epidermal-growth-factor and Nerve-Degeneration

Subacute combined degeneration (SCD) is a neuropathy due to cobalamin (Cbl) (vitamin B(12)) deficiency acquired in adult age. Hitherto, the theories advanced to explain the pathogenesis of SCD have postulated a causal relationship between SCD lesions and the impairment of either or both of two Cbl-dependent reactions. We have identified a new experimental model, the totally gastrectomized rat, to reproduce the key morphological features of the disease [spongy vacuolation, intramyelinic and interstitial edema of the white matter of the central nervous system (CNS), and astrogliosis], and found new mechanisms responsible for the pathogenesis of SCD: the neuropathological lesions in TGX rats are not only due to mere vitamin withdrawal but also to the overproduction of the myelinolytic tumor necrosis factor (TNF)-alpha and the reduced synthesis of the two neurotrophic agents, epidermal growth factor (EGF) and interleukin-6. This deregulation of the balance between TNF-alpha and EGF synthesis induced by Cbl deficiency has been verified in the sera of patients with pernicious anemia (but not in those with iron-deficient anemia), and in the cerebrospinal fluid (CSF) of SCD patients. These new functions are not linked to the coenzyme functions of the vitamin, but it is still unknown whether they involve genetic or epigenetic mechanisms. Low Cbl levels have also been repeatedly observed in the sera and/or CSF of patients with Alzheimer's disease or multiple sclerosis, but whether Cbl deficit plays a role in the pathogenesis of these diseases is still unclear.

Topics: Animals; Disease Models, Animal; Epidermal Growth Factor; Gastrectomy; Humans; Interleukin-6; Models, Biological; Nerve Degeneration; Rats; Tumor Necrosis Factor-alpha; Vitamin B 12; Vitamin B 12 Deficiency

Interleukin (IL)-1 beta , IL-2, IL-4, IL-6, epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha were measured for the first time in ventricular cerebrospinal fluid (VCSF) from control non-parkinsonian patients, patients with juvenile parkinsonism (JP) and patients with Parkinson's disease (PD) by highly sensitive sandwich enzyme immunoassays. All cytokines were detectable in VCSF from control and parkinsonian patients, and the concentrations were much higher than those in lumbar CFS. The concentrations of IL-1 beta, IL-2, IL-4 and TGF-alpha in VCSF were higher in JP than those in controls (P < 0.05). In contrast, the concentrations of IL-2 and IL-6 in VCSF from patients with PD were higher than those from control patients (P < 0.05). These results agree with our previous reports, in which the cytokine levels were elevated in the striatal dopaminergic region of the brain from patients with PD. Since VCSF is produced in the ventricles, the alteration of cytokines in VCSF may reflect the changes of cytokines in the brain. Because cytokines play an important role as mitogens and neurotrophic factors in the brain, the increases in cytokines as a compensatory response may occur in the brain of patients of JP or PD during the progress of neurodegeneration. Increase in cytokines may contribute not only as a compensatory response but as a primary initiating trigger for the neurodegeneration.

Topics: Adolescent; Adult; Age of Onset; Aged; Cerebral Ventricles; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Interleukins; Male; Middle Aged; Mitogens; Nerve Degeneration; Parkinson Disease; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Cobalamin-deficient (Cbl-D) central neuropathy in the rat is associated with a locally increased expression of neurotoxic tumour necrosis factor-alpha (TNF-alpha) and a locally decreased expression of neurotrophic epidermal growth factor (EGF). These recent findings suggest that cobalamin oppositely regulates the expression of TNF-alpha and EGF, and raise the possibility that these effects might be independent of its coenzyme function. Furthermore, adult Cbl-D patients have high levels of TNF-alpha and low levels of EGF in the serum and cerebrospinal fluid. Serum levels of TNF-alpha and EGF of cobalamin-treated patients normalize concomitantly with haematological disease remission. These observations suggest that cobalamin deficiency induces an imbalance in TNF-alpha and EGF levels in biological fluids that might have a role in the pathogenesis of the damage caused by pernicious anaemia.

Topics: Anemia, Pernicious; Animals; Central Nervous System; Disease Models, Animal; Epidermal Growth Factor; Gastrectomy; Humans; Nerve Degeneration; Nerve Growth Factor; Rats; Tumor Necrosis Factor-alpha; Vitamin B 12; Vitamin B 12 Deficiency

Since postnatal and adult mammalian brains have been shown to retain an ability to generate neurons from endogenous stem cells throughout life, these cells could play a central role in regeneration after neuronal loss. Therefore, we studied cell proliferation, glio- and neurogenesis respectively after brain injury in organotypic hippocampal slice cultures using a focal trauma by transecting Schaffer collaterals in the cornu ammonis (CA) 2 region mechanically. After determination of cell death using propidium iodide, neuroregenerative processes were quantitatively analyzed by various immunohistochemical techniques at different time points post injury. As this endogenous insult-induced neurogenesis is rather inefficient, we investigated if it can be enhanced by application of exogenous growth factors. Exogenous basic fibroblast growth factor (bFGF) enhanced neurogenesis significantly in the dentate gyrus (DG) region. A neutralizing antibody against endogenous bFGF revealed a significant decrease of basal and trauma-induced proliferation. Reverse transcription polymerase chain reaction (RT-PCR) studies exhibited a downregulation of FGF messenger ribonucleic acid (mRNA) transcription after the antibody treatment. In contrast, epidermal growth factor (EGF) increased proliferation, but not neurogenesis. A combination of bFGF and EGF displayed an EGF-like effect on proliferation and no effect on neurogenesis. These results demonstrate, that in our model bFGF but not EGF sustains neurogenesis, whereas together the two growth factors permit an increased proliferation but not neurogenesis in organic hippocampal slice cultures.

Topics: Animals; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Hippocampus; Nerve Degeneration; Neurons; Organ Culture Techniques; Rats; Rats, Wistar; RNA

The administration of growth factors (GFs) for treatment of experimental spinal cord injury (SCI) has shown limited benefits. One reason may be the mode of delivery to the injury site. We have developed a minimally invasive and safe drug delivery system (DDS) consisting of a highly concentrated collagen solution that can be injected intrathecally at the site of injury providing localized delivery of GFs. Using the injectable DDS, epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) were co-delivered in the subarachnoid space of Sprague-Dawley rats. The in vivo distribution of EGF and FGF-2 in both injured and uninjured animals was monitored by immunohistochemistry. Although significant differences in the distribution of EGF and FGF-2 in the spinal cord were evident, localized delivery of the GFs resulted in significantly less cavitation at the lesion epicenter and for at least 720 mum caudal to it compared to control animals without the DDS. There was also significantly more white matter sparing at the lesion epicenter in animals receiving the GFs compared to control animals. Moreover, at 14 days post-injection, delivery of the GFs resulted in significantly greater ependymal cell proliferation in the central canal immediately rostral and caudal to the lesion edge compared to controls. These results demonstrate that the injectable DDS provides a new paradigm for localized delivery of bioactive therapeutic agents to the injured spinal cord.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Drug Administration Routes; Ependyma; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Injections, Spinal; Mice; Microinjections; Nerve Degeneration; Nerve Regeneration; Neural Pathways; NIH 3T3 Cells; PC12 Cells; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Subarachnoid Space; Treatment Outcome

Recent development in stem cell biology has indicated a new possible approach for the treatment of neurological diseases. However, in spite of tremendous hope generated, we are still on the way to understand if the use of stem cells to repair mature brain and spinal cord is a reliable possibility. In particular, we know very little on the in situ regulation of adult neural stem, and this also negatively impact on cell transplant possibilities. In this chapter we will discuss issues concerning the role and function of stem cells in neurological diseases, with regard to the impact of features of degenerating neurons and glial cells on in situ stem cells. Stem cell location and biology in the adult brain, brain host reaction to transplantation, neural stem cell reaction to experimental injuries and possibilities for exogenous regulation are the main topics discussed.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Bisbenzimidazole; Blotting, Western; Brain; Brain Diseases; Cell Differentiation; Cell Division; Cells, Cultured; Cerebral Ventricle Neoplasms; Encephalomyelitis, Autoimmune, Experimental; Epidermal Growth Factor; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; In Vitro Techniques; Injections, Intraventricular; Intermediate Filament Proteins; Male; Microscopy, Confocal; Neoplasms; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Nestin; Neural Cell Adhesion Molecule L1; Neuroglia; Neurons; Rats; Sialic Acids; Stem Cell Transplantation; Stem Cells; Wound Healing

Hair cell losses in the mammalian cochlea following an ototoxic insult are irreversible. However, past studies have shown that amikacin treatment in rat cochleae resulted in the transient presence of atypical Deiters' cells (ACs) in the damaged organ of Corti. These ACs arise through a transformation of Deiters' cells, which produce, at their apical pole, densely packed microvilli reminiscent of early-differentiating stereociliary bundles. The ACs do not, however, express typical hair cell markers such as parvalbumin or calbindin. The present study was designed to determine whether specific growth factors could influence the survival and differentiation of these ACs and stimulate hair cell regeneration processes in vitro. Apical-medial segments of organ of Corti of juvenile amikacin-treated rats were established as organotypic cultures, and the effects of epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), transforming growth factor-alpha (TGFalpha), and retinoic acid were studied using morphological and molecular approaches. Our results indicate that TGFalpha supports the survival of the damaged organ of Corti and influences ACs differentiation in vitro, possibly acting through reorganization of the actin cytoskeleton. These effects could be directly mediated through activation of the EGF receptor, which is expressed by supporting cells in the mature organ of Corti. TGFalpha does not, however, allow the ACs to progress towards a hair cell phenotype.

Topics: Actins; Amikacin; Animals; Anti-Bacterial Agents; Bromodeoxyuridine; Cell Differentiation; Cell Division; Cell Survival; Epidermal Growth Factor; Hair Cells, Auditory; Immunohistochemistry; Insulin-Like Growth Factor I; Microscopy, Electron; Microscopy, Electron, Scanning; Nerve Degeneration; Nerve Regeneration; Neuroglia; Neurotoxins; Organ Culture Techniques; Rats; Rats, Wistar; Transforming Growth Factor alpha; Tretinoin

The ready availability of unlimited quantities of neural stem cells derived from the human brain holds great interest for basic and applied neuroscience, including therapeutic cell replacement and gene transfer following transplantation. We report here the combination of epigenetic and genetic procedures for perpetuating human neural stem cell lines. Thus we tested various culture conditions and genes for those that optimally allow for the continuous, rapid expansion and passaging of human neural stem cells. Among them, v-myc (the p110 gag-myc fusion protein derived from the avian retroviral genome) seems to be the most effective gene; we have also identified a strict requirement for the presence of mitogens (FGF-2 and EGF) in the growth medium, in effect constituting a conditional perpetuality or immortalization. A monoclonal, nestin-positive, human neural stem cell line (HNSC.100) perpetuated in this way divides every 40 h and stops dividing upon mitogen removal, undergoing spontaneous morphological differentiation and upregulating markers of the three fundamental lineages in the CNS (neurons, astrocytes, and oligodendrocytes). HNSC.100 cells therefore retain basic features of epigenetically expanded human neural stem cells. Clonal analysis confirmed the stability, multipotency, and self-renewability of the cell line. Finally, HNSC.100 can be transfected and transduced using a variety of procedures and genes encoding proteins for marking purposes and of therapeutic interest (e.g., human tyrosine hydroxylase I).

Topics: Blotting, Southern; Brain; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Line, Transformed; Central Nervous System; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation, Viral; Genes, gag; Genes, myc; Genetic Therapy; Glial Fibrillary Acidic Protein; Humans; Intermediate Filament Proteins; Microtubule-Associated Proteins; Nerve Degeneration; Nerve Regeneration; Nerve Tissue Proteins; Nestin; Neurons; Phenotype; Proliferating Cell Nuclear Antigen; Retroviridae; Stem Cells; Transfection; Tubulin; Vimentin

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), an EGF receptor ligand, was investigated in rat forebrain under basal conditions and after kainate-induced excitotoxic seizures. In addition, a potential neuroprotective role for HB-EGF was assessed in hippocampal cultures. In situ hybridization analysis of HB-EGF mRNA in developing rat hippocampus revealed its expression in all principle cell layers of hippocampus from birth to postnatal day (P) 7, whereas from P14 through adulthood, expression decreased in the pyramidal cell layer versus the dentate gyrus granule cells. After kainate-induced excitotoxic seizures, levels of HB-EGF mRNA increased markedly in the hippocampus, as well as in several other cortical and limbic forebrain regions. In the hippocampus, HB-EGF mRNA expression increased within 3 hr after kainate treatment, continued to increase until 24 hr, and then decreased; increases occurred in the dentate gyrus granule cells, in the molecular layer of the dentate gyrus, and in and around hippocampal pyramidal CA3 and CA1 neurons. At 48 hr after kainate treatment, HB-EGF mRNA remained elevated in vulnerable brain regions of the hippocampus and amygdaloid complex. Western blot analysis revealed increased levels of HB-EGF protein in the hippocampus after kainate administration, with a peak at 24 hr. Pretreatment of embryonic hippocampal cell cultures with HB-EGF protected neurons against kainate toxicity. The kainate-induced elevation of [Ca2+]i in hippocampal neurons was not altered in cultures pretreated with HB-EGF, suggesting an excitoprotective mechanism different from that of previously characterized excitoprotective growth factors. Taken together, these results suggest that HB-EGF may function as an endogenous neuroprotective agent after seizure-induced neural activity/injury.

Topics: Animals; Epidermal Growth Factor; Female; Heparin; Heparin-binding EGF-like Growth Factor; Hippocampus; Intercellular Signaling Peptides and Proteins; Kainic Acid; Male; Nerve Degeneration; Neuroprotective Agents; Prosencephalon; Rats; Rats, Sprague-Dawley; RNA, Messenger; Seizures

Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

Topics: Animals; Antineoplastic Agents; Arrestin; Blood Proteins; Cell Survival; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Nerve Degeneration; Neuroglia; Photoreceptor Cells, Vertebrate; Rats; Rats, Wistar; Receptors, Fibroblast Growth Factor; RNA, Messenger; Suramin; Tyrphostins

Neuronal degeneration was induced in cultured rat hippocampal neurons by a 20-min exposure to the glutamatergic agonist, N-methyl-D-aspartate (NMDA; 100 microM), and the neuroprotective activity of a set growth factors and cytokines was compared. During the early stages of degeneration, NMDA induced changes that were characteristic of neuronal necrosis, including swelling and darkening of the neuronal soma and swelling of neurites, leading to the formation of beaded varicosities ('blebs'). These changes were followed by nuclear pyknosis, formation of double-stranded DNA breaks and loss of membrane integrity. Only transforming growth factor-beta 1 (TGF-beta 1; 1-10 ng/ml) and tumor necrosis factor-alpha (TNF-alpha; 30 ng/ml) protected the hippocampal neurons against NMDA neurotoxicity after short-term (60 min) pre-treatments. Interleukin-1 beta (10-100 ng/ml) and fibroblast growth factor-2 (FGF-2; 50 ng/ml) were clearly effective when administered 24 h prior to the NMDA exposure, but not when given 60 min before the insult. Interestingly, the protective effect of interleukin-1 beta was significantly reduced in the presence of a neutralizing antibody to TGF-beta. Of note, short-term pre-treatment with brain-derived neurotrophic factor (BDNF; 5-50 ng/ml) significantly potentiated NMDA-induced neurodegeneration. These experiments demonstrate marked diversity in the actions of growth factors on NMDA-induced neuronal degeneration.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Culture Techniques; Epidermal Growth Factor; Excitatory Amino Acid Agonists; Fibroblast Growth Factor 2; Gene Expression; Hippocampus; Humans; Interleukin-1; N-Methylaspartate; Nerve Degeneration; Nerve Growth Factors; Neurons; Rats; Rats, Inbred F344; Transforming Growth Factor alpha; Transforming Growth Factor beta