Damien D. Pearse

cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show thatcAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.

Combining Schwann Cell Bridges and Olfactory-Ensheathing Glia Grafts with Chondroitinase Promotes Locomotor Recovery after Complete Transection of the Spinal Cord

Numerous obstacles to successful regeneration of injured axons in the adult mammalian spinal cord exist. Consequently, a treatment strategy inducing axonal regeneration and significant functional recovery after spinal cord injury has to overcome these obstacles. The current study attempted to address multiple impediments to regeneration by using a combinatory strategy after complete spinal cord transection in adult rats: (1) to reduce inhibitory cues in the glial scar (chondroitinase ABC), (2) to provide a growth-supportive substrate for axonal regeneration [Schwann cells (SCs)], and (3) to enable regenerated axons to exit the bridge to re-enter the spinal cord (olfactory ensheathing glia). The combination of SC bridge, olfactory ensheathing glia, and chondroitinase ABC provided significant benefit compared with grafts only or the untreated group. Significant improvements were observed in the Basso, Beattie, and Bresnahan score and in forelimb/hindlimb coupling. This recovery was accompanied by increased numbers of both myelinated axons in the SC bridge and serotonergic fibers that grew through the bridge and into the caudal spinal cord. Although prominent descending tracts such as the corticospinal and reticulospinal tracts did not successfully regenerate through the bridge, it appeared that other populations of regenerated fibers were the driving force for the observed recovery; there was a significant correlation between numbers of myelinated fibers in the bridge and improved coupling of forelimb and hindlimb as well as open-field locomotion. Our study tests how proven experimental treatments interact in a well-established animal model, thus providing needed direction for the development of future combinatory treatment regimens.

Transplantation of Schwann cells and/or olfactory ensheathing glia into the contused spinal cord: Survival, migration, axon association, and functional recovery

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have shown promise for spinal cord injury repair. We sought their in vivo identification following transplantation into the contused adult rat spinal cord at 1 week post‐injury by: (i) DNA in situ hybridization (ISH) with a Y‐chromosome specific probe to identify male transplants in female rats and (ii) lentiviral vector‐mediated expression of EGFP. Survival, migration, and axon‐glia association were quantified from 3 days to 9 weeks post‐transplantation. At 3 weeks after transplantation into the lesion, a 60–90% loss of grafted cells was observed. OEG‐only grafts survived very poorly within the lesion (<5%); injection outside the lesion led to a 60% survival rate, implying that the injury milieu was hostile to transplanted cells and or prevented their proliferation. At later times post‐grafting, p75+/EGFP− cells in the lesion outnumbered EGFP+ cells in all paradigms, evidence of significant host SC infiltration. SCs and OEG injected into the injury failed to migrate from the lesion. Injection of OEG outside of the injury resulted in their migration into the SC‐injected injury site, not via normal‐appearing host tissue but along the pia or via the central canal. In all paradigms, host axons were seen in association with or ensheathed by transplanted glia. Numerous myelinated axons were found within regions of grafted SCs but not OEG. The current study details the temporal survival, migration, axon association of SCs and OEG, and functional recovery after grafting into the contused spinal cord, research previously complicated due to a lack of quality, long‐term markers for cell tracking in vivo.

Specific pathophysiological functions of JNK isoforms in the brain

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c‐JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6‐hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment‐specific expression of JNK isoforms with 46‐kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6‐hydroxydopamine was transiently improved in JNK3 ko and c‐JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c‐JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid‐induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c‐Jun irrespective on the experimental set‐up. This comprehensive study provides novel insights into the context‐dependent physiological and pathological functions of JNK isoforms.

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.

Transplantation strategies to promote repair of the injured spinal cord.

This review describes the results of the transplantation of Schwann cells and olfactory ensheathing glia in combination with other interventions. The complete transection injury model was used to test the combination of Schwann cell bridges with methylprednisolone, neurotrophins, or olfactory ensheathing glia. The contusion injury model was used to compare Schwann cell and olfactory ensheathing glia transplantation and to examine the results of combining Schwann cell transplants with elevated levels of cyclic adenosine monophosphate. The combination strategies were more effective than cell transplantation alone. The improved regeneration response usually involved a reduction in secondary tissue loss, axonal regeneration from brainstem neurons, an increase in myelinated fibers in the transplant, the exit of regenerated fibers from the transplant into the contiguous cord, and an improvement in locomotor function.

TRANSDUCED SCHWANN CELLS PROMOTE AXON GROWTH AND MYELINATION AFTER SPINAL CORD INJURY

We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.

Modulation of the cAMP signaling pathway after traumatic brain injury

Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream target, protein kinase A, from 15 min to 48 h after moderate FPI. To determine if preventing hydrolysis of cAMP by administration of a phosphodiesterase (PDE) IV inhibitor would improve outcome after TBI, we treated animals intraperitoneally with rolipram (0.3 or 3.0 mg/kg) 30 min prior to TBI, and then once per day for 3 days. Rolipram treatment restored cAMP to sham levels and significantly reduced cortical contusion volume and improved neuronal cell survival in the parietal cortex and CA3 region of the hippocampus. Traumatic axonal injury, characterized by beta-amyloid precursor protein deposits in the external capsule, was also significantly reduced in rolipram-treated animals. Furthermore, levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), were significantly decreased with rolipram treatment. These results demonstrate that the cAMP-PKA signaling cascade is downregulated after TBI, and that treatment with a PDE IV inhibitor improves histopathological outcome and decreases inflammation after TBI.

Combination of engineered Schwann cell grafts to secrete neurotrophin and chondroitinase promotes axonal regeneration and locomotion after spinal cord injury.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

Transgenic inhibition of astroglial NF-κB leads to increased axonal sparing and sprouting following spinal cord injury

We previously showed that Nuclear Factor κB (NF‐κB) inactivation in astrocytes leads to improved functional recovery following spinal cord injury (SCI). This correlated with reduced expression of pro‐inflammatory mediators and chondroitin sulfate proteoglycans, and increased white matter preservation. Hence we hypothesized that inactivation of astrocytic NF‐κB would create a more permissive environment for axonal sprouting and regeneration. We induced both contusive and complete transection SCI in GFAP‐Inhibitor of κB‐dominant negative (GFAP‐IκBα‐dn) and wild‐type (WT) mice and performed retrograde [fluorogold (FG)] and anterograde [biotinylated dextran amine (BDA)] tracing 8 weeks after injury. Following contusive SCI, more FG‐labeled cells were found in motor cortex, reticular formation, and raphe nuclei of transgenic mice. Spared and sprouting BDA‐positive corticospinal axons were found caudal to the lesion in GFAP‐IκBα‐dn mice. Higher numbers of FG‐labeled neurons were detected immediately rostral to the lesion in GFAP‐IκBα‐dn mice, accompanied by increased expression of synaptic and axonal growth‐associated molecules. After transection, however, no FG‐labeled neurons or BDA‐filled axons were found rostral and caudal to the lesion, respectively, in either genotype. These data demonstrated that inhibiting astroglial NF‐κB resulted in a growth‐supporting terrain promoting sparing and sprouting, rather than regeneration, of supraspinal and propriospinal circuitries essential for locomotion, hence contributing to the improved functional recovery observed after SCI in GFAP‐IκBα‐dn mice.