Cortical AAV-CNTF Gene Therapy Combined with Intraspinal Mesenchymal Precursor Cell Transplantation Promotes Functional and Morphological Outcomes after Spinal Cord Injury in Adult Rats.

Ciliary neurotrophic factor (CNTF) promotes survival and enhances long-distance regeneration of injured axons in parts of the adult CNS. Here we tested whether CNTF gene therapy targeting corticospinal neurons (CSN) in motor-related regions of the cerebral cortex promotes plasticity and regrowth of axons projecting into the female adult F344 rat spinal cord after moderate thoracic (T10) contusion injury (SCI). Cortical neurons were transduced with a bicistronic adeno-associated viral vector (AAV1) expressing a secretory form of CNTF coupled to mCHERRY (AAV-CNTFmCherry) or with control AAV only (AAV-GFP) two weeks prior to SCI. In some animals, viable or nonviable F344 rat mesenchymal precursor cells (rMPCs) were injected into the lesion site two weeks after SCI to modulate the inhibitory environment. Treatment with AAV-CNTFmCherry, as well as with AAV-CNTFmCherry combined with rMPCs, yielded functional improvements over AAV-GFP alone, as assessed by open-field and Ladderwalk analyses. Cyst size was significantly reduced in the AAV-CNTFmCherry plus viable rMPC treatment group. Cortical injections of biotinylated dextran amine (BDA) revealed more BDA-stained axons rostral and alongside cysts in the AAV-CNTFmCherry versus AAV-GFP groups. After AAV-CNTFmCherry treatments, many sprouting mCherry-immunopositive axons were seen rostral to the SCI, and axons were also occasionally found caudal to the injury site. These data suggest that CNTF has the potential to enhance corticospinal repair by transducing parent CNS populations.

Significant changes in endogenous retinal gene expression assessed 1 year after a single intraocular injection of AAV-CNTF or AAV-BDNF.

Use of viral vectors to deliver therapeutic genes to the central nervous system holds promise for the treatment of neurodegenerative diseases and neurotrauma. Adeno-associated viral (AAV) vectors encoding brain-derived neurotrophic factor (BDNF) or ciliary derived neurotrophic factor (CNTF) promote the viability and regeneration of injured adult rat retinal ganglion cells. However, these growth-inducing transgenes are driven by a constitutively active promoter, thus we examined whether long-term AAV-mediated secretion of BDNF or CNTF affected endogenous retinal gene expression. One year after the intravitreal injection of AAV-green fluorescent protein (GFP), bi-cistronic AAV-BDNF-GFP or AAV-CNTF-GFP, mRNA was extracted and analyzed using custom 96 well polymerase chain reaction arrays. Of 93 test genes, 56% showed significantly altered expression in AAV-BDNF-GFP and/or AAV-CNTF-GFP retinas compared with AAV-GFP controls. Of these genes, 73% showed differential expression in AAV-BDNF versus AAV-CNTF injected eyes. To focus on retinal ganglion cell changes, quantitative polymerase chain reaction was undertaken on mRNA (16 genes) obtained from fixed retinal sections in which the ganglion cell layer was enriched. The sign and extent of fold changes in ganglion cell layer gene expression differed markedly from whole retinal samples. Sustained and global alteration in endogenous mRNA expression after gene therapy should be factored into any interpretation of experimental/clinical outcomes, particularly when introducing factors into the central nervous system that require secretion to evoke functionality.

The Acquisition of Target Dependence by Developing Rat Retinal Ganglion Cells

Similar to neurons in the peripheral nervous system, immature CNS-derived RGCs become dependent on target-derived neurotrophic support as their axons reach termination sites in the brain. To study the factors that influence this developmental transition we took advantage of the fact that rat RGCs are born, and target innervation occurs, over a protracted period of time. Early-born RGCs have axons in the SC by birth (P0), whereas axons from late-born RGCs do not innervate the SC until P4-P5. Birth dating RGCs using EdU allowed us to identify RGCs (1) with axons still growing toward targets, (2) transitioning to target dependence, and (3) entirely dependent on target-derived support. Using laser-capture microdissection we isolated ∼34,000 EdU(+) RGCs and analyzed transcript expression by custom qPCR array. Statistical analyses revealed a difference in gene expression profiles in actively growing RGCs compared with target-dependent RGCs, as well as in transitional versus target-dependent RGCs. Prior to innervation RGCs expressed high levels of BDNF and CNTFR α but lower levels of neurexin 1 mRNA. Analysis also revealed greater expression of transcripts for signaling molecules such as MAPK, Akt, CREB, and STAT. In a supporting in vitro study, purified birth-dated P1 RGCs were cultured for 24-48 h with or without BDNF; lack of BDNF resulted in significant loss of early-born but not late-born RGCs. In summary, we identified several important changes in RGC signaling that may form the basis for the switch from target independence to dependence.

Neurotrophic factors for spinal cord repair: Which, where, how and when to apply, and for what period of time?

A variety of neurotrophic factors have been used in attempts to improve morphological and behavioural outcomes after experimental spinal cord injury (SCI). Here we review many of these factors, their cellular targets, and their therapeutic impact on spinal cord repair in different, primarily rodent, models of SCI. A majority of studies report favourable outcomes but results are by no means consistent, thus a major aim of this review is to consider how best to apply neurotrophic factors after SCI to optimize their therapeutic potential. In addition to which factors are chosen, many variables need be considered when delivering trophic support, including where and when to apply a given factor or factors, how such factors are administered, at what dose, and for how long. Overall, the majority of studies have applied neurotrophic support in or close to the spinal cord lesion site, in the acute or sub-acute phase (0-14 days post-injury). Far fewer chronic SCI studies have been undertaken. In addition, comparatively fewer studies have administered neurotrophic factors directly to the cell bodies of injured neurons; yet in other instructive rodent models of CNS injury, for example optic nerve crush or transection, therapies are targeted directly at the injured neurons themselves, the retinal ganglion cells. The mode of delivery of neurotrophic factors is also an important variable, whether delivered by acute injection of recombinant proteins, sub-acute or chronic delivery using osmotic minipumps, cell-mediated delivery, delivery using polymer release vehicles or supporting bridges of some sort, or the use of gene therapy to modify neurons, glial cells or precursor/stem cells. Neurotrophic factors are often used in combination with cell or tissue grafts and/or other pharmacotherapeutic agents. Finally, the dose and time-course of delivery of trophic support should ideally be tailored to suit specific biological requirements, whether they relate to neuronal survival, axonal sparing/sprouting, or the long-distance regeneration of axons ending in a different mode of growth associated with terminal arborization and renewed synaptogenesis. This article is part of a Special Issue entitled SI: Spinal cord injury.

Differential effects of 670 and 830 nm red near infrared irradiation therapy: a comparative study of optic nerve injury, retinal degeneration, traumatic brain and spinal cord injury.

Red/near-infrared irradiation therapy (R/NIR-IT) delivered by laser or light-emitting diode (LED) has improved functional outcomes in a range of CNS injuries. However, translation of R/NIR-IT to the clinic for treatment of neurotrauma has been hampered by lack of comparative information regarding the degree of penetration of the delivered irradiation to the injury site and the optimal treatment parameters for different CNS injuries. We compared the treatment efficacy of R/NIR-IT at 670 nm and 830 nm, provided by narrow-band LED arrays adjusted to produce equal irradiance, in four in vivo rat models of CNS injury: partial optic nerve transection, light-induced retinal degeneration, traumatic brain injury (TBI) and spinal cord injury (SCI). The number of photons of 670 nm or 830 nm light reaching the SCI injury site was 6.6% and 11.3% of emitted light respectively. Treatment of rats with 670 nm R/NIR-IT following partial optic nerve transection significantly increased the number of visual responses at 7 days after injury (P ≤ 0.05); 830 nm R/NIR-IT was partially effective. 670 nm R/NIR-IT also significantly reduced reactive species and both 670 nm and 830 nm R/NIR-IT reduced hydroxynonenal immunoreactivity (P ≤ 0.05) in this model. Pre-treatment of light-induced retinal degeneration with 670 nm R/NIR-IT significantly reduced the number of Tunel+ cells and 8-hydroxyguanosine immunoreactivity (P ≤ 0.05); outcomes in 830 nm R/NIR-IT treated animals were not significantly different to controls. Treatment of fluid-percussion TBI with 670 nm or 830 nm R/NIR-IT did not result in improvements in motor or sensory function or lesion size at 7 days (P>0.05). Similarly, treatment of contusive SCI with 670 nm or 830 nm R/NIR-IT did not result in significant improvements in functional recovery or reduced cyst size at 28 days (P>0.05). Outcomes from this comparative study indicate that it will be necessary to optimise delivery devices, wavelength, intensity and duration of R/NIR-IT individually for different CNS injury types.