Neuronal Membrane Disruption Occurs Late Following Diffuse Brain Trauma in Rats and Involves a Subpopulation of NeuN Negative Cortical Neurons

Abstract

The repercussions of traumatic brain injury (TBI) endure years following the initial insult and involve chronic impairments/disabilities. Studies indicate that these morbidities stem from diffuse pathologies, however, knowledge regarding TBI-mediated diffuse pathologies, and in particular, diffuse neuronal membrane disruption, is limited. Membrane disruption has been shown to occur acutely following injury, primarily within neurons, however, the progression of TBI-induced membrane disruption remains undefined. Therefore, the current study investigated this pathology over a longer temporal profile from 6 h to 4 w following diffuse TBI induced using the central fluid percussion injury (CFPI) model in rats. To visualize membrane disruption, animals received an intracerebroventricular infusion of tagged cell-impermeable dextran 2 h prior to experimental endpoints at 6 h, 1 d, 3 d, 1 w, 2 w, or 4 w post-CFPI. The percentage of total neurons demonstrating dextran uptake, indicative of membrane disruption, was quantified within the lateral neocortex layers V and VI from 6 h to 4 w post-injury. We found that membrane disruption displayed a biphasic pattern, where nearly half of the neurons were membrane disrupted sub-acutely, from 6 h to 3 d post-TBI. At 1 w the membrane disrupted population was dramatically reduced to levels indistinguishable from sham controls. However, by 2 and 4 w following CFPI, approximately half of the neurons analyzed displayed membrane disruption. Moreover, our data revealed that a subset of these late membrane disrupted neurons were NeuN negative (NeuN-). Correlative western blot analyses, however, revealed no difference in NeuN protein expression in the lateral neocortex at any time following injury. Furthermore, the NeuN- membrane disrupted neurons did not co-label with traditional markers of astrocytes, microglia, oligodendrocytes, or NG2 cells. Immunohistochemistry against NeuN, paired with a hematoxylin and eosin counter-stain, was performed to quantify the possibility of overall NeuN+ neuronal loss following CFPI. A NeuN- population was observed consistently in both sham and injured animals regardless of time post-injury. These data suggest that there is a consistent subpopulation of NeuN- neurons within the lateral neocortex regardless of injury and that these NeuN- neurons are potentially more vulnerable to late membrane disruption. Better understanding of membrane disruption could provide insight into the mechanisms of diffuse pathology and lead to the discovery of novel treatments for TBI.

Buprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury.

“…As has been well characterized previously, CFPI (2.05±0.1 atmospheres) did not generate gross tissue pathology 24,25,27,28 . While there was apparent sub-arachnoid bleeding at 1d post-injury, there was no apparent damage to the underlying cortex.…”

“…Finally, as somatic neuronal damage, in the form of neuronal membrane disruption, is present acutely following both diffuse and focal TBI, and as this pathology is exacerbated by secondary insults, we investigated the potential effects of Bup on neuronal membrane disruption 24,25,28,[34][35][36] . As we and others have demonstrated previously, tagged 10kDa dextran, which is normally excluded from intact membranes, are a reliable way to evaluate neuronal plasmalemmal disruptions 24,25,28,35,37,38 . However, this method only allows assessment of the cortex, due to the variability of dextran diffusion within the thalamic domain.…”

“…A 2mm diameter burr hole was also drilled into the left parietal bone overlaying the left lateral ventricle (0.8 mm posterior, 1.3 mm lateral, and 2.5 to 3 mm ventral relative to bregma) through which a 25-gauge needle, connected to a pressure transducer and a micro infusion pump 11 Elite syringe pump (Harvard Apparatus) via PE50 tubing, was placed into the left ventricle. Appropriate placement of the infusion pump into the lateral ventricle was veri ed via a 2.3μl/min infusion of sterile saline within the closed uid pressure system during needle placement 24,25 . The needle was held in the lateral ventricle for at least 5 minutes to record preinjury ICP; then the needle was slowly removed.…”

“…Bone wax was used to seal the burr hole used for the ICP measurements before preparation for central uid percussion injury (CFPI). The procedures used to induce CFPI were consistent with those described previously [24][25][26][27] . Brie y, a Luer-Loc syringe hub was a xed to the craniotomy site with dental acrylic (methyl methacrylate; Hygenic, Akron, OH, USA) that was applied around the hub, including the area overlying the sealed burr hole and allowed to harden.…”

How Can a Punch Knock You Out?

“…For TBI in general, it has been suggested that a role may be played by several other mechanosensitive ion channels, including Piezo receptors, the NMDA receptor, TRP receptors, as well as voltage-sensitive sodium channels (21)(22)(23)(24)(25). Mechanosensitive ion channels have also been suggested to play a role in the later stages of TBI, where they may play a role in the development of secondary injury (26,27).…”