Trans-neuronal transduction of spinal neurons following cortical injection and anterograde axonal transport of a bicistronic AAV1 vector.

Adeno-associated viral (AAV) vectors are one of the most promising gene delivery systems to the central nervous system. We now report, that AAV1 can be used to express transgenes trans-neuronally in neurons distant from the injection site. Specifically, intracortical injection of a bicistronic AAV1 vector trans-neuronally transduced spinal neurons as shown by fluorescence microscopy, the presence of AAV genome and AAV transcript in the contralateral spinal cord. Prior pyramidotomy abolished spinal transduction, confirming anterograde axonal transport of AAV1 in the corticospinal tract. These observations demonstrate the potential of bicistronic AAV1 for trans-neuronal expression of therapeutic transgenes in neurological disorders or reporter genes in connectivity studies.

Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector.

The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.

Neuronal overexpression of tissue-type plasminogen activator does not enhance sensory axon regeneration or locomotor recovery following dorsal hemisection of adult mouse thoracic spinal cord.

CNS axons rarely regenerate spontaneously back to original targets following spinal cord injury (SCI). Neuronal expression of the serine protease tissue-type plasminogen activator (tPA) enhances axon growth in vitro and following PNS injury. Here we test the hypothesis that neuronal overexpression of tPA in adult transgenic mice promotes CNS axon regeneration and functional recovery following SCI. Adult wild-type and transgenic mouse spinal cords were subjected to dorsal hemisection at the level of the T10/T11 vertebrae. PCR confirmed incorporation of the transgene. Immunolabeling revealed overexpression of tPA in transgenic mice in neurons, including large-diameter neurons in lumbar dorsal root ganglia that contribute axons to the dorsal columns. Immunolabeling also revealed the presence of tPA protein within axons juxtaposing the injury site in transgenics but not wild types. In situ zymography revealed abundant enzymatic activity of tPA in gray matter of thoracic spinal cords of transgenics but not wild types. Rotorod locomotor testing revealed no differences between groups in locomotor function up to 21 days postinjury. Transganglionic tracer was injected into the crushed right sciatic nerve 28 days postinjury, and mice were killed 3 days later. There was no evidence for regrowth of ascending dorsal column sensory axons through or beyond the injury site. In conclusion, despite neuronal overexpression of tPA in injured neurons of transgenics, neither locomotor recovery nor regeneration of ascending sensory axons was observed following thoracic dorsal hemisection.

Motor enrichment sustains hindlimb movement recovered after spinal cord injury and glial transplantation.

PURPOSE: This study investigated whether enrichment improves hindlimb movement following complete spinal cord transection and transplantation of olfactory ensheathing glia (OEG), with or without a Schwann cell (SC) bridge. METHODS: Motor activity was encouraged through provision of motor enrichment housing (MEH); a multi-level cage containing ramps, textured surfaces and rewards. Hindlimb joint movement was assessed weekly for 22 weeks starting one week post-surgery, comparing rats housed in MEH to those in basic housing (BH). Transganglionic tracer was injected into the crushed right sciatic nerve three days prior to sacrifice, allowing sensory axons in the dorsal columns to be visualized by immunolabeling. Serotonergic axons and glial cells expressing low affinity nerve growth factor receptor were identified by immunolabeling. RESULTS: All rats, having received transplants, recovered some hindlimb movement. Rats housed in BH progressively lost recovered hindlimb function whereas recovered hindlimb movements were sustained in most rats in MEH. In rats transplanted with SCs and OEG, effects of MEH were first significant 14 weeks after injury. In rats transplanted with OEG, a trend was seen from 14 weeks after injury, but this did not reach significance. In all rats, traced sensory axons died back from sites of transplantation and did not regenerate rostrally. Further, in no rat were serotonergic axons observed regenerating into, around or beyond transplants. CONCLUSIONS: Transection and transplantation of SC/OEG or OEG induced recovery of hindlimb function. This recovered hindlimb movement was sustained in rats housed in MEH but was progressively lost in rats housed in BH. Because benefits of MEH were not observed until 14 weeks after injury, long-term assessment of behavior is recommended. BH conditions are not conducive to maintenance of recovered hindlimb function, and MEH should be used in studies of recovery of function following spinal cord injury.

Schwann cells for spinal cord repair.

The complex nature of spinal cord injury appears to demand a multifactorial repair strategy. One of the components that will likely be included is an implant that will fill the area of lost nervous tissue and provide a growth substrate for injured axons. Here we will discuss the role of Schwann cells (SCs) in cell-based, surgical repair strategies of the injured adult spinal cord. We will review key studies that showed that intraspinal SC grafts limit injury-induced tissue loss and promote axonal regeneration and myelination, and that this response can be improved by adding neurotrophic factors or anti-inflammatory agents. These results will be compared with several other approaches to the repair of the spinal cord. A general concern with repair strategies is the limited functional recovery, which is in large part due to the failure of axons to grow across the scar tissue at the distal graft-spinal cord interface. Consequently, new synaptic connections with spinal neurons involved in motor function are not formed. We will highlight repair approaches that did result in growth across the scar and discuss the necessity for more studies involving larger, clinically relevant types of injuries, addressing this specific issue. Finally, this review will reflect on the prospect of SCs for repair strategies in the clinic.

Schwann cells for spinal cord repair

The complex nature of spinal cord injury appears to demand a multifactorial repair strategy. One of the components that will likely be included is an implant that will fill the area of lost nervous tissue and provide a growth substrate for injured axons. Here we will discuss the role of Schwann cells (SCs) in cell-based, surgical repair strategies of the injured adult spinal cord. We will review key studies that showed that intraspinal SC grafts limit injury-induced tissue loss and promote axonal regeneration and myelination, and that this response can be improved by adding neurotrophic factors or anti-inflammatory agents. These results will be compared with several other approaches to the repair of the spinal cord. A general concern with repair strategies is the limited functional recovery, which is in large part due to the failure of axons to grow across the scar tissue at the distal graft-spinal cord interface. Consequently, new synaptic connections with spinal neurons involved in motor function are not formed. We will highlight repair approaches that did result in growth across the scar and discuss the necessity for more studies involving larger, clinically relevant types of injuries, addressing this specific issue. Finally, this review will reflect on the prospect of SCs for repair strategies in the clinic.

Inhibiting cell proliferation during formation of the glial scar: effects on axon regeneration in the CNS.

Following a CNS lesion many glial cell types proliferate and/or migrate to the lesion site, forming the glial scar. The majority of these cells express chondroitin sulphate proteoglycans (CS-PGs), previously shown to inhibit axonal growth. In this study, in an attempt to diminish glial scar formation and improve axonal regeneration, proliferating cells were eliminated from the lesion site. Adult rats received a continuous infusion of 2% cytosine-D-arabinofuranoside (araC) or saline for 7 days over the lesion site, immediately following a unilateral transection of the right medial forebrain bundle. Additional groups of rats that received subdural infusions prior to the lesion, and lesioned rats which received no infusion, were also compared in the analyses. Animals were killed at 4, 7, 12 or 18 days post-lesion (dpl) and immunohistochemistry was used to determine the effects of these treatments on tyrosine hydroxylase (TH)-lesioned axons, and on the injury response of glial cells. Almost complete elimination of NG2 oligodendrocyte progenitor cells from the lesion site was seen up to 7 dpl in araC-infused animals; reduced numbers of reactive CD11b microglia were also seen but no effects were seen on the injury response of GFAP astrocytes. Significantly more TH axons were seen distal to the lesion in araC-treated brains, but these numbers dwindled by 18 dpl.

Relationship between sprouting axons, proteoglycans and glial cells following unilateral nigrostriatal axotomy in the adult rat.

Proteoglycans may modulate axon growth in the intact and injured adult mammalian CNS. Here we investigate the distribution and time course of deposition of a range of proteoglycans between 4 and 14 days following unilateral axotomy of the nigrostriatal tract in anaesthetised adult rats. Immunolabelling using a variety of antibodies was used to examine the response of heparan sulphate proteoglycans, chondroitin sulphate proteoglycans and keratan sulphate proteoglycans. We observed that many proteoglycans became abundant between 1 and 2 weeks post-axotomy. Heparan sulphate proteoglycans were predominantly found within the lesion core (populated by blood vessels, amoeboid macrophages and meningeal fibroblasts) whereas chondroitin sulphate proteoglycans and keratan sulphate proteoglycans were predominantly found in the lesion surround (populated by reactive astrocytes, activated microglia and adult precursor cells). Immunolabelling indicated that cut dopaminergic nigral axons sprouted prolifically within the lesion core but rarely grew into the lesion surround. We conclude that sprouting of cut dopaminergic nigral axons may be supported by heparan sulphate proteoglycans but restricted by chondroitin sulphate proteoglycans and keratan sulphate proteoglycans.