David J. Barakat

Survival, Integration, and Axon Growth Support of Glia Transplanted Into the Chronically Contused Spinal Cord

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 μl of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 ± 6.8% of the SCs and 2.3 ± 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury–graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Inactivation of astroglial NF‐κB promotes survival of retinal neurons following ischemic injury

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF‐κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia‐reperfusion in wild‐type (WT) and in GFAP‐IκBα‐dn transgenic mice, where the NF‐κB classical pathway is suppressed specifically in astrocytes. The GFAP‐IκBα‐dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro‐inflammatory genes, encoding Tnf‐α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO‐synthase in the retinas of GFAP‐IκBα‐dn mice. These data suggest that certain NF‐κB‐regulated pro‐inflammatory and redox‐active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies.

Schwann cell transplantation improves reticulospinal axon growth and forelimb strength after severe cervical spinal cord contusion.

Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting modest supraspinal axon growth when the site of axon injury is closer to the cell body of the axotomized neuron.

Inhibition of tumour necrosis factor‐α by antisense targeting produces immunophenotypical and morphological changes in injury‐activated microglia and macrophages

Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial‐produced cytokine, tumour necrosis factor‐α (TNF‐α), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF‐α antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post‐contused rat spinal cord were large and rounded (86.3 ± 9.6%). They were GSA‐IB4‐positive (GSA‐IB4+) (Griffonia simplicifolia lectin, microglia specific; 94.8 ± 5.1%), strongly OX‐42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 ± 9.1%), ED‐1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 ± 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 ± 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF‐α (up to 436 pg/µg total protein, 16 h). Fluoroscein isothiocyanate‐labelled TNF‐α ASOs (5, 50 and 200 nm) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF‐α production by up to 92% (26.5 pg/µg total protein, 16 h, 200 nm TNF‐α ASOs). Furthermore, few of the treated cells at this time were round (5.4 ± 2.7%), having become predominantly spindle shaped (74.9 ± 6.3%) or stellate (21.4 ± 2.7%); immunophenotypically, although all of them remained GSA‐IB4 positive (91.6 ± 6.2%), many were weakly OX‐42 positive and few expressed either ED‐1 (12.9 ± 2.5%) or IIA (19.8 ± 7.4%). Thus, the secretion of TNF‐α early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF‐α.

Combining neurotrophin-transduced schwann cells and rolipram to promote functional recovery from subacute spinal cord injury.

Following spinal cord injury (SCI), both an inhibitory environment and lack of intrinsic growth capacity impede axonal regeneration. In a previous study, prevention of cyclic adenosine monophosphate (AMP) hydrolysis by the phosphodiesterase-4 inhibitor rolipram, in combination with Schwann cell (SC) grafts, promoted significant supraspinal and proprioceptive fiber growth and/or sparing and improved locomotion. In another study, transplanted SCs transduced to generate a bifunctional neurotrophin (D15A) led to significant increases in graft SCs and axons, including supraspinal and myelinated axons. Here we studied the growth and myelination of local and supraspinal axons and functional outcome following the combination of rolipram administration and neurotrophin-transduced SC implantation after SCI. Rolipram was administered subcutaneously for 4 weeks immediately after contusion at vertebral T8 (25.0-mm weight drop, MASCIS impactor). GFP or GFP-D15A-transduced SCs were injected into the injury epicenter 1 week after SCI. GFP-D15A SC grafts and GFP SC grafts with rolipram contained significantly more serotonergic fibers compared to GFP SCs. SC myelinated axons were increased significantly in GFP SC with rolipram-treated animals compared to animals receiving SCI alone. Rolipram administered with either GFP or GFP-D15A SCs significantly increased numbers of brain stem-derived axons below the lesion/implant area and improved hindlimb function. Compared to the single treatments, the combination led to the largest SC grafts, the highest numbers of serotonergic fibers in the grafts, and increased numbers of axons from the reticular formation below the lesion/implant area and provided the greatest improvement in hindlimb function. These findings demonstrate the therapeutic potential for a combination therapy involving the maintenance of cyclic AMP levels and neurotrophin-transduced SCs to repair the subacutely injured spinal cord.

Astroglial NF-κB mediates oxidative stress by regulation of NADPH oxidase in a model of retinal ischemia reperfusion injury.

J. Neurochem. (2012) 120, 586–597.

Differential gene expression profiling of large and small retinal ganglion cells

Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.