Schwann cell coculture improves the therapeutic effect of bone marrow stromal cells on recovery in spinal cord-injured mice

Studies of bone marrow stromal cells (MSCs) transplanted into the spinal cord-injured rat give mixed results: some groups report improved locomotor recovery while others only demonstrate improved histological appearance of the lesion. These studies show no clear correlation between neurological improvements and MSC survival. We examined whether MSC survival in the injured spinal cord could be enhanced by closely matching donor and recipient mice for genetic background and marker gene expression and whether exposure of MSCs to a neural environment (Schwann cells) prior to transplantation would improve their survival or therapeutic effects. Mice underwent a clip compression spinal cord injury at the fourth thoracic level and cell transplantation 7 days later. Despite genetic matching of donors and recipients, MSC survival in the injured spinal cord was very poor (~1%). However, we noted improved locomotor recovery accompanied by improved histopathological appearance of the lesion in mice receiving MSC grafts. These mice had more white and gray matter sparing, laminin expression, Schwann cell infiltration, and preservation of neurofilament and 5-HT-positive fibers at and below the lesion. There was also decreased collagen and chondroitin sulphate proteoglycan deposition in the scar and macrophage activation in mice that received the MSC grafts. The Schwann cell cocultured MSCs had greater effects than untreated MSCs on all these indices of recovery. Analyses of chemokine and cytokine expression revealed that MSC/Schwann cell cocultures produced far less MCP-1 and IL-6 than MSCs or Schwann cells cultured alone. Thus, transplanted MSCs may improve recovery in spinal cord-injured mice through immunosuppressive effects that can be enhanced by a Schwann cell coculturing step. These results indicate that the temporary presence of MSCs in the injured cord is sufficient to alter the cascade of pathological events that normally occurs after spinal cord injury, generating a microenvironment that favors improved recovery. © 2011 Cognizant Comm. Corp

CD11d antibody treatment improves recovery in spinal cord-injured mice

Acute administration of a monoclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. This has been chiefly attributed to the reduced infiltration of neutrophils into the injured spinal cord in treated rats. More recently, treating spinal cord-injured mice with a Ly-6G neutrophil-depleting antibody was demonstrated to impair neurological recovery. These disparate results could be due to different mechanisms of action utilized by the two antibodies, or due to differences in the inflammatory responses between mouse and rat that are triggered by SCI. To address whether the anti-CD11d treatment would be effective in mice, a CD11d mAb (205C) or a control mAb (1B7) was administered intravenously at 2, 24, and 48 h after an 8-g clip compression injury at the fourth thoracic spinal segment. The anti-CD11d treatment reduced neutrophil infiltration into the injured mouse spinal cord and was associated with increased white matter sparing and reductions in myeloperoxidase (MPO) activity, reactive oxygen species, lipid peroxidation, and scar formation. These improvements in the injured spinal cord microenvironment were accompanied by increased serotonin (5-HT) immunoreactivity below the level of the lesion and improved locomotor recovery. Our results with the 205C CD11d mAb treatment complement previous work using this anti-integrin treatment in a rat model of SCI. © 2012, Mary Ann Liebert, Inc

The role of BDNF in the injured/regenerating sensory neuron

Peripheral nerve injury induces a robust regenerative state in sensory neurons that includes elevated expression of injury/regeneration-associated genes. The molecular signal(s) underlying the transition to the regenerating state are largely unknown. Brain-derived neurotrophic factor (BDNF) is the sole identified neurotrophin that is upregulated in sensory neurons following peripheral nerve injury. As members of the neurotrophin family exert a profound influence on the intact phenotype of sensory neurons, I hypothesize that injury-associated alterations in BDNF expression play a similar role in the injured/regenerating response. Antagonizing endogenous BDNF with a function-blocking antibody prevented increases in injury/regeneration-associated gene expression and decreased the growth capabilities of the injured sensory neurons. However, BDNF was not important for maintaining this cell body response in injured neurons. The elevation of BDNF expression in injured sensory neurons either through intrathecal infusion or electrical stimulation was associated with increased injury/regeneration-associated gene expression in a dose dependent manner and the latter corresponded to increased sensory axonal regeneration. Though BDNF was able to induce and enhance the intrinsic cell body response of injured sensory neurons, exogenous BDNF was not sufficient to induce an injury phenotype in intact sensory neurons. Thus, additional signals are likely induced by the injury response. In conclusion, BDNF plays a critical role in inducing the regenerative state in sensory neurons following injury and strategies aimed at elevating levels of BDNF available to the injured sensory neuron during the inductive phase improve the cell body response

Anti-Chondroitin Sulfate Proteoglycan Strategies in Spinal Cord Injury: Temporal and Spatial Considerations Explain the Balance between Neuroplasticity and Neuroprotection

Loss of function after spinal cord injury (SCI) results from the primary injury that causes interruption of axonal tracts, neuronal and glial cell death, and the secondary injury in which inflammation drives lesion expansion, and further loss of gray and white matter. There are two main therapeutic strategies for the treatment of SCI: pro -reparative sprouting strategies that aim to promote functional recovery through enhancing the plasticity of spared axons and neuroprotection/anti-inflammatory strategies that aim to decrease secondary injury. Chondroitin sulfate proteoglycans (CSPGs) are matrix molecules that are major constituents of the glial scar at the SCI epicenter and of perineuronal nets found throughout the central nervous system. In this review, we summarize the wealth of literature describing the application of anti-CSPG strategies that target either CSPG synthesis or degradation or signalling after SCI. The weight of the evidence suggests that the balance between neuroprotection and neuroplasticity achieved by any one anti-CSPG strategy depends on the when and the where of its application

Sox9 knockout mice have improved recovery following stroke

The partial recovery that can occur after a stroke has been attributed to structural and functional plasticity that compensate for damage and lost functions. This plasticity is thought to be limited in part by the presence of growth inhibitors in the central nervous system. Blocking or reducing signals from inhibitors of axonal sprouting such as Nogo and chondroitin sulfate proteoglycans (CSPGs) increases post-stroke axonal sprouting and improves recovery. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and demonstrated that conditional Sox9 ablation leads to increased axonal sprouting and improved recovery after spinal cord injury. In the present study we evaluate the effect of conditional Sox9 ablation in a transient middle cerebral artery occlusion (MCAO) model of stroke. We demonstrate that conditional Sox9 ablation leads to reduced CSPG levels, increased tissue sparing and improved post-stroke neurological recovery. Anterograde tract tracing studies demonstrate that in the Sox9 conditional knockout mice corticorubral and corticospinal projections from the contralateral, uninjured cortex increase projections to targets in the midbrain and spinal cord denervated by the injury. These results suggest that targeting SOX9 is a viable strategy to promote reparative axonal sprouting, neuroprotection and recovery after stroke

Alternate approaches supporting BDNF regulation of injury-associated PACAP expression.

<p>(A) Impact of BDNF siRNA infusion on PACAP mRNA expression in injured DRG neurons. Darkfield photomicrographs of L5 DRG sections processed for in situ hybridization to detect PACAP mRNA in intact sensory neurons (Intact), in association with 3d sciatic spinal nerve injury (Injury), 3d injury + infusion of either non-targeting control siRNA (Injury + NT siRNA) or siRNA targeting BDNF mRNA (Injury + siBDNF) as indicated. Note, there is no detectable difference in injury-induced PACAP expression after infusion of non-targeting control siRNA as compared to the Injury alone group, whereas animals treated with siBDNF, display decreased neuronal PACAP expression. Scale bar 100 µm. (B) qRT-PCR analysis of mRNA samples extracted from L4,5 DRG having undergone unilateral sciatic spinal nerve injury with or without either immediate or one week delayed 3 day intrathecal infusion of anti-BDNF or control IgG (Ctrl IgG) as indicated (performed in triplicate). Note that immediate anti-BDNF treatment significantly impairs the induction of injury-associated PACAP expression, while control IgG infusion has no effect. Delayed anti-BDNF does not significantly impact maintenance of PACAP expression in injured sensory neurons (***p<0.001).</p

Incidence, level and distribution of PACAP mRNA expression in DRG neurons after immediate antibody infusion.

<p>Representative scatterplots depicting relative changes in PACAP mRNA hybridization signal over individual neurons in relation to neuronal size after sciatic spinal nerve transection and immediate intrathecal control IgG (A), or anti-BDNF (B) infusion. Each dot represents signal intensity over an individual neuron as a function of neuronal size. Solid lines divide the plots into unlabeled (shaded), and labeled (≥6 X background labeling) populations, and dashed lines separate moderately from highly labeled (≥32 X background) neuronal populations. (C, D) STEP I -incidence of PACAP expression was determined for all neurons analysed for the Control IgG infused group (C; n = 4 animals or 1369 neurons) or anti-BDNF infused group (D; n = 4 animals or 1168 neurons). STEP II –impact of treatment on the incidence of PACAP expression in small (<35 µm) versus medium-large (>35 µm) DRG neurons was determined for immediate intrathecal infusions of either Control IgG (C; n = 2 or 482 neurons) or anti-BDNF (D; n = 2 or 416 neurons) for the animals that had undergone computer-assisted image analysis. Note: the injury-induced increase in PACAP mRNA expression is significantly prevented by immediate intrathecal anti-BDNF infusion (p<0.001, chi-square test). A reduction is observed both in the number of small and medium-large diameter DRG neurons expressing detectable PACAP mRNA, as well as at the level of hybridization signal/neuron.</p

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and T alpha 1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced. (C) 2009 Elsevier Inc. All rights reserved

Incidence, level and distribution of PACAP mRNA expression in DRG neurons after delayed antibody infusion.

<p>Representative scatterplots depicting relative changes in PACAP mRNA hybridization signal over individual neurons in relation to neuronal size after delayed control IgG (A) or anti-BDNF (B) infusions. Each dot represents hybridization signal intensity over a quantified neuron. Solid lines divide the plots into unlabeled (shaded) and labeled (≥6X background labeling) populations, and dashed lines separate moderately from highly labeled populations (≥32 X background). Numbers and percentages of DRG neurons expressing detectable PACAP mRNA after delayed intrathecal control (C) or anti-BDNF (D) antibody treatment for 3 days following 7 days of injury (n = 6, 3 animals analysed per treatment group). Note, delayed intrathecal anti-BDNF infusion did not have any significant effect on the injury-induced increase in PACAP mRNA expression after axotomy.</p

PACAP mRNA expression is reduced in injured sensory and motor neurons after immediate anti-BDNF infusion.

<p>Darkfield photomicrographs of PACAP mRNA hybridization signal over sensory neurons in L5 DRG sections (A, B) and spinal cord motor neurons (C, D) in response to 3 days sciatic spinal nerve transection in conjunction with immediate 3 days infusion of intrathecal control IgG (A, C) or anti-BDNF (B, D) infusions. The increase in PACAP mRNA expression observed after nerve transection is markedly reduced by anti-BDNF infusion. Both the incidence of neurons expressing detectable PACAP mRNA and the level of expression in individual neurons are decreased. Scale bar (A, B) 100 µm, (C, D) 200 µm.</p