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.

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.

Evidence that intracellular cyclophilin A and cyclophilin A/CD147 receptor-mediated ERK1/2 signalling can protect neurons against in vitro oxidative and ischemic injury.

We previously reported that cyclophilin A protein is up-regulated in cortical neuronal cultures following several preconditioning treatments. In the present study, we have demonstrated that adenoviral-mediated over-expression of cyclophilin A in rat cortical neuronal cultures can protect neurons from oxidative stress (induced by cumene hydroperoxide) and in vitro ischemia (induced by oxygen glucose deprivation). We subsequently demonstrated that cultured neurons, but not astrocytes, express the recently identified putative cyclophilin A receptor, CD147 (also called neurothelin, basigin and EMMPRIN), and that administration of purified cyclophilin A protein to neuronal cultures induces a rapid but transient phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2. Furthermore, administration of purified cyclophilin A protein to neuronal cultures protects neurons from oxidative stress and in vitro ischemia. Interestingly, we detected up-regulation of cyclophilin A mRNA, but not protein in the hippocampus following a 3-min period of sublethal global cerebral ischemia in the rat. Despite our in vivo findings, our in vitro data show that cyclophilin A has both intracellular- and extracellular-mediated neuroprotective mechanisms. To this end, we propose cyclophilin A's extracellular-mediated neuroprotection occurs via CD147 receptor signalling, possibly by activation of ERK1/2 pro-survival pathways. Further characterization of cyclophilin A's neuroprotective mechanisms may aid the development of a neuroprotective therapy.

Magnesium sulfate fails to reduce infarct volume following transient focal cerebral ischemia in rats.

Studies on the neuroprotective effect of magnesium treatment in animal models of focal and global cerebral ischemia have produced inconsistent results. Nevertheless, two magnesium acute stroke phase III trials (IMAGES and FAST-MAG) have either been completed or are planned. Therefore, we decided to re-evaluate the efficacy of magnesium following focal cerebral ischaemia in rats. Two experiments were carried out in two independent laboratories based in Australia. Both used the intraluminal thread method to induce focal cerebral ischemia in the rat. In the Perth study the middle cerebral artery (MCA) was occluded for 45 min and body temperature was controlled during and after ischemia. In the Canberra laboratory the MCA was occluded for 2 h and body temperature was only controlled during surgery. Three different doses (180, 360, or 720 micromol/kg) of MgSO4 in the Perth study and two different MgSO4 doses (370 or 740 micromol/kg) in the Canberra study were intravenously or intra-arterially administered immediately before ischemia. Control animals were given an equal volume of normal saline just before ischemia in both studies. Twenty-four or 72 h post-ischemia, infarct volume was determined following 2',3',5'-triphenyl-2H-tetrazolium chloride (TTC) staining. No significant differences (P > 0.05) in total, cortical and striatal infarct volumes between saline and MgSO4 treated animals were observed in either study. We conclude MgSO4 does not reduce infarct volume when administered before focal cerebral ischemia in rats.

Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death.

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.