Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.

Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

Molecular and Cellular Mechanisms of Sporadic Alzheimer's Disease: Studies on Rodent Models in vivo.

In this review, recent data are presented on molecular and cellular mechanisms of pathogenesis of the most widespread (about 95%) sporadic forms of Alzheimer's disease obtained on in vivo rodent models. Although none of the available models can fully reproduce the human disease, several key molecular mechanisms (such as dysfunction of neurotransmitter systems, especially of the acetylcholinergic system, ß-amyloid toxicity, oxidative stress, neuroinflammation, mitochondrial dysfunction, disturbances in neurotrophic systems) are confirmed with different models. Injection models, olfactory bulbectomy, and senescence accelerated OXYS rats are reviewed in detail. These three approaches to in vivo modeling of sporadic Alzheimer's disease have demonstrated a considerable similarity in molecular and cellular mechanisms of pathology development. Studies on these models provide complementary data, and each model possesses its specific advantages. A general analysis of the data reported for the three models provides a multifaceted and the currently most complete molecular picture of sporadic Alzheimer's disease. This is highly relevant also from the practical viewpoint because it creates a basis for elaboration and preclinical studies of means for treatment of this disease.

[Effect of Draconis Sanguis-containing serum on NGF, BDNF, CNTF, LNGFR, TrkA, GDNF, GAP-43 and NF-H expressions in Schwann cells].

OBJECTIVE: To observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration. METHOD: SD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later. RESULT: Most of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA. CONCLUSION: Traditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

Effects of Jinmaitong Capsule () on ciliary neurotrophic factor in sciatic nerves of diabetes mellitus rats.

OBJECTIVE: To study the effects of the Chinese medicine Jinmaitong Capsule (, JMT) on the pathomorphology of sciatic nerves, ciliary neurotrophic factor (CNTF), and the mRNA expressions of CNTF in rats with streptozotocin-induced diabetes mellitus (STZ-DM). METHODS: The animal model was established by one time intraperitoneal injection of streptozotocin. The rats were simply divided by random into 5 groups including model group, low-dose JMT group (JL), medium-dose JMT group (JM), high-dose JMT group (JH) and neurotropin group. For each of the above 5 groups, a group of 10 normal Wistar rats matched in body weight, age and gender were set as normal group. Intragastric administrations were started after the animal model established. The JL group were administered with five times the JMT dose recommended for a human adult; the JM group were administered with ten times the JMT dose recommended for a human adult; the JH group were administered with twenty times the JMT dose recommended for a human adult. The neurotropin group was administered with ten times the neurotropin dose recommended for a human adult. All rats were given intragastric administration for 16 weeks and then killed. In the 4th, 8th, 12th, 16th week, body weight and blood glucose level were detected before and after the intervention. The morphologic changes of the sciatic nerves were observed by optical microscope and transmission electron microscope. The CNTFmRNA expressions were detected by real-time fluorescent quantitative polymerase chain protein, and the CNTF protein expressions were detected by immunohistochemical method. RESULTS: The blood glucose levels of the STZ-DM rats were much higher than normal group (P<0.01), and there was no apparent difference between any treatment groups and the model group (P>0.05). Before and after the intervention in the 4th, 8th, 12th, 16th week, there were no significant differences in the body weight among all the groups (P>0.05). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. The levels of CNTF and CNTF-mRNA expressions in the STZ-DM rats were both significantly decreased (P<0.01). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. CONCLUSION: JMT could improve the pathomorphology of sciatic nerves by increasing CNTF's and CNTF-mRNA expressions in sciatic nerve tissues, and promote the repair and regeneration of damaged nerve fibers.

Neuroprotective effect of transcorneal electrical stimulation on light-induced photoreceptor degeneration.

Direct electrical stimulation of neural tissues is a strategic approach to treat injured axons by accelerating their outgrowth [Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602-2608] and promoting their regeneration [Geremia, N.M., Gordon, T., Brushart, T.M., Al-Majed, A.A., Verge, V.M.K., 2007. Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp. Neurol. 205, 347-359]. Recently, transcorneal electrical stimulation (TCES), a novel less invasive method, has been shown to rescue axotomized and damaged retinal ganglion cells [Morimoto, T., Miyoshi, T., Matsuda, S., Tano, Y., Fujikado, T., Fukuda, Y., 2005. Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest. Ophthalmol. Vis. Sci. 46(6), 2147-2155]. Here, we investigated the neuroprotection of TCES on light-induced photoreceptor degeneration and the underlying mechanism. Adult male Sprague-Dawley (SD) rats received TCES before (pre-TCES) or after (post-TCES) intense light exposure. After fourteen days of light exposure, retinal histology and electroretinography were performed to evaluate the neuroprotective effect of TCES. The mRNA and protein levels of apoptotic-associated genes including Bcl-2, Bax, Caspase-3 as well as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the retinas were determined by real-time PCR and Western blot analysis. The localization of these gene products in the retinas was examined by immunohistochemistry. Both pre- and post-TCES ameliorated the progressive photoreceptor degeneration. The degree of rescue depended on the strength of the electric charge. Post-TCES showed a relatively better and longer-term protective effect than pre-TCES. Real-time PCR and Western blot analysis revealed an upregulation of Bcl-2, CNTF, and BDNF and a downregulation of Bax in the retinas after TCES. Immunohistochemical studies showed that Bcl-2 and CNTF were selectively upregulated in Müller cells. These findings provide a new therapeutic method to prevent or delay photoreceptor degeneration through activating the intrinsic survival system.

Ciliary neurotrophic factor infused intracerebroventricularly shows reduced catabolic effects when linked to the TAT protein transduction domain.

Ciliary neurotrophic factor (CNTF) regulates the differentiation and survival of a wide spectrum of developing and adult neurons, including motor neuron loss after injury. We recently described a cell-penetrant recombinant human CNTF (rhCNTF) molecule, formed by fusion with the human immunodeficiency virus-1 transactivator of transcription (TAT) protein transduction domain (TAT-CNTF) that, upon subcutaneous administration, retains full neurotrophic activity without cytokine-like side-effects. Although the CNTF receptor is present in hypothalamic nuclei, which are involved in the control of energy, rhCNTF but not TAT-CNTF stimulates signal transducers and activators of transcription 3 phosphorylation in the rat hypothalamus after subcutaneous administration. This could be due limited TAT-CNTF distribution in the hypothalamus and/or altered intracellular signaling by the fusion protein. To explore these possibilities, we examined the effect of intracerebroventricular administration of TAT-CNTF in male adult rats. TAT-CNTF-induced weight loss, although the effect was smaller than that seen with either rhCNTF or leptin (which exerts CNTF-like effects via its receptor). In contrast to rhCNTF and leptin, TAT-CNTF neither induced morphological changes in adipose tissues nor increased uncoupling protein 1 expression in brown adipose tissue, a characteristic feature of rhCNTF and leptin. Acute intracerebroventricular administration of TAT-CNTF induced a less robust phosphorylation of signal transducers and activators of transcription 3 in the hypothalamus, compared with rhCNTF. The data show that fusion of a protein transduction domain may change rhCNTF CNS distribution, while further strengthening the utility of cell-penetrating peptide technology to neurotrophic factor biology beyond the neuroscience field.

Aging exacerbates intracerebral hemorrhage-induced brain injury.

Aging may be an important factor affecting brain injury by intracerebral hemorrhage (ICH). In the present study, we investigated the responses of glial cells and monocytes to intracerebral hemorrhage in normal and aged rats. ICH was induced by microinjecting autologous whole blood (15 microL) into the striatum of young (4 month old) and aged (24 month old) Sprague-Dawley rats. Age-dependent relations of brain tissue damage with glial and macrophageal responses were evaluated. Three days after ICH, activated microglia/macrophages with OX42-positive processes and swollen cytoplasm were more abundantly distributed around and inside the hemorrhagic lesions. These were more dramatic in aged versus the young rats. Western blot and immunohistochemistry analyses showed that the expression of interleukin-1beta protein after ICH was greater in aged rats, whereas the expression of GFAP and ciliary neurotrophic factor protein after ICH was significantly lower in aged rats. These results suggest that ICH causes more severe brain injury in aged rats most likely due to overactivation of microglia/macrophages and concomitant repression of reactive astrocytes.

Differential modulation of energy balance by leptin, ciliary neurotrophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression.

Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.

Dose-dependent neuroprotective effect of ciliary neurotrophic factor delivered via tetracycline-regulated lentiviral vectors in the quinolinic acid rat model of Huntington's disease.

The ability to regulate gene expression constitutes a prerequisite for the development of gene therapy strategies aimed at the treatment of neurologic disorders. In the present work, we used tetracycline (Tet)-regulated lentiviral vectors to investigate the dose-dependent neuroprotective effect of human ciliary neurotrophic factor (CNTF) in the quinolinic acid (QA) model of Huntington's disease (HD). The Tet system was split in two lentiviruses, the first one containing the CNTF or green fluorescent protein (GFP) cDNAs under the control of the Tet-response element (TRE) and a second vector encoding the transactivator (tTA). Preliminary coinfection study demonstrated that 63.8% +/- 2.0% of infected cells contain at least two viral copies. Adult rats were then injected with CNTF- and GFP-expressing viral vectors followed 3 weeks later by an intrastriatal administration of QA. A significant reduction of apomorphine-induced rotations was observed in the CNTF-on group. In contrast, GFP-treated animals or CNTF-off rats displayed an ipsilateral turning behavior in response to apomorphine. A selective sparing of DARPP-32-, choline acetyltransferase (ChAT)-, and NADPH-d-positive neurons was observed in the striatum of CNTF-on rats compared to GFP animals and CNTF-off group. Enzyme-linked immunosorbent assay (ELISA) performed on striatal samples of rats sacrificed at the same time point indicated that this neuroprotective effect was associated with the production of 15.5 +/- 4.7 ng CNTF per milligram of protein whereas the residual CNTF expression in the off state (0.54 +/- 0.02 ng/mg of protein) was not sufficient to protect against QA toxicity. These results establish the proof of principle of neurotrophic factor dosing for neurodegenerative diseases and demonstrate the feasibility of lentiviral-mediated tetracycline-regulated gene transfer in the brain.

Alpha-lipoic acid protects the retina against ischemia-reperfusion.

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

The regrowth of axons within tissue defects in the CNS is promoted by implanted hydrogel matrices that contain BDNF and CNTF producing fibroblasts.

In this study we demonstrate the potential for combining biocompatible polymers with genetically engineered cells to elicit axon regrowth across tissue defects in the injured CNS. Eighteen- to 21-day-old rats received implants of poly N-(2-hydroxypropyl)-methacrylamide (HPMA) hydrogels containing RGD peptide sequences that had been infiltrated with control (untransfected) fibroblasts (n = 8), fibroblasts engineered to express brain-derived neurotrophic factor (BDNF) (n = 5), ciliary neurotrophic factor (CNTF) (n = 5), or a mixture of BDNF and CNTF expressing fibroblasts (n = 11). Fibroblasts were prelabeled with Hoechst 33342. Cell/polymer constructs were inserted into cavities made in the left optic tract, between thalamus and superior colliculus. After 4-8 weeks, retinal projections were analyzed by injecting right eyes with cholera toxin (B-subunit). Rats were perfused 24 h later and sections were immunoreacted to visualize retinal axons, other axons (RT97 antibody), host astrocytes and macrophages, donor fibroblasts, and extracellular matrix molecules. The volume fraction (VF) of each gel that was occupied by RT97(+) axons was quantified. RT-PCR confirmed expression of the transgenes prior to, and 5 weeks after, transplantation. Compared to control rats (mean VF = 0.02 +/- 0.01% SEM) there was increased ingrowth of RT97(+) axons into implants in CNTF (mean VF = 0.33 +/- 0.19%) and BDNF (mean VF = 0.62 +/-0.19%) groups. Axon growth into hydrogels in the mixed BDNF/CNTF group (mean VF = 3.58 +/- 0.92%) was significantly greater (P < 0.05) than in the BDNF or CNTF fibroblast groups. Retinal axons exhibited a complex branching pattern within gels containing BDNF or BDNF/CNTF fibroblasts; however, they regrew the greatest distances within implants containing both BDNF and CNTF expressing cells.