Acute up-regulation of brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family of trophic factors, has multiple functions including a role in the promotion of neuronal survival and nerve fiber elongation in both the central and the peripheral nervous systems. We assessed the expression of endogenous BDNF following an experimentally induced compression injury to the spinal cord. Expression of BDNF mRNA was increased following the spinal cord injury; reaching maximum levels 24 h after the injury. Expression of BDNF mRNA returned to the levels observed in sham-operated control animals within 3 days of the injury. Using the in situ hybridization technique, we observed a wide distribution of BDNF expression among the different cell types in the spinal cord, including motor and sensory neurons, and in glia cells, including astrocytes. We also observed expression of BDNF in putative macrophages and/or microglia; however, this effect was not observed until day 7 following spinal cord injury. These results suggest that BDNF is synthesized in both neurons and astrocytes during the acute response to injury to the spinal cord, functioning in a mainly neuroprotective role. This is followed by a later phase of expression in which BDNF is produced by macrophages and/or microglia, apparently functioning in a restorative capacity.

Analysis of effects and pharmacokinetics of subcutaneously administered BDNF.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

Neuroprotective effects of brain-derived neurotrophic factor in eyes with NMDA-induced neuronal death.

PURPOSE: To determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced cell death in retina. METHODS: NMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system. RESULTS: Morphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection. CONCLUSIONS: Exogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration.

Brain-derived neurotrophic factor regulates glucose metabolism by modulating energy balance in diabetic mice.

We previously reported that brain-derived neurotrophic factor (BDNF) regulates both food intake and blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. To elucidate the effect of BDNF on glucose metabolism, we designed a novel pellet pair-feeding apparatus to eliminate the effect of appetite alteration on glucose metabolism. The apparatus was used to synchronize food intake precisely between BDNF-treated and vehicle-treated db/db mice. It was shown using this pellet pair-feeding apparatus that BDNF administered daily (20 mg x kg(-1) x day(-1)) to db/db mice significantly lowered blood glucose compared with pellet pair-fed db/db mice. To evaluate the effect of BDNF on insulin action, we used streptozotocin-induced type 1 diabetic mice. In this case, BDNF did not lower blood glucose concentration but rather enhanced the hypoglycemic action of insulin. In hyperglycemic db/db mice, pancreatic insulin content was reduced and glucagon content was increased compared with normoglycemic db/m mice. BDNF administered to db/db mice significantly restored both pancreatic insulin and glucagon content. Histological observations of aldehyde-fuchsin staining and immunostaining with anti-insulin indicated that insulin-positive pancreatic beta-cells were extensively regranulated by BDNF administration. We also studied the effect of BDNF on KK mice, normoglycemic animals with impaired glucose tolerance. In these mice, BDNF administration improved insulin resistance in the oral glucose tolerance test. To elucidate how blood glucose was metabolized in BDNF-treated animals, we investigated the effect of BDNF on the energy metabolism of db/db mice. Body temperature and oxygen consumption of the pellet pair-fed vehicle-treated mice were remarkably lower than the ad libitum-fed vehicle-treated mice. Daily BDNF administration for 3 weeks completely ameliorated both of the reductions. Finally, to clarify its action mechanism, the effect of intracerebroventricular administration of BDNF on db/db mice was examined. Here, a small dose of BDNF was found to be effective in lowering blood glucose concentration. This indicates that BDNF regulates glucose metabolism by acting directly on the brain.

Intermittent administration of brain-derived neurotrophic factor ameliorates glucose metabolism in obese diabetic mice.

We have previously shown that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, interacts with the endocrine system in obese diabetic mice, and systemic peripheral administration of BDNF regulates glucose metabolism in this model. Results from the present study show that the hypoglycemic effect induced by 2 weeks' daily administration of BDNF (20 mg/kg/d) to db/db mice lasts for several weeks after treatment cessation, irrespective of food reduction. On the other hand, the antidiabetic agent, metformin had no lasting effect. This duration of the BDNF hypoglycemic action prompted us to examine the efficacy of BDNF intermittent administration on glucose metabolism. BDNF administered once or twice per week (70 mg/kg/wk) to db/db mice for 3 weeks significantly reduced blood glucose concentrations and hemoglobin A(1c), (HbA(1c)) as compared with ad libitum-fed phosphate-buffered saline (PBS)-treated and pair-fed PBS-treated groups. This suggests that BDNF not only temporarily reduced blood glucose concentrations but also ameliorated systemic glucose balance in this obese diabetic mouse model during the experimental period. Our results indicate that BDNF could be a novel hypoglycemic agent with an exceptional ability to normalize glucose metabolism even with treatment as infrequently as once per week.

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration.

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

BDNF diminishes caspase-2 but not c-Jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia.

PURPOSE: Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury. METHODS: After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the GCL were counted. Recombinant human BDNF (5 microg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared. RESULTS: Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1- and caspase-3-positive cells were fewer than c-Jun- or caspase-2-positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2-positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P<0.01). The number of c-Jun-positive cells in the treated retinas was not altered by the treatment. CONCLUSIONS: Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.

BDNF attenuates retinal cell death caused by chemically induced hypoxia in rats.

PURPOSE: To investigate the neuroprotective effects of brain-derived neurotrophic factor (BDNF) against potassium cyanide (KCN)-induced retinal damage. METHODS: Rats were injected intravitreally with iodinated BDNF. Two days later, eyeballs were dissected into various parts, and the level of radioactivity in each part was measured. Retinal damage was induced by incubating rat eyeballs with 5 mM KCN. BDNF was injected intravitreally 2 days before KCN treatment, and subsequent morphometric analysis was carried out to evaluate the retinal cell damage. To elucidate the mechanisms of BDNF's neuroprotective effects, the intravitreal concentrations of amino acids and the expression of calretinin were investigated. RESULTS: Intravitreally injected BDNF was distributed evenly throughout the eyes, and the incorporation of iodinated BDNF into the retina was three times higher than in other ocular tissues. Immunohistochemical analysis demonstrated that exogenous BDNF diffused throughout the retina and was especially concentrated in the inner (INL) and outer nuclear layer. Morphometric analysis showed that the number of INL cells of the posterior area, 880 microm from the optic nerve head, was 190 +/- 4 with KCN treatment and 284 +/- 9 in control animals. Cell death appeared to be necrotic. When eyes injected with either phosphate-buffered saline (PBS) or BDNF were subjected to treatment with KCN, the number of INL cells was 186 +/- 5 in the PBS-treated controls and 253 +/- 8 in eyes treated with BDNF. Also, BDNF increased the number of calretinin-positive cells in the INL and reduced the KCN-induced elevation of intravitreal glutamate levels. CONCLUSIONS: BDNF injected intravitreally reaches the retina and attenuates the INL cell death caused by KCN-induced metabolic insult. The neuroprotective effects of BDNF are partly ascribed to the upregulation of a calcium-binding protein and the attenuation of glutamate release into the vitreous body.

Bleomycin enhances random integration of transfected DNA into a human genome.

In mammalian cells, nonhomologous (illegitimate) recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin (BLM), on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake and/or accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies.

Brain-derived neurotrophic factor reduces blood glucose level in obese diabetic mice but not in normal mice.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. However, it is not yet known if BDNF works on the endocrine system itself. Here we report that BDNF improves hyperglycemia in obese diabetic animals. BDNF reduced the blood glucose level in obese db/db diabetic mice in which the effect of BDNF was age-dependent and high under the condition of hyperinsulinemia, while BDNF showed no effect on non-diabetic db/m mice. These results suggest that BDNF ameliorates insulin resistance by enhancing insulin action in peripheral tissues. Furthermore, BDNF was found to reduce the plasma insulin level in db/db mice. Among the neurotrophin family, NT-3 also reduced the blood glucose level in db/db mice. These results provide a novel insight that neurotrophin functions on the endocrine system as well as the nervous system.

Construction and characterization of ciliary neurotrophic factor (CNTF) antagonists: microenvironmental difference in the CNTF receptor between rat and chicken cells for recognizing the D1 cap region.

Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.

BDNF prevents and reverses adult rat motor neuron degeneration and induces axonal outgrowth.

To assess the therapeutic potential of brain-derived neurotrophic factor (BDNF) in clinics, we extensively investigated the effects of BDNF on adult motor neurons in a rat spinal root avulsion model. Intrathecal administration of BDNF immediately after the spinal root avulsion greatly protected against the motor neuron cell death. BDNF also showed a protective effect on the atrophy of soma and on the reduction of transmitter-related enzymes such as choline acetyl transferase and acetylcholine esterase. Very interestingly, BDNF induced axonal outgrowth of severely damaged motor neurons at the avulsion site. The BDNF administration following 2-week treatment with phosphate-buffered saline after avulsion prevented further augmentation of cell death and reversed cholinergic transmitter-related enzyme deficiency. BDNF was demonstrated to possess a wide variety of biological effects on survival, soma size, cholinergic enzymes, and axonal outgrowth of adult motor neurons. These results provide a rationale for BDNF treatment in motor neuron diseases such as spinal cord injury and amyotrophic lateral sclerosis.

V170 area plays an essential role in the biological activity of human ciliary neurotrophic factor.

The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.

Activating mechanism of CNTF and related cytokines.

Ciliary neurotrophic factor (CNTF) shares structural and functional properties with members of the hematopoietic cytokine family. It is composed of a four-helix bundle structure and shares the transmembrane signal transducing proteins, glycoprotein-130 (gp130) and leukemia inhibitory factor receptor (LIF-R). Structure-function analysis showed that the gp130-interactive proteins bind in a similar manner to that of growth hormone (site I and II). In addition, gp130-interactive proteins and granulocyte colony-stimulating factor (G-CSF) utilize another binding site (site III) at the boundary between CD loop and helix D. CNTF triggers the association of receptor components, resulting in activation of a signal transduction cascade mediated by specific intracellular protein tyrosine kinases. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and Ras/mitogen-activated protein kinase (MAPK) signaling pathways have been characterized in terms of gp130-interactive protein, and there should be other pathways and some crosstalk between them to enhance, prolong, or specify the signals.

Resident gynecologists and total hysterectomy.

We examined the specific number of surgeries necessary for a three-year obstetrics and gynecology resident to acquire proficiency in two types of hysterectomies. Improvement in the technical skills of the residents was assessed using surgical time and blood loss, and resected tumor weight was chosen as the factor representing the difficulty of the surgery. Regarding abdominal total hysterectomy (ATH), early residents (less than 25 ATH experience) performed relatively easier surgeries, and improvement in technical skill was manifested as reduced blood loss by mid residents (25 to 49 ATH) and as shortened surgical time by later residents (75 or more ATH). Regarding vaginal total hysterectomy (VTH), blood loss for earlier residents (less than 15 VTH) was greater than that for the staff, and there was a significant difference between staff surgical time and that for each resident group. These data suggest that performing more than 75 ATH during the residency period of three years is adequate to establish proficiency in this type of surgery, but that the execution of 25 VTH is insufficient and that residents require more training to learn VTH.

D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

Monoclonal antibodies recognizing 2-oxo acid dehydrogenase components in granular structures in neurons.

Monoclonal antibodies (MAbs) were raised against the hippocampal homogenate of young rats and classified into three types by immunohistochemical analysis: (1) MAbs specific for a granular structure observed within neurons, (2) MAbs specific for neuronal cell surface and cell body, and (3) MAbs specific for both neurons and astroglial cells. One MAb (2D11-7) specifically reacted with granular structures observed in neurons. A specific protein antigen was purified from rat homogenate by immunoadsorbent assay with MAb 2D11-7. Amino acid sequencing followed by lysyl endopeptidase digestion of the proteins in the eluate demonstrated that the antigens recognized by MAb 2D11-7 were E2 components of the 2-oxoglutarate dehydrogenase complex and pyruvate dehydrogenase complex. The cell specificity and age dependency of these proteins are also discussed.