Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

University of Illinois Urbana-Champaign

Few proteins are known to be selectively expressed in retinal ganglion cells (RGCs), the neurons directly affected by optic nerve stroke and glaucoma. In addition, subsets of RGCs are reported to project to various CNS areas via the retinohypothalamic pathway in rodents and primates. Many of these areas exhibit immunoreactivity for the brain-expressed X-linked (Bex) proteins Bex1 and Bex2. This prompted us to evaluate expression of these proteins in retina.

Analysis of optic nerve stroke by retinal Bex expression.

Nonarteritic anterior ischemic optic neuropathy (NAION) is an optic nerve infarct involving axons of retinal ganglion cell (RGC) neurons. The rodent NAION model (rAION) can use transgenic mouse strains to reveal unique characteristics about the effects of sudden optic nerve ischemia on RGCs and their axons. The impact of rAION on RGC stress patterns, RGC loss, and their axons after axonal infarct were evaluated.

Neuron stress and loss following rodent anterior ischemic optic neuropathy in double-reporter transgenic mice.

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.

A primate model of nonarteritic anterior ischemic optic neuropathy.

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.

Rodent anterior ischemic optic neuropathy (rAION) induces regional retinal ganglion cell apoptosis with a unique temporal pattern.

This technical note describes a modification to a fundus camera that permits simultaneous recording of pattern electroretinograms (pERGs) and pattern visual evoked potentials (pVEPs). The modification consists of placing an organic light-emitting diode (OLED) in the split-viewer pathway of a fundus camera, in a plane conjugate to the subject's pupil. In this way, a focused image of the OLED can be delivered to a precisely known location on the retina. The advantage of using an OLED is that it can achieve high luminance while maintaining high contrast, and with minimal degradation over time. This system is particularly useful for animal studies, especially when precise retinal positioning is required.

A simple integrated system for electrophysiologic recordings in animals.

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.

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

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-Δ(12,14)-prostaglandin J2 (PGJ(2)), an analog of the metabolically active PGD(2) metabolite. We evaluated PGJ(2)'s effects and mechanisms using rodent anterior ischemic optic neuropathy (rAION); an in vivo white matter ischemia model. PGJ(2) 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 PGJ(2) improves perfusion at 1 day post-infarct by reducing tissue edema. Our results suggest that PGJ(2) acts by reducing NFκB signaling through preventing p65 nuclear localization and inhibiting inflammatory gene expression. Importantly, PGJ(2) 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. PGJ(2) 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.

PGJ(2) provides prolonged CNS stroke protection by reducing white matter edema.

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-CSF's effects after ON ischemia in the NAION rodent model (rAION).

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

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.

Bernard J. Slater

Neuroscience Program

University of Illinois Urbana-Champaign

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Bernard J. Slater

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Neuroscience Program

University of Illinois Urbana-Champaign

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Neuroscience Program