Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury - PubMed (original) (raw)

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

Min D Tang-Schomer et al. Exp Neurol. 2012 Jan.

Abstract

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

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Figures

Figure 1

Figure 1. In vitro Model: Dynamic Stretch Injury of Axons

Pulsed air pressure introduces uniaxial stretch on patterned longitudinally arranged axon tracts, simulating stretch injury of CNS axons during head trauma. (a) The injury device consists of a culture well sealed within a pressure chamber into which a controlled air pulse is delivered. The culture well contains a central deformable membrane onto which primary cortical neuronal cells are plated (b). Two separate populations of cells are separated by a lithography fabricated micro-patterned barrier. Axonal processes extend through the 2mm microchannels to integrate with the opposing population of neurons, thus creating a unique axon-only region that can be placed over the slit in the device (c). Upon controlled delivery of air into the sealed chamber, the subsequent pressure change within the chamber allows the regionally specific deflection of the axon-only region.

Figure 2

Figure 2. Microtubule Displacement and Undulation Formation Immediately After Dynamic Stretch Injury of Axons

(a) Representative images displaying multiple axons with an undulating morphology and immunoreactive for βIII-tubulin immediately (≤2 minutes) following dynamic stretch injury (Scale bar approx. 5μm). (b-c) TEM images showing a normal region of axon with intact microtubules aligned in parallel cables. (d-i) TEM images within 2 minutes following dynamic stretch injury showing widening of the spaces between microtubules near the peak of axon undulations accompanied by compaction of the polymers. Microtubules are also no longer linear and aligned in parallel but rather appear distorted and twisted with discrete breaks (arrows). Breaking and twisting of microtubules was observed near the peak of axon undulations where newly generated free-ends at disconnection points failed to align. Scale bars: 500 nm.

Figure 3

Figure 3. Immunocytochemical Staining of Axons Following Dynamic Stretch Injury

Immunofluorescent images of injured axons (3hr post-injury) displaying a series of swellings along the axonal length like beads on a string. Swellings display accumulations of (a) tubulin, (b) the microtubule-associated protein tau, (c) amyloid precursor protein (APP), and (d) neurofilament (NF200). Scale bar, 10 μm.

Figure 4

Figure 4. Microtubule Breakage and Loss in Varicose Axonal Swellings 3 Hours After Dynamic Stretch Injury of Axons

(a-d) TEM images of axons at 3hr post-injury were stitched together to reconstruct a panoramic image of an axon segment. Individual microtubules display selective breakage within axon swellings (a,c,d arrows). While breakage or loss of microtubules can be seen in association with swelling, other microtubules can be observed traversing the swollen region intact. Specifically, (b) shows a solitary intact microtubule traversing a large swelling (arrow) whereas multiple microtubules can be observed in the adjacent non-swollen region from which it emerged (arrow). This sole remaining microtubule feeds into a subsequent but separate swelling. Scale bars: 500 nm.

Figure 5

Figure 5. Undulations and Varicosities Following Acute Traumatic Brain Injury in Humans at Post-Mortem

(a) An absence of APP immunoreactivity in normal white matter within the corpus callosum of an 18F with no history of TBI who died as a result of haematological malignancy (case #5). Scale bar: 25μm (b) APP Immunoreactivity within the corpus callosum of an 18M who died 10 hours following acute severe traumatic brain injury caused by an assault (blunt force trauma to the head) (case #1). Axons display an undulating morphology akin to what is observed in vitro following dynamic stretch injury. Scale bar: 25μm (c) APP Immunoreactivity within the corpus callosum, also in case #1, displaying a classic varicose morphology with multiple individual swellings along the length of an individual axon can be observed. Scale bar: 30μm.

Figure 6

Figure 6. Varicose Axons within the Corpus Callosum of 4 Human Cases of Acute Severe TBI Examined at Post-Mortem via APP Immunohistochemistry

(a) Extensive axonal pathology with classic varicosities and axonal bulb formation within the corpus callosum of an 18F who died 22 hours following a MVC (case #3). Axons with an undulating morphology can be observed within the same field (arrow). Scale bar: 100μm (b and c) High magnification axonal varicosities in the corpus callosum of a 41 year-old male who died 16 hours following a fall (case #2). Note the serial swellings indicating multiple points of partial transport interruption. Scale bars (b):100μm, (c)30μm. (d) Extensive axonal pathology with classic varicosities and axonal bulb formation within the corpus callosum of an 18M who died 10 hours following blunt force trauma to the head (case #1). Scale bar: 100μm (e) High magnification of a single axon accumulating APP in a 25 year old male following a MVC (case #4). Note the multiple points of swelling along the visible axon length culminating in a large axonal swelling (axonal bulb) at the disconnected axon terminal. Scale bar: 90μm (f) Individual axon displaying a varicose morphology with isolated serial swellings, aslso from case #3. Scale bar: 50μm

Figure 7

Figure 7. Proposed Mechanism of Varicosity Formation after Traumatic Axonal Injury

(a) Illustration displaying two illustrated microtubules (MT1 and MT2) within an intact axon (pre-injury). (b) Following injury, mechanical breaking occurs at different sites in both microtubule 1 and microtubule 2. Misalignment of broken microtubules causes deformation of the axon observed as two discrete undulations. (c) Shortly afterward, catastrophic depolymerization from the broken ends of the microtubules allows the undulations to collapse and the axon recovers its linear morphology. (d) Microtubule breakage leads to impairment of axonal transport and subsequent accumulation of transported cargos near the microtubule breaking point. By contrast, axon transport on the intact microtubules remains normal. This ‘partial transport impairment’ may account for the formation of serial swellings that give axons a varicose appearance following traumatic axonal injury.

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