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Gerald Wolf

Publications and source records attributed to Gerald Wolf.

At least 55 records · Page 3Linked to original sources
PubMed2003

Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia.

Nitric oxide (NO) is produced in mammals by different isoforms of NO synthase (NOS), including the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochondria. We show that mitochondrially derived NO mediates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the impaired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L-NAME. These observations suggest that mitochondria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochondrial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.

Animals↗
PubMed2003

An ultrastructural demonstration of NADPH-diaphorase/nitric oxide synthase activity in the rat striatal astroglia.

In the present study we have used electron microscopical NADPH-diaphorase (NADPH-d) histochemistry as a visualization procedure for nitric oxide synthase (NOS) to examine patterns of activity in the subcellular distribution of NADPH-d in the rat striatal astroglia. Electron microscopical examination revealed deposition of the NADPH-d reaction product in a nuclear envelope, fragments of endoplasmic reticulum and mitochondria. Predominantly mitochondria of astrocytic "end feet" were labelled. Our ultrastructural observations promote the possibility that astroglial NADPH-d/NOS is involved in adaptation of the local blood flow in the striatal microenvironment.

Animals↗
PubMed2002

Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts1.

BACKGROUND: A nerve gap must be bridged by autologous nerve grafts that serve as scaffold and consist of viable Schwann cells that promote regeneration. Owing to the necessary immunosuppression, nerve allografts remain limited to special cases. Alternatively, tissue engineering of peripheral nerves focuses on the implantation of cultured Schwann cells into suitable scaffolds. We established grafts from Schwann cells and basal lamina from acellular muscles. These grafts offer a regeneration that is comparable to autologous nerve grafts. METHODS: Using a rat model (DALEW.1W strain), the present study evaluates the host response to acellular muscle allografts by assessing cellular reaction major histocompatability (MHC) class I and II, lymphocytes, macrophages. The results were compared to untreated muscle allografts. RESULTS: Macroscopically, the untreated muscles showed a strong inflammatory reaction as a sign of rejection, whereas the acellular muscle offered only minor reactions in the periphery of the graft. Expression of MHC I and II and invasion of CD4/CD8 positive cells and macrophages was pronounced after grafting the untreated muscles. Only a moderate reaction was noted for these parameters after acellular grafting. CONCLUSIONS: The acellular muscle graft is not completely free of cellular response; however the reaction is considered to be moderate and is located only in the periphery. To date, synthetic scaffolds that represent endoneurial tube-like structures and allow sufficient adhesion of Schwann cells and axonal regeneration are not available. The decreased response to acellular muscle allografts offers at least a basis for further experiments.

Animals↗
PubMed2002

NOS-mediated differences in peripheral nerve graft revascularization and regeneration.

Dependence of peripheral nerve revascularization on endogenous nitric oxide supply and consequences for nerve regeneration were investigated using a sciatic nerve graft model in mice lacking one of the nitric oxide synthase (NOS) isoforms. Wild type mice and mice lacking neuronal or inducible NOS exhibited similar revascularization patterns. Perfusion was consistently established 3 days after nerve reconstruction. Mice lacking endothelial NOS showed a delay in revascularization of about 2 days. The regeneration outcome did not reflect these differences. In mice lacking endothelial NOS, axon counts, myelination and recovery of sensory and motor function were comparable to wild type mice, whereas in mice lacking neuronal or inducible NOS a disturbed regeneration was found. Present results demonstrate, that the disturbance of nerve revascularization as result of reduced endothelial NOS-mediated NO supply can be tolerated, possibly by enhanced phagocytic capacity of Schwann cells and/or resident endoneurial macrophages.

Animals↗
PubMed2002

Release patterns of excitatory and inhibitory amino acids within the hypothalamic supraoptic nucleus in response to direct nitric oxide administration during forced swimming in rats.

The effect of direct intrahypothalamic nitric oxide (NO) administration on the release of selected amino acids in the hypothalamic supraoptic nucleus (SON) with and without concomitant forced swimming was investigated. Adult male Wistar rats were chronically fitted with U-shaped microdialysis probes in the SON and forced to swim for 10-min in 20-degree C warm water. Thirty-min microdialysis samples were collected before, during and after the forced swimming period while NO was administered into the SON via microdialysis. The results show that NO administration in combination with forced swimming affects the release of aspartate, glutamate, taurine, and gamma aminobutyric acid (GABA) in different patterns. Whereas the release of the excitatory amino acids aspartate and glutamate was triggered only during NO administration and forced swimming, the inhibitory amino acids GABA and taurine were found in the extracellular fluid in increased concentrations also after the treatment. These data indicate that NO administration differently affects the release of excitatory and inhibitory amino acids within the SON. Thus, SON neurons which contain high concentrations of neuronal NO synthase, might contribute to the regulation of their own secretory activity by releasing NO that controls the orchestrated release of excitatory and inhibitory amino acids from axon terminals of afferences and interneurons as well as release from glial cells.

Animals↗
PubMed2002

Differences in peripheral nerve degeneration/regeneration between wild-type and neuronal nitric oxide synthase knockout mice.

Nitric oxide (NO), a unique biological messenger molecule, is synthesized by three isoforms of the enzyme NO synthase (NOS) and diffuses from the site of production across cellular membranes. A postulated role for NO in degeneration and regeneration of peripheral nerves has been explored in a sciatic nerve model comparing wild-type mice and mice lacking neuronal NOS after transection and microsurgical repair. In NOS knockout mice, regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). In the regenerated nerve, pruning of uncontrolled sprouts was disturbed, leading to an enhanced number of axons, whereas remyelination seemed to be less affected. Delayed regeneration was associated with a delayed recovery of sensor and motor function. In such a context, possible NO targets are neurofilaments and myelin sheaths of the interrupted axon, filopodia of the growth cone, newly formed neuromuscular endplates, and Schwann cells in the distal nerve stump. The results presented suggest that 1) local release of NO following peripheral nerve injury is a crucial factor in degeneration/regeneration, 2) success of fiber regeneration in the peripheral nervous system depends on a regular WD, and 3) manipulation of NO supply may offer interesting therapeutic options for treatment of peripheral nerve lesions.

Animals↗
PubMed2002

[SAPHO syndrome: case report of an 18-year-old man and review of the literature].

We report about an 18-year-old man, suffering from acne on his face and trunk and complaining about indefinite bone pains--without any trauma. SAPHO-syndrome was diagnosed which consists of synovitis, acne, pustulosis, hyperostosis and osteitis. Five types of this syndrome can be differentiated: spondarthritis hyperostotica pustulo-psoriatica, chronic recurrent multifocal osteomyelitis, inflammatory chest wall syndrome, isolated sternocostoclavicular hyperostosis, and the combined form of acne-associated spondarthritis and chronic recurrent osteomyelitis. Although the sapho-syndrome is very rare, it should be considered a possible diagnosis in the case of indefinite bone pains in combination with acne, pustulosis and hyperostosis.

Acne Vulgaris↗
PubMed2002

Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide.

4,5-diaminofluorescein diacetate (DAF-2DA) is widely used as a fluorescent probe to detect endogenously produced nitric oxide (NO). Recent reports that refer to the high sensitivity of DAF-2 toward NO prompted us to test its efficiency and specificity in a mixed murine primary glial culture model, in which the NO-synthesizing enzyme inducible nitric oxide synthase (iNOS) is expressed by stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cultures were loaded with DAF-2DA and the fluorescence was measured using confocal microscopy. NO production in the cultures was determined using the ozone/chemiluminescence technique. Due to the extremely high photosensitivity of DAF-2, low laser intensities were used to avoid artifacts. No difference in DAF-2 fluorescence was observed in NO-producing cultures compared to control cultures, whereas the NO/peroxynitrite-sensitive dye 2,7-dihydrodichlorofluorescein (DCF) showed a significant fluorescence increase specifically in microglia cells. A detectable gain in fluorescence was seen when NO-containing buffer was added to the DAF-2DA-loaded cells with a minimum NO concentration at 7.7 microM. An additional gain of DAF-2 fluorescence was obtained when the cells were depleted of glutathione (GSH) with L-buthionine S,R-sulfoximine (BSO). Hence, we monitored the change in DAF-2 fluorescence intensity in the presence of NO and O(-*)(2) in a cell-free solution. The fluorescence due to NO was indeed larger when O(-*)(2) was added, implying a higher sensitivity of DAF-2 for peroxynitrite. Nevertheless, our results also indicate that measurement of DCF fluorescence is a better tool for monitoring intracellular changes in the levels of NO and/or peroxynitrite than DAF-2.

Animals↗
PubMed2002

Differential distribution of spermidine/spermine-like immunoreactivity in neurons of the adult rat brain.

The polyamines spermidine and spermine are small, widely distributed polycations. In the brain, they confer rectification properties upon inwardly rectifying potassium channels and Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptors and also modify functional properties of N-methyl-D-aspartate receptors. Therefore, functional roles of spermidine/spermine in the adult brain will depend on the colocalization of the spermidine/spermine-sensitive receptors/channels and the polyamines either in the same or in closely associated cell types. We previously immunocytochemically demonstrated a prominent localization of spermidine/spermine in glial cells, especially astrocytes (Laube and Veh [ 1997] Glia 19:171-179). In contrast to the commonly accepted assumption of a ubiquitous distribution of polyamines in various cell types, in neurons of the rat brain, we detected a highly diverse spermidine/spermine-like immunoreactivity. The immunoreactivity in neurons and neuropil throughout the rat brain is listed according to intensity in arbitrary groups. The strongest neuronal staining was observed in the hypothalamic paraventricular, supraoptic, and accessory neurosecretory nuclei. Strong cytoplasmic staining was also evident in some motor and somatosensory areas such as the Me5 nucleus of the mesencephalic trigeminal tract, the nucleus ruber, and the large motor neurons of the spinal cord ventral horn. In contrast, in most cortical and hippocampal regions spermidine/spermine-like immunoreactivity in neurons was relatively weak, whereas in these areas, the labeling pattern was dominated by a diffuse neuropil labeling. In addition to spermidine/spermine immunocytochemistry, ornithine decarboxylase labeling was performed and the resulting labeling patterns were compared. The prominent localization of spermidine/spermine in neurosecretory neurons might point to a functional role different from channel/receptor modification. In these neurons, polyamines might be involved in secretory processes.

Animals↗
PubMed2002

Life imaging of peroxynitrite in rat microglial and astroglial cells: Role of superoxide and antioxidants.

Free radicals, such as superoxide and nitric oxide, are known to play a role in a number of inflammatory and degenerative brain diseases, in which resident microglia upregulate the inducible nitric oxide synthase (iNOS) and thus produce large amounts of nitric oxide. Simultaneously, microglia generate superoxide mainly via NADPH-oxidase, which reacts at a diffusion-limited rate with nitric oxide to form the powerful oxidant peroxynitrite. We used mixed astroglial/microglial cultures to study the effects of iNOS induction by lipopolysaccharide and interferon-gamma on free radical formation. Using the fluorogenic compound 2,7-dihydrodichlorofluorescein diacetate, we monitored cellular peroxynitrite formation by confocal laser microscopy. Peroxynitrite formation in continuously nitric oxide-producing microglial cells was rather limited. However, activation of the superoxide-generating enzyme NADPH-oxidase dramatically increased DCF fluorescence within a few minutes. We conclude that superoxide is the limiting factor for peroxynitrite formation. Since the formation and oxidant activity of peroxynitrite depends strongly on the availability of cellular antioxidants, we investigated the capacity of several compounds to influence peroxynitrite formation. Among the substances under investigation in this study, glutathione and the synthetic compound ebselen had a major effect on preventing peroxynitrite formation, whereas ascorbate failed to decrease peroxynitrite levels.

Animals↗
PubMed2002

Reconstruction of peripheral nerves using acellular nerve grafts with implanted cultured Schwann cells.

The bridging of nerve gaps is still one of the major problems in peripheral nerve surgery. The present experiment describes our attempt to engineer different biologic nerve grafts in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into 2-cm autologous acellular nerve grafts or autologous predegenerated nerve grafts. Autologous nerve grafts and predegenerated or acellular nerve grafts without implanted Schwann cells served as controls. The regenerated nerves were assessed histologically and morphometrically after 6 weeks. Predegenerated grafts showed results superior in regard to axon count and histologic appearance in comparison to standard grafts and acellular grafts. The acellular nerve grafts showed the worst histologic picture, but axon counts were in the range of standard grafts. The implantation of Schwann cells did not yield significant improvements in any group. In conclusion, the status of activation of Schwann cells and the stadium of Wallerian degeneration in a nerve graft might be key factors for regeneration, rather than total number of Schwann cells. Predegenerated nerve grafts are therefore superior to standard grafts in the rat model. Acellular grafts are able to bridge nerve gaps of up to 2 cm in the rat model, but even the addition of cultivated Schwann cells did not lead to results as good as in the group with autologous nerve grafts.

Animals↗
PubMed2002

Influence of insulin-like growth factor-I (IGF-I) on nerve autografts and tissue-engineered nerve grafts.

To overcome the problems of limited donor nerves for nerve reconstruction, we established nerve grafts made from cultured Schwann cells and basal lamina from acellular muscle and used them to bridge a 2-cm defect of the rat sciatic nerve. Due to their basal lamina and to viable Schwann cells, these grafts allow regeneration that is comparable to autologous nerve grafts. In order to enhance regeneration, insulin-like growth factor (IGF-I) was locally applied via osmotic pumps. Autologous nerve grafts with and without IGF-I served as controls. Muscle weight ratio was significantly increased in the autograft group treated with IGF-I compared to the group with no treatment; no effect was evident in the tissue-engineered grafts. Autografts with IGF-I application revealed a significantly increased axon count and an improved g-ratio as indicator for "maturity" of axons compared to autografts without IGF-I. IGF-I application to the engineered grafts resulted in a decreased axon count compared to grafts without IGF-I. The g-ratio, however, revealed no significant difference between the groups. Local administration of IGF-I improves axonal regeneration in regular nerve grafts, but not in tissue-engineered grafts. Seemingly, in these grafts the interactive feedback mechanisms of neuron, glial cell, and extracellular matrix are not established, and IGF-I cannot exert its action as a pleiotrophic signal.

Animals↗
PubMed2002

Nitric oxide affects the phosphorylation state of microtubule-associated protein 2 (MAP-2) and neurofilament: an immunocytochemical study in the brain of rats and neuronal nitric oxide synthase (nNOS)-knockouts.

Alterations in function and specific cellular location of cytoskeletal elements are characterized by changes in their phosphorylation state. On this background we studied immunocytochemically the distribution pattern of neurofilament (NF) in its phosphorylated (P-NF) and nonphosphorylated (NP-NF) form and of microtubule-associated protein-2 (MAP-2) in the rat and mouse brain. Neurons that are strongly positive for neuronal nitric oxide synthase (nNOS)-immunoreactivity (IR) showed, interestingly, neither P-NF- nor MAP-2-IR. In contrast, nNOS-negative neuronal cell elements exhibited an intense IR and specific location for both antigens throughout the brain. As a model we chose the dorsolateral tegmental nucleus (LDT) of knockout (nNOS(-/-)) mice in which the main splice isoform nNOSalpha is lacking, but a low nNOS-activity persists, apparently due to the splice isoforms nNOSbeta and gamma. The principal neurons of such nNOS(-/-)-mice, which are equivalent to the nNOS-containing neurons in the LDT of wild-type mice exhibited a decreased nitrotyrosine-IR and an increased phosphotyrosine-IR if compared to those of wild-type mice. The same neurons failed to show NF-IR and MAP-2-IR, though. When the residual nNOS activity in nNOS(-/-)-mice was inhibited by treatment with N-omega-nitro-L-arginine methyl ester (L-NAME) the principal neurons displayed a moderate MAP-2 and NF-staining. NO and NO-derived oxygen species are suggested to modulate the balance between the activities of kinases and phosphatases, thus changing phosphorylation levels for NF, MAP-2, and, possibly, other proteins in neurons and adjacent cell elements.

Animals↗
PubMed2002

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy.

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.

Animals↗
PubMed2002

XY99-5038 promotes long-term survival of cultured retinal neurons.

We have previously reported that XY99-5038, a preparation from a specific formula of Traditional Chinese Medicine, could effectively inhibit hydrogen peroxide-induced retinal cell death. In the present study, we investigated the possibility of XY99-5038 to prolong neuronal survival in a long-term retinal neuronal culture. Basic fibroblast survival factor (bFGF), a potent neurotrophic factor, was employed as comparable agent. Retinas of 0-2 days old Sprague-Dawley rats were isolated and dissociated. The cells were maintained in tissue culture for up to 9 weeks in a synthetic serum-free media. XY99-5038 (100 ng/ml) or a vehicle was added to culture every 3-4 days, starting at the first week of culturing. The number of cells were counted and compared for each time point and treatment. Cell viability was also determined by MTT assay, whereas apoptotic cell death was evaluated by the TUNEL assay. XY99-5038 treatment significantly reduced cell loss, increased cell viability, and inhibited apoptosis in this long-term retinal neuronal culture. Our data also show that the protective effect of XY99-5038 is more potent than that of bFGF. Our data suggest that XY99-5038 could be beneficial to the prolongation of neuron survival.

Aging↗
PubMed2002

Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons.

Overproduction of nitric oxide by NMDA receptor stimulation is implicated in calcium deregulation and neurodegeneration of striatal neurons. We investigated the involvement of nitric oxide (NO) in inducing intracellular calcium release and in modifying calcium transients evoked by NMDA. NO application (4-10 microM) reversibly and repeatedly increased the intracellular calcium concentration [Ca2+]i in Fura-2- or fluo-3-loaded cultured mouse striatal neurons. NO-induced [Ca2+]i responses persisted in the absence of extracellular calcium, indicating that Ca2+ was released from intracellular stores. The source of calcium was distinct from [Ca2+]i-activated (ruthenium red and ryanodine sensitive) or IP3-activated (thapsigargin-sensitive) Ca2+ stores and was not dependent on cGMP production because a cell permeant analog, 8-bromo-cGMP, did not increase basal [Ca2+]i. Glucose removal potentiated the NO-induced release of [Ca2+]i. In contrast, pretreatment with either the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone or cyclosporin A, a blocker of the mitochondrial permeability transition pore, prevented the [Ca2+]i increase after NO. The rise in [Ca2+]i during NO exposure was preceded by a decrease in mitochondrial membrane potential that was partly reversible during washout. Repeated applications of NMDA induced irreversible [Ca2+]i responses in a subpopulation of striatal cells that were greatly reduced by the NOS inhibitor N omega-nitro-l-arginine. Calcium transients were prolonged by conjoint application of NMDA and NO. We conclude that NMDA-evoked [Ca2+]i transients are modulated by endogenous NO production, which leads to release of calcium from the mitochondrial pool. An NO-activated mitochondrial permeability transition pore may lead to cell death after overstimulation of NMDA receptors.

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