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K Wewetzer

Publications and source records attributed to K Wewetzer.

At least 19 recordsLinked to original sources
PubMed2005

Axonal branching and recovery of coordinated muscle activity after transection of the facial nerve in adult rats.

Facial nerve surgery inevitablyleads to pareses, abnormally associated movements, and pathologically altered reflexes. The reason for this "post-paralytic syndrome" is the misdirected reinnervation of targets, which consists of two major components. First, due to malfunctioning axonal guidance, a muscle gets reinnervated by a "foreign" axon, that has been misrouted along a "wrong" fascicle. Second, the supernumerary collateral branches emerging from all transected axons simultaneously innervate antagonistic muscles and cause severe impairment of coordinated activity. Since it is hardly possible to influence the first major component and improve the guidance of several thousands of axons, we concentrated on the second major component and tried to reduce the collateral axonal branching. The efficiency of various treatments was evaluated in rats by determining: (1) the degree of post-operative axonal branching as estimated by the number of double-or triple-labeled perikarya after application of crystalline DiI, Fluoro-Gold (FG), and Fast Blue (FB) to the zygomatic, buccal, and marginal mandibular branch of the facial nerve respectively; (2) the accuracy of reinnervation as estimated by the number of double-labeled perikarya innervating the whisker pad muscles before and after surgery as shown by intramuscular injections of FG and FB respectively; (3) the recovery of vibrissal motor performance, estimated by a video based motion analysis. So far, we have tried to reduce branching by alteration of the afferent trigeminal input to the axotomized facial motoneurons and by focal application of: (1) neurite outgrowth fostering ECM proteins; (2) neutralizing antibodies to NGF, BDNF, CNTF, GDNF, IGF-I, and FGF-II; (3) suspensions of olfactory ensheathing cells, Schwann cells, and bone marrow stroma cells; and (4) pieces of autologous olfactory mucosa to the transection site. Although most of these manipulations do influence peripheral nerve regeneration to some extent, only the application of autologous olfactory mucosa yielded a major improvement, i.e., better function.

Animals↗
PubMed2001

In vitro expression and regulation of ciliary neurotrophic factor and its alpha receptor subunit in neonatal rat olfactory ensheathing cells.

During development and in the adult, ciliary neurotrophic factor (CNTF) is expressed at high levels in the olfactory system. In the present study, we asked whether neonatal rat olfactory ensheathing cells (OECs) express CNTF- and CNTF receptoralpha (CNTFRalpha)-mRNA in vitro and studied the regulation of both transcripts in response to growth factor and forskolin (FSK) treatment. We show here that OECs in vitro express CNTF and CNTFRalpha-mRNA under control conditions. Administration of FSK increased the expression of CNTFRalpha while lowering the levels of CNTF. Contrary to fibroblast growth factor-2, CNTF did not stimulate the proliferation of OECs. The observation that OECs express both the ligand and part of its receptor complex may indicate that CNTF exerts paracrine and/or autocrine effects in vivo, which apparently do not include the regulation of cell division.

Animals↗
PubMed2001

Regeneration of a transected peripheral nerve by transplantation of spinal cord encapsulated in a vein.

The objective was to investigate the regeneration of a transected peripheral nerve after transplantation of fragmented embryonic (E14-15) spinal cord cells which were encapsulated within a vein cavity. After 3 months transplantation, axonal regeneration was observed by staining with HE and antibody to neurofilament subtypes in six of 10 rats. In all six animals compound muscle action potentials to electrical stimulation could be recorded and indicated incomplete reinnervation of the fibular and tibial nerve, respectively. A chronic inflammation process around the transplant and a negative result of staining neurofilaments within the vein cavity and the transected nerve were found in animals lacking electrophysiological response to stimulation.

Action Potentials↗
PubMed2001

Exposure to dimethyl sulfoxide at 37 degrees C prior to freezing significantly improves the recovery of cryopreserved hybridoma cells.

Standard cryopreservation protocols recommend the use of dimethyl sulfoxide (Me2SO) at moderate temperatures only (room temperature, 4 degrees C) due to its toxicity which appears to be potentiated by warm temperatures. In the present study, we asked whether a transient increase in temperature during membrane sealing of cryovials affects the cell viability. We show here that the cell viability of hybridoma cells and Schwann cells was not reduced following membrane sealing of cryotubes. On the contrary, incubation of cells at 37 degrees C in Me2SO-containing medium prior to freezing significantly stimulated the viability of cryopreserved hybridoma cells, whereas the viability of Schwann cells remained unaltered. We conclude that the exposure of cells to Me2SO at elevated temperatures does not necessarily reduce cell viability and that contrary to this, cell type-specific, beneficial effects of Me2SO could be observed.

Animals↗
PubMed2001

Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons.

Axon regrowth after CNS and PNS injury is only the first step toward complete functional recovery which depends largely on the specificity of the newly formed nerve-target projections. Since most of the studies involving the application of glial cells to the lesioned nervous system have focused primarily on the extent of neurite outgrowth, little is known regarding their effects on the accompanying processes of axonal sprouting and pathfinding. In this study, we analyzed the effects of transplanted olfactory ensheathing cells (OECs) on axonal sprouting of adult facial neurons by using triple fluorescent retrograde tracing and biometrical analysis of whisking behavior. We found that 2 months after facial nerve axotomy and immediate implantation of OECs in between both nerve stumps fixed in a silicon tube, the total number of labeled neurons was increased by about 100%, compared to animals with simple facial nerve suture or entubulation in an empty conduit. This change in the number of axon sprouts was not random. The highest increase in axon number was observed in the marginal mandibular branch, whereas no changes were detected in the zygomatic branch. This increased sprouting did not improve the whisking behavior as measured by biometric video analysis. Our results demonstrate that OECs are potent inducers of axonal sprouting in vivo. Hence OEC-filled nerve conduits may be a powerful tool to enforce regeneration of a peripheral nerve under adverse conditions, e.g., after long delay between injury and surgical repair. In mixed nerves, increased axonal sprouting will improve specificity since inappropriate nerve-target connections are pruned off during preferential motor innervation. In pure motor nerves, however, OEC-mediated axonal sprouting may result in polyneuronal innveration of target muscles.

Animals↗
PubMed2001

Immunohistochemical characterization of axonal sprouting and reactive tissue changes after long-term implantation of a polyimide sieve electrode to the transected adult rat sciatic nerve.

The development of artificial microstructures suited for interfacing of peripheral nerves is not only relevant for basic neurophysiological research but also for future prosthetic approaches. Aim of the present study was to provide a detailed analysis of axonal sprouting and reactive tissue changes after implantation of a flexible sieve electrode to the proximal stump of the adult rat sciatic nerve. We report here that massive neurite growth after implantation, steadily increasing over a period of 11 months, was observed. Parallel to this increase was the expression of myelin markers like Po, whereas non-myelin-forming Schwann cells did not change. Compared to five weeks post-implantation. where both Schwann-cell phenotypes were intermingled with each other, non-myelin-forming Schwann cells occupied a peripheral position in each microfascicle after 11 months. After an initial increase, hematogenous macrophages were down-regulated in number but maintained close contact with the implant. However, at no time were signs of its degradation observed. It is concluded that the introduced flexible polyimide electrode is suitable for contacting peripheral nerves since it permits substantial neurite growth and offers excellent long-term stability.

Animals↗
PubMed2000

Expression of interleukin-6 and its receptor in the sciatic nerve and cultured Schwann cells: relation to 18-kD fibroblast growth factor-2.

Expression of interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2) in Schwann cells is modulated by external stimuli. To study possible interactions of both factors we have analyzed mutual effects of exogenous IL-6 and FGF-2 on the expression of each other and the corresponding receptor (R) molecules IL-6R and FGFR1 after peripheral nerve lesion in vivo and in vitro using cultured Schwann cells. Using rat Schwann cells we found that IL-6 did not exert any effects on the expression of FGF-2 and FGF receptor type 1 (R1) whereas exogenously applied 18-kD FGF-2 strongly increased the expression of the mRNAs of IL-6 and its receptor. In addition, immortalized Schwann cells over-expressing the 18-kD FGF-2 isoform showed elevated levels of IL-6 and IL-6R whereas immortalized Schwann cells over-expressing the high-molecular-weight isoforms (21 kD and 23 kD) displayed unaltered IL-6 and IL-6R expression levels. According to in situ hybridization studies of intact and crushed sciatic nerves in vivo, Schwann cells seems to be the main source of IL-6 and IL-6R. Following sciatic nerve crush, the FGF-2 and the IL-6 system are upregulated after the first hours. Furthermore, we showed that the early increase of the FGF-2 protein is mainly confined to the 18-kD isoform. These results are consistent with the idea of a functional coupling of FGF-2 and the IL-6 system in the early reaction of Schwann cells to nerve injury.

Animals↗
PubMed2000

The high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt and 23,000 mol. wt) mediate neurotrophic activity on rat embryonic mesencephalic dopaminergic neurons in vitro.

Basic fibroblast growth factor is expressed in different isoforms which display tissue and species specificity and are differentially regulated during development and after experimental interventions. The differential regulation of the fibroblast growth factor-2 isoforms may indicate specific activities and functions of these molecules. The characterization of fibroblast growth factor-2 effects, however, is almost exclusively based on studies including the 18,000 mol. wt isoform. It is not yet known whether the high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt, 23,000 mol. wt) exert similar or distinct activities in the nervous system. In the present study, we investigated the effects of the high molecular weight isoforms on dissociated rat mesencephalic dopaminergic neurons. For this purpose, recombinant fibroblast growth factor-2 isoforms, prepared in a histidine expression system, were administered on dopaminergic neurons in vitro, and Schwann cells over-expressing the high molecular weight isoforms were co-cultured with dopaminergic neurons. This is the first demonstration to show that the high molecular weight isoforms mediate a neurotrophic activity. Exogenous high molecular weight fibroblast growth factor-2 isoforms stimulated the survival of embryonic mesencephalic dopaminergic neurons and protected them from 6-hydroxydopamine neurotoxicity. In addition, co-culture of dopaminergic neurons with high molecular weight fibroblast growth factor-2 over-expressing Schwann cells revealed an increased survival and neurite formation of the mesencephalic dopaminergic neurons. These results suggest that the high molecular weight fibroblast growth factor-2 isoforms may serve as a new tool for the treatment of Parkinson's disease.

Animals↗
PubMed2000

Hyaluronan in the nuchal skin of chromosomally abnormal fetuses.

Nuchal skin oedema at 10-14 weeks gestation, observed by ultrasonography as increased nuchal translucency (NT), is found in approximately 70% of fetuses with trisomies 21, 18 and 13 as well as those with Turner's syndrome. This study investigates the possibility that one mechanism for increased translucency is an altered composition of the skin with a higher concentration of hyaluronan; large amounts of hyaluronan can lead to excessive hydration of the extracellular matrix. We isolated the hyaluronic acid binding region (HABR) from aggrecan in the extracellular matrix of hyaline cartilage and used it in a biotinylated form in combination with a fluorescent probe as a marker for hyaluronan. Immunohistochemistry was then used to examine the nuchal skin of chromosomally abnormal and normal fetuses, obtained after termination of pregnancy. In fetuses with trisomy 21 there was a substantial increase in hyaluronan, whereas in trisomies 18 and 13 and Turner's syndrome the amount was similar to that in chromosomally normal controls. This finding suggests that hyaluronan may be implicated in the pathogenesis of increased NT in fetuses with trisomy 21, but the common phenotypic expression of increased translucency in different chromosomal abnormalities may be the consequence of other mechanisms.

Chromosome Aberrations↗
PubMed1999

Macrowell cultures identify a subpopulation of neonatal rat dorsal root ganglionic neurons displaying nerve growth factor independent survival.

Dorsal root ganglionic (DRG) neurons of the newborn rat in vitro die by apoptosis within 24-48 h unless nerve growth factor (NGF) is added. Using a novel cell culture system (macrowell), we identified a neuronal subpopulation displaying NGF-independent survival in vitro. Neurons were grown on glass coverslips at standard cell density in different volumes of defined medium (standard: 500 microl; macrowell: 10 ml). In standard culture, 40% of neurons survived in the presence of NGF whereas there was no survival under control conditions. In macrowell culture, however, about 15% of neurons survived even in the absence of NGF. Addition of NGF to these cultures increased survival up to 65%. Neurons surviving independent of NGF in macrowell culture were heterogeneous in size and were lacking the low-affinity NGF receptor.

Animals↗
PubMed1999

The monoclonal antibody 23E9 defines a novel developmentally-regulated Schwann cell surface antigen.

The present study describes the identification and partial characterization of a novel Schwann cell surface molecule by means of a monoclonal antibody (23E9). The 23E9 antigen was found in association with Schwann cells of the peripheral nerve but not with sensory neurons and satellite cells of the dorsal root ganglion. The expression of the antigen in the sciatic nerve starts after birth, is high around postnatal day 8 and becomes down-regulated towards the adult stage. This suggests that it may be involved in the induction of myelin formation. On Western blots, the antibody identified two major bands of approximately 27 and 42 kDa. Treatment of cultured Schwann cells with forskolin, an agent known to mimic neuronal contact in vitro, stimulated the up-regulation of the antigen. This implies that the expression of 23E9 is induced and maintained by axon-derived signals in vivo. Comparison of the presented data with the literature suggests that we have identified a novel cell surface molecule not previously characterized in the context of Schwann cell biology. To clarify the molecular identity of the antigen and define its physiological relevance, the antibody will be used in future studies for immunoprecipitation and functional in vitro assays.

Aging↗
PubMed1998

Over-expression of the 18 kD and 21/23 kD fibroblast growth factor-2 isoforms in PC12 cells and Schwann cells results in altered cell morphology and growth.

Basic fibroblast growth factor (FGF-2) occurs in different isoforms which represent alternative translation products from a single mRNA. The question of whether the presence of multiple FGF-2 isoforms has physiological implications is compelling but unresolved so far. However, it has been shown recently that the FGF-2 isoforms are differentially regulated in sensory ganglia and peripheral nerve following nerve injury and, moreover, in the adrenal medulla during postnatal development and after hormonal stimuli suggesting that the isoforms may serve different physiological functions. To investigate isoform-specific effects we have established immortalized Schwann cells and PC12 cells stably over-expressing the 18 kD and the HMW isoforms. We found that the over-expression of the different isoforms alters morphology and growth of the Schwann cells. PC12 cells over-expressing the 18 kD FGF-2 were found to differentiate towards the neuronal phenotype whereas over-expression of the HMW isoforms resulted in a stabilization of the endocrine phenotype. Taken together, these data corroborate the idea of FGF-2 isoform-specific functions.

Animals↗
PubMed1998

Cell death of neonatal rat sensory neurons is prevented by culture at clonal density.

We studied the trophic requirements of neonatal rat sensory neurons in single neuron and standard culture (9400 neurons/cm2). In agreement with previous studies, we found that survival of sensory neurons in standard culture is strictly dependent on nerve growth factor (NGF). Contrary to this, however, no difference between NGF-treated and untreated cultures with respect to survival and neurite formation was evident when neurons were plated as single cells. Approximately 70% of singly-seeded sensory neurons survived for 2 days independent of supply with NGF. Survival of large, mid-sized, and small sensory neurons in single neuron culture demonstrated that this phenomenon is not confined to a neuronal subpopulation. We speculate that survival in single neuron culture is due to the absence or inactivation of yet unidentified cell death-inducing factors that in standard culture are overridden or suppressed by NGF.

Animals↗
PubMed1997

Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures.

In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2-4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2-4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity-the presumed chick ciliary neurotrophic factor (CNTF)-stimulated the expression of opsin in retinal cultures (EC50; 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures.

Animals↗
PubMed1997

Identification and characterization of differentiation-dependent Schwann cell surface antigens by novel monoclonal antibodies: introduction of a marker common to the non-myelin-forming phenotype.

In an attempt to identify and characterize novel Schwann cell surface molecules with putative functions during development, maintenance, and regeneration of the peripheral nervous system (PNS), we have produced monoclonal antibodies against viable neonatal rat Schwann cells. Using a sensitive live cell ELISA protocol, three monoclonal antibodies reactive with cultured Schwann cells, designated 27B10, 26F2, and 27C7 were isolated. The 27B10 and 26F2 antibodies specifically labelled forskolin-stimulated secondary Schwann cells in vitro as determined by live cell ELISA implying that the expression of the antigens in situ is regulated by axonal contact. The observation that the antigens seemed to be associated with both Schwann cell phenotypes clearly discriminated them from the well characterized myelin proteins as well as from molecules known to be confined to the non-myelin-forming phenotype. Interestingly, both antigens were found to be concentrated at the nodes of Ranvier. Further studies therefore have to show whether the identified antigens share structural or functional homology with adhesion or channel molecules, which display a similar distribution. Following transection of the adult sciatic nerve, the 26F2 antigen was rapidly down-regulated in the distal nerve stump. The 27C7 antibody reacted with an 80 kDa cell surface molecule common to non-myelin-forming Schwann cells. No differences in expression of the antigen between forskolin-treated and untreated Schwann cells in vitro were found, suggesting that the antigen is expressed independently from axonal contact. Two weeks after nerve transection in the absence of myelinating Schwann cells, the antigen was associated with S-100-positive Schwann cells of the distal nerve stump. The antigen was found to be expressed also by non-neuronal tissues, the level of the protein declined towards the adult stage. Comparison of the 27C7 antigen with previously described marker molecules suggests that we have identified a novel Schwann cell surface antigen of the non-myelin-forming phenotype.

Animals↗
PubMed1997

Expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 messenger RNAs in spinal ganglia and sciatic nerve: regulation after peripheral nerve lesion.

In order to determine functional roles of basic fibroblast growth factor (FGF-2) in the peripheral nervous system we have analysed the expression of FGF-2 and FGF receptor 1 (FGFR1) in spinal ganglia and the sciatic nerve under normal conditions and after nerve crush using RNAse protection assay and in situ hybridization. In intact spinal ganglia, both FGF-2 and FGFR1 messenger RNAs are expressed, albeit at different levels. In situ hybridization identifies satellite cells as the source of FGF-2 and sensory neurons as the source of FGFR1 suggesting a paracrine mode of action of FGF-2 on sensory neurons. One day after crush lesion FGF-2 is significantly up-regulated in sensory ganglia L4-L6. Highest levels are found at day 7; control levels are approached after 28 days. FGFR1 messenger RNA, which is strongly expressed in intact spinal ganglia, displays no significant change after lesion. In the intact sciatic nerve, FGFR1 messenger RNA is detected at higher levels than FGF-2 messenger RNA. After injury, both transcripts display a time-dependent up-regulation in both the proximal and distal nerve stump. Schwann cells, as a putative source of the sciatic nerve-derived FGF-2, express both FGF-2 and FGFR1 messenger RNAs in vitro. The FGFR1 transcript level is increased in the presence of forskolin. FGF-2 does not affect expression of FGFR1 messenger RNA but stimulates its own expression. These results show that during peripheral nerve regeneration FGF-2 is up-regulated in both the crushed nerve and the respective spinal ganglia suggesting a possible physiological function of FGF-2 during the regeneration process.

Animals↗
PubMed1996

An improved cell-ELISA for the differential screening of antibodies against cell surface molecules of viable adherent Schwann cells.

In this report we describe a highly sensitive large-scale screening assay that uses viable adherent cells. The newly developed test design combines the advantages of live cell immunocytochemistry with the versatility of a conventional ELISA technique. Culturing of target cells as well as incubation with antibodies is done in the same microtiter plate. No processing of cells prior to incubation with the antibodies, such as fixation or enzymatic detachment of the cells, is performed. This eliminates possible factors that might interfere with antibody-antigen recognition and results in a dramatically increased sensitivity that is comparable to cell-free ELISA procedures with an assay detection limit reproducibly between 0.004 and 0.002 micrograms/ml. Furthermore, this test design is convenient for the rapid identification of antibodies against differentiation-dependent antigens. Incubation in parallel with the same antibody of different cell types (forskolin-stimulated Schwann cells or fibroblasts) or cells grown under different conditions (forskolin-stimulated and non-stimulated Schwann cells) facilitates the selection of antibodies displaying genotypic or phenotypic specificity. Since culture of target cells and the detection of antibodies is done on one microtiter plate, it is not only possible to detect cell surface molecules but also secreted molecules that remain bound to the cell surface or the culture substrate. Compared to other previously introduced cell-ELISAs, our protocol offers the following advantages: (i) increased sensitivity, (ii) convenience for large-scale screening, (iii) optimal identification of antibodies reacting with native molecules, and (iv) identification of differentiation-dependent antigens. Since this assay could also be used in studies examining the regulation of cell surface molecules at a semiquantitative level it may be of general relevance.

Animals↗