Neuroaxonal Regeneration is More Pronounced in Early Multiple Sclerosis than in Traumatic Brain Injury Lesions

The extent of irreversible neuroaxonal damage is the key determinant of permanent disability in traumatic and inflammatory conditions of the central nervous system (CNS). Structural damage is nevertheless in part compensated by neuroplastic events. However, it is unknown whether the same kinetics and mechanisms of neuroaxonal de‐ and regeneration take place in inflammatory and traumatic conditions. We analyzed neuroaxonal degeneration and plasticity in early multiple sclerosis (MS) lesions and traumatic brain injury (TBI). Neuroaxonal degeneration identified by the presence of SMI31+ chromatolytic neurons and SMI32+ axonal profiles were characteristic features of leukocortical TBI lesions. Axonal transport disturbances as determined by amyloid precursor protein (APP)+ spheroids were present in both TBI and MS lesions to a similar degree. Neurons expressing growth‐associated protein 43 (GAP43) and synaptophysin (Syn) were found under both pathological conditions. However, axonal swellings immunopositive for GAP43 and Syn clearly prevailed in subcortical MS lesions, suggesting a higher regenerative potential in MS. In this context, GAP43+/APP+ axonal spheroid ratios correlated with macrophage infiltration in TBI and MS lesions, supporting the idea that phagocyte activation might promote neuroplastic events. Furthermore, axonal GAP43+ and Syn+ swellings correlated with prolonged survival after TBI, indicating a sustained regenerative response.     

A lack of amyloid beta plaques despite persistent accumulation of amyloid beta in axons of long-term survivors of traumatic brain injury

Traumatic brain injury (TBI) is a risk factor for developing Alzheimer's disease (AD). Additionally, TBI induces AD-like amyloid β (Aβ) plaque pathology within days of injury potentially resulting from massive accumulation of amyloid precursor protein (APP) in damaged axons. Here, progression of Aβ accumulation was examined using brain tissue from 23 cases with post-TBI survival of up to 3 years. Even years after injury, widespread axonal pathology was consistently observed and was accompanied by intra-axonal co-accumulations of APP with its cleavage enzymes, beta-site APP cleaving enzyme and presenilin-1 and their product, Aβ. However, in marked contrast to the plaque pathology noted in short-term cases post TBI, virtually no Aβ plaques were found in long-term survivors. A potential mechanism for Aβ plaque regression was suggested by the post-injury accumulation of an Aβ degrading enzyme, neprilysin. These findings fail to support the premise that progressive plaque pathology after TBI ultimately results in AD.     

Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons

Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In Caenorhabditis elegans following laser‐induced axotomy, the axon still attached to the cell body is able to regrow and reconnect with its separated distal fragment. Here we show that reconnection of separated axon fragments during regeneration of C. elegans mechanosensory neurons occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re‐establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. We also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re‐growing axon recognizing its own separated distal fragment. Thus, efficient axonal regeneration can occur by selective reconnection and fusion of separated axonal fragments beyond an injury site, with restoration of the damaged neuronal tract. Developmental Dynamics 240:1365–1372, 2011. © 2011 Wiley‐Liss, Inc.     

Age-dependent changes in noradrenergic innervations of the frontal cortex in F344 rats

Noradrenergic innervations of the frontal cortex with advancing age (9, 13 and 25 months) in male F344 rats were quantified by immunohistochemistry for dopamine-beta-hydroxylase (DBH), which is a marker enzyme for noradrenergic axons. The density of DBH-positive axons, varicosities (swellings along an axon from which noradrenaline is released), and the number of varicosities per unit length of axon were measured in the frontal cortex. We found that the density of axons and varicosities significantly decreased at an earlier stage of aging (9-13 months), but not at a later stage (13-25 months). On the other hand, the number of varicosities per unit length of axon did not change with age. The result shows that the density of varicosities, which represent the synapses of noradrenergic neurons, decrease in the frontal cortex in the early aging process.     

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

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

Three‐Dimensional Visualization of Developing Neurovascular Architecture in the Craniofacial Region of Embryonic Mice

Recent studies have highlighted the mechanism of vascular and axonal guidance to ensure proper morphogenesis and organogenesis. We aimed to perform global mapping of developing neurovascular networks during craniofacial development of embryonic mice. To this end, we developed histology‐based three‐dimensional (3D) reconstructions using paraffin‐embedded serial sections obtained from mouse embryos. All serial sections were dual‐immunolabeled with Pecam1 and Pgp9.5/Gap43 cocktail antibodies. All immunolabeled serial sections were digitized with virtual microscopy to acquire high spatial resolution images. The 3D reconstructs warranted superior positional accuracy to trace the long‐range connectivity of blood vessels and individual cranial nerve axons. It was feasible to depict simultaneously the details of angiogenic sprouting and axon terminal arborization and to assess quantitatively the locoregional proximity between blood vessels and cranial nerve axons. Notably, 3D views of the craniofacial region revealed the following: Branchial arch arteries and blood capillary plexi were formed without accompanying nerves at embryonic day (E) 9.5. Cranial nerve axons began to grow into the branchial arches, developing a labyrinth of small blood vessels at E10.5. Vascular remodeling occurred, and axon terminals of the maxillary, mandibular, chorda tympani, and hypoglossal nerve axons had arborized around the lateral lingual swellings at E11.5. The diverged patterning of trigeminal nerves and the arterial branches from the carotid artery became congruent at E11.5. The overall results support the advantage of dual‐immunolabeling and 3D reconstruction technology to document the architecture and wiring of the developing neurovascular networks in mouse embryos. Anat Rec, 298:1824–1835, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural observations of organelle accumulation in the equine recurrent laryngeal nerve

Summary The left recurrent laryngeal nerves from five horses with sub-clinical neuropathy were examined by light and electron microscopy in a study designed to examine accumulation of axonal organelles at paranodal and internodal locations. Transverse sections of the nerve showed scattered fibres with split myelin sheaths and axonal accumulation of organelles. On longitudinal sections these collections were seen to result from an axonal outpouching in which dense lamellar bodies and mitochondria had accumulated. These paranodal collections, which could be found on both sides of the node, were often associated with infoldings of the terminal loops of myelin and with occasional paranodal demyelination. The fact that many of the organelles in the outpouches were lysosomal in nature was confirmed by their positive staining for cathepsin D activity. Longitudinal sections demonstrated a number of axons which were swollen over a long distance and which contained focal accumulations of similar organelles. In places, however, there was a clear separation between these organelles and the cytoskeletal proteins. In each case these swollen axons were surrounded by Schwann cell nuclei and their processes, forming well-ordered onion bulbs. The possibility that these two types of changes, i.e. the paranodal accumulations and the axonal swellings could result from a disturbance in axonal transport in this distal axonopathy is discussed.     

Injury-induced remodelling and regeneration of the ribbon presynaptic terminalin vitro

Summary The neuronal response to axonal injury may relate to the type of insult incurred. Recently, neuritic and presynaptic varicosity regeneration by isolated adult salamander photoreceptors was demonstrated. We have used this system to compare the rod photoreceptor response to two types of injury:denervation/detargeting, the removal of pre-and postsynaptic partners from the axon terminal, andaxotomy, the removal of the axon terminal itself. Cells were followed with time-lapse video microscopy for 24–48h in culture and immunolabelled for SV2 or synaptophysin to identify synaptic vesicle-containing varicosities. Although all injured cells responded with regenerative growth, denervated/detargeted photoreceptors (i.e. neurons which retain their axon terminal) grew 80% more processes and fourfold more presynaptic varicosities than axotomized neurons. In cells which retained their original axon and terminal, varicosity formation generally began with axon retraction. Retraction was followed by elaboration of a lamellipodium and, by 48 h, development of varicosity-bearing neurites from the lamellipodium. Synaptic vesicle protein localization in denervated/detargeted cells paralleled axon terminal reorganization. Axotomized cells, in contrast, lacked synaptic vesicle protein immunoreactivity during this period. To detect synaptic protein synthesis, photoreceptors were examined for colocalization of synaptic vesicle protein with rab6, a Golgi marker, by confocal microscopy. As expected, synaptic vesicle protein staining was present in the Golgi complex during regeneration; however, in cells with an axon, new synaptic vesicle protein-labelled varicosities were found at early stages, prior to the appearance of immunolabel in the Golgi complex. The data demonstrate remarkable plasticity in the ribbon synapse, and suggest that in adult rod cells with an intact axon terminal, synaptic vesicle protein synthesis is not a prerequisite for the formation of new presynaptic-like terminals. We propose that preexisting axonal components are reutilized to expedite presynaptic renewal as an early response to denervation/detargeting.     

Calcium signaling is implicated in the diffuse axonal injury of brain stem

Evaluating diffuse axonal injury (DAI) remains challenging in clinical sciences since the physiopathologic mechanism of DAI is still unclear. The calcium overload in the axoplasm is considered to be crucial for secondary axonal injury. The present study use calcium channel blocker, nimodipine, to explore the influence of Ca²⁺ in the pathogenesis of rat DAI. In the DAI group, the expressions of β-APP and NF-L in axons were increased from 12 to 72 h. The ultrastructural observation indicated the axon and vessel injury appeared at 12 h post-injury and severely aggravated from 24 to 72 h. The expression of vWF and brain water content was increased at 12 h after injury and further increased at 24 h. Nimodipine decreased the expression of β-APP, NF-L and vWF, and also attenuated the ultrastructural damage of vascular wall and axons. Furthermore, Ca-dependent enzyme, the calcineurin activity were increased in DAI and nimodipine suppressed the activity of calcineurins (CaN). However, the amount of CaN expression was not changed. Our results showed that disturbances of axonal calcium homeostasis play an important role in the secondary damage of the axon, neuron and capillary vessel which may be related with activating CaN during the acute phase of DAI. Nimodipine can alleviate the secondary damage by suppressing the calcineurin activity.     

Axon collaterals of mossy fibers from the pontine nucleus in the cerebellar dentate nucleus.

1. Single axons of pontine nucleus neurons (PN axons) receiving cerebral input were stained intra-axonally with horseradish peroxidase (HRP) in the cerebellum of cats. The axonal trajectory of single PN axons was reconstructed from serial sections of the cerebellum and the brain stem. 2. Axons were penetrated in the white matter near the dentate nucleus, and, after electrophysiological identification, PN axons were injected iontophoretically with HRP. The identification criteria for the PN axons were 1) their direct responses to stimulation of the contralateral pontine nucleus (PN), 2) their synaptic activation from the contralateral cerebral cortex, and 3) the decrease in threshold for evoking direct spikes in stimulation of the PN by conditioning stimuli applied in the cerebral cortex. 3. Two hundred thirty-three axons were electrophysiologically identified as PN axons receiving the input from the cerebral cortex. Ninety-six of them were stained successfully with HRP, and reconstructions were made from 40 well-stained PN axons. All of them gave rise to mossy fibers and terminated in the granular layer of the cerebellar cortex as typical mossy fiber rosettes. Out of these, 22 gave axon collaterals to the dentate nucleus. Virtually all of the axon branches observed in the dentate nucleus were axon collaterals of mossy fibers from the PN to the cerebellar cortex. In 7 of these 22 PN axons, cell bodies were retrogradely labeled with HRP, and all of them were found in the contralateral PN. 4. The stained-stem axons arising from the PN ran medially in the pons, crossed the midline, and then ascended dorsocaudally in the branchium pontis. After passing in the white matter anterior to or lateral to the dentate nucleus, they entered into the cerebellar cortex. On their way, one to three axon collaterals were given off from parent axons to the dentate nucleus. The diameter of these collaterals was very thin (mean, 0.6 microns), compared with the large diameter of the parent axons (mean, 2.1 microns). 5. Some axon collaterals were very simple and had only one terminal branch with or without short branchlets, whereas others were more complex, and single axon collaterals ramified before forming a terminal arborization. Axon collaterals of single PN axons mainly spread mediolaterally or dorsoventrally in the frontal plane but had a very narrow rostrocaudal extension. 6. Terminal branches usually bore swellings en passant along their length and one terminal swelling at their end. The number of swellings per axon collateral ranged 23-180 (116 +/- 52, mean +/- SD).(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

Summary Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.     

The impact of myelination on axon sparing and locomotor function recovery in spinal cord injury assessed using diffusion tensor imaging

The dysmyelinated axons of shiverer mice exhibit impaired conduction characteristics, similar to early postnatal axons before myelination, whereas the patterns of neuronal activity and connectivity are relatively comparable with those of wild‐type myelinated axons. This unique dysmyelination pattern is exploited in the present study to determine the role of compact myelin in the loss and recovery of function following traumatic spinal cord injury (SCI). We applied in vivo diffusion tensor imaging (DTI) and post‐mortem immunohistochemistry analysis to examine changes in myelin and axonal integrity, and evaluated these changes in concert with the analysis of locomotor function from 1 to 4 weeks following a mid‐thoracic contusion injury in homozygous shiverer and heterozygous littermate mice. The DTI biomarkers, axial and radial diffusivities, are noninvasive indicators of axon and myelin integrity in response to SCI of both myelinated and dysmyelinated spinal cord. We show that myelin is critical for normal hind limb function in open field locomotion. However, when the functional outcome is limited during chronic SCI, the extent of recovery is associated with residual axonal integrity and independent of the extent of intact myelin at the lesion epicenter. Copyright © 2013 John Wiley & Sons, Ltd.     

Reticular endings of Purkinje cell axons in the rat cerebellar nuclei: scanning electron microscopic observation of the pericellular plexus of Cajal.

The pericellular plexus of Purkinje cell axons in the cerebellar nuclei was first recorded by Cajal (1911), using the Golgi method. The present study observed the axonal plexus in the rat by scanning electron microscopy after the plexus' detachment from the soma of target neurons by NaOH maceration. The pericellular plexus revealed numerous axons with ellipsoidal and moniliform swellings. They branched and crossed with each other to form, as a whole, a reticulum which enveloped the target neuron instead of multiple isolated boutons. Some axon terminals were separated from each other by thin glial processes, while others lacked such septa, thus being directly juxtaposed. Immunohistochemistry for spot 35 protein, a Purkinje cell-specific protein, detected similar beaded and reticular axons terminating on the neuronal somata, confirming their identification as Purkinje cells. Transmission electron microscopic observation showed that most of the axon terminals in question contained elliptical vesicles, characteristic for Purkinje cells. The vesicles were not accumulated toward small patchy areas of synaptic specialization but disseminated along the entire length of the terminal portion of axons.     

Diffusion tensor imaging detects axonal injury in a mouse model of repetitive closed-skull traumatic brain injury

Mild traumatic brain injuries (TBI) are common in athletes, military personnel, and the elderly, and increasing evidence indicates that these injuries have long-term health effects. However, the difficulty in detecting these mild injuries in vivo is a significant impediment to understanding the underlying pathology and treating mild TBI. In the following experiments, we present the results of diffusion tensor imaging (DTI) and histological analysis of a model of mild repetitive closed-skull brain injury in mouse. Histological markers used included silver staining and amyloid precursor protein (APP) immunohistochemistry to detect axonal injury, and Iba-1 immunohistochemistry to assess microglial activation. At 24h post-injury, before silver staining or microglial abnormalities were apparent by histology, no significant changes in any of the DTI parameters were observed within white matter. At 7 days post-injury we observed a reduction in axial and mean diffusivity. Relative anisotropy at 7 days correlated strongly with the degree of silver staining. Interestingly, APP was not observed at any timepoint examined. In addition to the white matter alterations, mean diffusivity was elevated in ipsilateral cortex at 24h but returned to sham levels by 7 days. Altogether, this demonstrates that DTI is a sensitive method for detecting axonal injury despite a lack of conventional APP pathology. Further, this reflects a need to better understand the histological basis for DTI signal changes in mild TBI.     

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

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

Early and Sustained Activation of Autophagy in Degenerating Axons after Spinal Cord Injury

Axonal degeneration is one of the initial steps in many neurological disorders and has been associated with increased autophagic activity. Although there are increasing data on the regulation of autophagy proteins in the neuronal soma after spinal cord injury (SCI), their characterization in the axon is scarce. Here, we examined the regulation of autophagy during axonal degeneration in a rat model of SCI following a lesion at Th 8. We analyzed the morphological and ultrastructural changes in injured axons by immunohistochemical evaluation of autophagy‐related proteins and electron microscopy at different time points following SCI. The expression of ULK1, Atg7 and Atg5 in damaged axons was rapidly upregulated within hours after SCI. The number of axonal LC3‐positive autophagosomes was also rapidly increased after SCI and remained at an increased level for up to 6 weeks. Ultrastructural analysis showed early signs of axonal degeneration and increased autophagy. In conclusion, we show that autophagy is increased early and for a sustained period in degenerating axons after SCI and that it might be an important executive step involved in axonal degeneration. Therefore, autophagy may represent a promising target for future therapeutic interventions in the treatment of axonal degeneration in traumatic central nervous system disorders.     

Diverse changes in microglia morphology and axonal pathology during the course of 1 year after mild traumatic brain injury in pigs

Over 2.8 million people experience mild traumatic brain injury (TBI) in the United States each year, which may lead to long‐term neurological dysfunction. The mechanical forces that are caused by TBI propagate through the brain to produce diffuse axonal injury (DAI) and trigger secondary neuroinflammatory cascades. The cascades may persist from acute to chronic time points after injury, altering the homeostasis of the brain. However, the relationship between the hallmark axonal pathology of diffuse TBI and potential changes in glial cell activation or morphology have not been established in a clinically relevant large animal model at chronic time points. In this study, we assessed the tissue from pigs subjected to rapid head rotation in the coronal plane to generate mild TBI. Neuropathological assessments for axonal pathology, microglial morphological changes, and astrocyte reactivity were conducted in specimens out to 1‐year post‐injury. We detected an increase in overall amyloid precursor protein pathology, as well as periventricular white matter and fimbria/fornix pathology after a single mild TBI. We did not detect the changes in corpus callosum integrity or astrocyte reactivity. However, detailed microglial skeletal analysis revealed changes in morphology, most notably increases in the number of microglial branches, junctions, and endpoints. These subtle changes were most evident in periventricular white matter and certain hippocampal subfields, and were observed out to 1‐year post‐injury in some cases. These ongoing morphological alterations suggest persistent change in neuroimmune homeostasis. Additional studies are needed to characterize the underlying molecular and neurophysiological alterations, as well as potential contributions to neurological deficits.     

Axonal injury following mild traumatic brain injury is exacerbated by repetitive insult and is linked to the delayed attenuation of NeuN expression without concomitant neuronal death in the mouse

Mild traumatic brain injury (mTBI) affects brain structure and function and can lead to persistent abnormalities. Repetitive mTBI exacerbates the acute phase response to injury. Nonetheless, its long‐term implications remain poorly understood, particularly in the context of traumatic axonal injury (TAI), a player in TBI morbidity via axonal disconnection, synaptic loss and retrograde neuronal perturbation. In contrast to the examination of these processes in the acute phase of injury, the chronic‐phase burden of TAI and/or its implications for retrograde neuronal perturbation or death have received little consideration. To critically assess this issue, murine neocortical tissue was investigated at acute (24‐h postinjury, 24hpi) and chronic time points (28‐days postinjury, 28dpi) after singular or repetitive mTBI induced by central fluid percussion injury (cFPI). Neurons were immunofluorescently labeled for NeuroTrace and NeuN (all neurons), p‐c‐Jun (axotomized neurons) and DRAQ5 (cell nuclei), imaged in 3D and quantified in automated manner. Single mTBI produced axotomy in 10% of neurons at 24hpi and the percentage increased after repetitive injury. The fraction of p‐c‐Jun+ neurons decreased at 28dpi but without neuronal loss (NeuroTrace), suggesting their reorganization and/or repair following TAI. In contrast, NeuN+ neurons decreased with repetitive injury at 24hpi while the corresponding fraction of NeuroTrace+ neurons decreased over 28dpi. Attenuated NeuN expression was linked exclusively to non‐axotomized neurons at 24hpi which extended to the axotomized at 28dpi, revealing a delayed response of the axotomized neurons. Collectively, we demonstrate an increased burden of TAI after repetitive mTBI, which is most striking in the acute phase response to the injury. Our finding of widespread axotomy in large fields of intact neurons contradicts the notion that repetitive mTBI elicits progressive neuronal death, rather, emphasizing the importance of axotomy‐mediated change.