J. R. Perez-Polo

Acute and chronic changes in aquaporin 4 expression after spinal cord injury

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

Early Postnatal Development of Rat Brain : In Vivo Diffusion Tensor Imaging

Perinatal hypoxia is a major cause of neurodevelopmental deficits. Neuronal migration patterns are particularly sensitive to perinatal hypoxia/ischemia and are associated with the clinical deficits. The rat model of hypoxia/ischemia at P7 mimics that of perinatal injury in humans. Before assessing the effects of postnatal injury on brain development, it is essential to determine the normal developmental trajectories of various brain structures in individual animals. In vivo longitudinal diffusion tensor imaging (DTI) was performed from postnatal day 0 (P0) to P56 on Wistar rats. The DTI metrics, mean diffusivity (MD), fractional anisotropy (FA), axial (λl) and radial (λt) diffusivities, were determined for four gray matter and eight white matter structures. The FA of the cortical plate and the body of corpus callosum decreased significantly during the first 3 weeks after birth. The decrease in the cortical plates FA value was associated mainly with an increase in λt. The initial decrease in FA of corpus callosum was associated with a significant decrease in λl. The FA of corpus callosum increased during the rest of the observational period, which was mainly associated with a decrease in λt. The FA of gray matter structures, hippocampus, caudate putamen, and cortical mantle did not show significant changes between P0 and P56. In contrast, the majority of white matter structures showed significant changes between P0 and P56. These temporal changes in the DTI metrics were related to the neuronal and axonal pruning and myelination that are known to occur in the developing brain.

Increased expression of brain‐derived neurotrophic factor but not neurotrophin‐3 mRNA in rat brain after cortical impact injury

Levels of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3) mRNA expression were measured in a rodent model of traumatic brain injury (TBI) following unilateral injury to the cerebral cortex. To obtain reliable data on the co‐expression of neurotrophin genes, adjacent coronal sections from the same rat brains were hybridized in situ with BDNF and NT3 cRNA probes. BDNF mRNA increased at 1, 3, and 5 hr after unilateral cortical injury in the cortex ipsilateral to the injury site and bilaterally in the dorsal hippocampus. NT3 mRNA did not change significantly following injury. Our results suggest that TBI produces rapid increases in BDNF mRNA expression in rat brain without changes in NT3 mRNA expression, a finding which differs from studies of ischemia and seizures. It is possible that increased levels of BDNF mRNA rather than NT3 are important components of pathophysiological responses to TBI.

DNA microarray analysis of the contused spinal cord: Effect of NMDA receptor inhibition

Spinal cord injury (SCI)‐induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post‐traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI‐induced N‐methyl‐D‐aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with ‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]‐cyclohepten‐5,10‐imine hydrogen maleate [MK‐801]) reversed the effect of SCI on about 50% of the SCI‐affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up‐regulation of inflammatory factors. Therefore, SCI‐induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK‐801 effects on the SCI‐induced mRNA changes reported here are novel. Additionally, we found that the MK‐801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Exogenous Bcl-xl fusion protein spares neurons after spinal cord injury

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl‐xL, Bcl‐2, and Bcl‐w, using Affymetrix DNA microarrays. Because Bcl‐xL is the most robustly expressed antiapoptotic Bcl‐2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl‐xL expression. We found Bcl‐xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl‐xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl‐xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl‐xL to neuronal death, we augmented endogenous Bcl‐xL levels by administering Bcl‐xL fusion protein (Bcl‐xL FP) into injured spinal cords. Bcl‐xL FP significantly increased neuronal survival, suggesting that SCI‐induced changes in Bcl‐xL contribute considerably to neuronal death. Because Bcl‐xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.

Liposome-mediated NGF gene transfection following neuronal injury: Potential therapeutic applications

We have systematically investigated the therapeutic potential of cationic liposome-mediated neurotrophic gene transfer for treatment of CNS injury. Following determination of optimal transfection conditions, we examined the effects of dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) liposome-mediated NGF cDNA transfection in injured and uninjured primary septo-hippocampal cell cultures and rat brains. In in vitro studies, we detected an increase of NGF mRNA in cultures 1 day after transfection. Subsequent ELISA and PC12 cell biological assays confirmed that cultured cells secreted soluble active NGF into the media from day 2 after gene transfection. Further experiments showed that such NGF gene transfection reduced the loss of chol- ine acetyltransferase (ChAT) activity in cultures following calcium-dependent depolarization injury. In in vivo studies, following intraventricular injections of NGF cDNA complexed with DC-Chol liposomes, ELISA detected nine- to 12-fold increases of NGF in rat CSF. Further studies showed that liposome/NGF cDNA complexes could attenuate the loss of cholinergic neuronal immunostaining in the rat septum after traumatic brain injury (TBI). Since deficits in cholinergic neurotransmission are a major consequence of TBI, our studies demonstrate for the first time that DC-Chol liposome-mediated NGF gene transfection may have therapeutic potential for treatment of brain injury.

Aquaporin 1 - a novel player in spinal cord injury.

The role of water channel aquaporin 1 (AQP‐1) in uninjured or injured spinal cords is unknown. AQP‐1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP‐1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four‐ to eightfold increases in AQP‐1 levels at the site of injury (T10) persisting up to 11 months post‐contusion, a novel finding. Delayed AQP‐1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP‐1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI‐induced AQP‐1 increases and that hypoxia inducible factor‐1α was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP‐1 increases after SCI. Interestingly; AQP‐1 levels were not affected by long‐lasting hypertonicity that significantly increased astrocytic AQP‐4, suggesting that the primary role of AQP‐1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP‐1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP‐1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP‐1 in melatonin‐treated SCI rats correlated with decreased AQP‐1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP‐1 and chronic neuropathic pain after SCI.

p75 and TrkA Receptor Signaling Independently Regulate Amyloid Precursor Protein mRNA Expression, Isoform Composition, and Protein Secretion in PC12 Cells

Abstract: The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose‐dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain‐derived neurotrophic factor abolished NGFs effects on neurite outgrowth and APP metabolism, whereas addition of brain‐derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2‐ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR‐activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross‐linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors—in contrast to p75NTR‐associated signaling—stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.

Effect of nerve growth factor on AP-1, NF-kappa B, and Oct DNA binding activity in apoptotic PC12 cells: extrinsic and intrinsic elements.

Both intrinsic signals, such as serum and neurotrophic factor deprivation, and extrinsic events or agents, such as oxidative stress and glucose deprivation, can induce cell death in pheochromocytoma (PC12) cells. Also, treatment with nerve growth factor (NGF) reduces cell death due to the treatments mentioned. Serumless‐induced cell death, as a model of apoptosis, has been intensively investigated in PC12 cells. In the present study, we investigated the molecular components of H2O2‐induced cell death and compared it with serumless‐induced cell death. Exposure of PC12 cells to intermediate concentrations of H2O2 (100 μM) induced nuclear condensation and DNA fragmentation, indicating that there is an apoptotic component in H2O2‐induced cell death. Since transcription factors have been shown to play an essential role in the control of cellular proliferation, differentiation, and survival, we measured changes in the DNA binding activities of the transcription factors activator protein‐1 (AP‐1), nuclear factor kappa B (NF‐κB), and octamer‐binding protein (Oct) by electrophoretic mobility shift assay (EMSA) after H2O2 treatment and serum deprivation, both in the absence and presence of exogenous NGF in PC12 cells. AP‐1 DNA binding activity transiently increased during apoptosis due to serum deprivation, and NGF treatment further stimulated AP‐1 DNA binding activity in a more persistent fashion. NF‐κB DNA binding activity only increased slightly after serum deprivation, and NGF treatment of PC12 cells decreased NF‐κB binding activity in the late stages of serum deprivation. Oct DNA binding activity decreased after serum deprivation, while NGF had an opposite effect. AP‐1 DNA binding activity also transiently increased after H2O2 treatment, as did NF‐κB DNA binding activity. Our results suggest that AP‐1 is likely to be a common component of signaling pathways associated with both the induction or suppression of apoptosis induced by intrinsic or extrinsic stimuli.

Neurotrophin regulation of energy homeostasis in the central nervous system.

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimers, and Parkinsons diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.