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Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

Aix Marseille University

Oxidative stress (OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 (HO-1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/Akt may modulate HO-1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO-1 in OS-induced apoptosis of human aortic smooth muscle cells (HASMCs).

Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT.

Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein, microtubule-associated protein2a/b (MAP2a/b); some had GAP-43-immunoreactive terminals; and nearly all had morphological features typical of axons. Dendrites of other CINs did not give rise to de novo axons. These CINs did, however, each have a long axon-like process (L-ALP) that projected directly from the soma or a very proximal dendrite. L-ALPs were devoid of MAP2a/b immunoreactivity. Some of these L-ALPs projected through the lesion and formed bouton-like swellings. These results suggest that proximally axotomized spinal interneurons have the potential to form new connections via de novo axons that emerge from distal dendrites. Others may be capable of regeneration of their original axon.

Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy.

It is well established that long, descending axons of the adult mammalian spinal cord do not regenerate after a spinal cord injury (SCI). These axons do not regenerate because they do not mount an adequate regenerative response and growth is inhibited at the injury site by growth cone collapsing molecules, such as chondroitin sulfate proteoglycans (CSPGs). However, whether axons of axotomized spinal interneurons regenerate through the inhibitory environment of an SCI site remains unknown. Here, we show that cut axons from adult mammalian spinal interneurons can regenerate through an SCI site and form new synaptic connections in vivo. Using morphological and immunohistochemical analyses, we found that after a midsagittal transection of the adult feline spinal cord, axons of propriospinal commissural interneurons can grow across the lesion despite a close proximity of their growth cones to CSPGs. Furthermore, using immunohistochemical and electrophysiological analyses, we found that the regenerated axons conduct action potentials and form functional synaptic connections with motoneurons, thus providing new circuits that cross the transected commissures. Our results show that interneurons of the adult mammalian spinal cord are capable of spontaneous regeneration after injury and suggest that elucidating the mechanisms that allow these axons to regenerate may lead to useful new therapeutic strategies for restoring function after injury to the adult CNS.

Spinal interneuron axons spontaneously regenerate after spinal cord injury in the adult feline.

We recently reported that some, but not all, axotomized propriospinal commissural interneurons (PCI) of the adult mammal can regenerate through spinal midsagittal transection injury sites (Fenrich and Rose [2009] J Neurosci 29:12145-12158). In this model, regenerating axons grow through a lesion site surrounded by a dense deposition of chondroitin sulfate proteoglycans (CSPG), which are typically inhibitory to regenerating axons. However, the mechanisms that lead some regenerating axons to grow through spinal cord injury (SCI) sites while others remain trapped in the CSPG zones or retract to their soma remain unknown. As a first step toward elucidating these mechanisms, here we show that the ability of PCI axons to regenerate across a SCI site depends on the branching patterns of their distal terminals. Using 3D reconstruction techniques through multiple serial sections and immunohistochemical analyses, we found that at 7 days postinjury a majority of PCI axons terminated in CSPG zones ipsilateral of the spinal midline. Conversely, at 9 days postinjury some PCI axons had regenerated across the midline, but others terminated outside the CSPG zones near their soma. Furthermore, we show that the most successful regenerators were those with the most extensive branching patterns, whereas those that terminated outside the CSPG zones had terminal regions indistinguishable from dystrophic terminals. Our results demonstrate that the morphological characteristics of regenerating axons play an important role in their ability to regenerate across SCI sites, and that the branching patterns of some regenerating axons are more extensive and have a far greater complexity than previously reported.

Axons with highly branched terminal regions successfully regenerate across spinal midline transections in the adult cat.

Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a ‘pin-prick' spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.

Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows.

The input-output properties of motoneurons are dynamically regulated. This regulation depends, in part, on the relative location of excitatory and inhibitory synapses, voltage-dependent and -independent channels, and neuromodulatory synapses on the dendritic tree. The goal of the present study was to quantify the number and distribution of synapses from two powerful neuromodulatory systems that originate from noradrenergic (NA) and serotonergic (5-HT) neurons. Here we show that the dendritic trees of motoneurons innervating a dorsal neck extensor muscle, splenius, in the adult cat are densely, but not uniformly innervated by both NA and 5-HT boutons. Identified splenius motoneurons were intracellularly stained with Neurobiotin. Using 3D reconstruction techniques we mapped the distributions of contacts formed by NA and 5-HT boutons on the reconstructed dendritic trees of these motoneurons. Splenius motoneurons received an average of 1,230 NA contacts (range = 647-1,507) and 1,582 5-HT contacts (range = 1,234-2,143). The densities of these contacts were 10 (NA) to 6 (5-HT)-fold higher on small compared to large-diameter dendrites. This relationship largely accounts for the bias of NA and 5-HT contacts on distal dendrites and is partially responsible for the higher density of NA contacts on dendrites located more than 200 μm dorsal to the soma. These results suggest that the neuromodulatory actions of NA and 5-HT are compartmentalized and regulate the input-output properties of motoneurons according to precisely arranged interactions with voltage-dependent and -independent channels that are primarily located on small-diameter dendrites.

Nonuniform distribution of contacts from noradrenergic and serotonergic boutons on the dendrites of cat splenius motoneurons.

After spinal cord injury (SCI), resident and peripheral myelomonocytic cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris, and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown, and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two-photon microscopy of adult mice with SCI, here we show that infiltrating lysozyme M (LysM(+)) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles, and exhibit changes in morphology, motility, phagocytic activity and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons, and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI.

Keith K. Fenrich

Developmental Biology Institute of Marseille-Luminy (IBDML)

Aix Marseille University

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Keith K. Fenrich

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Developmental Biology Institute of Marseille-Luminy (IBDML)

Aix Marseille University

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Developmental Biology Institute of Marseille-Luminy (IBDML)