Bernard J. Slater

A Primate Model of Nonarteritic Anterior Ischemic Optic Neuropathy

PURPOSE Nonarteritic anterior ischemic optic neuropathy (NAION) is an optic nerve (ON) stroke and a leading cause of sudden ON-related vision loss. A primate (p)NAION model is crucial to further understanding of the clinical disorder and can provide information regarding the pathophysiology of other central nervous system (CNS) ischemic axonopathies. In the current study, a primate model of NAION was developed, and short-and long-term responses to this condition were characterized. METHODS pNAION was induced with a novel photoembolic mechanism. Short-and long-term responses were evaluated by minimally invasive testing (electrophysiology, fundus photography, indocyanine green and fluorescein angiography, and magnetic resonance imaging) and compared with histologic and immunohistochemical findings. RESULTS Optic disc edema, similar to that observed in cases of human NAION was seen 1 day after induction, with subsequent resolution associated with the development of optic disc pallor. Magnetic resonance imaging (MRI) performed 3 months after induction revealed changes consistent with ON atrophy. Electrophysiological studies and vascular imaging suggest an ON-limited infarct with subsequent axonal degeneration and selective neuronal loss similar to that seen in human NAION. ON inflammation was evident 2 months after induction at the site of the lesion and at distant sites, suggesting that inflammation-associated axonal remodeling continues for an extended period after ON infarct. CONCLUSIONS pNAION resembles human NAION in many respects, with optic disc edema followed by loss of cells in the retinal ganglion cell (RGC) layer and ON remodeling. This model should be useful for evaluating neuroprotective and other treatment strategies for human NAION as well as for other ischemic processes that primarily affect CNS white-matter tracts.

Rodent Anterior Ischemic Optic Neuropathy (rAION) Induces Regional Retinal Ganglion Cell Apoptosis with a Unique Temporal Pattern

PURPOSE Nonarteritic anterior ischemic optic neuropathy (NAION) results in optic nerve damage with retinal ganglion cell (RGC) loss. An NAION model, rodent anterior ischemic optic neuropathy (rAION), was used to determine AION-associated mechanisms of RGC death and associated regional retinal changes. METHODS rAION was induced in male Wistar rats, and the retinas analyzed at various times after induction. RGCs were positively identified by both retrograde fluorogold labeling and brain-expressed X-linked protein-1/2 (Bex1/2) immunoreactivity. RGC death was analyzed by fluorescein-tagged annexin-V labeling (FITC-annexin-V), as well as by terminal nucleotide nick-end labeling (TUNEL). Retinal flatmount preparations enabled regional retinal analysis of labeled dying cells. Apoptosis pathway activation was confirmed by Western analysis, with an antibody that recognizes cleaved caspase-3. RESULTS Post-rAION, RGCs die by apoptosis over a longer period than previously recognized. Cleaved caspase-3 immunoreactivity was greatest between 11 and 15 days. rAION-induced RGC death occurs regionally, with sparing of large contiguous regions of RGCs. CONCLUSIONS rAION results in later RGC death than in traumatic optic nerve damage models. Apoptosis, measured by FITC-annexin, occurs maximally in the second to third week after infarct. Cleaved caspase-3 activation confirms that after rAION, RGCs undergo apoptosis by the caspase activation pathway. The regional pattern in dying RGCs after rAION implies that a measure of retinotopic organization occurs in the rodent optic nerve. The prolonged period from insult to death suggests that the window for successful treatment after ON infarct may be longer than previously recognized.

Axonal degeneration, regeneration and ganglion cell death in a rodent model of anterior ischemic optic neuropathy (rAION)

Using laser-induced photoactivation of intravenously administered rose Bengal in rats, we generated an ischemic infarction of the intrascleral portion of the optic nerve (ON) comparable to that which occurs in humans to investigate optic nerve axon degenerative events following optic nerve infarct and the potential for axon re-growth. Animals were euthanized at different times post infarct. Axon degeneration was evaluated with SMI312 immunolabeling, and GAP-43 immunostaining was used to identify axon regeneration. Terminal dUTP nick end labeling (TUNEL) was used to evaluate retinal ganglion cell (RGC) death. There was significant axon structural disruptinot ion at the anterior intrascleral portion of the ON by 3d post-infarct, extending to the posterior ON by 7d post-stroke. Destruction of normal axon structure and massive loss of axon fibers occurred by 2 weeks. GAP-43 immunoreactivity occurred in the anterior ON by 7d post-infarct, lasting 3-4 weeks, without extension past the primary ischemic lesion. TUNEL-positive cells in the RGC layer appeared by 7d post-insult. These results indicate that following induction of ischemic optic neuropathy, significant axon damage occurs by 3d post-infarct, with later neuronal death. Post-stroke adult rat retinal ganglion cells attempt to regenerate their axons, but this effort is restricted to the unmyelinated region of the anterior ON. These responses are important in understanding pathologic process that underlies human non-arteritic anterior ischemic optic neuropathy (NAION) and may guide both the appropriate treatment of NAION and the window of opportunity for such treatment.

PGJ2 Provides Prolonged CNS Stroke Protection by Reducing White Matter Edema

Few clinically effective approaches reduce CNS-white matter injury. After early in-vivo white matter infarct, NFκB-driven pro-inflammatory signals can amplify a relatively small amount of vascular damage, resulting in progressive endothelial dysfunction to create a severe ischemic lesion. This process can be minimized by 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), an analog of the metabolically active PGD2 metabolite. We evaluated PGJ2s effects and mechanisms using rodent anterior ischemic optic neuropathy (rAION); an in vivo white matter ischemia model. PGJ2 administration systemically administered either acutely or 5 hours post-insult results in significant neuroprotection, with stereologic evaluation showing improved neuronal survival 30 days post-infarct. Quantitative capillary vascular analysis reveals that PGJ2 improves perfusion at 1 day post-infarct by reducing tissue edema. Our results suggest that PGJ2 acts by reducing NFκB signaling through preventing p65 nuclear localization and inhibiting inflammatory gene expression. Importantly, PGJ2 showed no in vivo toxicity structurally as measured by optic nerve (ON) myelin thickness, functionally by ON-compound action potentials, on a cellular basis by oligodendrocyte precursor survival or changes in ON-myelin gene expression. PGJ2 may be a clinically useful neuroprotective agent for ON and other CNS infarcts involving white matter, with mechanisms of action enabling effective treatment beyond the currently considered maximal time for intervention.

EVIDENCE FOR LAYER-SPECIFIC DIFFERENCES IN AUDITORY CORTICOCOLLICULAR NEURONS

Recent data suggest that there may be distinct processing streams emanating from auditory cortical layers 5 and 6 that influence the auditory midbrain. To determine whether these projections have different physiological properties, we injected rhodamine-tagged latex tracer beads into the inferior colliculus of >30-day-old mice to label these corticofugal cells. Whole-cell recordings were performed on 62 labeled cells to determine their basic electrophysiological properties and cells were filled with biocytin to determine their morphological characteristics. Layer 5 auditory corticocollicular cells have prominent I(h)-mediated sag and rebound currents, have relatively sluggish time constants, and can generate calcium-dependent rhythmic bursts. In contrast, layer 6 auditory corticocollicular cells are non-bursting, do not demonstrate sag or rebound currents and have short time constants. Quantitative analysis of morphology showed that layer 6 cells are smaller, have a horizontal orientation, and have very long dendrites (>500 μm) that branch profusely both near the soma distally near the pia. Layer 5 corticocollicular cells are large pyramidal cells with a long apical dendrite with most branching near the pial surface. The marked differences in physiological properties and dendritic arborization between neurons in layers 5 and 6 make it likely that each type plays a distinct role in controlling auditory information processing in the midbrain.

Open-loop organization of thalamic reticular nucleus and dorsal thalamus: a computational model

The thalamic reticular nucleus (TRN) is a shell of GABAergic neurons that surrounds the dorsal thalamus. Previous work has shown that TRN neurons send GABAergic projections to thalamocortical (TC) cells to form reciprocal, closed-loop circuits. This has led to the hypothesis that the TRN is responsible for oscillatory phenomena, such as sleep spindles and absence seizures. However, there is emerging evidence that open-loop circuits are also found between TRN and TC cells. The implications of open-loop configurations are not yet known, particularly when they include time-dependent nonlinearities in TC cells such as low-threshold bursting. We hypothesized that low-threshold bursting in an open-loop circuit could be a mechanism by which the TRN could paradoxically enhance TC activation, and that enhancement would depend on the relative timing of TRN vs. TC cell stimulation. To test this, we modeled small circuits containing TC neurons, TRN neurons, and layer 4 thalamorecipient cells in both open- and closed-loop configurations. We found that open-loop TRN stimulation, rather than universally depressing TC activation, increased cortical output across a broad parameter space, modified the filter properties of TC neurons, and altered the mutual information between input and output in a frequency-dependent and T-type calcium channel-dependent manner. Therefore, an open-loop model of TRN-TC interactions, rather than suppressing transmission through the thalamus, creates a tunable filter whose properties may be modified by outside influences onto the TRN. These simulations make experimentally testable predictions about the potential role for the TRN for flexible enhancement of cortical activation.

An auditory colliculothalamocortical brain slice preparation in mouse

Key questions about the thalamus are still unanswered in part because of the inability to stimulate its inputs while monitoring cortical output. To address this, we employed flavoprotein autofluorescence optical imaging to expedite the process of developing a brain slice in mouse with connectivity among the auditory midbrain, thalamus, thalamic reticular nucleus, and cortex. Optical, electrophysiological, anatomic, and pharmacological tools revealed ascending connectivity from midbrain to thalamus and thalamus to cortex as well as descending connectivity from cortex to thalamus and midbrain and from thalamus to midbrain. The slices were relatively thick (600-700 μm), but, based on typical measures of cell health (resting membrane potential, spike height, and input resistance) and use of 2,3,5-triphenyltetrazolium chloride staining, the slices were as viable as thinner slices. As expected, after electrical stimulation of the midbrain, the latency of synaptic responses gradually increased from thalamus to cortex, and spiking responses were seen in thalamic neurons. Therefore, for the first time, it will be possible to manipulate and record simultaneously the activity of most of the key brain structures that are synaptically connected to the thalamus. The details for the construction of such slices are described herein.

Visualizing Dynamic Brain Networks Using an Animated Dual-Representation

Dynamic network visualization has been a challenging topic for dynamic networks analysis, especially for spatially embedded networks like brain networks. In this paper, we present an animated interactive visualization design that combines enhanced node-link diagrams and distance matrix layouts to assist neuroscientists in their exploration of dynamic brain networks and that enables them to understand how functional connections relate to the spatial structure of the brain. Our visualization also provides the ability to observe the evolution of a network, the change in the community identities, and node behavior over time.

Optic nerve inflammation and demyelination in a rodent model of nonarteritic anterior ischemic optic neuropathy.

PURPOSE Optic nerve (ON) ischemia associated with nonarteric anterior ischemic optic neuropathy (NAION) results in axon and myelin damage. Myelin damage activates the intraneural Ras homolog A (RhoA), contributing to axonal regeneration failure. We hypothesized that increasing extrinsic macrophage activity after ON infarct would scavenge degenerate myelin and improve postischemic ON recovery. We used the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to upregulate ON macrophage activity, and evaluated GM-CSFs effects after ON ischemia in the NAION rodent model (rAION). METHODS Following rAION induction, GM-CSF was administered via intraventricular injection. Retinal ganglion cell (RGC) stereologic analysis was performed 1 month postinduction. The retinae and optic nerve laminae of vehicle- and GM-CSF-treated animals were examined immunohistochemically and ultrastructurally using transmission electron microscopy (TEM). RhoA activity was analyzed using a rhotekin affinity immunoanalysis and densitometry. Isolated ONs were analyzed functionally ex vivo by compound action potential (CAP) analysis. RESULTS Rodent NAION produces ON postinfarct demyelination and myelin damage, functionally demonstrable by CAP analysis and ultrastructurally by TEM. Granulocyte-macrophage colony-stimulating factor increased intraneural inflammation, activating and recruiting endogenous microglia, with only a moderate amount of exogenous macrophage recruitment. Treatment with GM-CSF reduced postinfarct intraneural RhoA activity, but did not neuroprotect RGCs after rAION. CONCLUSIONS Sudden ON ischemia results in previously unrecognized axonal demyelination, which may have a clinically important role in NAION-related functional defects and recovery. Granulocyte-macrophage colony-stimulating factor is not neuroprotective when administered directly to the optic nerve following ON ischemia, and does not improve axonal regeneration. It dramatically increases ON-microglial activation and recruitment.

Blocking elevated VEGF-A attenuates non-vasculature Fragile X syndrome abnormalities

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, ). One of the most prominent regulators of vascular growth is VEGF‐A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF‐A expression. In addition to its role in vascular regulation, VEGF‐A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF‐A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF‐A in brain. These studies suggest that increased VEGF‐A expression in FXS is contributing to non‐vascular FXS abnormalities. To explore the role of VEGF‐A in mediating non‐vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF‐A. Bevacizumab treatment was found to decrease FXS Synapsin‐1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF‐A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF‐A is elevated in FXS brain and suggest that its expression promotes non‐vascular FXS abnormalities.