Victoria P.A. Johnstone

Cortical Hypoexcitation Defines Neuronal Responses in the Immediate Aftermath of Traumatic Brain Injury

Traumatic brain injury (TBI) from a blow to the head is often associated with complex patterns of brain abnormalities that accompany deficits in cognitive and motor function. Previously we reported that a long-term consequence of TBI, induced with a closed-head injury method modelling human car and sporting accidents, is neuronal hyper-excitation in the rat sensory barrel cortex that receives tactile input from the face whiskers. Hyper-excitation occurred only in supra-granular layers and was stronger to complex than simple stimuli. We now examine changes in the immediate aftermath of TBI induced with same injury method. At 24 hours post-trauma significant sensorimotor deficits were observed and characterisation of the cortical population neuronal responses at that time revealed a depth-dependent suppression of neuronal responses, with reduced responses from supragranular layers through to input layer IV, but not in infragranular layers. In addition, increased spontaneous firing rate was recorded in cortical layers IV and V. We postulate that this early post-injury suppression of cortical processing of sensory input accounts for immediate post-trauma sensory morbidity and sets into train events that resolve into long-term cortical hyper-excitability in upper sensory cortex layers that may account for long-term sensory hyper-sensitivity in humans with TBI.

Heterogeneous distribution of axonal cytoskeleton proteins in the human optic nerve.

PURPOSE Cytoskeleton proteins play a critical role in maintaining retinal ganglion cell structure, viability, and function. This study documents the distribution of cytoskeleton protein subunits in the various regions of the normal human optic nerve and identifies important relationships among mitochondria, myelin, and neurofilament proteins. METHODS Twenty-three optic nerves from human cadavers were used. Confocal microscopy was used to examine the distribution of neurofilament light, neurofilament medium, neurofilament heavy (phosphorylated and unphosphorylated), neurofilament heavy (phosphorylated only), actin, and microtubule associated protein (MAP)-1 along the sagittal plane of the optic nerve. Comparisons were made among superior, middle, and inferior regions and also among temporal, central, and nasal portions of the optic nerve. Colocalization of neurofilament light, mitochondrial cytochrome c oxidase (COX), and myelin was also performed. RESULTS There are significant differences in the pattern and distribution of neurofilament protein subunits, actin, and MAP-1 along the sagittal plane of the optic nerve. Cytoskeleton proteins and COX mitochondria are found in highest concentrations in the prelaminar and lamina cribrosa regions. COX and neurofilament light occurs predominantly in unmyelinated nerve, with a significant decrease in concentration occurring on optic nerve myelination. CONCLUSIONS The heterogeneous distribution of cytoskeleton proteins along the sagittal plane may be an important functional adaptation that reflects the nonuniform nature of the physiological and structural environment of the optic nerve. The heterogeneous distribution of cytoskeleton proteins may also partly account for the asymmetric pattern of optic nerve damage after intraocular pressure elevation.

Histomorphometric measurements in human and dog optic nerve and an estimation of optic nerve pressure gradients in human.

Intraocular pressure and cerebrospinal fluid (CSF) pressure are important determinants of the trans-laminar pressure gradient which is believed to be important in the pathogenesis of glaucomatous optic nerve degeneration. Computational models and finite element calculations of optic nerve head biomechanics have been previously used to predict pressures and stresses in the human optic nerve. The purpose of this report is to morphometrically compare the optic nerve laminar and pia mater structure between humans and dogs, and to use previously reported tissue pressure measurements in the dog optic nerve to estimate individual-specific human optic nerve pressures and pressure gradients. High resolution light microscopy was used to acquire quantitative histological measurements from sagittal sections taken from the middle of the optic nerve in 34 human cadaveric eyes and 10 dog eyes. Parameters measured included the pre-laminar and lamina cribrosa thickness, distance from posterior boundary of lamina cribrosa to inner limiting membrane (ILM), shortest distance between anterior lamina cribrosa surface and subarachnoid space, shortest distance between ILM and inner surface of pia mater in contact with the subarachnoid space and optic nerve diameter. Pia mater thickness in the proximal 4 mm of post-laminar nerve was also determined. There was no significant difference in lamina cribrosa thickness between dog and human eyes (P = 0.356). The distance between the intraocular and subarachnoid space was greater in dogs (P < 0.001). Pia mater thickness was greatest at the termination of subarachnoid space in both species. In humans, pia mater thickness decreased over the proximal 500 mum to reach a constant value of approximately 60 mum. In dogs this decrease occurred over 1000 mum to reach a constant diameter of approximately 30 mum. Using previous measurements of optic nerve pressures and pressure gradients in dogs we estimate that at an IOP of 15 mmHg and a CSF pressure of 0 mmHg the mean pressure difference across the human pia mater will be 4.8 +/- 2.2 mmHg. If we assume that the pressure difference between the intraocular space and post-laminar tissue falls across the entire thickness of the human lamina cribrosa then an estimate of the trans-laminar pressure gradient is 2.0 +/- 0.8 mmHg/100 mum. If we assume that this pressure difference only occurs across the dense collagenous plates of the posterior lamina cribrosa then a trans-laminar pressure gradient high estimate of 3.3 +/- 1.4 mmHg/100 mum is calculated. Changes in tissue pressure gradients in the optic nerve may be an important factor in the pathogenesis of glaucomatous optic neuropathy.

A Protein Synthesis and Nitric Oxide-Dependent Presynaptic Enhancement in Persistent Forms of Long-Term Potentiation.

Long-term potentiation (LTP) is an important process underlying learning and memory in the brain. At CA3-CA1 synapses in the hippocampus, three discrete forms of LTP (LTP1, 2, and 3) can be differentiated on the basis of maintenance and induction mechanisms. However, the relative roles of pre- and post-synaptic expression mechanisms in LTP1, 2, and 3 are unknown. Neurotransmitter release in the expression of LTP1, 2, and 3 was measured via FM 1-43 destaining from CA3 terminals in hippocampal slices from male Wistar rats (7-8 wk). No difference in vesicle turnover rate was observed for LTP1 up to 160 min following induction by one train of theta-burst stimulation (1TBS). A presynaptic enhancement was found for LTP2 at 160 min after induction by 4TBS, and for LTP3 at both 80 and 160 min after induction by 8TBS. Inhibition of nitric oxide (NO) signaling blocked both LTP2 and LTP3 maintenance and the associated enhanced release. LTP2 maintenance and its presynaptic expression were dependent on protein synthesis, but not gene transcription. LTP3 maintenance was dependent on both translation and transcription, but like LTP2, the enhanced release only required translation. These data considerably strengthen the mechanistic separation of LTP1, 2, and 3, supporting a model of multiple, discrete forms of LTP at CA3-CA1 synapses rather than different temporal phases.

Experimental Traumatic Brain Injury Results in Long-Term Recovery of Functional Responsiveness in Sensory Cortex but Persisting Structural Changes and Sensorimotor, Cognitive, and Emotional Deficits

Traumatic brain injury (TBI) is a leading cause of death worldwide. In recent studies, we have shown that experimental TBI caused an immediate (24-h post) suppression of neuronal processing, especially in supragranular cortical layers. We now examine the long-term effects of experimental TBI on the sensory cortex and how these changes may contribute to a range of TBI morbidities. Adult male Sprague-Dawley rats received either a moderate lateral fluid percussion injury (n=14) or a sham surgery (n=12) and 12 weeks of recovery before behavioral assessment, magnetic resonance imaging, and electrophysiological recordings from the barrel cortex. TBI rats demonstrated sensorimotor deficits, cognitive impairments, and anxiety-like behavior, and this was associated with significant atrophy of the barrel cortex and other brain structures. Extracellular recordings from ipsilateral barrel cortex revealed normal neuronal responsiveness and diffusion tensor MRI showed increased fractional anisotropy, axial diffusivity, and tract density within this region. These findings suggest that long-term recovery of neuronal responsiveness is owing to structural reorganization within this region. Therefore, it is likely that long-term structural and functional changes within sensory cortex post-TBI may allow for recovery of neuronal responsiveness, but that this recovery does not remediate all behavioral deficits.

Postsynaptic protein synthesis is required for presynaptic enhancement in persistent forms of long-term potentiation

Long-term potentiation (LTP) in the hippocampus is a fundamental process underlying learning and memory in the brain. At CA3-CA1 synapses, three discrete forms of LTP (LTP1, 2, and 3) have been differentiated on the basis of their persistence, maintenance mechanisms, Ca2+ signaling pathways, expression loci, and electrophysiological requirements. We previously showed that LTP2 and LTP3 involve a presynaptic expression component that is established in a translation-dependent manner. Here we investigate the locus of translation required for presynaptic expression. Neurotransmitter release rate was estimated via FM 1-43 destaining from CA3 terminals in hippocampal slices from male Wistar rats (6–8 weeks). Destaining was measured at sites making putative contact with CA1 dendritic processes in stratum radiatum that had been filled with a membrane impermeable translation inhibitor and a fluorescent indicator. Our results suggest that inhibition of postsynaptic translation eliminates the enhanced release ordinarily observed at 160 min post-LTP induction, and that this effect is limited to sites closely apposed to the filled postsynaptic cell. We conclude that postsynaptic translation is required for the presynaptic component of LTP2 and LTP3 expression. These data considerably strengthen the mechanistic separation of LTP1, 2, and 3 and provide evidence for an expanded repertoire of communication between synaptic elements.

Characterising effects of impact velocity on brain and behaviour in a model of diffuse traumatic axonal injury

The velocity of impact between an object and the human head is a critical factor influencing brain injury outcomes but has not been explored in any detail in animal models. Here we provide a comprehensive overview of the interplay between impact velocity and injury severity in a well-established weight-drop impact acceleration (WDIA) model of diffuse brain injury in rodents. We modified the standard WDIA model to produce impact velocities of 5.4, 5.85 and 6.15 m/s while keeping constant the weight and the drop height. Gradations in impact velocity produced progressive degrees of injury severity measured behaviourally, electrophysiologically and anatomically, with the former two methods showing greater sensitivity to changes in impact velocity. There were impact velocity-dependent reductions in sensorimotor performance and in cortical depth-related depression of sensory cortex responses; however axonal injury (demonstrated by immunohistochemistry for β-amyloid precursor protein and neurofilament heavy-chain) was discernible only at the highest impact velocity. We conclude that the WDIA model is capable of producing graded axonal injury in a repeatable manner, and as such will prove useful in the study of the biomechanics, pathophysiology and potential treatment of diffuse axonal injury.

Diffuse traumatic brain injury and the sensory brain.

In this review we discuss the consequences to the brains cortex, specifically to the sensory cortex, of traumatic brain injury. The thesis underlying this approach is that long‐term deficits in cognition seen after brain damage in humans are likely underpinned by an impaired cortical processing of the sensory information needed to drive cognition or to be used by cognitive processes to produce a response. We take it here that the impairment to sensory processing does not arise from damage to peripheral sensory systems, but from disordered brain processing of sensory input.

Nanoparticle-Mediated Dual Delivery of an Antioxidant and a Peptide against the L-Type Ca2+ Channel Enables Simultaneous Reduction of Cardiac Ischemia-Reperfusion Injury

Increased reactive oxygen species (ROS) production and elevated intracellular Ca(2+) following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca(2+) channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca(2+) channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca(2+) channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca(2+) channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.

Progesterone Exacerbates Short-Term Effects of Traumatic Brain Injury on Supragranular Responses in Sensory Cortex and Over-Excites Infragranular Responses in the Long Term

Progesterone (P4) has been suggested as a neuroprotective agent for traumatic brain injury (TBI) because it ameliorates many post-TBI sequelae. We examined the effects of P4 treatment on the short-term (4 days post-TBI) and long-term (8 weeks post-TBI) aftermath on neuronal processing in the rodent sensory cortex of impact acceleration-induced diffuse TBI. We have previously reported that in sensory cortex, diffuse TBI induces a short-term hypoexcitation that is greatest in the supragranular layers and decreases with depth, but a long-term hyperexcitation that is exclusive to the supragranular layers. Now, adult male TBI-treated rats administered P4 showed, in the short term, even greater suppression in neural responses in supragranular layers but a reversal of the TBI-induced suppression in granular and infragranular layers. In long-term TBI there were only inconsistent effects of P4 on the TBI-induced hyperexcitation in supragranular responses but infragranular responses, which were not affected by TBI alone, were elevated by P4 treatment. Intriguingly, the effects in the injured brain were almost identical to P4 effects in the normal brain, as seen in sham control animals treated with P4: in the short term, P4 effects in the normal brain were identical to those exercised in the injured brain and in the long term, P4 effects in the normal brain were rather similar to what was seen in the TBI brain. Overall, these results provide no support for any protective effects of P4 treatment on neuronal encoding in diffuse TBI, and this was reflected in sensorimotor and other behavior tasks also tested here. Additionally, the effects suggest that mechanisms used for P4 effects in the normal brain are also intact in the injured brain.