions in which wounds fail to heal, as occurs in physiological conditions.
... Mechanoporation refers to the formation of pores in the cell membrane induced by mechanical forces [46][47][48]. These pores form in milliseconds because of membrane stretching, which leads to the inhibition of action potential generation [49]. ...
Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality. Consequences vary from mild cognitive impairment to death and, no matter the severity of subsequent sequelae, it represents a high burden for affected patients and for the health care system. Brain trauma can cause neuronal death through mechanical forces that disrupt cell architecture, and other secondary consequences through mechanisms such as inflammation, oxidative stress, programmed cell death, and, most importantly, excitotoxicity. This review aims to provide a comprehensive understanding of the many classical and novel pathways implicated in tissue damage following TBI. We summarize the preclinical evidence of potential therapeutic interventions and describe the available clinical evaluation of novel drug targets such as vitamin B12 and ifenprodil, among others.
... In fact, approximately 80% of TBI cases are mild to moderate such that cognitive, motor, and somatosensory symptoms such as dizziness, headaches, attention loss may go un-noticed however, somatic and memory deficits are common and may progress further if left untreated due to alterations in signaling pathways (Bazarian et al., 2005;Langlois et al., 2006;Laskowski et al., 2015). For example, mild TBI can cause axonal injury due to focal, microscopic mechanoporation (Pettus et al., 1994;Pettus and Povlishock, 1996) leading to abnormal intra-axonal Ca 2+ influx (Büki et al., 1999). Furthermore, oral administration of a commercially available 10% CBD was also effective against chronic pain associated with anxious and aggressive behavior, depression, and impaired social interaction behavior in TBI mice (Belardo et al., 2019). ...
Despite the high incidence of traumatic brain injury (TBI), there is no universal treatment to safely treat patients. Blunt brain injuries destroy primary neural tissue that results in impaired perfusion, excessive release of glutamate, inflammation, excitotoxicity, and progressive secondary neuronal cell death. We hypothesized that administration of cannabidiol (CBD) directly to a brain contusion site, will optimize delivery to the injured tissue which will reduce local neural excitation and inflammation to spare neural tissue and improve neurological outcome following TBI. CBD was infused into a gelfoam matrix forming an implant (CBDi), then applied over the dura at the contusion site as well as delivered systemically by injection (CBD.IP). Post-injury administration of CBDi+IP greatly reduced defecation scores, lesion volume, the loss of neurons in the ipsilateral hippocampus, the number of injured neurons of the contralateral hippocampus, and reversed TBI-induced glial fibrillary acidic protein (GFAP) upregulation which was superior to either CBD.IP or CBDi treatment alone. Vestibulomotor performance on the beam-balance test was restored by 12 days post-TBI and sustained through 28 days. CBDi+IP treated rats exhibited preinjury levels of spontaneous alternation on the spontaneous alternation T-maze. In the object recognition test, they had greater mobility and exploration of novel objects compared to contusion or implant alone consistent with reduced anxiety and restored cognitive function. These results suggest that dual therapy by targeting the site of injury internally with a CBD-infused medical carrier followed by systemic supplementation may offer a more effective countermeasure than systemic or implant treatment alone for the deleterious effects of penetrating head wounds.
... Experimental observations have so far led to the formulation of two main theories regarding the cellular primary injury mechanism. Disruption of the axolemma (Pettus and Povlishock, 1996;LaPlaca and Thibault, 1997;Fitzpatrick et al., 1998), or alternatively cytoskeletal damage (Tang-Schomer et al., 2012) has been suggested mainly as injury trigger. However, using a purely mechanical approach we discarded microtubule damage as injury trigger and revealed instead high level of strains on the axonal membrane (Montanino and Kleiven, 2018). ...
Around half of the traumatic brain injuries are thought to be axonal damage. Disruption of the cellular membranes, or alternatively cytoskeletal damage has been suggested as possible injury trigger. Here, we have used molecular models to have a better insight on the structural and mechanical properties of axon sub-cellular components. We modelled myelin sheath and node of Ranvier as lipid bilayers at a coarse grained level. We built ex-novo a model for the myelin. Lipid composition and lipid saturation were based on the available experimental data. The model contains 17 different types of lipids, distributed asymmetrically between two leaflets. Molecular dynamics simulations were performed to characterize the myelin and node-of-Ranvier bilayers at equilibrium and under deformation and compared to previous axolemma simulations. We found that the myelin bilayer has a slightly higher area compressibility modulus and higher rupture strain than node of Ranvier. Compared to the axolemma in unmyelinated axon, mechanoporation occurs at 50% higher strain in the myelin and at 23% lower strain in the node of Ranvier in myelinated axon. Combining the results with finite element simulations of the axon, we hypothesizes that myelin does not rupture at the thresholds proposed in the literature for axonal injury while rupture may occur at the node of Ranvier. The findings contribute to increases our knowledge of axonal sub-cellular components and help to understand better the mechanism behind axonal brain injury.
... For example, increased numbers of mitochondria have been observed in axons belonging to patients with MS than equivalent axons in healthy controls (Mahad et al., 2009), as well as experimentally demyelinated axons in cats (Mutsaers and Carroll, 1998) and larval zebrafish (?). Furthermore, measurement of cytoskeletal morphological properties using TEM images is an established readout of axonal degeneration during development (Lyons et al., 2005) and following injury (Fournier et al., 2015;Pettus and Povlishock, 1996). The described analysis should be performed prior to assigning future behavioural phenotypes solely to the absence of myelination. ...
Activity-mediated myelination, the adjustment of myelin morphology in response to
neuronal activity, has been proposed as a novel mechanism of central nervous system
(CNS) plasticity. As a key regulator of conduction velocity, adaptations in myelin structure
have the potential to exert spatiotemporal control over action potentials across
neurons of a given circuit, in turn influencing circuit function and behaviour. Despite
a breadth of evidence supporting this hypothesis, a defi�nitive conclusion has been hindered
by the technical difficulties of assessing circuit activity in parallel with myelin
morphology and behaviour in vivo.
Using larval zebra�sh as a model, this study investigated the effect of disrupting
the normal program of CNS myelination on the development of locomotor behaviour.
CRISPR/Cas9 mutagenesis of Myelin Regulatory Factor (Myrf) gene, encoding for a
transcription factor vital for CNS myelination, was used to create a CNS specifi�c model
of hypomyelination. Larvae from Myrf heterozygous in-crosses were then tested across
a suite of behavioural assays, allowing the measurement of detailed kinematic parameters
during spontaneous and stimulus-driven responses.
Myrf homozygous mutants displayed a 66% reduction in the number of myelinated
axons in the spinal cord along with reduced gene expression of myelin basic protein
(Mbp). Unexpectedly, heterozygous animals exhibited precocious myelination of small
caliber axons, resulting in a 53% increase in the number of myelinated axons. This
fi�nding was associated with a subtle upregulation of Mbp gene expression. Subsequent
behavioural analysis revealed that Myrf homozygous mutants demonstrated a significant
delay in the latency to perform acoustic startle responses. Interestingly, both
homozygous and heterozygous mutants exhibited an increase in the frequency of high
velocity swim bouts performed during spontaneous swimming, driven by subtle adjust-
ments in tail kinematics.
The fi�ndings of this study support a role for myelination in the control of action potential
timing across de�ned circuits of the CNS and suggest that a balance of myelination
is important for the function of more complex circuits such as those controlling swim
speed. Future work, using in vivo electrophysiology and functional imaging, will interrogate
how neuronal activity is altered in the circuits underlying these behaviours.
Together, these fi�ndings will advance our understanding of the role that CNS myelination
plays in circuit function and behaviour.
... Pore formation in a cell membrane induced by mechanical forces is referred to as mechanoporation, which primarily has been investigated by using molecular markers to evaluate cell membrane permeability following experimental brain trauma (28,29). Briefly, the procedure starts by injecting a marker which floods the cerebral extracellular space. ...
Several hypotheses have been put forth over time to explain how consciousness can be so rapidly lost, and then spontaneously regained, following mechanical head trauma. The knockout punch in boxing is a relatively homogenous form of traumatic brain injury and can thus be used to test the predictions of these hypotheses. While none of the hypotheses put forth can be considered fully verified, pore formation following stretching of the axonal cell membrane, mechanoporation, is a strong contender. We here argue that the theoretical foundation of mechanoporation can be strengthened by a comparison with the experimental method electroporation.
... [16][17][18] Nonspecific mechanoporation may contribute to many of the acute cellular responses following a mechanical insult, such as a loss in ion homeostasis, influx of extracellular molecules, and leakage of cytosolic constituents. 10,[19][20][21][22] Furthermore, plasma membrane damage has been associated with conduction block, 23 aberrant cell signaling, 24 protease activation, 9 alterations in cellular morphology and cytoskeletal integrity, 7,8,25 and delayed cell death. 6,15 The mechanical threshold for membrane damage in brain is not fully understood; however, there is evidence for a relationship between loading intensity and degree of membrane damage in many settings. ...
The acute response of neurons subjected to traumatic loading involves plasma membrane disruption, yet the mechanical tolerance for membrane compromise, time course, and mechanisms for resealing are not well understood. We have used an in vitro traumatic neuronal injury model to investigate plasma membrane integrity immediately following a high-rate shear injury. Cell-impermeant fluorescent molecules were added to cortical neuronal cultures prior to insult to assess membrane integrity. The percentage of cells containing the permeability marker was dependent on the molecular size of the marker, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were positively correlated with insult loading rate. Membrane disruption was transient, evidenced by a membrane resealing within the first minute after the insult. In addition, chelation of either extracellular Ca ²⁺ or intracellular Ca ²⁺ limited membrane resealing. However, injury following chelation of both extracellular and intracellular Ca ²⁺ caused diminished permeability as well as a greater resealing ability compared to chelation of extracellular or intracellular Ca ²⁺ alone. Treatment of neuronal cultures with jasplakinolide, which stabilizes filamentous actin, reduced permeability increases, while latrunculin-B, an actin depolymerizing agent, both reduced the increase in plasma membrane permeability and promoted resealing. This study gives insight into the dynamics of neuronal membrane disruption and subsequent resealing, which was found to be calcium dependent and involve actin in a role that differs from non-neuronal cells. Taken together, these data will lead to a better understanding of the acute neuronal response to traumatic loading.
... Increases in the concentration of intracellular calcium trigger a cascade that leads to the collapse of the neurofilament with disassembly of the microtubules. This results in axonal damage, known as secondary axotomy, which might be part of the pathobiology of mTBI [20,21]. The neuroinflammation after TBI might induce excitability of the central nervous system. ...
Background: Oftentimes, persistent post traumatic headache (PPTH) and migraine are phenotypically similar and the only clinical feature that differentiate them is the presence of a mild or moderate traumatic brain injury (mTBI). The aim of this study is to describe the differences in brain area and in biochemical cascade after concussion and to define the efficacy and safety of treatments in use. Methods: Sources were chosen in according to the International Classification of Headache Disorder (ICHD) criteria. Results: The articles demonstrated a significant difference between PPTH and migraine regarding static functional connectivity (sFC) and dynamic functional connectivity (dFC) in brain structure that could be used for exploring the pathophysiological mechanisms in PPTH. Many studies described a cascade of neuro-metabolic changes that occur after traumatic brain injury. These variations are associated to the mechanism occurring when developing a PPTH. Conclusions: The state of art of this important topic show how although the mechanisms underlying the development of the two different diseases are different, the treatment of common migraine is efficacious in patients that have developed a post traumatic form.
... It is also important to stress that our model represents a generic portion within the distal axon of an unmyelinated neuron. Several studies have so far proposed specific axonal sites such as the nodal, paranodal, internodal segments as primary sites of injury (109)(110)(111). Few studies have also highlighted the susceptibility to injury of the axon initial segment (AIS), the parasomatic region where action potentials are initiated (112)(113)(114). ...
Traumatic brain injuries are a leading cause of morbidity and mortality worldwide. With almost 50% of traumatic brain injuries being related to axonal damage, understanding the nature of cellular level impairment is crucial. Experimental observations have so far led to the formulation of conflicting theories regarding the cellular primary injury mechanism. Disruption of the axolemma, or alternatively cytoskeletal damage has been suggested mainly as injury trigger. However, mechanoporation thresholds of generic membranes seem not to overlap with the axonal injury deformation range and microtubules appear too stiff and too weakly connected to undergo mechanical breaking. Here, we aim to shed a light on the mechanism of primary axonal injury, bridging finite element and molecular dynamics simulations. Despite the necessary level of approximation, our models can accurately describe the mechanical behavior of the unmyelinated axon and its membrane. More importantly, they give access to quantities that would be inaccessible with an experimental approach. We show that in a typical injury scenario, the axonal cortex sustains deformations large enough to entail pore formation in the adjoining lipid bilayer. The observed axonal deformation of 10–12% agree well with the thresholds proposed in the literature for axonal injury and, above all, allow us to provide quantitative evidences that do not exclude pore formation in the membrane as a result of trauma. Our findings bring to an increased knowledge of axonal injury mechanism that will have positive implications for the prevention and treatment of brain injuries.
... Neurofilaments are the most abundant cytoskeletal filaments in myelinated axons and are an indication of axonal caliber [22,56]. Compaction of neurofilament are thought to be responsible for impaired transport. ...
Traumatic brain injury leads to delayed secondary injury events consisting of cellular and molecular cascades, that exacerbate the initial injury. Human umbilical cord perivascular cells (HUCPVC) secrete neurotrophic and pro-survival factors. Here we examined the effects of HUCPVC in sympathetic axon and cortical axon survival models and sought to determine whether HUCPVC provide axonal survival cues. We then examined the effects of the HUCPVC in an in vivo fluid percussion injury (FPI) model of traumatic brain injury (TBI). Our data indicate that HUCPVC express neurotrophic and neural survival factors. They also express and secrete relevant growth and survival proteins when cultured alone, or in the presence of injured axons. Co-culture experiments indicate that HUCPVC interact preferentially with axons when co-cultured with sympathetic neurons and reduce axonal degeneration. NGF withdrawal in axonal compartments resulted in 66 ± 3 % axon degeneration whereas HUCPVC co-culture rescued axon degeneration, to 35 ± 3 %. Inhibition of Akt (LY294002) resulted a significant increase in degeneration compared to HUCPVC co-cultures (48 ± 7% degeneration). Under normoxic conditions, control cultures showed 39 ± 5% degeneration. OGD resulted in 58 ± 3% degeneration and OGD HUCPVC co-cultures reduced degeneration to 34 ± 5% (p<0.05). In an in vivo model of TBI, immunohistochemical analysis of NF200 showed improved axon morphology in HUCPVC-treated animals compared to injured animals. The data presented here indicate an important role for perivascular cells in protecting axons from injury and a potential cell-based therapy to treat secondary injury after TBI.
... Repetitive head trauma, such as concussive and subconcussive impact injury, produces primary axonal injury through a physical stress and cytoskeletal disruption [37][38][39][40][41][42][43][44]. Axonal injury also triggers molecular signaling pathways that result in axonal degeneration (so called secondary axotomy) [45]. ...
Chronic traumatic encephalopathy (CTE) is a distinct neurodegenerative disease that associated with repetitive head trauma. CTE is neuropathologically defined by the perivascular accumulation of abnormally phosphorylated tau protein in the depths of the sulci in the cerebral cortices. In advanced CTE, hyperphosphorylated tau protein deposits are found in widespread regions of brain, however the mechanisms of the progressive neurodegeneration in CTE are not fully understood. In order to identify which proteomic signatures are associated with CTE, we prepared RIPA-soluble fractions and performed quantitative proteomic analysis of postmortem brain tissue from individuals neuropathologically diagnosed with CTE. We found that axonal guidance signaling pathwayrelated proteins were most significantly decreased in CTE. Immunohistochemistry and Western blot analysis showed that axonal signaling pathway-related proteins were down regulated in neurons and oligodendrocytes and neuron-specific cytoskeletal proteins such as TUBB3 and CFL1 were reduced in the neuropils and cell body in CTE. Moreover, oligodendrocyte-specific proteins such as MAG and TUBB4 were decreased in the neuropils in both gray matter and white matter in CTE, which correlated with the degree of axonal injury and degeneration. Our findings indicate that deregulation of axonal guidance proteins in neurons and oligodendrocytes is associated with the neuropathology in CTE. Together, altered axonal guidance proteins may be potential pathological markers for CTE.
... The loss of viscoelasticity in axons that avoid disconnection post-SRC is particularly relevant in the context of RHIE (Christman, Grady, Walker, Holloway, & Povlishock, 1994), in which subsequent impacts of lesser force may be sufficient for causing primary or secondary axotomy. Secondary cell damage resulting from disrupted axonal transport systems and neurofilament accumulation precipitates microstructural cell damage (Giza & Hovda, 2014;Pettus & Povlishock, 1996). Giza and Hovda (2014) consolidated findings from preclinical research and described the pathophysiology associated with concussion, commonly referred to as the "neurometabolic cascade." ...
Concussions occur frequently among the general population, with athletes and certain
military personnel being particularly vulnerable. Population differences (demographics,
risk factors, injury frequency, injury severity, etc.) require that both clinicians and
researchers carefully consider individual patient or participant characteristics. Recent
evidence suggests that the physiological alterations associated with concussion may
outlast the symptoms that manifest, although current concussion management is almost
exclusively dictated by clinical presentation. Accordingly, there has been a rapid rise in
research aiming to better characterize the neurophysiological effects of concussion and
time course of neurobiological recovery after injury, particularly through employment of
advanced neuroimaging metrics and fluid-based biomarkers. Concussion prevention
efforts, particularly in athletic settings, range from limiting head impact exposure to
mitigating recovery time (e.g., post-concussion syndrome) through identification of both
neurobiological and psychosocial risk factors. Recommendations for extended periods of
complete physical and cognitive rest after concussion have been replaced with guidelines
for symptom-limited activity much earlier in the recovery process. Ultimately,
improvements in both physiological and clinical injury identification, as well as
concussion rehabilitation, may reduce the long-term impact of single-event or multiple
concussions. The current literature on the relationship between concussion history and
late-life risk for neurodegeneration or dementia is mixed. Several important topics
related to the acute, subacute, and long-term outcomes of concussion require further
study and clarification. Ongoing large-scale research initiatives hold the promise of
accelerating access to essential, yet complex, answers.
... Axonal stretch results in mechanoporation that allows ionic flux directly into the axoplasm. Influx of calcium has been demonstrated to result in neurofilament side-arm cleavage and microtubule collapse (Buki and Povlishock, 2006;Pettus and Povlishock, 1996). This disruption of axonal cytoskeletal integrity impairs critical functions such as axonal transport, resulting in accumulation of molecules within the axon and axonal blebbing, demonstrated both experimentally and in humans (Johnson et al., 2013;Tang-Schomer et al., 2012). ...
Traumatic brain injury is the leading cause of death and disability in the United States, and may be associated with long lasting impairments into adulthood. The multitude of ongoing neurobiological processes that occur during brain maturation confer both considerable vulnerability to TBI but may also provide adaptability and potential for recovery. This review will examine and synthesize our current understanding of developmental neurobiology in the context of pediatric TBI. Delineating this biology will facilitate more targeted initial care, mechanism-based therapeutic interventions and better long-term prognostication and follow-up.
... For example, dynamic stretch injury of neuronal cultured cells induced axon degeneration, which was inhibited by taxol (Tang-Schomer et al., 2010). This strengthens earlier research in this area, suggesting that cytoskeletal changes begin as early as 5 min post fluid percussive injury (Pettus and Povlishock, 1996), indicating that changes to microtubules could be one of the earliest events to occur after initiating axonal injury and degeneration. Another study which investigated alpha tubulin levels after optic nerve stretch injury found decreased alpha tubulin levels between 0.5 and 4 h postinjury, and a secondary decline after 72 h post-injury (Serbest et al., 2007). ...
Axon degeneration has been implicated as a pathological process in several neurodegenerative diseases and acquired forms of neural injury. We have previously shown that stabilizing microtubules can protect axons against excitotoxin-induced fragmentation, however, the alterations of microtubules following excitotoxicity that results in axon degeneration are currently unknown. Hence, this study investigated whether excitotoxicity affects the post-translational modifications of microtubules and microtubule-associated proteins, and whether reversing these changes has the potential to rescue axons from degeneration. To investigate microtubule alterations, primary mouse cortical neurons at 10 days in vitro were treated with 10 or 25 μM kainic acid to induce excitotoxicity and axon degeneration. Post-translational modifications of microtubules and associated proteins were examined at 6 h following kainic acid exposure, relative to axon degeneration. While there were no changes to tyrosinated tubulin or MAP1B, acetylated tubulin was significantly (p < 0.05) decreased by 40% at 6 h post-treatment. To determine whether increasing microtubule acetylation prior to kainic acid exposure could prevent axon fragmentation, we investigated the effect of reducing microtubule deacetylation with the HDAC6 inhibitor, trichostatin A. We found that trichostatin A prevented kainic acid-induced microtubule deacetylation and significantly (p < 0.05) protected axons from fragmentation. These data suggest that microtubule acetylation is a potential target for axonal protection where excitotoxicity may play a role in neuronal degeneration.
... Despite the identification of axonal injury as a predominant cellular pathology in pediatric TBI, surprisingly few studies have focused on attenuating axonal degeneration. Proposed secondary mechanisms underlying traumatic axonal injury include ionic dysregulation, impaired axonal transport, calpain-mediated proteolysis, calcineurin-mediated dephosphorylation, microtubule degradation, and neurofilament compaction (Pettus et al., 1994;Pettus and Povlishock, 1996;Saatman et al., 1996;Okonkwo et al., 1998;Buki et al., 1999;Stone et al., 2001;Reeves et al., 2007). Calpain activation has been documented within injured axons in the immature rat (Huh et al., 2006 although administration of calpain inhibitor III (MDL28,170) at doses of either 30 mg/kg or 60 mg/kg over the first 24 h following TBI in the juvenile rat reduced calpain activation but not axonal function deficits (unpublished observations). ...
Pediatric traumatic brain injury (TBI) remains one of the leading causes of morbidity and mortality in children. Experimental and clinical studies demonstrate that the developmental age, the type of injury (diffuse vs. focal) and sex may play important roles in the response of the developing brain to a traumatic injury. Advancements in acute neurosurgical interventions and neurocritical care have improved and led to a decrease in mortality rates over the past decades. However, survivors are left with life-long behavioral deficits underscoring the need to better define the cellular mechanisms underlying these functional changes. A better understanding of these mechanisms some of which begin in the acute post-traumatic period may likely lead to targeted treatment strategies. Key considerations in designing pre-clinical experiments to test therapeutic strategies in pediatric TBI include the use of age-appropriate and pathologically-relevant models, functional outcomes that are tested as animals age into adolescence and beyond, sex as a biological variable and the recognition that doses and dosing strategies that have been demonstrated to be effective in animal models of adult TBI may not be effective in the developing brain.
This article is part of the Special Issue entitled “Novel Treatments for Traumatic Brain Injury”.
... Traumatically-induced plasma membrane damage-mechanoporation-in both axons and somata, has been observed across many experimental models of traumatic central nervous system (CNS) injury and is considered a part of the acute injury response (Pettus et al., 1994, LaPlaca et al., 1997, Geddes et al., 2003a, 2003b, Singleton and Povlishock, 2004, Stone et al., 2004, Farkas et al., 2006, Cho et al., 2007, Kilinc et al., 2008, Whalen et al., 2008, LaPlaca and Prado, 2010, Cullen et al., 2011. Plasmalemma compromise is associated with calcium influx and general ion dysregulation, as well as rapid onset of faulty signaling and degradative cellular processes (Geddes et al., 2003a(Geddes et al., , 2003bKilinc et al., 2009;LaPlaca et al., 1997;Okonkwo et al., 1998;Pettus and Povlishock, 1996;Singleton and Povlishock, 2004;Whalen et al., 2008). Whether membrane permeability sets the course for cell death, however, likely depends on the level of membrane damage, resealing capacity of the cell, and course of secondary injury (Farkas et al., 2006;Mbye et al., 2012;Whalen et al., 2008). ...
Background:
An increases in plasma membrane permeability is part of the acute pathology of traumatic brain injury and may be a function of excessive membrane force. This membrane damage, or mechanoporation, allows non-specific flux of ions and other molecules across the plasma membrane, and may ultimately lead to cell death. The relationships among tissue stress and strain, membrane permeability, and subsequent cell degeneration, however, are not fully understood.
Methods:
Fluorescent molecules of different sizes were introduced to the cerebrospinal fluid space prior to injury and animals were sacrificed at either 10 min or 24 h after injury. We compared the spatial distribution of plasma membrane damage following controlled cortical impact in the rat to the stress and strain tissue patterns in a 3-D finite element simulation of the injury parameters.
Findings:
Permeable cells were located primarily in the ipsilateral cortex and hippocampus of injured rats at 10 min post-injury; however by 24 h there was also a significant increase in the number of permeable cells. Analysis of colocalization of permeability marker uptake and Fluorojade staining revealed a subset of permeable cells with signs of degeneration at 24 h, but plasma membrane damage was evident in the vast majority of degenerating cells. The regional and subregional distribution patterns of the maximum principal strain and shear stress estimated by the finite element model were comparable to the cell membrane damage profiles following a compressive impact.
Interpretation:
These results indicate that acute membrane permeability is prominent following traumatic brain injury in areas that experience high shear or tensile stress and strain due to differential mechanical properties of the cell and tissue organization, and that this mechanoporation may play a role in the initiation of secondary injury, contributing to cell death.
... Previous models have employed either uniaxial [24][25][26] or biaxial [27][28][29] loading modes. Biaxial loading induces channel-independent calcium influx via mechanoporation of the cell membrane, which is a widely accepted injury mechanism [30][31][32] . Uniaxial loading isolates calcium influx due to activation of stretch-sensitive ion channels 33 . ...
Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types. © 2018, Journal of Visualized Experiments. All rights reserved.
... The earliest change in spared axons following traumatic insult is swelling of axonal mitochondria 62 . High intracellular concentrations of Ca 2+ result in sequestering of these ions into mitochondria, leading to mitochondrial failure and reduced ATP production 63 , generation of free radicals and apoptotic cell death 64 . ...
Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.
... Organic materials begin to accumulate along the axon creating periodic swellings (Christman et al., 1994;Povlishock and Becker, 1985;Smith et al., 1999). These varicosities signal a breakdown in cellular structure ( Pettus and Povlishock, 1996;Povlishock and Pettus, 1996;Saatman et al., 2003). Axons may even- tually disconnect from the cell body and deteriorate, a process known as Wallerian degeneration ( Wang et al., 2012). ...
Standard MRI methods are often inadequate for identifying mild traumatic brain injury (TBI). Advances in diffusion tensor imaging now provide potential biomarkers of TBI among white matter fascicles (tracts). However, it is still unclear which tracts are most pertinent to TBI diagnosis. This study ranked fiber tracts on their ability to discriminate patients with and without TBI. We acquired diffusion tensor imaging data from military veterans admitted to a polytrauma clinic (Overall n = 109; Age: M = 47.2, SD = 11.3; Male: 88%; TBI: 67%). TBI diagnosis was based on self-report and neurological examination. Fiber tractography analysis produced 20 fiber tracts per patient. Each tract yielded four clinically relevant measures (fractional anisotropy, mean diffusivity, radial diffusivity, and axial diffusivity). We applied receiver operating characteristic (ROC) analyses to identify the most diagnostic tract for each measure. The analyses produced an optimal cutpoint for each tract. We then used kappa coefficients to rate the agreement of each cutpoint with the neurologist's diagnosis. The tract with the highest kappa was most diagnostic. As a check on the ROC results, we performed a stepwise logistic regression on each measure using all 20 tracts as predictors. We also bootstrapped the ROC analyses to compute the 95% confidence intervals for sensitivity, specificity, and the highest kappa coefficients. The ROC analyses identified two fiber tracts as most diagnostic of TBI: the left cingulum (LCG) and the left inferior fronto-occipital fasciculus (LIF). Like ROC, logistic regression identified LCG as most predictive for the FA measure but identified the right anterior thalamic tract (RAT) for the MD, RD, and AD measures. These findings are potentially relevant to the development of TBI biomarkers. Our methods also demonstrate how ROC analysis may be used to identify clinically relevant variables in the TBI population.
... TBI results from an initial primary injury induced by an external force to the brain (e.g., direct impact, acceleration/deceleration, blast). The primary injury goes on to initiate a secondary pathophysiological cascade, which is characterized by excitotoxicity, the generation of free radicals, lipid peroxidation (Hall, Vaishnav, & Mustafa, 2010), mitochondrial dysfunction (Mustafa, Wang, Carrico, & Hall, 2011), the swelling and loss of astrocytes (Meaney, Margulies, & Smith, 2001), and axonal (Pettus & Povlishock, 1996) and neuronal injury (Clark et al., 2000). Secondary injury induced after TBI is associated with a marked inflammatory response and alterations in both cerebral blood flow and metabolism (Kochanek et al., 2000), which leads to the parenchymal accumulation of tau and amyloid beta protein (Johnson, Stewart, & Smith, 2012), demyelination (Ng &, 1994), axonal degeneration (Smith et al., 1999), and apoptotic neuronal death via caspase-3 activation (Clark et al., 2000). ...
Traumatic brain injuries (TBIs) pose a massive burden of disease and continue to be a leading
cause of morbidity and mortality throughout the world. A major obstacle in developing effective
treatments is the lack of comprehensive understanding of the underlying mechanisms that mediate
tissue damage and recovery after TBI. As such, our work aims to highlight the development of a
novel experimental platform capable of fully characterizing the underlying pathobiology that
unfolds after TBI. This platform encompasses an empirically optimized multiplex immunohistochemistry
staining and imaging system customized to screen for a myriad of biomarkers required
to comprehensively evaluate the extent of neuroinflammation, neural tissue damage, and repair in
response to TBI. Herein, we demonstrate that our multiplex biomarker screening platform is capable
of evaluating changes in both the topographical location and functional states of resident and
infiltrating cell types that play a role in neuropathology after controlled cortical impact injury to
the brain in male Sprague–Dawley rats. Our results demonstrate that our multiplex biomarker
screening platform lays the groundwork for the comprehensive characterization of changes that
occur within the brain after TBI. Such work may ultimately lead to the understanding of the governing
pathobiology of TBI, thereby fostering the development of novel therapeutic interventions
tailored to produce optimal tissue protection, repair, and/or regeneration with minimal side effects,
and may ultimately find utility in a wide variety of other neurological injuries, diseases, and disorders
that share components of TBI pathobiology.
... In addition, the TBI-induced oxidative stress can reduce the levels of brain-derived neurotrophic factor (BDNF) and synaptic protein which are required for maintaining synaptic contacts and function (Ansari et al. 2008, Wu et al. 2006. It is also noteworthy that the TBI-induced oxidative stress can aggravate the increase of axolemmal permeability, leading to compaction of axonal neurofilament, reduction in microtubule density, and degradation of subaxolemmal spectrin (Buki et al. 1999, Lewen and Hillered 1998, Pettus and Povlishock 1996. These observations together corroborate that oxidative stress is tightly linked to TBI-caused secondary axonal injury. ...
A number of neurological disorders such as epidural hematoma can cause compression of cerebral cortex. We here tested the hypothesis that sustained compression of primary somatosensory cortex may affect stellate neurons and thalamocortical afferent (TCA) fibers. A rat model with barrel cortex subjected to bead epidural compression was used. Golgi-Cox staining analyses showed the shrinkage of dendritic arbors and the stripping of dendritic spines of stellate neurons for at least 3 months post-lesion. Anterograde tracing analyses exhibited a progressive decline of TCA fiber density in barrel field for 6 months post-lesion. Due to the abrupt decrease of TCA fiber density at 3 days after compression, we further used electron microscopy to investigate the ultrastructure of TCA fibers at this time. Some TCA fiber terminal profiles with dissolved or darkened mitochondria and fewer synaptic vesicles were distorted and broken. Furthermore, the disruption of mitochondria and myelin sheath was observed in some myelinated TCA fibers. In addition, expressions of oxidative markers 3-nitrotyrosine and 4-hydroxynonenal were elevated in barrel field post-lesion. Treatment of antioxidant ascorbic acid or apocynin was able to reverse the increase of oxidative stress and the decline of TCA fiber density, rather than the shrinkage of dendrites and the stripping of dendritic spines of stellate neurons post-lesion. Together, these results indicate that sustained epidural compression of primary somatosensory cortex affects the TCA fibers and the dendrites of stellate neurons for a prolonged period. In addition, oxidative stress is responsible for the reduction of TCA fiber density in barrels rather than the shrinkage of dendrites and the stripping of dendritic spines of stellate neurons. © 2017, Nencki Institute of Experimental Biology. All rights reserved.
... In the first case, an increase in IOP could cause a subthreshold insult to the axons (Quigley, 1999;Buckingham et al., 2008). Although axons are capable of sustaining minor membrane deformations, they are relatively rigid structures within an elastic extracellular surround (Javid et al., 2014), and deleterious mechanical stress experienced by IOP rises could induce plasmalemmal instability and cytoskeletal disassembly (Pettus and Povlishock, 1996;Singleton and Povlishock, 2004). ATP has been suggested as one such signaling molecule (Ahmed et al., 2000;Verderio and Matteoli, 2001;Neary et al., 2003Neary et al., , 2005; it can be released from axons of the white matter (Fields and Stevens, 2000;Hamilton et al., 2008;Fields and Ni, 2010). ...
Reactive remodeling of optic nerve head astrocytes is consistently observed in glaucoma and other optic nerve injuries. However, it is unknown whether this reactivity is beneficial or harmful for visual function. In this study, we used the Cre recombinase (Cre)– loxP system under regulation of the mouse glial fibrillary acidic protein promoter to knock out the transcription factor signal transducer and activator of transcription 3 (STAT3) from astrocytes and test the effect this has on reactive remodeling, ganglion cell survival, and visual function after experimental glaucoma and nerve crush. After injury, STAT3 knockout mice displayed attenuated astrocyte hypertrophy and reactive remodeling; astrocytes largely maintained their honeycomb organization and glial tubes. These changes were associated with increased loss of ganglion cells and visual function over a 30-day period. Thus, reactive astrocytes play a protective role, preserving visual function. STAT3 signaling is an important mediator of various aspects of the reactive phenotype within optic nerve astrocytes.
... Importantly, even neurons that avoid disconnection may be less elastic and more brittle after injury, leaving them potentially susceptible to outright disconnection if reinjured in subsequent shear-strain events. Mechanical deformation precipitates the excitotoxic release of neurotransmitters, which researchers indicate can cause secondary damage to cell microstructure (Pettus and Povlishock 1996). Microtubule damage from stretch mechanisms has also been linked to axon transport dysfunction, which may lead to proteomic and neurochemical abnormalities at the synaptic junction (Büki and Povlishock 2006). ...
This review seeks to summarize diffusion tensor imaging (DTI) studies that have evaluated structural changes attributed to the mechanisms of mild traumatic brain injury (mTBI) in adult civilian, military, and athlete populations. Articles from 2002 to 2016 were retrieved from PubMed/MEDLINE, EBSCOhost, and Google Scholar, using a Boolean search string containing the following terms: “diffusion tensor imaging”, “diffusion imaging”, “DTI”, “white matter”, “concussion”, “mild traumatic brain injury”, “mTBI”, “traumatic brain injury”, and “TBI”. We added studies not identified by this method that were found via manually-searched reference lists. We identified 86 eligible studies from English-language journals using, adult, human samples. Studies were evaluated based on duration between injury and DTI assessment, categorized as acute, subacute/chronic, remote mTBI, and repetitive brain trauma considerations. Since changes in brain structure after mTBI can also be affected by other co-occurring medical and demographic factors, we also briefly review DTI studies that have addressed socioeconomic status factors (SES), major depressive disorder (MDD), and attention-deficit hyperactivity disorder (ADHD). The review describes population-specific risks and the complications of clinical versus pathophysiological outcomes of mTBI. We had anticipated that the distinct population groups (civilian, military, and athlete) would require separate consideration, and various aspects of the study characteristics supported this. In general, study results suggested widespread but inconsistent differences in white matter diffusion metrics (primarily fractional anisotropy [FA], mean diffusivity [MD], radial diffusivity [RD], and axial diffusivity [AD]) following mTBI/concussion. Inspection of study designs and results revealed potential explanations for discrepant DTI findings, such as control group variability, analytic techniques, the manner in which regional differences were reported, and the presence or absence of persistent functional disturbances. DTI research in adult mTBI would benefit from more standardized imaging and analytic approaches. We also found significant overlap in white matter abnormalities reported in mTBI with those commonly affected by SES or the presence of MDD and ADHD. We conclude that DTI is sensitive to a wide range of group differences in diffusion metrics, but that it currently lacks the specificity necessary for meaningful clinical application. Properly controlled longitudinal studies with consistent and standardized functional outcomes are needed before establishing the utility of DTI in the clinical management of mTBI and concussion.
... Previous models have employed either uniaxial [24][25][26] or biaxial [27][28][29] loading modes. Biaxial loading induces channel-independent calcium influx via mechanoporation of the cell membrane, which is a widely accepted injury mechanism [30][31][32] . Uniaxial loading isolates calcium influx due to activation of stretch-sensitive ion channels 33 . ...
Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.
... Accomplishing these tasks requires T lymphocytes to undergo large deformations, both passively as they move through narrow capillaries during their patrols (Fung, 2013), and actively upon extravasation at a site of inflammation or injury (Carman and Springer, 2004;Carman, 2009;Carman and Martinelli, 2015). In certain cell types, for instance neural cells during brain trauma, excessive strain can cause membrane damage both in vitro (Geddes et al., 2003) and in vivo (Pettus et al., 1994;Pettus and Povlishock, 1996). To our knowledge, however, there have been no reports of T lymphocyte membrane damage in vivo despite the large deformations that these cells undergo. ...
T lymphocytes in the human body routinely undergo large deformations, both passively when going through narrow capillaries and actively when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T lymphocytes maintain a constant volume while increasing their apparent membrane surface area upon aspiration. Human resting T lymphocytes, T lymphoblasts and the leukemic Jurkat T cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur.
... While it was once thought that traumatically injured axons are mechanically severed at the time of injury, it is now known that primary axotomy rarely occurs (Maxwell et al., 1997), at least after lateral fluid percussion injury (Singleton et al., 2002). Rather, in the majority of cases, the primary mechanical insult provokes secondary biochemical processes involving a transient, focal disruption of the axolemma allowing for calcium influx and the initiation of an enzyme cascade resulting in further damage (Maxwell et al., 1995(Maxwell et al., ,1999Pettus and Povlishock, 1996). The resulting mitochondrial swelling and/or injury and disruption of axoplasmic transport can ultimately lead to disconnection of swollen axons from their distal segment (Buki et al., 1999b(Buki et al., ,2000Maxwell et al., 1997;Okonkwo et al., 1998), with either cell death, or simply neuronal atrophy in more mild types of injury (Greer et al., 2011). ...
Description
The latest volume in this ongoing series enhances your understanding of both the injuries incurred in the game of ice hockey and the techniques used to decrease the risk of these injuries.
Twenty-three peer-reviewed papers address a diverse range of topics from the fields of sports science, sports medicine, athletic training, biomechanics, risk factor management, epidemiology, sports psychology, injury surveillance, sports equipment, physical conditioning, behavioral factors in sports, as well as case reports from individuals associated with national sports governing bodies, playing facilities, officiating, and playing rules.
Equally important, this new publication also discusses strategies of prevention, including protective equipment; different approaches to managing the conduct of players, coaches and parents, and better implementation of training and conditioning.
Four sections cover
This volume is a valuable resource for hockey equipment manufacturers, biomechanical engineers, hockey coaches and administrators, sports medicine physicians, and athletic trainers.
Traumatic axonal injury (TAI) is one of the most common pathological features of severe traumatic brain injury (TBI). Our previous study using proteomics suggested that peripherin (PRPH) should be a potential candidate as a biomarker for TAI diagnosis. This study is to further elucidate the role and association of PRPH with TAI. In the animal study, we performed immunohistochemistry, ELISA and morphological analysis to evaluate PRPH level and distribution following a severe impact. PRPH‐positive regions were widely distributed in the axonal tract throughout the whole brain. Axonal injuries with PRPH inclusion were observed post‐TBI. Besides, PRPH was significantly increased in both cerebral spinal fluid and plasma at the early phase post‐TBI. Colocalization analysis based on microscopy revealed that PRPH represents an immunohistological biomarker in the neuropathological diagnosis of TAI. Brain samples from patients with TBI were included to further test whether PRPH is feasible in the real practice of neuropathology. Immunohistochemistry of PRPH, NFH, APP and NFL on human brain tissues further confirmed PRPH as an immunohistological biomarker that could be applied in practice. Collectively, we conclude that PRPH mirrors the cytoskeleton injury of axons and could represent a neuropathological biomarker for TAI.
Damage to the axonal white matter tracts within the brain is a key cause of neurological impairment and long-term disability following traumatic brain injury (TBI). Understanding how axonal injury develops following TBI requires gyrencephalic models that undergo shear strain and tissue deformation similar to the clinical situation and investigation of the effects of post-injury insults like hypoxia. The aim of this study was to determine the effect of post-traumatic hypoxia on axonal injury and inflammation in a sheep model of TBI. Fourteen male Marino sheep were allocated to receive a single TBI via a modified humane captive bolt animal stunner, or sham surgery, followed by either a 15 minute period of hypoxia or maintenance of normoxia. Head kinematics were measured in injured animals. Brains were assessed for axonal damage, microglia and astrocyte accumulation and inflammatory cytokine expression at 4 hrs following injury. Early axonal injury was characterised by calpain activation, with significantly increased SNTF immunoreactivity, a proteolytic fragment of alpha-II spectrin, but not with impaired axonal transport, as measured by amyloid precursor protein (APP) immunoreactivity. Early axonal injury was associated with an increase in GFAP levels within the CSF, but not with increases in IBA1 or GFAP+ve cells, nor in levels of TNFα, IL1β or IL6 within the cerebrospinal fluid or white matter. No additive effect of post-injury hypoxia was noted on axonal injury or inflammation. This study provides further support that axonal injury post-TBI is driven by different pathophysiological mechanisms, and detection requires specific markers targeting multiple injury mechanisms. Treatment may also need to be tailored for injury severity and timing post-injury to target the correct injury pathway.
The optic nerve is a complex biomechanical structure. It is subjected to various mechanical loadings including intraocular pressure, cerebrospinal fluid pressure, eye movements, and compression from tumours etc. In this chapter, we firstly reviewed the optic nerve structure, mechanical behaviours of the optic nerve tissues, and mechanisms of optic nerve fiber injury. Next, we discussed ocular responses to optic nerve traction during eye movements and its potential links to glaucoma and myopia. The cerebrospinal fluid dynamics of the optic nerve is also briefly introduced. Finally, biomechanics of optic chiasmal compression and its application in explaining the mechanisms of bitemporal hemianopia is presented. This chapter is not an extensive review of optic nerve related biomechanics but rather to discuss some key aspects of optic nerve biomechanics that are involved in pathophysiology of several important ocular diseases.
Traumatic brain and spinal cord injury cause stretch-induced damage to cell membranes along the axon. Here, we discuss the role that ionic changes may play that underlie such injury, in particular discussing the potentially pivotal role that sodium dysregulation may play in the ionic changes that occur after traumatic axon injury. Cellular axon stretch models show axonal stretch injuries trigger a series of ionic and proteolytic cascades with disruption of normal cellular ionic homeostasis. There is frank membrane mechanoporation as well as transcriptional upregulation of sodium channels with ensuing increases in intracellular sodium and calcium. Increased intracellular calcium can further result in cellular dysfunction and damage including cytoskeletal and membrane disruptions. Animal models show these post-injury ionic cascades can be mitigated with the application of sodium channel blockade, further supporting the central role of sodium in mediating ion dysregulation and related damage after TBI. Recent work using sodium magnetic resonance imaging (MRI) in human subjects reveals sodium MRI signal differences in vivo in patients with mild TBI as compared to healthy controls.
The promise of gene therapy is alluring not only for CNS disorders but also for other pathological conditions. Gene therapy employs the insertion of a healthy gene into the identified genome to replace or replenish genes responsible for pathological disorder or damage due to trauma. The last decade has seen a sea change in the understanding of vital aspects of gene therapy. Despite the complexity of traumatic brain injury (TBI), the advent of gene therapy in various neurodegenerative disorders has reinforced the ongoing efforts of alleviating TBI-related outcomes with gene therapy. The review highlights the genes modulated in response to TBI and evaluates their impact on the severity and duration of the injury. We reviewed strategies that pinpointed the most relevant gene targets to restrict debilitating events of brain trauma and utilize vector of choice to deliver the gene of interest at the appropriate site. We attempted to summarize the long-term neurobehavioral consequences of TBI due to numerous pathometabolic perturbations associated with a plethora of genes. Herein, we shed light on the basic pathological mechanisms of brain injury, genetic polymorphism in individuals susceptible to severe outcomes, modulation of gene expression due to TBI, and identification of genes for their possible use in gene therapy. The review also provided insights on the use of vectors and challenges in translations of this gene therapy to clinical practices.
Sports deserve a special place in human life to impart healthy and refreshing wellbeing. However, sports activities, especially contact sports, renders athlete vulnerable to brain injuries. Athletes participating in a contact sport like boxing, rugby, American football, wrestling, and basketball are exposed to traumatic brain injuries (TBI) or concussions. The acute and chronic nature of these heterogeneous injuries provides a spectrum of dysfunctions that alters the neuronal, musculoskeletal, and behavioral responses of an athlete. Many sports-related brain injuries go unreported, but these head impacts trigger neurometabolic disruptions that contribute to long-term neuronal impairment. The pathophysiology of post-concussion and its underlying mechanisms are undergoing intense research. It also shed light on chronic disorders like Parkinson's disease, Alzheimer's disease, and dementia. In this review, we examined post-concussion neurobehavioral changes, tools for early detection of signs, and their impact on the athlete. Further, we discussed the role of nutritional supplements in ameliorating neuropsychiatric diseases in athletes.
Post-traumatic headache (PTH) is a highly disabling secondary headache disorder and one of the most common sequelae of mild traumatic brain injury, also known as concussion. Considerable overlap exists between PTH and common primary headache disorders. The most common PTH phenotypes are migraine-like headache and tension-type-like headache. A better understanding of the pathophysiological similarities and differences between primary headache disorders and PTH could uncover unique treatment targets for PTH. Although possible underlying mechanisms of PTH have been elucidated, a substantial void remains in our understanding, and further research is needed. In this Review, we describe the evidence from animal and human studies that indicates involvement of several potential mechanisms in the development and persistence of PTH. These mechanisms include impaired descending modulation, neurometabolic changes, neuroinflammation and activation of the trigeminal sensory system. Furthermore, we outline future research directions to establish biomarkers involved in progression from acute to persistent PTH, and we identify potential drug targets to prevent and treat persistent PTH.
Primary objective: Secondary axotomy is more prevalent than the primary axotomy and involves subtle intraaxonal changes in response to the injury leading to cytoskeletal disruptions including neurofilament (NF) misalignment and compaction, which is associated with the genesis of impaired axoplasmic transport (IAT). Recent studies have reported two differential axonal responses to injury, one associated with the cytoskeletal collapse and another with the IAT. The objective of this study was to determine the extent of IAT and early NF changes in axons that were subjected to a stretch of various degrees at different strain rates.
Research design and methods: Fifty-six L5 dorsal spinal nerve roots were subjected to a predetermined strain at a specified displacement rate (0.01 and 15 mm/second) only once. The histological changes were determined by performing standard immunohistochemical procedures using beta amyloid precursor protein (β APP) and NF-68 kDa antibodies.
Results and conclusions: No significant differences in the occurrence rate of either of the staining in the axons were observed when subjected to similar loading conditions, and the occurrence rate of both β APP and NF68 staining was strain and rate-dependent.
Traumatic brain and spinal cord injury manifests following structural compromise of the tissue, including neurons and their axons, glial cells, blood vessels and extracellular components, and subsequent secondary injury cascades. The degree of primary injury depends on the cellular orientation within the tissue, local tissue mechanical properties, strain experienced at the cell level, and rate at which the insult is delivered. The plasma membrane may be directly stretched to a point of failure in either a permanent (lethal) or transient (initially sublethal) manner. The acute evidence of membrane damage is a nonspecific increase in membrane permeability immediately following a traumatic insult. Increased membrane permeability has been observed in several in vitro and in vivo models of traumatic neural injury; therefore, understanding the events that occur in the acute and subacute time periods may provide a preclinical basis for protective and/or reparative therapeutic targets. The implementation of a strategy to repair damaged membranes in the injured nervous system, however, requires continued study of the temporal and spatial extents of membrane damage, biomolecules that are acutely damaged, role of ongoing membrane damage due to free radical attacks and other degradative processes, and potential for endogenous repair. Experimental procedures to detect the presence and mechanisms of plasma membrane damage and repair, therefore, should be established in order to scrutinize the link between primary insult parameters and membrane damage and the balance between secondary membrane damage and endogenous repair attempts. The use of various markers to label cells with compromised membranes in TBI models is described, as well as limitations and considerations for future studies.
Concussion is one of the most common neurological conditions to occur during childhood. Since 1997, the incidence of concussion has doubled, likely due to increased reporting of events by parents, schools, and team officials. Concussion occurs after an impact to the body or head causes a rotational force on the brain sufficient to disturb consciousness. A complex neurochemical cascade ensues, sometimes accompanied by physical damage to neuronal structures. Patients can suffer from a multitude of somatic and cognitive complaints, although they resolve in the majority of cases after 7–10 days. There is no proven treatment, and the most commonly prescribed remedy is a combination of physical and cognitive rest. Children should only return to playing sports when symptoms have resolved, and this return should occur in a stepwise manner from light exercise, to sports-specific exercise, to noncontact drills, and finally to full-contact practice and return to play after clearance by a licensed medical professional. The management of chronic symptoms and likely temporary cognitive deficits should be dealt with on a case-by-case basis, and academic accommodation should be utilized when necessary.
The neuronal cytoskeleton is integral to neuronal function and structure. Neuronal cytoskeletal proteins interact via crosslinks to each other and to other cell organelles, the nuclear envelope and the plasma membrane. Cytoskeletal proteins are involved in cytoplasmic and axonal transport of organelles and macromolecules and in maintaining structural integrity of the neuron. Alterations or reorganization of cytoskeletal proteins are necessary for normal neuronal functions such as differentiation and movement of secretory granules (Burgoyne 1991), reflecting the dynamic nature of the cytoskeleton. However, abnormal organization or destruction of components of the cytoskeleton may lead to impaired intracellular transport and aberrant neuronal structure. This chapter will review data from studies of human and experimental traumatic brain injury (TBI) that are providing increasing evidence that multiple neurocytoskeletal elements sustain damage as a result of trauma, potentially contributing to neuronal dysfunction. Some of the major neurocytoskeletal proteins include neurofilaments (NFs), tubulin, microtubule-associated proteins (MAPs) such as MAP2 and tau, actin, and spectrin. Because cytoskeletal integrity is a critical determinant of neuronal viability, it is important to understand neurocytoskeletal dynamics following TBI in the ultimate hope of preventing or attenuating harmful changes, while promoting events that might be reparative or adaptive.
We assayed calpain activity in 27 human brain regions from adult (43–65 years of age) and aged (66–83 years of age) postmortem tissue samples. Calpain I (M Ca-requiring) activity was 10% or less of the total activity; it was below detectable levels in a number of areas, and so data are are expressed as total (M+mM Ca-dependent) calpain activity. The distribution of the enzyme was regionally heterogeneous. Highest activity was found in the spinal cord, followed by the amygdala, and levels in mesencephalic areas and in cerebellar grey matter were also high. Levels in cerebellar white matter, tegmentum, pons, and putamen were low, and activity in cortical areas was also relatively low. Although in some areas activity seemed higher with aging, the differences were not statistically significant. We previously found that the regional distribution of cathepsin D in human and in rat brain is similar, this seems to be true for calpain activity as well. The increase of protease activity with age found in rat brain is not found in human areas, as was shown previously with cathepsin D, and in the present study with calpain.
Purified cold-stable microtubules from the rat brain are insensitive to podophyllotoxin and to millimolar concentrations of free calcium. However, in the presence of calmodulin at concentrations substoichiometric to that of tubulin, calcium causes rapid microtubule disassembly. The half-maximal effective calcium concentration in the presence of calmodulin is 100 microM. With 800 microM free calcium, the half-maximal effective concentration of calmodulin is 1.0 microM (or one-tenth the tubulin concentration). Calmodulin is without effect in the absence of calcium. Troponin C is approximately one-fifth as effective as calmodulin, and parvalbumin is totally ineffective. Troponin I partially inhibits the calcium/calmodulin-induced disassembly of microtubules in the crude extract and blocks the calcium/calmodulin effect on purified cold-stable microtubules. A 5-fold excess of trifluoperazine does not inhibit the calcium/calmodulin-induced disassembly.
The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation-dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150-microns level and further increased a total of threefold by the 1,200-microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl-terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network.
Previous studies in the hypomyelinating mouse mutant Trembler have suggested that demyelinating axons are smaller in caliber compared to normal axons, and that there are differences in the organization of axonal neurofilaments. In the normal PNS, however, the relationship between neurofilament organization and myelination has not been investigated extensively. In normal axons, only the initial segments, the nodes of Ranvier (approximately 1 micron), and the terminals are not covered by myelin. We took advantage of an unusual feature of the primary sensory neurons in the dorsal root ganglion, the relatively long nonmyelinated stem process (up to several hundred micrometers), to determine if the presence of myelination correlates with differences in cytoskeletal organization and neurofilament phosphorylation. Axonal caliber and neurofilament numbers were substantially greater in the myelinated internodes than in the stem process or nodes of Ranvier. Neurofilament spacing, assessed by measuring the nearest-neighbor neurofilament distance, was 25-50% less in the stem processes and nodes of Ranvier than in the myelinated internodes. In the myelinated internodes, neurofilaments had greater immunoreactivity for phosphorylated epitopes than those in the stem process. These findings indicate that interactions with Schwann cells modulate neurofilament phosphorylation within the ensheathed axonal segments, and that increased phosphorylation within myelinated internodes leads to greater interfilament spacing. Lastly, the myelinated internodes had three fold more neurofilaments, but the same number of microtubules. Both the increased neurofilament spacing and the increase in neurofilament numbers in myelinated internodes contribute to a greater axonal caliber in the myelinated internodes.
The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation-dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150-microns level and further increased a total of threefold by the 1,200-microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl-terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network.
Anterograde axonal transport of horseradish peroxidase (HRP) in selected cerebral and cerebellar efferents was studied in cats subjected to minor head injury. After trauma, the animals were allowed to survive from one to 24 hours, when they were perfused with aldehydes and processed for the light and electron microscopic visualization of the peroxidase reaction product. By light microscopy, the brain injury elicited an initial intra-axonal peroxidase pooling. With longer post-traumatic survival, HRP pooling increased in size, demonstrated frequent lobulation, and ultimately formed large ball- or club-like swellings which suggested frank axonal separation from the distal axonal segment. Ultrastructural examination revealed that the initial intra-axonal peroxidase pooling was associated with organelle accumulation which occurred without any other form of axonal change or related parenchymal or vascular damage. This accumulation of organelles increased with time and was associated with conspicuous axonal swelling. Ultimately these organelle-laden swellings lost continuity with the distal axonal segment and the axonal swelling was either completely invested by a thin myelin sheath or protruded without myelin investment into the brain parenchyma. This study suggests that axonal change is a consistent feature of minor head injury. Since these axonal changes occurred without any evidence of focal parenchymal or vascular damage, minor brain injury may ultimately disrupt axons without physically shearing or tearing them.
The number of neurofilaments and microtubules present in nerve fibers was determined for sciatic nerves from adult mice and from rats of three different ages. More microtubules than neurofilaments were found in nonmyelinated fibers; the ratio of tubules/filaments was reversed in myelinated fibers and was found to change with axon caliber independent of the presence of a myelin sheath. A series of regression analyses indicated that axon caliber correlates best with the sum of the number of neurofilaments and microtubules per fiber. This correlation was only slightly better than that for neurofilaments alone. Axon caliber also correlated better with the filament-tubular material than with the thickness of the myelin sheath. The results were similar for both rats and mice, and age differences were not apparent in the samples of nerves analyzed.
Protein uptake and transport within the brain stem vasculature of mechanically brain injured cats was studied by means of both light and electron microscopy utilizing intravenously injected horseradish peroxidase as the protein tracer. In animals sustaining low grade head injuries not of sufficient intensity to elicit either microscopic, intraparenchymal hemorrhages or subtle, neuropathological responses, peroxidase extravasation was noted both in the vascular walls and in the surrounding parenchyma of the ventromedial aspect of the brain stem. At the ultrastructural level as early as 3 min after brain injury, occasional arterioles, venules and capillaries displayed peroxidase leakage. In serial sections large endothelial segments of these vessels revealed the peroxidase reaction product within numerous vesicles which often shared continuity with tubular and vacuolar profiles. Such vesicular activity apparently moved the peroxidase from the luminal surface to extrude it into the basal lamina. From the perivascular basal lamina, the reaction product flooded the interstices of the surrounding brain stem parenchyma where occasional neural, glial and pericytic elements incorporated the peroxidase within coated invaginations, vesicles, tubules and vacuoles. In that protein leakage was consistently observed despite the apparent integrity of both the endothelial tight junctions and their cell membranes, it is concluded that the vesicular transport of horseradish peroxidase across the endothelia of the brain stem vasculature represents a possible mechanism of blood-brain barrier dysfunction in mechanical brain injury.
Mechanical brain injury was produced in 36 cats with a fluid-percussion model in which brain damage or dysfunction is produced by a single, brief, hydraulically-induced pressure transient that is conducted through the brain. Fluid-percussion injury induce elastic deformation of the brain resembling the brain deformation known to occur following head impact. Physiological responses and pahtological changes following injury were expressed as a function of peak pressure. Macroscopic central nervous system lesions concentrated at the pontomesencephalic junction, cervicomedullary junction, and in the cerebellar tonsils were consistently observed at and above 2.6 atmospheres (atm). At higher levels of injury (greater than or equal to 3.2 atm) there was extensive basal subarachnoid hemorrhage. At very high levels of injury (greater than 4.0 atm) hemorrhagic contusions were noted at the cerebral hemisphere impact site. A spectrum of neuronal alterations was identified in the damaged areas. Computer analysis showed correlation of electroencephalographic (EEG) changes with the neuropathological changes, since EEG recovery became severely impaired above 2.6 atm. No EEG changes were noted below 1.5 atm. From 1.5 to 2.2 atm there was a physiological response to injury but no significant changes were seen on neuropathological examination. This range of injury should permit further studies of the more subtle changes following mechanical brain injury without intraparenchymal hemorrhage or subarachnoid hemorrhage. The fluid-percussion model relates brain deformation following mechanical loading to a single pressure transient that is easily measured and controlled. Further quantitative investigation into the pathobiology of mechanical brain injury following graded brain deformation is thus made possible.
This work reviews the pathobiology of traumatically induced axonal injury. Drawing upon literature gleaned from the experimental and clinical setting, this review attempts to emphasize that, other than the most destructive insults, traumatic brain injury does not typically cause direct mechanical disruption of the axon. Rather, this review documents that with traumatic injury focal, subtle axonal change occurs, and that over time, such change leads to impaired axoplasmic transport, continued axonal swelling, and ultimate disconnection. The initial intra-axonal events that trigger the above described sequence of reactive axonal change are considered with focus on the possibility of either traumatically altered axolemmal permeability, direct cytoskeletal damage/perturbation, or more overt metabolic/functional disturbances. Not only does this review focus on the sequence of traumatically induced axonal change, but also, it considers its attendant consequences in terms of Wallerian degeneration and subsequent deafferentation. The concept that traumatically induced diffuse axonal injury leads to diffuse deafferentation is emphasized together with its pathobiological implications for morbidity and recovery. The potential for either adaptive or maladaptive neuroplasticity subsequent to such diffuse deafferentation is considered in the context of mild, moderate and severe traumatic brain injury.
Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies.
Reactive axonal change has long been recognized as a feature of traumatic brain injury. To date, the histological methods used to identify reactive axons have been of limited utility, and they have not provided insight into the initial intraaxonal event that triggers reactive change. In this investigation, monoclonal antibodies to the 68, 150, and 200 kilodalton (kD) neurofilament subunits have been used to follow the progression of reactive axonal change. Anesthetized rats and cats were subjected to moderate traumatic brain injury. One to 72 hours (h) postinjury, their brains were processed for the light (LM) and electron (EM) microscopic immunocytochemical visualization of the various neurofilament subunits. Although all of the chosen antibodies revealed some degree of immunoreactivity within the reactive axon, the 68 kD antibody revealed a dramatic increase in immunoreactivity following injury. Within one h of injury, intensely 68 kD-immunoreactive axonal segments were observed with LM, and parallel EM microscopic analyses demonstrated that this increased immunoreactivity was associated with an increased number of 68 kD-immunoreactive neurofilaments, the majority of which coursed in an axis parallel to the axon's course. Over 2-6 h postinjury, these 68 kD-immunoreactive filaments demonstrated increasingly disordered alignment in relation to the axon's long axis, withdrawing from the focus of injury while becoming encompassed by an expanding organelle cap. It is posited that this increased 68 kD immunoreactivity is associated with a traumatically-induced increase in subunit exchange which contributes to cytoskeletal dysfunction leading to organelle accumulation, focal swelling and ultimate axonal detachment.
Dynamic remodeling of cytoskeleton architecture is necessary for axonal growth and guidance, signal transduction and other fundamental aspects of neuron function. Protein phosphorylation plays a key part in these remodeling processes. Since neurofilaments are major cytoskeletal constituents and are among the most highly phosphorylated neuronal proteins, the control of their behavior serves as a possible model for understanding how phosphorylation regulates the many other phosphoproteins in the cytoskeleton. Recent studies show that neurofilament protein subunits are phosphorylated on both their amino-terminal head domains and carboxy-terminal tails by different protein kinases. This review considers the implications of this complex regulation for neurofilament function in normal neurons and in disease states characterized by neurofibrillary pathology.
The relationship between the degree of nodal narrowing and the changes in the structure of the axonal cytoskeleton was studied in 53 fibres of mouse sciatic nerve. Nodal narrowing increased with increasing fibre calibre to reach about 20% of the internodal area in the thicker fibres. The narrowing corresponded quantitatively to a decreased number of nodal neurofilaments. Nodal microtubule numbers varied greatly, and a majority of fibres had considerably (approximately 55%) more microtubules in their nodal profile than in the internode. Nodal profiles of different calibre showed an increase in the number of filaments and of microtubules with nodal calibre, although at rates different from those in the internode. The degree of observed axon non-circularities had no discernible effect on the restructuring of the axonal cytoskeleton at the node. A transnodal transport of the axonal cytoskeleton can occur with: (1) accelerated transnodal transport of filaments, (2) stationary internodal fraction of filaments, (3) depolymerization of filaments proximal to the node and repolymerization distally, or (4) different nodal and internodal polymerization equilibria.
Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.
Based upon recent clinical findings, evidence exists that severe traumatic brain injury causes widespread axonal damage. In the clinical setting, it has been assumed that such axonal damage is the immediate consequence of traumatically induced tearing. However, in laboratory studies of minor head injury, evidence for primary traumatically induced axonal tearing has not been found. Rather the traumatic event has been linked to the onset of subtle axonal abnormalities, which become progressively severe over time (i.e., 12-24 h). In the light of these discrepant findings, we investigated, in the present study, whether progressive axonal change other than immediate tearing occurs with severe traumatic brain injury. Anesthetized cats were subjected to high intensity fluid-percussion brain injury. Prior to injury all animals received cortical implants of horseradish peroxidase (HRP) conjugated to what germ agglutinin to anterogradely label the major motor efferent pathways. Such an approach provided a sensitive probe for detecting traumatically induced axonal abnormality via both light microscopy (LM) and transmission electron microscopy (TEM). The animals were followed over a 1- to 6-h posttraumatic course, and processed for the LM and TEM visualization of HRP. Through such an approach no evidence of frank traumatically induced tearing was found. Rather, with LM, an initial intra-axonal peroxidase pooling was observed. With time, unilobular HRP-containing pools increased in size and progressed to bi- or multilobulated profiles. Ultimately, these lobulated configurations separated. Ultrastructurally, the initial unilobular pool was associated with organelle accumulation and focal axolemmal distention without frank disruption. Over time, such organelle accumulations increased in size and sequestered into multiple pools reminiscent of the bi- and multilobulated structures seen with LM. Ultimately, these organelle accumulations became detached, resulting in physically separated proximal and distal organelle-laden swellings surrounded by a distended axolemma and thinned myelin sheath. The findings reject the hypothesis that axons are immediately torn upon impact.
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.
A new model of traumatic axonal injury has been developed by causing a single, rapid, controlled elongation (tensile strain) in the optic nerve of the albino guinea pig. Electron microscopy demonstrates axonal swelling, axolemmal blebs, and accumulation of organelles identical to those seen in human and experimental brain injury. Quantitative morphometric studies confirm that 17% of the optic nerve axons are injured without vascular disruption, and horseradish peroxidase (HRP) studies confirm alterations in rapid axoplasmic transport at the sites of injury. Since 95% to 98% of the optic nerve fibers are crossed, studies of the cell bodies and terminal fields of injured axons can be performed in this model. Glucose utilization was increased in the retina following injury, confirming electron microscopic changes of central chromatolysis in the ganglion cells and increased metabolic activity in reaction to axonal injury. Decreased activity at the superior colliculus was demonstrated by delayed HRP arrival after injury. The model is unique because it produces axonal damage that is morphologically identical to that seen in human brain injury and does so by delivering tissue strains of the same type and magnitude that cause axonal damage in the human. The model offers the possibility of improving the understanding of traumatic damage of central nervous system (CNS) axons because it creates reproducible axonal injury in a well-defined anatomical system that obviates many of the difficulties associated with studying the complex morphology of the brain.
Diffuse axonal injury is one of the most important types of brain damage that can occur as a result of non-missile head injury, and it may be very difficult to diagnose post mortem unless the pathologist knows precisely what he is looking for. Increasing experience with fatal non-missile head injury in man has allowed the identification of three grades of diffuse axonal injury. In grade 1 there is histological evidence of axonal injury in the white matter of the cerebral hemispheres, the corpus callosum, the brain stem and, less commonly, the cerebellum; in grade 2 there is also a focal lesion in the corpus callosum; and in grade 3 there is in addition a focal lesion in the dorsolateral quadrant or quadrants of the rostral brain stem. The focal lesions can often only be identified microscopically. Diffuse axonal injury was identified in 122 of a series of 434 fatal non-missile head injuries--10 grade 1, 29 grade 2 and 83 grade 3. In 24 of these cases the diagnosis could not have been made without microscopical examination, while in a further 31 microscopical examination was required to establish its severity.
Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.
There are two types of calcium-activated neutral protease (CANP), m-CANP and mu-CANP, following the nomenclature of Suzuki et al to show that each requires mM and microM Ca2+, respectively, for its activation. We found mu-CANP activity in a crude CANP fraction extracted from the peripheral nerve, which degraded the neurofilament (Nf) triplet (200 K, 160 K, 68 K), especially the 160 K component, at Ca2+ concentrations of 50 microM and 0.1 mM. The triplet was degraded in the order of the 160 K, 68 K, and 200 K components, respectively. In addition, the effects of partially purified mu-CANP of rabbit skeletal muscle, purified natural mu-CANP of bovine liver, derived mu-CANP prepared by autodigestion of chicken muscle m-CANP, m-CANP of chicken skeletal muscle, and cathepsin B of rat liver on the Nf were examined. Among the triplet components, the 160 K component was most rapidly degraded by all proteases so far tested. The difference in the effect of mu-CANP and m-CANP or cathepsin B on susceptibility of the 200 K component to degradation might be due to the difference of the relative amounts of enzymes to Nf.
Minor head injury or concussion was produced in experimental animals by an acceleration-deceleration non-impact injury. The animals sustained a brief loss of consciousness and no sequelae were observed. The brains were examined at 7 days by means of the Nauta and Fink-Heimer techniques. Degenerating axons were noted in the inferior colliculus, pons, and dorsolateral medulla. Degeneration was not seen in the subcortical white matter, thus suggesting a primary brain-stem locus for concussion. These findings also suggest that, in some instances, minor head injury or concussion can be associated with organic damage to the central nervous system.
A blow to the head will impart rotational velocity to the brain and, depending on its magnitude, will produce effects ranging from concussion to profound neurological dysfunction. Resultant shear strains distort and rupture axons, blood vessels and major fibre tracts. Thirty-seven patients with head injury that was not complicated by significant hemorrhage or superficial laceration of the brain had coma or severe dementia, spastic quadriparesis, incontinence and autonomic dysfunction. These patients survived 24 hours to 243 days. Gross pathological examination revealed little, but there was microscopic evidence of axonal and small vessel injury in all; this was localized to the basal and midsagittal areas of the diencephalon and mesencephalon, particularly in those less severely injured. Such changes represent the basic pathology of all head injury. Data from this study suggest that concussion depends upon varying degrees of damage to the axon as well as the neuron. The current definition of concussion-immediate loss of consciousness with rapid and complete recovery of cerebral function-should not exclude the fact that a small number of neurons may have been permanently disconnected or have perished.
Although considerable gross damage to the brain may be found, on surgical intervention or at autopsy, in many head injuries, it is often slight, and usually insufficient to explain either the patient's death or even his neurological symptoms. Consequently, several workers (1, 2) have postulated the existence of a diffuse lesion involving both the nerve cells and their pathways throughout the brain. Indeed, microscopic examination may reveal widespread damage to the white matter following a head injury which does not necessarily produce hemorrhage, contusion or laceration (3, 4). The degeneration is thought to be due to the stretching and tearing of nerve fibers at the moment of the accident. Patients showing this microscopic change, however, usually have been severely incapacitated and have survived for a long time following the injury. It seems possible that lesser degrees of white matter damage may occur in all patients with head injury, but the frequency is difficult to estimate as too few acute head injuries have been investigated histologically. This paper describes the neuropathological changes in the white matter in 40 consecutive head injuries.
A calcium-dependent protease fully active with 0.2 mM Ca2+ was found associated with the neurofilament-enriched cytoskeleton of the rat spinal cord prepared by the treatment with Triton X-100. The enzyme preferentially degrades the 160-kDa component of the neurofilament triplet. In addition, a soluble calcium-dependent protease activity was found in the supernatant from the spinal cord, which degraded a variety of cytoskeletal proteins including the neurofilament triplet, glial fibrillary acidic (GFA) protein, actin, tubulin, and a high molecular weight protein associated with microtubules. The possibility that the cytoskeleton-bound activity is an artefactual association of the soluble enzyme to the cytoskeleton seems to be negated on the basis of the following dissociation and reassociation experiments. The protease activity remained associated with the cytoskeleton in the physiological ionic strength, and was not completely dissociated from it until the KC1 concentration was raised to 0.6 M. When the 0.6 M KCl-extract was dialysed against salt-free buffer to remove KC1, and added back to the protease-free cytoskeletal pellet, proteolytic activity was partially restored. Full activity returned only when the extract and the protease-free cytoskeletal pellet were first combined in the presence of 0.6 M KC1, and then slowly reassociated by dialysis against salt-free buffer. Dissociated enzyme was rapidly inactivated at 37 degrees C in the presence of Ca2+. These results suggest the structural association of the protease with the cytoskeleton under the physiological condition.
Forty-five cases of diffuse axonal injury (DAI) brought about by nonmissile head injury in humans are analyzed and compared with 132 cases of fatal head injury without DAI. All cases were subjected to a comprehensive neuropathological study. In the patients with DAI a statistically significant lower incidence of lucid interval, fracture of the skull, cerebral contusions, intracranial hematoma, and evidence of high intracranial pressure were found, with a higher incidence of head injury due to road traffic accident. Brain swelling and hypoxic brain damage were not statistically different in the two groups. The features of DAI in humans are compared with the DAI that has been produced in subhuman primates by pure inertial loading brought about by angular acceleration of the head. The available evidence indicates that DAI in human beings occurs at the time of head injury and is not due to complicating factors such as hypoxia, brain swelling, or raised intracranial pressure.
We have examined the mechanism of calcium induced microtubule dissasembly by combined kinetic and steady state analysis. Our results indicate that calcium induces microtubule disassembly by binding directly to the wall of the microtubule and promoting dissociation of subunits from the ends.
Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.
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 perturbation of the axon results in axonal swelling and disconnection at a minimum of 2 h after injury.
In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the "rod" domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament "sidearm" domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rod-specific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1-2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons.
The spinal cord of the larval sea lamprey contains identified giant axons that readily regenerate following spinal transection. In this study, we used serial light and electron microscopy to analyze the early ultrastructural consequences of axotomy in the proximal stumps of these axons near the lesion site. Axotomy results in two types of striking ultrastructural changes: 1) changes associated with the degeneration of axoplasm and subsequent retraction of the cut axon from the lesion and 2) changes associated with the early stages of axonal regeneration. Degenerative changes include the disruption of mitochondria to form large vacuoles, the collapse of neurofilaments into closely packed masses (condensed filamentous cores; CFCs), and the appearance of amorphous electron-dense bodies (dense granular masses; DGMs). Events associated with regeneration include the disappearance of vacuoles, DGMs, and CFCs and the appearance of small, sprout-like projections from the axon stump. Thus, we show that degenerative and regenerative events can be clearly separated from one another in identified axons, unlike the situation in the central nervous systems of amniote vertebrates such as mammals.
Recent studies have suggested that severe forms of traumatic brain injury (TBI) can be associated with direct alterations of the axolemma. The present study evaluated whether injuries of mild to moderate severity are associated with comparable change. To this end, we used extracellular horseradish peroxidase (HRP) to determine if altered axolemmal permeability occurred following the traumatic event. Adult cats received intrathecal infusions of peroxidase and then were prepared for mild to moderate fluid percussion injury. At intervals ranging from 5 min to 3 h, animals were perfused with aldehydes and prepared for the histochemical visualization of the peroxidase, in addition to the immunocytochemical visualization of the neurofilament 68 kD subunit, a long recognized marker of reactive axonal change. The histochemically and immunocytochemically prepared tissue was examined at both the light and electron microscopic level. With mild TBI, the injured animals displayed a repertoire of neurofilament misalignment and axonal swelling consistent with that previously described in our laboratories, yet these changes were not associated with the passage of peroxidase from the extracellular to the intraaxonal compartment. With moderate injury, on the other hand, focal axolemmal permeability change to the extracellularly confined peroxidase was recognized. This peroxidase passage was associated with local mitochondrial abnormalities in addition to an increased packing of the neurofilaments. Over a 3 h course, these neurofilaments began to disassemble, showing a delayed progression of reactive axonal change. Collectively, the results of this investigation suggest that traumatically induced axonal injury involves complex subsets of pathobiology, one evoking rapid primary neurofilamentous change and misalignment, the other eliciting altered membrane permeability concomitant with rapid neurofilament compaction, leading to a delayed progression of reactive axonal change.
Axonal injury is a common feature of human traumatic brain injury. Typically, damaged axons cannot be recognized unless a patient survives the injury by at least 10-12 hours (h). Limitations associated with the use of the traditional silver methods have been linked with this inability to recognize early posttraumatic reactive axonal change. Recently, we reported that antibodies targeting the neurofilament subunits proved useful in recognizing early traumatically induced axonal change in traumatically brain-injured animals. Accordingly, in the present communication, we employed antibodies to detect at the light microscopic level the 68 kD Nf-L and 170-200 kD Nf-H neurofilament subunits in head-injured patients who survived the traumatic event for periods ranging from 6 h to 59 days. Antibodies targeting all of the above-described subunits revealed a progression of reactive axonal change. Antibodies to the 68 kD subunit proved most useful, as they were not complicated by concomitant immunoreactivity in surrounding nuclei and/or dendritic and somatic elements. These immunocytochemical strategies revealed, at 6 h postinjury, focally swollen axons which appeared intact. By 12 h, this focal swelling had progressed to disconnection, with the immunoreactive swelling undergoing further expansion over 1 week postinjury. These findings demonstrate the utility of the previously described immunocytochemical strategies for detecting reactive axonal change in brain-injured humans, particularly in the early posttraumatic course. More importantly, these methods also demonstrate in humans that reactive axonal change is not necessarily caused by traumatically induced tearing.
Recent studies have demonstrated that delayed or secondary axotomy is a consistent feature of traumatic brain injury in both animals and man. Moreover, these studies have shown that the pathogenesis of this secondary axotomy involves various forms of initiating pathology, with the suggestion that, in some cases, only the axonal cytoskeleton is perturbed, while, in other cases, both the axonal cytoskeleton and related axolemma manifest traumatically induced perturbations. In the current communication, we continue in our investigation of the significance of these traumatically induced alterations in axolemmal permeability and their relation to any related intra-axonal cytoskeletal change. This was accomplished in cats which received intrathecal infusions of peroxidase, an agent normally excluded by the intact axolemma. These animals were subjected to traumatic brain injury, and sites showing altered axolemmal permeability to the peroxidase were assessed at the light and electron microscopic level. Through this approach, we recognized that a traumatic episode of moderate severity evoked changes in axolemmal permeability which surprising endured for up to 5 hrs postinjury. At such focal sites of altered permeability, the related cytoskeleton showed a statistically significantly neurofilament compaction, with the strong suggestion of concomitant neurofilament sidearm loss, microtubular dispersion, and mitochondrial abnormality. Over time, these events led to further disorganization of the axonal cytoskeleton which translated into impaired axoplasmic transport and secondary axotomy. Most likely, these alterations in axolemmal permeability result in either the direct or indirect effects upon the axonal cytoskeleton that precipitate the damaging sequences resulting in delayed axotomy.
Recently we reported that traumatic brain injury evokes local changes in the axolemma's permeability, in concert with local cytoskeletal changes involving neurofilament (NF) compaction and sidearm loss, all of which contribute to the genesis of reactive axonal change. Since it was of concern that these events may be either injury model- or species-specific, we sought to address these phenomena in a different but well-characterized animal model and species. Further, to provide more compelling insight into the potential for NF compaction and sidearm alteration, we also employed antibodies specific for the NF rod domains, which are readily visualized only when the NF sidearms are disturbed. Rats were subjected to impact acceleration injury. To assess the potential for altered axolemmal permeability, 5 animals received intrathecal horseradish peroxidase (HRP), normally excluded by the intact axolemma. To assess the potential for NF sidearm alteration, another 14 animals were processed for the visualization of antibodies targeting the NF rod domain at 5 minutes (min) to 24 hours (h) postinjury. All animals were evaluated at the LM and EM levels. Those animals receiving intrathecal HRP showed immediate focal alterations in the axolemma's permeability to the normally excluded tracer. Over a 2 h period, these axons demonstrated NF compaction. Antibodies targeted to the rod domains revealed focal intra-axonal immunoreactivity in sites closely correlated with those showing altered axolemmal permeability. These same sites also demonstrated evidence of NF compaction and sidearm loss/perturbation. Collectively, these findings suggest that occurrence of altered axolemmal permeability and concomitant cytoskeletal change are features common to traumatic brain injury in various animal models and species. Further, these studies underscore the utility of antibodies targeting the rod domain for the early detection of traumatically induced reactive change.
Microtubular changes in axons after stretch injury
- Maxwell
Maxwell, W.L., Microtubul~: changes in axons after stretch injury, J. Neurotrauma, 12 (1995) 3,53 (Abstract).
Vascular permeability alterations to horseradish peroxidase in experimental brain injury
- Povlishock
Diffuse axonal injury in head injury
- Adams