Bernadette T. Majda

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 As extracellular-mediated neuroprotection occurs via CD147 receptor signalling, possibly by activation of ERK1/2 pro-survival pathways. Further characterization of cyclophilin As neuroprotective mechanisms may aid the development of a neuroprotective therapy.

Intravenous administration of magnesium is only neuroprotective following transient global ischemia when present with post-ischemic mild hypothermia

We hypothesized that post-ischemic hypothermia plays an important role in magnesium mediated neuroprotection following global cerebral ischemia. To test this hypothesis, we subjected rats to 8 min of global cerebral ischemia and magnesium treatment with and without post-ischemic body temperature maintenance. In Group 1, rats received an intravenously administered loading dose (LD) of 360 micromol/kg MgSO4 immediately before ischemia followed by a 48-h intravenous infusion (IVI) at either 60, 120 or 240 micromol/kg/h. Animal body temperature was kept at 37+/-0.2 degrees C during ischemia and between 36.6 and 37.8 degrees C for 6 h after ischemia. In Group 2, rats received a 360 micromol/kg MgSO4 LD followed by a 48-h IVI of either 120 or 240 micromol/kg/h MgSO4. In this group, body temperature following ischemia was monitored but not regulated. Control animals in Groups 1 and 2 received normal saline. Seven days after ischemia, hippocampal CA1 neurons were histologically examined. All Group 1 MgSO4-treated and control animals demonstrated less than 6% hippocampal CA1 neuronal survival. In Group 2, the rectal temperature of MgSO4-treated and control animals spontaneously dropped as low as 35.4 degrees C during the 6-h post-ischemia monitoring period. In addition, Group 2 animals that received the LD followed by an IVI of 120 or 240 micromol/kg/h MgSO4 demonstrated 34% (p<0.05) and 20% (p=0.936) CA1 neuronal survival, respectively. The CA1 neuronal survival in saline-treated control animals in both groups was less than 6%. Our data demonstrate only the combination of MgSO4 treatment and post-ischemic mild hypothermia is neuroprotective following global ischemia.

Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats.

OBJECTIVE We aimed to determine an effective dose schedule for intravenously administered magnesium, to establish its neuroprotective efficacy in both pre- and postischemic treatment paradigms, and to compare the neuroprotective properties of MgSO4 and MgCl2. METHODS Rats that had been subjected to the bilateral carotid artery occlusion plus hypotension model of transient forebrain cerebral ischemia received either an intravenously administered loading dose (LD) of 360 &mgr;mol/kg MgSO4 only or an intravenously administered LD of 360 &mgr;mol/kg followed by a 48-hour intravenous infusion of MgSO4 at either 60, 120, 240, or 480 &mgr;mol/kg/h. For evaluation of the efficacy of MgSO4 after ischemia, the dose (LD, 360 &mgr;mol/kg; infusion, 120 &mgr;mol/kg/h) that provided maximal neuroprotection before ischemia was administered 4, 8, 12, or 24 hours after ischemia. MgCl2 (LD, 360 &mgr;mol/kg; infusion, 120 &mgr;mol/kg/h) was administered before and 8 hours after ischemia. At 7 days after ischemia, hippocampal CA1 neurons were histologically examined for protection. RESULTS Animals that received the LD only demonstrated 33% hippocampal CA1 neuronal survival. Animals that received the LD followed by continuous infusion of MgSO4 at either 60, 120, 240, or 480 &mgr;mol/kg/h demonstrated 30, 80, 16, and less than 5% CA1 neuronal survival, respectively. MgSO4 treatment commencing at 4, 8, 12, or 24 hours resulted in 82, 71, 52, and 33% CA1 neuronal survival, respectively. Preischemic and 8-hour postischemic administration of MgCl2 resulted in 50% and less than 5% CA1 neuronal survival, respectively. CONCLUSION These results demonstrate a neuroprotective intravenous dose of MgSO4, which is effective when administered before or late after ischemia, and a previously uncharacterized dose-response curve for MgSO4.

Establishment of neuronal in vitro models of ischemia in 96-well microtiter strip-plates that result in acute, progressive and delayed neuronal death

Using 96-well microtiter strip-plates we established in vitro ischemia models with acute, progressive and delayed neuronal death onset. In vitro ischemia was induced by washing neuronal cultures with a balanced salt solution with (acute/delayed models) or without (progressive model) 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and washing and/or adding Dulbeccos modified Eagle medium containing N2 supplement. Acute neuronal death resulted in cell swelling during in vitro ischemic incubation with the majority of neurons appearing swollen and necrotic within 3 h post-insult. Progressive neuronal death was characterized by cell shrinkage during and immediately following in vitro ischemia with increasing neuronal degeneration resembling both necrosis and apoptosis over a 24-h period post-in vitro ischemia. Delayed neuronal death was induced by glutamate-receptor blockade during in vitro ischemia. Neurons appeared morphologically normal immediately following and up to 6 h after in vitro ischemia and then started to degenerate over the next 42 h by a process resembling apoptosis. We monitored oxygen consumption during in vitro ischemia and found it to be similar for the three models and have shown that plastic culture wells store oxygen. The establishment of acute, progressive and delayed in vitro models of ischemia using 96-well microtiter strip-plates will provide useful tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput system to evaluate potential neuroprotective agents. Oxygen storage in plastic culture wells is likely to contribute to the extended oxygen- and oxygen-glucose-deprivation times required to induce significant neuronal injury in vitro.

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.

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.

Suppression subtraction hybridization and northern analysis reveal upregulation of heat shock, trkB, and sodium calcium exchanger genes following global cerebral ischemia in the rat

Driver (sham-operated) and tester (ischemic) hippocampal cDNAs were subtracted, and the resulting ischemia-induced upregulated gene expression was verified by northern analysis. cDNAs isolated corresponded to (1) genes known to be upregulated following ischemia, (hsc70, hsp90, hsp105 and trkB) and (2) a gene not previously implicated with cerebral ischemia, sodium calcium exchanger (ncx). Furthermore, upregulation of these genes was demonstrated following preconditioning transient global ischemia.

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.

Na+/Ca2+ exchanger subtype (NCX1, NCX2, NCX3) protein expression in the rat hippocampus following 3 min and 8 min durations of global cerebral ischemia

There is increasing evidence that the sodium-calcium exchanger (NCX) subtypes, NCX1, NCX2 and NCX3 play an important role in intracellular calcium homeostasis/dysregulation following cerebral ischemia. In the present study we examined NCX1, NCX2 and NCX3 protein levels in the rat hippocampus at 3, 6, 12, 18, 24 and 48 h following a 3 min and 8 min duration of global cerebral ischemia. We observed that NCX1 protein levels were significantly increased by 22.3% and 20.6% at the 6 and 12 h respective time points following a 3 min duration of global ischemia, while NCX2 and NCX3 protein levels remained relatively constant. Following a 8 min duration of global ischemia, NCX1 protein levels remained relatively constant, while NCX2 protein levels were down-regulated by 6.9%, 10.8%, 14.4% and 10.3% at the 6, 18, 24 and 48 h respective time points, and NCX3 protein levels were up-regulated by 22.1% at the 18 h time point. Taken together, our results show that NCX subtype protein expression is sensitive to cerebral ischemia, and indicates that changes in NCX activity may be playing an important role in calcium maintenance and neuronal outcome following ischemia.

Tectal tissue grafted to the midbrain of newborn rats: effect of donor age on the survival, growth and connectivity of transplants.

Tectal tissue from fetal (E15, E18, E20) and newborn (P0) rats was transplanted to the midbrain of newborn rats. Graft survival and size decreased with increasing donor age. Host retinal input was found in E15, E18 and E20 grafts; the specific pattern of retinal innervation was similar for all fetal donor ages.