Biomedical subjects

W Tetzlaff

Publications and source records attributed to W Tetzlaff.

At least 55 records · Page 3Linked to original sources

PubMed1993

Differential expression of immediate early genes in rubrospinal neurons following axotomy in rat.

Many immediate early genes are rapidly and transiently expressed in the central nervous system following a variety of stimuli. Damage to the axons of peripheral and certain central neurons has been shown to result in a long-term increase in expression of c-Jun in the parent cell bodies. In the peripheral nervous system this increased expression of c-Jun protein and mRNA develops over 24 h following sciatic nerve section and is maintained if the damaged nerve is ligated, but returns to basal levels if the peripheral nerve is allowed to regenerate. Here, we report on the response of rubrospinal neurons to spinal cord hemisection at levels C3 and T10. c-Jun expression was first seen at 12 h post-lesion in a limited number of rubral neurons. The number of positively stained neurons increased up to 10 days post-lesion and then declined over the following weeks. By 7 weeks post-lesion there was still evidence of c-Jun immunoreactivity in both large and other clearly atrophic rubrospinal neurons. c-Fos immunoreactivity was seen only at 12-48 h in a small number of rubrospinal neurons. Evidence from retrograde tracing experiments following fluorogold application to the hemisected cord suggested that all c-Jun-positive neurons projected into the spinal cord. No c-Jun response was seen following a lesion at T10.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1993

Developmental study of the expression of B50/GAP-43 in rat retina.

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons-bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule.

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PubMed1993

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

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PubMed1992

Elevated gene expression in the red nucleus after spinal cord compression injury.

Since the mechanism of injury in the majority of human traumatic spinal cord injuries involves rapid cord compression due to bone displacement or fracture-dislocation, we have used a compression injury model to examine the response of adult rat rubrospinal neurons to traumatic spinal cord injury. We have applied in situ hybridization techniques to examine levels of mRNA for cytoskeletal and growth-associated proteins. We report a population of magnocellular red nucleus neurons with elevated levels of T alpha 1 tubulin and GAP-43 up to four weeks post-lesion. In control animals, both probes had very low hybridization signals indistinguishable from background. These and other findings suggest that kinetic compression injuries of the adult spinal cord can result in regeneration-associated gene expression in intrinsic CNS neurons.

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PubMed1992

A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes.

The mitogenic actions of epidermal growth factor (EGF) were examined in low-density, dissociated cultures of embryonic day 14 mouse striatal primordia, under serum-free defined conditions. EGF induced the proliferation of single progenitor cells that began to divide between 5 and 7 d in vitro, and after 13 d in vitro had formed a cluster of undifferentiated cells that expressed nestin, an intermediate filament present in neuroepithelial stem cells. In the continued presence of EGF, cells migrated from the proliferating core and differentiated into neurons and astrocytes. The actions of EGF were mimicked by the homolog transforming growth factor alpha (TGF alpha), but not by NGF, basic fibroblast growth factor, platelet-derived growth factor, or TGF beta. In EGF-generated cultures, cells with neuronal morphology contained immunoreactivity for GABA, substance P, and methionine-enkephalin, three neurotransmitters of the adult striatum. Amplification of embryonic day 14 striatal mRNA by using reverse transcription/PCR revealed mRNAs for EGF, TGF alpha, and the EGF receptor. These findings suggest that EGF and/or TGF alpha may act on a multipotent progenitor cell in the striatum to generate both neurons and astrocytes.

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PubMed1991

Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43.

Neurons confined within the mammalian CNS usually do not regenerate after axonal injury, while axonal regeneration is the rule in the PNS. It has been hypothesized that this may be related to differences in the microenvironment of the PNS versus CNS and to differences in the neuronal response to injury. In order to test the latter hypothesis, we compared changes in gene expression after axotomy in two populations of neurons: rat facial motoneurons and rat rubrospinal neurons. In situ hybridization with cDNA probes for the medium and light neurofilament protein revealed a reduced mRNA content in both facial and rubrospinal neurons at all times investigated (i.e., 1, 2, and 3 weeks after axotomy). On the other hand, mRNAs for actin and tubulin were increased in both neuronal populations during the first week after axotomy. While this increase was sustained in facial motoneurons for several weeks, total tubulin mRNA and actin mRNA were decreased in rubrospinal neurons at 2 and 3 weeks after axotomy, coincident with their atrophy. The developmentally regulated T alpha 1 tubulin mRNA, which was previously shown to be reexpressed in facial motoneurons after axotomy, was elevated severalfold in axotomized rubrospinal neurons, and increased levels persisted in some rubrospinal neurons as late as 7 weeks after axotomy. Similarly, the developmentally regulated GAP-43 mRNA increased in both axotomized facial and rubrospinal neurons, and increased levels were sustained in some axotomized rubrospinal neurons for at least 7 weeks. The response of rubrospinal neurons to axotomy in the cervical spinal cord is, in the first week, qualitatively similar to the response of facial motoneurons. However, by 2 weeks after axotomy there is a generalized reduction in mRNA levels for all three cytoskeletal proteins that is associated with neuronal atrophy. During this period, mRNA levels for the two specific markers of the growth state, T alpha 1 tubulin and GAP-43, remain elevated. Thus, axotomy of rubrospinal neurons appears to set in motion two independent events. First, an axotomy signal initiates a cell-body reaction similar to that of PNS neurons, including increased mRNA levels for T alpha 1 tubulin and GAP-43. Later, a generalized cellular atrophy and decrease in mRNA levels occur without reversing the specific responses of T alpha 1 and GAP-43 to axotomy. We conclude that the failure of rubrospinal neurons to regenerate is not due to a failure to initiate gene-expression changes characteristic of regenerating peripheral neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗

PubMed1990

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system.

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.

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PubMed1990

Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons.

To analyze the possible influence of nerve growth factor (NGF) on neurofilament synthesis in primary sensory neurons, adjacent cryostat sections of lumbar dorsal root ganglia (DRG) from adult rats were processed for either NGF-receptor radioautography or in situ hybridization with a neurofilament cDNA probe. Labeling by both procedures was quantified with computer assistance for approximately 300 neurons in each of selected ganglia. For uninjured neurons, no correction was detected between NGF binding and neurofilament mRNA, even after infusion of NGF into the lumbar subarachnoid space for 1 week. One or 3 weeks after sciatic nerve transection, neurofilament labeling densities in large DRG neurons were sharply reduced and the normal bimodal pattern in frequency histograms had become unimodal. Intrathecal infusion of NGF counteracted this injury-induced reduction of neurofilament mRNA but only in neurons with high-affinity NGF receptors. To explain the effects of NGF on axotomized neurons and the normal diversity of neurofilament gene expression among neurons with NGF receptors, we postulate that NGF permits NGF-sensitive DRG neurons to respond differentially to a second factor stimulating neurofilament synthesis.

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PubMed1990

Correlation between GAP43 and nerve growth factor receptors in rat sensory neurons.

In mature rat sensory neurons, expression of the gene for the growth-associated protein, GAP43, was studied by in situ hybridization with a cDNA probe. Among neurons in normal lumbar dorsal root ganglia, labeling for GAP43 mRNA was heterogeneous, approximately one-half of the neurons being densely labeled. To characterize the latter population, individual neurons were examined in adjacent sections processed either for GAP43 hybridization or NGF-receptor radioautography. Virtually all neurons with high-affinity NGF binding sites had high basal levels of GAP43 mRNA and most GAP43-positive neurons bore NGF receptors. Another NGF-responsive population, sympathetic neurons in the superior cervical ganglion, also had high basal concentrations of GAP43 mRNA. Further co-localization studies in dorsal root ganglia were performed with immunohistochemistry for somatostatin and enzyme histochemistry for acid phosphatase. The latter 2 groups of sensory neurons have been previously shown to lack high-affinity receptors and were here shown to have low basal concentrations of GAP43 mRNA. From this and earlier studies, it can be assumed that substance P-immunoreactive neurons and strongly positive CGRP neurons synthesize GAP43 at high basal rate. One week following peripheral nerve transection, almost all neurons had high concentrations of GAP43 mRNA without correlation with NGF binding. Intrathecal infusion of NGF after the sciatic nerve was cut did not strongly influence this post-traumatic elevation in GAP mRNA. In normal dorsal root ganglia, neurons that have high-affinity NGF binding sites and are therefore potentially responsive to NGF also have high basal rates of synthesis of GAP43.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1989

Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro.

In order to study microglial cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a microglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ.

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PubMed1989

Neurofilament elongation into regenerating facial nerve axons.

Immunocytochemistry was used to show that neurofilaments advance into regenerating facial nerve axons at 2.5 mm/day, which is less than the rate of axonal elongation (4.3 mm/day), measured from the transport of radiolabeled protein into the axons. Thus, the distal region of the newly-regenerated axons is deficient in neurofilaments, and this was confirmed by electron microscopy. These neurofilament-free regenerating axons could also be detected by immunocytochemistry using antibody to protein B50 (GAP43), a component of growth-cones. Immunoblots of nerve segments, incubated with monoclonal antibodies against the three neurofilament proteins, showed that all three proteins were present in the neurofilaments elongating into the regenerating axons, and confirmed the more distal extensions of B50 immunoreactivity. These results show that neurofilament immunocytochemistry underestimates the extent of axonal regeneration, and it is suggested that this technique should be employed with caution in regeneration studies. When the facial nerve received a conditioning lesion 7 days prior to a test lesion, axonal regeneration rate increased to 6.0 mm/day, and there was a proportional increase in neurofilament elongation rate to 4.4 mm/day. This occurred in spite of the reduction in cell body neurofilament protein synthesis induced by the lesions. It is concluded that the rate of neurofilament extension into regenerating axons is not governed by cell body synthesis but by local interactions with other cytoskeletal materials which support the increased regeneration rate of conditioned axons.

Animals↗

PubMed1989

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat.

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.

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PubMed1989

Rapid induction of the major embryonic alpha-tubulin mRNA, T alpha 1, during nerve regeneration in adult rats.

The mRNAs for 2 isotypes of alpha-tubulin, termed T alpha 1 and T26, are differentially regulated in the developing rat nervous system. T alpha 1 alpha-tubulin mRNA is expressed at high levels when neurons extend processes whereas T26 mRNA is expressed constitutively (Miller et al., 1987b). We have examined the expression of these 2 alpha-tubulin mRNAs in regenerating facial and sciatic motor neurons of the rat using Northern blot and in situ hybridization analyses. T alpha 1 alpha-tubulin mRNA is rapidly induced in axotomized motor neurons of the facial nerve: increased levels of mRNA are detectable 4 hr after a lesion is made 1.5 cm distal to the neuronal cell bodies. T alpha 1 mRNA levels are highest from 3-7 d postcrush and decline slowly to control levels following functional reinnervation of facial muscles. In contrast, T26 mRNA levels remain constant throughout the regeneration process. Total alpha-tubulin mRNA levels do not change until 1 d postaxotomy; otherwise the changes in expression are similar to T alpha 1 mRNA, although the relative increase is not as great. Enhanced T alpha 1 alpha-tubulin mRNA expression also occurs in motor neurons of crushed or tied sciatic nerve. Ligature or crush of the sciatic nerve leads to approximately the same peak in the expression of T alpha 1 mRNA at 7-15 d postaxotomy. Following the facial nerve transection, under conditions in which reinnervation is prevented, T alpha 1 alpha-tubulin mRNA levels remain elevated significantly longer than when the nerve is crushed. Taken together, the data indicate that T alpha 1 alpha-tubulin mRNA is rapidly induced following neuronal axotomy, remains elevated during the period of axonal regrowth, and is subsequently down-regulated at the approximate time of target contact. These results are reminiscent of changes in T alpha 1 mRNA that occur during neuronal development. This growth-associated pattern of T alpha 1 gene expression can be modified by inhibiting appropriate regeneration of the damaged nerve.

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PubMed1988

Retrograde changes in transglutaminase activity after peripheral nerve injuries.

It has been previously demonstrated that transglutaminase activity in rat superior cervical ganglion is rapidly and transiently increased minutes after nerve injury. The present series of experiments sought to determine: (1) whether or not similar changes are expressed by other peripheral neuronal systems, and (2) if injury-induced changes in the enzyme activity can be detected along the injured nerve, and if so do they occur in axons or in non-neuronal cells. In the nodose ganglion transglutaminase activity increased (approximately 40%) 48 h after the vagus nerve was crushed 25 mm from the ganglion. In the vagus nerve the activity was transiently increased (approximately 100%) within 1 h, followed by a second increase (approximately 140%) after 3 h. This occurred only in the proximal nerve stump close to the injury site and not in the section of nerve closer to the ganglion. Comparable enzyme activity was found in unoperated vagus nerve and in distal stumps of previously ligated vagus nerves. In dorsal root ganglia no changes were found for up to 24 h after the sciatic nerve was crushed 40 mm from the ganglion. In the facial nucleus a transient increase was observed after the facial nerve was crushed about 14 mm distally with a peak (approximately 300) at 3 days and a decline within 14 days. A second lesion of the facial nerve made 12 days following a conditioning lesion led to a rebound of enzyme activity in the facial nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1988

Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy.

Transection of the facial nerve leads to a glial response within its central nucleus of origin. Concomitant with a proliferation of satellite microglial cells an astrocytic reaction is also seen. In the present study light and electron microscopic autoradiography were performed in order to clarify whether only microglial cells undergo mitosis following facial nerve axotomy or if astrocytes also divide. Our results provide the first electron microscopical autoradiographic evidence for the labelling of endogenous microglial cells. We suggest that microglial cells are the only proliferating element during this process in the rat facial nucleus.

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PubMed1988

Increased glial fibrillary acidic protein synthesis in astrocytes during retrograde reaction of the rat facial nucleus.

Glial fibrillary acidic protein (GFAP) increases in astrocytes following axotomy of facial motoneurons. In the present study we quantified GFAP synthesis both in regenerating facial nuclei after nerve crush and in nonregenerating facial nuclei after nerve resection. An increase in GFAP synthesis during regeneration occurs as early as 24 h after the axotomy. Thus, the increase in the astrocytic GFAP synthesis seems to be the earliest glial response to retrograde changes in facial motoneurons.

Animals↗

PubMed1988

Axonal transport of 16S acetylcholinesterase is increased in regenerating peripheral nerve in guinea-pig, but not in rat.

The axonal transport of the molecular forms of acetylcholinesterase was investigated in regenerating facial nerves of guinea-pig and rat. Four forms were separated by velocity sedimentation corresponding to 16S (A12), 10S (G4), 6S (G2) and 4S (G1) acetylcholinesterase. They displayed species-specific changes, which are in good accordance with those previously found in the neuronal perikarya. In the rat, axonal transport decreased for all forms. In the guinea-pig, however, the molecular forms showed differential changes. Whereas after transection, the nerve content of 10S acetylcholinesterase decreased, 16S activity was considerably increased. Anterograde transport of 16S acetylcholinesterase was found to be enhanced, whilst transport of the 10S from decreased. The two lighter forms showed only minor changes. Similar results were obtained for the guinea-pig sciatic nerve. Changes in the localization of acetylcholinesterase activity were investigated by electron microscopical cytochemistry. In the normal facial nerve of both species, activity was located intra-axonally in tubular membraneous structures and on the outer surface of the axonal membrane. In the regenerating facial nerve of the rat, intra-axonal as well as axolemmal activity decreased. Axonal sprouts at the end of the proximal nerve stump showed no activity. In the guinea-pig, however, activity of the axonal membrane increased. This was especially prominent on the surface of axonal sprouts. Strong activity was found also in the extracellular space between the sprouting axons and in the endoneurial space filled by collagen fibres. Biochemical analysis of this region revealed that the histochemical activity was mainly due to the A12 form. Thus it was concluded that, in the guinea-pig, axonal sprouts represent a target for axonally transported A12 acetylcholinesterase, which may also be secreted to extracellular sites.

Acetylcholinesterase↗

PubMed1988

Changes in cytoskeletal proteins in the rat facial nucleus following axotomy.

Changes in L-35S-methionine incorporation into cytoskeletal proteins of the facial nucleus of the rat were studied at various times after unilateral crush or resection of the facial nerve by using 2-dimensional gel electrophoresis and fluorography. We found an increase in labeling of actin and tubulin and a decrease in the 68 kDa and 150 kDa neurofilament polypeptides (200 kDa was not studied). The increase in actin and decrease in neurofilament polypeptide labeling was already significant by 24 hr after nerve resection. These changes were more pronounced after nerve resection than after nerve crush on day 7; actin labeling increased to 270%, and tubulin to 205% of contralateral normal nuclei after resection, whereas both proteins increased to only 165% after crush. Neurofilament labeling decreased to 28% of the contralateral side after resection and to 50% after crush. Immunocytochemistry with a monoclonal antibody to the 150 kDa neurofilament component revealed decreased immunoreactivity in the axotomized facial axons at the inner facial genu, 1 cm proximal to the crush. In contrast, neurofilament immunoreactivity was not decreased in the axotomized perikarya of the facial motoneurons. All changes returned to normal 3 weeks after crush. When axonal regeneration was impeded by nerve resection, incorporation into tubulin remained elevated and into neurofilament proteins remained depressed. Actin returned to normal after either nerve resection or crush. We conclude that the synthesis of tubulin and neurofilament proteins following axotomy is regulated by successful axonal regeneration and/or target contact. Actin synthesis seems to be regulated independently of target-derived factors.

Actins↗