Linda L. Phillips

Presynaptic excitability changes following traumatic brain injury in the rat.

Pathological processes affecting presynaptic terminals may contribute to morbidity following traumatic brain injury (TBI). Posttraumatic widespread neuronal depolarization and elevated extracellular potassium and glutamate are predicted to alter the transduction of action potentials in terminals into reliable synaptic transmission and postsynaptic excitation. Evoked responses to orthodromic single‐ and paired‐pulse stimulation were examined in the CA1 dendritic region of hippocampal slices removed from adult rats following fluid percussion TBI. The mean duration of the extracellularly recorded presynaptic volley (PV) increased from 1.08 msec in controls to 1.54 msec in slices prepared at 1 hr postinjury. There was a time‐dependent recovery of this injury effect, and PV durations at 2 and 7 days postinjury were not different from controls. In slices removed at 1 hr postinjury, the initial slopes of field excitatory postsynaptic potentials (fEPSPs) were reduced to 36% of control values, and input/output plots revealed posttraumatic deficits in the transfer of excitation from pre‐ to postsynaptic elements. Manipulating potassium currents with 1.0 mM tetraethylammonium or elevating potassium ion concentration to 7.5 mM altered evoked responses but did not replicate the injury effects to PV duration. Paired‐pulse facilitation of fEPSP slopes was significantly elevated at all postinjury survivals: 1 hr, 2 days, and 7 days. These results suggest two pathological processes with differing time courses: 1) a transient impairment of presynaptic terminal functioning affecting PV durations and the transduction of afferent activity in the terminals to reliable synaptic excitation and 2) a more protracted deficit to the plasticity mechanisms underlying paired‐pulse facilitation. J. Neurosci. Res. 60:370–379, 2000

Traumatic Brain Injury-Induced Changes in Gene Expression and Functional Activity of Mitochondrial Cytochrome C Oxidase

Traumatic brain injury (TBI) is documented to have detrimental effects on CNS metabolism, including alterations in glucose utilization and the depression of mitochondrial oxidative phosphorylation. Studies on mitochondrial metabolism have also provided evidence for reduced activity of the cytochrome oxidase complex of the electron transport chain (complex IV) after TBI and an immediate (lhr) reduction in mitochondrial state 3 respiratory rate, which can persist for up to 14 days postinjury. Using differential display methods to screen for differences in gene expression, we have found that cytochrome c oxidase II (COII), a mitochondrial encoded subunit of complex IV, is upregulated following TBI. Since COII carries a binding site for cytochrome c in the respiratory chain, and since it is required for the passage of chain electrons to molecular oxygen, driving the production of ATP, we hypothesized that metabolic dysfunction resulting from TBI alters COII gene expression directly, perhaps influencing the synaptic plasticity that occurs during postinjury recovery processes. To test this hypothesis, we documented COII mRNA expression and complex IV (cytochrome c oxidase) functional activity at 7 days postinjury, focusing on the long-term postinjury period most closely associated with synaptic reorganization. Both central fluid percussion TBI and combined TBI and bilateral entorhinal cortical lesion were examined. At 7 days survival, differential display, RT-PCR, and Northern blot analysis of hippocampal RNA from both TBI and combined insult models showed a significant induction of COII mRNA. This long-term elevation in COII gene expression was supported by increases in COII immunobinding. By contrast, cytochrome oxidase histochemical activity within tissue sections from injured brains suggested a reduction of complex IV activity within the TBI cases, but not within animals subjected to the combined insult. These differences in cytochrome c oxidase activity were supported by in vitro assay of complex IV using cerebral cortical and hippocampal tissues. Our present results support the hypothesis that COII is selectively vulnerable to TBI and that COII differences may indicate the degree of metabolic dysfunction induced by different pathologies. Taken together, such data will better define the role of metabolic function in long-term recovery after TBI.

Antiproliferative effect of c-myc antisense phosphorothioate oligodeoxynucleotides in malignant glioma cells

OBJECTIVEnTo improve the prognosis for primary malignant tumors of the central nervous system, new therapeutic strategies are needed. Antisense oligodeoxynucleotides (ODNs) offer the potential to block the expression of specific genes within cells. The proto-oncogene c-myc has long been implicated in the control of normal cell growth and its deregulation in the development of neoplasia. We therefore reasoned that a strategy using ODNs complementary to c-myc messenger ribonucleic acid would be a potent inhibitor of glioma cell proliferation.nnnMETHODSnA variety of antisense, sense, and scrambled (15-mer) phosphorothioate ODNs targeted to rat and human c-myc messenger ribonucleic acid were synthesized and added to the media of cultured RT-2 cells (a rat glioblastoma cell line). Cell growth was assessed by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide dye assay 1 to 5 days after adding the ODNs. c-Myc protein expression was analyzed by Western blot analysis. The stability of the ODNs was confirmed by gel electrophoresis.nnnRESULTSnCompared with cultures containing standard media, two of three antisense ODNs significantly inhibited the growth of glioma cells, whereas sense and scrambled sequence ODNs did not significantly affect cell growth at the concentrations tested. A human c-myc antisense sequence, which differed from the rat sequence by one base substitution, also had an inhibitory effect on RT-2 cells. Western blot analysis demonstrated that expression of immunoreactive c-Myc protein was also greatly reduced in the rat antisense ODN-treated cells (and not in sense-, scrambled-, or control-treated cells). The degree of reduction of c-Myc protein expression correlated well with the decrease in cell growth observed with several antisense ODNs. Phosphorothioate ODNs were stable in cell culture media for at least 5 days.nnnCONCLUSIONnThese results suggest that c-Myc plays a critical role in glioma cell proliferation and demonstrate that antisense ODNs can suppress proto-oncogene expression and inhibit the proliferation of glioma cells. Our results indicate that the antiproliferative activity of these ODNs was mediated predominantly through sequence-specific antisense mechanisms, but that sequence-specific nonantisense effects may also contribute to the strongest effects demonstrated. These findings support a potential role for antisense strategies designed to inhibit c-myc expression in the treatment of malignant gliomas.

Increased MMP-3 and CTGF expression during lipopolysaccharide-induced dopaminergic neurodegeneration

Accumulating evidence indicates that neuroinflammation contributes significantly to progressive dopaminergic (DA) neurodegeneration in Parkinsons disease (PD). Altered matrix metalloproteinase-3 (MMP-3) expression has been reported in several neuroinflammatory paradigms; however, its relationship to inflammation-induced DA neurotoxicity has not been explored. To this end, we investigated the temporal expression pattern of MMP-3 and one of its downstream targets, connective tissue growth factor (CTGF), following lipopolysaccharide (LPS)-induced DA neurodegeneration. LPS was directly injected into the substantia nigra of male Sprague-Dawley rats. Lesion formation was confirmed with immunohistochemistry 48 h post-injection. MMP-3 and CTGF were measured by western blot 12, 24, and 48 h post-injection. In association with neurodegeneration, MMP-3 expression and activation was significantly increased 24 and 48 h after LPS injection. In addition, CTGF expression increased 5-fold at the 24h time point. The temporal changes in MMP-3 and CTGF expression corresponded to the neurodegenerative phase of this model, suggesting that these two proteins may participate in neuroinflammation-induced DA neurotoxicity.

Osteopontin expression in acute immune response mediates hippocampal synaptogenesis and adaptive outcome following cortical brain injury

Traumatic brain injury (TBI) produces axotomy, deafferentation and reactive synaptogenesis. Inflammation influences synaptic repair, and the novel brain cytokine osteopontin (OPN) has potential to support axon regeneration through exposure of its integrin receptor binding sites. This study explored whether OPN secretion and proteolysis by matrix metalloproteinases (MMPs) mediate the initial degenerative phase of synaptogenesis, targeting reactive neuroglia to affect successful repair. Adult rats received unilateral entorhinal cortex lesion (UEC) modeling adaptive synaptic plasticity. Over the first week postinjury, hippocampal OPN protein and mRNA were assayed and histology was performed. At 1-2d, OPN protein increased up to 51 fold, and was localized within activated, mobilized glia. OPN transcript also increased over 50 fold, predominantly within reactive microglia. OPN fragments known to be derived from MMP proteolysis were elevated at 1d, consistent with prior reports of UEC glial activation and enzyme production. Postinjury minocycline immunosuppression attenuated MMP-9 gelatinase activity, which was correlated with the reduction of neutrophil gelatinase-associated lipocalin (LCN2) expression, and reduced OPN fragment generation. The antibiotic also attenuated removal of synapsin-1 positive axons from the deafferented zone. OPN KO mice subjected to UEC had similar reduction of hippocampal MMP-9 activity, as well as lower synapsin-1 breakdown over the deafferented zone. MAP1B and N-cadherin, surrogates of cytoarchitecture and synaptic adhesion, were not affected. OPN KO mice with UEC exhibited time dependent cognitive deficits during the synaptogenic phase of recovery. This study demonstrates that OPN can mediate immune response during TBI synaptic repair, positively influencing synapse reorganization and functional recovery.

Time dependent integration of matrix metalloproteinases and their targeted substrates directs axonal sprouting and synaptogenesis following central nervous system injury

Over the past two decades, many investigators have reported how extracellular matrix molecules act to regulate neuroplasticity. The majority of these studies involve proteins which are targets of matrix metalloproteinases. Importantly, these enzyme/substrate interactions can regulate degenerative and regenerative phases of synaptic plasticity, directing axonal and dendritic reorganization after brain insult. The present review first summarizes literature support for the prominent role of matrix metalloproteinases during neuroregeneration, followed by a discussion of data contrasting adaptive and maladaptive neuroplasticity that reveals time-dependent metalloproteinase/substrate regulation of postinjury synaptic recovery. The potential for these enzymes to serve as therapeutic targets for enhanced neuroplasticity after brain injury is illustrated with experiments demonstrating that metalloproteinase inhibitors can alter adaptive and maladaptive outcome. Finally, the complexity of metalloproteinase role in reactive synaptogenesis is revealed in new studies showing how these enzymes interact with immune molecules to mediate cellular response in the local regenerative environment, and are regulated by novel binding partners in the brain extracellular matrix. Together, these different examples show the complexity with which metalloproteinases are integrated into the process of neuroregeneration, and point to a promising new angle for future studies exploring how to facilitate brain plasticity.

Subtle alterations in NMDA-stimulated cyclic GMP levels following lateral fluid percussion brain injury

This study examined whether NMDA-stimulated cyclic GMP levels were altered at two different time points following lateral fluid percussion injury. At 60 min and 15 days postinjury, the left and right hippocampi were dissected and chopped into mini-prisms. Each hippocampus was divided into five equal parts and incubated with either the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine, 500 microM) alone, IBMX and N-methyl-D-aspartic acid (NMDA) OR IBMX, NMDA, and glycine (10 MM). Two concentrations of NMDA were used: 500 or 1,000 microM. Tissues were then assayed for levels of cyclic GMP. Results indicated that there were no changes in basal levels of cyclic GMP at either postinjury time point. At 60 min postinjury, there were no significant main effects for injury or drug concentration. There was a significant injury x side interaction effect with increased levels of NMDA-stimulated cyclic GMP in the hippocampus ipsilateral to the injury impact and decreased cyclic GMP levels in the contralateral hippocampus. There were no significant alterations in NMDA-stimulated cyclic GMP levels at 15 days postinjury. The data from this study indicated that NMDA-stimulated cyclic GMP accumulation is differentially altered in the hippocampus ipsilateral and contralateral to the site of the injury at 1 h after injury, but is normalized by 15 days postinjury. These findings implicate NMDA-mediated intracellular signaling processes in the acute excitotoxic response to injury.

Investigation of left and right lateral fluid percussion injury in C57BL6/J mice: In vivo functional consequences

Although rodent models of traumatic brain injury (TBI) reliably produce cognitive and motor disturbances, behavioral characterization resulting from left and right hemisphere injuries remains unexplored. Here we examined the functional consequences of targeting the left versus right parietal cortex in lateral fluid percussion injury, on Morris water maze (MWM) spatial memory tasks (fixed platform and reversal) and neurological motor deficits (neurological severity score and rotarod). In the MWM fixed platform task, right lateral injury produced a small delay in acquisition rate compared to left. However, injury to either hemisphere resulted in probe trial deficits. In the MWM reversal task, left-right performance deficits were not evident, though left lateral injury produced mild acquisition and probe trial deficits compared to sham controls. Additionally, left and right injury produced similar neurological motor task deficits, impaired righting times, and lesion volumes. Injury to either hemisphere also produced robust ipsilateral, and modest contralateral, morphological changes in reactive microglia and astrocytes. In conclusion, left and right lateral TBI impaired MWM performance, with mild fixed platform acquisition rate differences, despite similar motor deficits, histological damage, and glial cell reactivity. Thus, while both left and right lateral TBI produce cognitive deficits, laterality in mouse MWM learning and memory merits consideration in the investigation of TBI-induced cognitive consequences.

Unmyelinated and Myelinated Axons Exhibit Differential Injury and Treatment Responses Following Traumatic Injury

This chapter summarizes evidence that after traumatic brain injury (TBI) unmyelinated axons undergo functional and structural alterations which are distinct from those of myelinated axons. A theoretical framework is presented which predicts an elevated risk to TBI, in the unmyelinated fibers, due to a disproportionately high membrane-to-cytoplasm ratio and extensive axolemmal exposure to aberrant extracellular conditions prevailing after injury. Experimental results focus on time-dependent changes in axons, induced using a rodent model of fluid percussion TBI, which leads to ultrastructural, functional, and molecular alterations without extensive cell death or axonal degeneration. The fiber-type-specific pathological processes, observed here in surviving but compromised axons, are likely to also be present in more severe injuries, affecting some axons while others undergo the Wallerian-type degeneration seen in diffuse axonal injury (DAI). Axons which survive the primary injury phase form the substrate for subsequent functional recovery, and further experimental findings are presented indicating that remaining unmyelinated and myelinated axons differentially respond to neuroprotective compounds (immunophilin ligands and matrix metalloproteinase inhibitors). In view of recent stereological evidence showing that unmyelinated fibers comprise the numerical majority of forebrain axons, it is essential that theories of white matter damage assimilate injury responses unique to these fibers. Efficient development of novel neuroprotective strategies is likewise dependent on comprehensive models of axon injury and recovery.