Keyi Yang

Hypothermia attenuates the loss of hippocampal microtubule-associated protein 2 (MAP2) following traumatic brain injury

Traumatic brain injury (TBI) produces a tissue-specific decrease in protein levels of microtubule-associated protein 2 (MAP2), an important cross-linking component of the neuronal cytoskeleton. Because moderate brain hypothermia (30°C) reduces certain neurobehavioral deficits produced by TBI, we examined the efficacy of moderate hypothermia (30°C) in reversing the TBI-induced loss of MAP2 protein. Naive, sham-injured, and moderate (2.1 atm) fluid percussion-injured rats were assessed for MAP2 protein content 3 h post injury using quantitative immunoreactivity measurements. Parallel groups of sham-injured and fluid percussion-injured animals were maintained in moderate hypothermia (30°C), as measured by temporalis muscle temperature, for MAP2 quantitation 3 h post injury. No difference in MAP2 levels was observed between naive and sham-injured normothermic animals. Hypothermia alone had no effect on soluble MAP2 levels in sham-injured animals compared with normothermic sham-injured controls (88.0 ± 7.3%; p > 0.10). Fluid percussion injury dramatically reduced MAP2 levels in he normothermic group (44.3 ± 5.9%; p < 0.0005) compared with normothermic sham-injured controls. No significant reduction of MAP2 was seen in the hypothermic injured group (95.2 ± 4.6%; compared with hypothermic sham-injured controls, p > 0.20). Although it is premature to infer any causal link, the data suggest that the attenuation of injury-induced MAP2 loss by hypothermia may contribute to its overall neuroprotective action.

Increased cortical nuclear factor-κB (NF-κB) DNA binding activity after traumatic brain injury in rats

Nuclear factor-kappa B (NF-kappa B) DNA binding factor is an inducible transcription factor that responds to various cellular signals. Levels of cortical NF-kappa B DNA binding activity were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Using electrophoretic mobility shift assays (EMSAs), we found that NF-kappa B DNA binding activity in cerebral cortex ipsilateral to the injury site increased at 1, 3, 5 and 7 days after injury. Binding activity peaked at 3 days after injury and subsided by 10 days after injury. These data indicate that TBI produces transient increases in NF-kappa B DNA binding activity. Further insights into the role of NF-kappa B in TBI may provide new therapeutic opportunities for head trauma.

Basal and scopolamine-evoked release of hippocampal acetylcholine following traumatic brain injury in rats

This study employed in vivo microdialysis in awake, freely-moving Sprague-Dawley rats to examine acetylcholine (ACh) release in the dorsal hippocampus at 14 days following lateral controlled cortical impact. Extracellular levels of ACh were measured prior to and after an intraperitoneal administration of scopolamine (1 mg/kg), which evokes ACh release by blocking autoreceptors. At 14 days post injury there were no differences in basal ACh levels. However, injury produced a significant reduction in scopolamine-evoked ACh release. The data suggest that cholinergic neurotransmission remains chronically compromised, and thus may contribute to previously documented post traumatic spatial memory deficits.

Calpain Inhibitors Protect Against Depolarization‐induced Neurofilament Protein Loss of Septo‐hippocampal Neurons in Culture

We examined the effect of a 6 min depolarization with 60 mM KCl and 1.8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF‐H), medium (NF‐M) and low (NF‐L) molecular weight in primary septo‐hippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to ˜80% of control values in the absence of cell death. Calcium‐dependent losses of the neurofilament proteins correlated with calcium‐dependent increases in calpain 1‐mediated breakdown products of alpha‐spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to ˜14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Optimizing liposome-mediated gene transfer in primary rat septo-hippocampal cell cultures

Although liposomes have been widely employed to transfect DNA into a variety of cell types, no previous studies have systematically examined conditions producing optimal liposomal-mediated transfection of DNA into central nervous system (CNS) cells. Thus, we used the beta-galactosidase (beta-gal) reporter gene to examine factors influencing the efficiency of liposome-mediated gene transfection in CNS cell cultures. Our results indicate that without increasing the amounts of DNA, increased liposome concentrations within certain limits enhanced transfection efficiency. However, higher liposome levels could produce cell lysis. Without increasing liposome concentrations, increased amounts of DNA did not improve transfection efficiency. Employing the optimal concentration (1 microgram DNA/3 microliters liposomes/well), beta-gal gene expression was sustained for at least two weeks after transfection in primary septo-hippocampal cultures.

DC-Chol liposome-mediated gene transfer in rat spinal cord

We examined the potential of non-viral vector-mediated gene transfection in the rat spinal cord. Reporter gene (beta-gal) or brain-derived neurotrophic factor (BDNF) cDNA containing a pCMV promoter complexed with DC-Chol liposomes was injected into the intact rat spinal cord gray matter. RT-PCR confirmed the increased expression of BDNF mRNA in the injection areas. X-gal staining demonstrated the localized expression of beta-gal reporter genes. No overt tissue damage caused by DC-Chol liposome/DNA complex injections was detected. These results suggest that cationic liposome-mediated delivery can be a practical method for gene transfer in spinal cord.

Sustained expression of functional nerve growth factor in primary septo-hippocampal cell cultures by liposome-mediated gene transfer

We examined liposome-mediated gene transfection of nerve growth factor (NGF) in primary central nervous system cultures. RT-PCR analyses detected increased expression of NGF mRNA one day after liposome-mediated NGF gene transfection. ELISA studies detected large increases in NGF protein in cells and in culture medium after NGF gene transfection. Cells continued to secrete NGF into the medium for at least 2 weeks. NGF bioassays confirmed that the NGF secreted after gene transfection was biologically active.

Rescue of injury-induced neurofilaments loss by BDNF gene transfection in primary septo-hippocampal cell cultures

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Liposome-mediated BDNF cDNA transfer in intact and injured rat brain.

We examined the temporal profile of the expression of brain-derived neurotrophic factor (BDNF) cDNA containing a viral promoter following the injection of liposome cDNA complexes into the intact and traumatically injured rat brain. In situ hybridization and PCR confirmed the presence of injected BDNF cDNA for at least 6 days after injection. A similar profile of BDNF cDNA was observed when it was injected following cortical impact injury. mRNA was also localized around the injection areas. These results suggest that liposome-mediated delivery of neurotrophin cDNA may be a practical gene transfer method for treating traumatic brain injury.

Activation of phosphatidylinositol 3-Kinase by brain-derived neurotrophic factor gene transfection in septo-hippocampal cultures

Brain‐derived neurotrophic factor (BDNF) has therapeutic potential for treatment of the injured central nervous system. BDNF induces both differentiation and survival of neurons by binding to trkB receptors. This interaction stimulates the intrinsic tyrosine kinase activity of trkB, initiating a signal cascade involving the phosphorylation of intracellular protein on tyrosine, serine, and threonine residues. The purpose of this investigation was to examine the effects of cationic lipid‐mediated gene transfection of BDNF on phosphatidylinositol 3 (PI3)‐kinase activity in primary septo‐hippocampal cell cultures. Thirty‐six hours after BDNF gene transfection in the primary CNS cell culture, PI3‐kinase activity was significantly increased. The increased PI3‐kinase activity was inhibited by wortmannin, a selective and irreversible inhibitor of PI3‐kinase. In addition, wortmannin blocked neurofilament increases induced by BDNF gene transfection. This result suggests a possible role of PI3‐kinase activation in neuroprotective effects produced by BDNF gene transfection. J. Neurosci. Res. 52:192–200, 1998. © 1998 Wiley‐Liss, Inc.