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The inhibition of neuronal apoptosis in acute traumatic and ischemic injuries as well as in long term neurodegenerative disorders like spinal muscular atrophy and possibly Alzheimer's disease is a fundamental requirement for a therapeutic strategy. In this study we used an established in vivo model system of induction of neuronal apoptosis in the CNS to evaluate the properties of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit secondary cell death after axonal lesions. We used adenoviral vectors to transduce retinal ganglion cells after axotomy of the optic nerve of adult rats. Vector application was performed at the optic nerve stump so that only the lesioned retinal neurons could be transduced. We found XIAP to be as effective as the viral broad spectrum caspase inhibitor protein p35. These findings suggest that axotomized RGCs degenerate through class II caspase activity and furthermore offer the possibility of using mammalian XIAP protein to inhibit neuronal apoptosis as a basis for a regenerative therapy in the CNS.

Programmed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programmed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programmed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.

Rat optic nerve (ON) transection leads to mainly apoptotic cell death of about 85% of the retinal ganglion cell (RGC) population within 14 days after lesion. In the present study, we tested the effect of adenovirally delivered CNTF (Ad-CNTF) on survival and regeneration of axotomized adult RGCs in vivo. Single intravitreal Ad-CNTF injection led to stable CNTF mRNA and protein expression for at least 18 days and significantly enhanced RGC survival by 155% when compared to control animals 14 days after ON transection. ON stump application of Ad-CNTF also resulted in an increased number of surviving RGCs. Ad-CNTF injection led to better preservation of intraretinal RGC axons but did not support regeneration of axotomized RGCs into a peripheral nerve graft. Thus, adenovirus-mediated neurotrophic factor supply is a suitable approach for reducing axotomy-induced RGC death in vivo and may constitute a relevant strategy for clinical treatment of traumatic brain injury., (Copyright 2000 Academic Press.)

Recently we have shown that the majority of retinal ganglion cells (RGCs) dies via activation of caspase-3 after transection of the optic nerve (ON) in the adult rat. In the present study we investigated whether insulin-like growth factor-I (IGF-I), an important factor in retinal development, prevents secondary death of RGCs after axotomy. Moreover, we studied potential intracellular mechanisms of IGF-mediated neuroprotection in more detail. Our results indicate that intraocular application of IGF-I protects RGCs from death after ON transection in a dose-dependent manner. We show reduced caspase-3 activity as one possible neuroprotective mechanism of IGF-I treatment in vivo. Caspase-3 mRNA expression remained unchanged. Because caspase inhibition can be mediated by Akt in vitro, we examined phosphorylation of Akt after axotomy and under IGF treatment. Western blot analysis revealed decreased Akt phosphorylation after axotomy without treatment and an increased phosphorylation of Akt under treatment with IGF-I. This strong increase could be reduced by simultaneous injection of wortmannin (WM), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K). To prove the pathway suggested by these experiments as relevant for the in vivo situation, we assessed the number of RGCs 14 d after ON transection under a combined treatment strategy of IGF-I and WM. As expected, WM significantly reduced the neuroprotective effects of IGF-I. In summary, we show for the first time in vivo that IGF is neuroprotective via PI3-K-dependent Akt phosphorylation and by inhibition of caspase-3.

The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: Exogenously applied BDNF has been shown to rescue rat retinal ganglion cells (RGCs) from axotomy-induced apoptotic death, presumably via activation of its high affinity receptor TrkB. Since both TrkB and BDNF are endogenously expressed in RGCs, auto- or para-crine neurotrophic loops in the retina may be involved. In the present study, we investigated whether expression levels of BDNF, TrkA, TrkB, TrkC and p75 protein in RGCs are specifically regulated following axonal lesion and during regeneration of optic fibres in the adult rat., Methods: By double labelling retinal cryosections with Fluorogold and respective antibodies we determined the percentage of RGCs expressing the above-mentioned markers. In addition, mRNA levels of BDNF and TrkB were measured using quantitative RT-PCR., Results: Compared to controls the number of BDNF-positive RGCs increased twofold 2 days after axotomy and the percentage of RGCs expressing TrkB was elevated by 50 %. Correspondingly, mRNA levels of BDNF increased about twofold 2 days after axotomy. During regen-eration, the percentage of BDNF-immunoreactive RGCs was further elevated compared to axotomy alone. The number of TrkA-positive RGCs doubled after axotomy, whereas no significant change in TrkC expression was observed. P75 expression was not detected in adult rat RGCs., Conclusion: Our results suggest that intrinsic rescue mechanisms may contribute to short term neuronal survival and axonal regeneration of RGCs after axonal lesions.

Purpose: Exogenously applied BDNF has been shown to rescue rat retinal ganglion cells (RGCs) from axotomy-induced apoptotic death, presumably via activation of its high affinity receptor TrkB. Since both TrkB and BDNF are endogenously expressed in RGCs, auto- or para-crine neurotrophic loops in the retina may be involved. In the present study, we investigated whether expression levels of BDNF, TrkA, TrkB, TrkC and p75 protein in RGCs are specifically regulated following axonal lesion and during regeneration of optic fibres in the adult rat. Methods: By double labelling retinal cryosections with Fluorogold and respective antibodies we determined the percentage of RGCs expressing the above-mentioned markers. In addition, mRNA levels of BDNF and TrkB were measured using quantitative RT-PCR. Results: Compared to controls the number of BDNF-positive RGCs increased twofold 2 days after axotomy and the percentage of RGCs expressing TrkB was elevated by 50 %. Correspondingly, mRNA levels of BDNF increased about twofold 2 days after axotomy. During regen-eration, the percentage of BDNF-immunoreactive RGCs was further elevated compared to axotomy alone. The number of TrkA-positive RGCs doubled after axotomy, whereas no significant change in TrkC expression was observed. P75 expression was not detected in adult rat RGCs. Conclusion: Our results suggest that intrinsic rescue mechanisms may contribute to short term neuronal survival and axonal regeneration of RGCs after axonal lesions.

Neuronal damage in the central nervous system leads to primary cell death, induced directly by the trauma, and delayed secondary death of neurons, the latter depending on environmental changes, lack of metabolic and trophic supply, and altered gene transcription. While primary death of neurons occurring within a short time after trauma is not a realistic target for therapy, secondary cell death might be prevented by new neuroprotective strategies. Although there are increasing data concerning cell rescue after ischemic and traumatic brain injury through the last decade, the mechanisms that underlie secondary death of neurons following lesion are still incompletely understood and are now the subject of a more detailed investigation. In this review, we want to give an overview on what is known about the molecular mechanisms of delayed ischemic and traumatic neuronal death in vivo and about promising neuroprotective treatment strategies that might be of future clinical relevance or have already entered clinical trials.

Glioblastoma multiforme is a lethal neoplasm refractory to radiochemotherapy. Although glioma cells undergo apoptosis when exposed to the death ligand, CD95 (Fas/APO-1) ligand, the therapeutic use of CD95L is considered impossible because of lethal side effects. Here, we report that the locoregional application of Apo2 ligand (Apo2L) exerts strong antitumor activity on preestablished intracranially growing human U87MG glioma xenografts in athymic mice. Two repetitive intratumoral injections of 2 microg Apo2L resulted in long-term survival of mice (>100 days), whereas the median survival of mock-treated mice was 36 days. The assessment of tumor volumes at 21 and 35 days after inoculation showed complete eradication of glioma xenografts in Apo2L-treated mice. Histology and TUNEL assay confirmed the induction of apoptosis by Apo2L in glioma cells in vivo. Importantly, the intracerebral injection of Apo2L does not result in acute or delayed neurotoxicity. We propose that a phase 1 trial of intralesional Apo2L therapy for human glioblastoma multiforme is warranted., (Copyright 1999 Academic Press.)

Although neurotrophins are best known for their trophic functions, growing evidence suggests that neurotrophins can also be neurotoxic, for instance by enhancing excitotoxic insults. We have shown recently that brain-derived neurotrophic factor (BDNF) limits its neuroprotective action on axotomized rat retinal ganglion cells (RGCs) by upregulating nitric oxide synthase (NOS) activity (Klöcker et al., 1998). The aim of the present study was to investigate this interaction of BDNF and NOS in the lesioned adult rat retina in more detail. We used NOS immunohistochemistry and NADPH-diaphorase (NADPH-d) reaction to characterize morphologically retinal NOS expression and activity. Using reverse transcription-PCR and Western blot analysis, we were able to identify the NOS isoforms being regulated. Six days after optic nerve lesion, we observed an increase in neuronal NOS (NOS-I) mRNA and protein expression in the inner retina. This did not lead to a marked increase in overall retinal NOS activity. Only RGC axons displayed strong de novo NADPH-d reactivity. In contrast, intraocular injection of BDNF resulted in a marked upregulation of NOS activity in NOS-I-immunoreactive structures, leaving the level of NOS-I expression unchanged. In addition, an induction of inducible NOS (NOS-II) was found after BDNF treatment. We identified microglial cells increasing in number and being activated by BDNF, which could serve as the cellular source of NOS-II. In summary, our data suggest that BDNF upregulates retinal NOS activity by both a post-translational regulation of NOS-I activity and an induction of NOS-II. These findings might be useful for developing pharmacological strategies to improve BDNF-mediated neuroprotection.

Axotomy of the rat optic nerve leads to apoptotic cell death of retinal ganglion cells (RGCs). We have used adenoviral vectors to transduce RGCs from the cut optic nerve stump, a paradigm in which only those neurons are transduced which are directly affected by the axonal lesion. Transgenes encoded by the vectors were p35 and CrmA, which are potent intracellular anti-apoptotic proteins. We found that p35, but not CrmA exerted significant rescue effects on RGCs 14 days after axotomy. Expression of the transgenes was driven by the murine CMV (MCMV) promoter. The respective mRNAs were detectable 7 days but not 14 days after transduction. Since surviving RGCs were present beyond the time-point of detectable transcription of the p35 transgene, we conclude that apoptosis has been efficiently inhibited. In addition, we observed that transduction with two control vectors without a transgene in E1 also resulted in a minor but significant RGC rescue, implicating neuroprotective effects due to adenoviral transduction itself. This system will be useful in dissecting the pathways leading to neuronal cell death after axonal lesions and in the evaluation of the important question whether the cellular suicide program can be reverted to survival by therapeutic gene delivery.

Decorin gene therapy for experimental malignant glioma is thought to involve antagonism of immunosuppression induced by glioma-derived transforming growth factor-beta (TGF-beta). TGF-beta is chemotactic for cells of the monocyte macrophage lineage but inhibits their functional activity in many in vitro paradigms. Here, we examined changes in the patterns of microglial infiltration of rat C6 gliomas expressing a decorin transgene. We find that the number of OX42/ED-1-positive microglial cells is reduced rather than enhanced in the presence of decorin. Decorin-expressing gliomas contain lower numbers of MHC class II antigen-expressing microglial cells whereas the relative frequency of MHC I immunoreactivity among microglial cells is increased. Interestingly, the reduction of TGF-beta levels in the tumors by decorin is associated with the de novo expression of inducible nitric oxide synthase (iNOS) in a minority of microglial cells. These data suggest that microglial cells do not participate in the regression of decorin-expressing rat C6 gliomas.

Following transection of the optic nerve (ON), retinal ganglion cells (RGCs) upregulate Bax protein expression and undergo apoptosis. The present study aimed at reducing Bax expression in order to test whether Bax plays a causative role in the induction of secondary RGC apoptosis. Following injection into the vitreous, fluoresceinated oligonucleotides transfected RGCs in vivo at the injection site in the temporal superior retina. Following ON lesion, and repeated injections of a partially phosphorothioated Bax antisense oligonucleotide, but not following injection of control oligonucleotides, expression of Bax protein was locally inhibited, and the number of surviving RGCs was increased in Bax antisense treated rats 8 days after axotomy. Our results indicate that Bax induction is a prerequisite for the execution of RGC apoptosis following ON axotomy. While the Bax antisense strategy offers an exciting perspective to inhibit secondary neuronal degeneration in vivo, both limited transfection efficacy, and the temporal restriction of this effect currently limit the use of this approach with respect to clinical applications for the treatment of neurodegeneration.

During early postnatal development, a widespread ipsilateral projection to the superior colliculus is secondarily restricted to a small topographically defined region by elimination of ipsilaterally projecting retinal ganglion cells. Brain-derived neurotrophic factor (BDNF) has been proposed as the target-derived neurotrophic factor for retinal ganglion cells in several studies. Here we investigated the long-term effects of excess BDNF in the retinal ganglion cell target on naturally occurring retinal ganglion cell (RGC) elimination and on the restriction of the ipsilateral projection. To this end, sustained overexpression of BDNF was achieved in the postnatal superior colliculus using an adenoviral vector. While the total number of retinal ganglion cells in the adenovirus-BDNF treated animals was unchanged, a much higher proportion of RGCs retained a projection to the ipsilateral superior colliculus. We conclude that an excess of target-derived BDNF does not reduce the net amount of naturally occurring cell death in the retino-collicular system, but prevents the negative selection of retinal ganglion cells making inappropriate topographic connections., (Copyright 1999 Academic Press.)

There is growing evidence that caspase inhibition exerts neuroprotective effects in various models of neuronal injury in vivo. However, whether caspase inhibition provides long-term neuroprotection is not known yet. In the present study, we therefore investigated the effects of prolonged caspase inhibition on the survival of adult rat retinal ganglion cells (RGCs) following optic nerve (ON) transection. Four weeks following ON transection the number of surviving RGCs in untreated animals declined to 11% of controls. Treatment for the initial 2 weeks with z-DEVD-cmk, an irreversible inhibitor of ced 3-like caspases, increased the number of surviving RGCs 4 weeks postlesion to 24%. Z-DEVD-cmk treatment over the entire experimental period of 4 weeks had no additional effect. Thus, we still found a neuroprotective effect of caspase inhibition on axotomized RGCs after extended survival time. However, in comparison to our recent observations 2 weeks after optic nerve transection, in which z-DEVD-cmk rescued 46% of RGCs (P. Kermer, N. Klöcker, M. Labes, and M. Bähr, 1998, J. Neurosci. 18(12), 4656-4662) the positive effect clearly decreased. In conclusion, our results indicate that the therapeutical approach presented here results in a significant delay of secondary death rather than providing a permanent and complete rescue of axotomized RGCs.

Recently, we have shown that inhibition of caspase-3-like caspases is the most effective treatment strategy to protect adult rat retinal ganglion cells from secondary death following optic nerve transection. In the present study, we localized active caspase-3 in axotomized retinal ganglion cells in vivo and demonstrated a co-localization of the active p20 fragment and TUNEL-staining in some of these cells. In line with this, we detected an enhanced cleavage and activity of caspase-3 protein in retinal tissue after lesion, while caspase-3 mRNA expression remained unchanged. These data suggest caspase-3 as an important mediator of secondary retinal ganglion cell death following axotomy in vivo.

Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.

Regeneration in the adult central nervous system (CNS) is thought to be hampered by the lesion-induced activation of astrocytes and meningeal cells and the consecutive formation of a glial scar. The substrate properties of reactive astrocytes differ significantly from their neonatal counterparts, which promote axon growth, but in spite of intensive studies the underlying molecular changes are still not fully understood. We have used two cell culture systems to compare the expression of certain surface molecules on neonatal astrocytes, reactive astrocytes and meningeal cells in vitro. Both, neonatal and reactive adult astrocytes exhibited a very similar expression of growth promoting molecules (NCAM, L1, laminin, fibronectin, DSD-1 proteoglycan) and potential inhibitors (tenascinC, chondroitin sulfate, and NG2-proteoglycan), whereas we could not detect the inhibitory keratan sulfate on either astrocyte population. In contrast, meningeal cells expressed considerable levels of keratan sulfate, but only minimal amounts of NCAM. In addition, the much higher expression of extracellular fibronectin around meningeal cells implies an excess formation of extracellular matrix (ECM). In coculture experiments, embryonic retinal ganglion cell (RGC) axons clearly avoided meningeal cells and instead preferred even reactive adult astrocytes. Our results suggest that the expression of inhibitory keratan sulfate proteoglycans together with a lack of NCAM and an excess production of ECM may be responsible for the non-permissiveness of meningeal cells. Compared to reactive astrocytes, meningeal cells are even worse a substrate for growing axons. None of the molecules investigated, however, seems to account for the different substrate properties of neonatal and reactive adult astrocytes.

Background: X linked dominant Charcot-Marie-Tooth disease (CMT1X) is an inherited motor and sensory neuropathy that mainly affects the peripheral nervous system. CMT1X is associated with mutations in the gap junction protein connexin 32 (Cx32). Cx32 is expressed in Schwann cells and oligodendrocytes in the peripheral (PNS) and in the (CNS) respectively., Methods: A CMT1X family with a Cx32 mutation was examined clinically and electrophysiologically to determine whether PNS, or CNS, or both pathways were affected., Results: In a CMT1X family a novel mutation (Asn205Ser) was found in the fourth transmembrane domain of Cx32. The patients showed typical clinical and electrophysiological abnormalities in the PNS, but in addition visual, acoustic, and motor pathways of the CNS were affected subclinically. This was indicated by pathological changes in visually evoked potentials (VEPs), brainstem auditory evoked potentials (BAEPs), and central motor evoked potentials (CMEPs)., Conclusions: These findings underscore the necessity of a careful analysis of CNS pathways in patients with CMT and Cx32 mutations. Abnormal electrophysiological findings in CNS pathway examinations should raise the suspicion of CMTX and a search for gene mutations towards Cx32 should be considered.

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

In the central nervous system (CNS) of mammals axonal regeneration is limited by two main factors: first, the low intrinsic regenerative potential of adult CNS neurons and second, inhibitory influences of the glial and extracellular environment. Myelin-associated inhibitors of neurite growth as well as some properties of so called "reactive astrocytes" contribute to the non-permissive of CNS tissue for axonal growth. In contrast, the peripheral nervous system (PNS) environment is supportive of regeneration because Schwann cells provide suitable substrates for regrowing axons. Purified PNS myelin, however, inhibits growth of PNS and CNS axons to a similar extent as does CNS myelin. The molecular basis of glial substrate properties has been studied intensively in the recent years and a large number of molecules have been recognized which might play a role in the regulation of axonal growth. Although the exact mechanisms are still not fully understood, accumulating data shed light on the complex interactions between neurons and glia that are required to establish, maintain, and regenerate axonal connections in the nervous system. In the following chapter we review the role of glial cells in the CNS and PNS during processes of de- and regeneration with respect to our own work.

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons., (Copyright 1998 Academic Press.)

To test the hypothesis whether a failure to express neurotrophins or a neurotrophin receptor might underlie the pathology observed in mutant mice with degeneration of regionally distinct subpopulations of neurons, the expression of BDNF, NT-3, TrkB, TrkC and synaptophysin mRNA was examined in the cerebellum of mutant lurcher (lc/+) and weaver (wv/+)/(wv/wv) mice. To identify the expression patterns of individual neurons, we used in situ hybridization with digoxigenin labeled ribonucleotide probes. RT-PCR of cerebellar mRNA for BDNF, NT-3, TrkB and TrkC (GAPDH as internal standard) was performed in parallel. Although especially in homozygous (wv/wv) weaver mice the normal anatomical order and number of the cerebellar neurons is grossly disturbed, residual Purkinje and granule neurons of both mutants displayed a normal expression pattern of the neurotrophins examined. Thus, the affected animals showed no significant signal decrease compared to healthy littermates or C3H mice. Our results suggest that the loss of specific neuron populations in the cerebellum of either mutant occurs via mechanisms either independent or downstream of the neurotrophins examined in this study., (Copyright 1998 Elsevier Science B.V.)

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain-derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival-promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad-BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad-BDNF administration and systemic application of the free radical scavenger, N-tert-butyl-(2-sulphophenyl)-nitrone (S-PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced RGC death. Further, as shown for S-PBN, therapeutic regimens that combine local virus-mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.

The majority of retinal ganglion cells (RGCs) degenerate and die after transection of the optic nerve (ON) in the adult rat. This secondary cell death can primarily be ascribed to apoptosis. Recent work strongly suggests a decisive role for a family of cysteine proteases, termed caspases, as mediators of neuronal apoptosis. In this study, we investigated whether activation of caspases contributes to delayed death of RGCs after axotomy. Intraocular application of various caspase inhibitors rescued up to 34% of RGCs that would otherwise have died 14 d after ON transection. Using a modified affinity-labeling technique, we detected a 17 kDa protease subunit upregulated after axotomy. Upregulation was prevented by caspase inhibitor treatment. The 17 kDa protein was identified as a CPP32-like protease by Western blot analysis and affinity labeling with biotinylated acetyl-Asp-Glu-Val-Asp-aldehyde, which specifically inhibits CPP32-like caspases. In vivo application of the irreversible caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone revealed CPP32-like proteases to be major mediators of caspase-induced apoptosis in axotomized RGCs, because this inhibitor showed an even higher neuroprotective potential than the irreversible wide-range inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. In summary, the data presented here provide further insight into the mechanisms of injury-induced neuronal apoptosis and could give rise to more effective therapeutic intervention strategies in CNS trauma and neurodegenerative diseases.

We examined whether regenerating axons from adult rat ganglion cells are able to recognize their appropriate target region in vitro. Explants from adult rat retina were cocultured with embryonic sagittal midbrain slices in Matrigel. The midbrain sections contained the superior colliculus, the main target for retinal ganglion cell axons in rats, and the inferior colliculus. We observed a statistically significant preference of both temporal and nasal retinal axons to grow toward their appropriate target region (anterior and posterior superior colliculus, respectively). No preferential growth of retinal ganglion cell axons was detected in controls, for which retinal explants were cultured on their own. When retinal ganglion cell axons were given a choice between superior colliculus and inferior colliculus, axons from nasal retina preferentially grew toward the posterior superior colliculus and avoided the inferior colliculus. In contrast, temporal axons in the same assay did not show preference for either of the colliculi. These findings suggest that regenerating axons from adult rat retina are able to recognize target-specific guidance cues released from embryonic midbrain targets in vitro.

Brain-derived neurotrophic factor (BDNF) partially promotes the survival of axotomized retinal ganglion cells (RGCs). In analogy with in vitro experiments (; ), we tested whether neuroprotection by BDNF is limited by adverse effects as a consequence of excessive free radical formation. First, we investigated whether BDNF and the free radical scavenger N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN) cooperate in protecting RGCs from axotomy-induced death. Although systemic S-PBN treatment alone did not influence RGC survival after axotomy, it potentiated the neuroprotective effects of BDNF significantly. Single BDNF treatment rescued 27% of the RGCs, which otherwise would have died 14 d after optic nerve transection, whereas a combined treatment of BDNF and S-PBN improved this rescue rate up to 68%. We then investigated whether the adverse effects of BDNF could be ascribed to activation of nitric oxide synthase (NOS). We found colocalization of NOS and the BDNF receptor TrkB in the retina. NADPH-diaphorase reactivity, a reliable marker for NOS in the rat retina, increased after chronic BDNF treatment in vivo. Systemic application of the NOS-inhibitor N-omega-nitro-L-arginine-methylester (L-NAME) potentiated the neuroprotective action of BDNF (55% rescue rate). We conclude that activation of NOS is a pathological consequence of BDNF application, which reduces its neuroprotective potential. The observation that this adverse effect can be antagonized by systemic application of free radical scavengers could be of relevance for clinical applications of neurotrophins in human neurodegenerative diseases.

Focal ischemia in the parietal cortex of the rat results in massive neuronal death in the infarct zone and penumbra between 12 hours and 6 days after photothrombosis. To examine a possible role of Bcl-2 family proteins in this process of cell death, we investigated their expression by immunoblot assays and immunocytochemistry, and correlated expression patterns with TUNEL as well as morphological signs indicative of apoptosis. In the center of the lesion Bax immunostaining was increased in many degenerating neurons between 4 hours and 3 days after the induction of photothrombosis. At all time points examined, Bcl-2 and Bcl-X protein levels were markedly reduced in injured neurons as compared to the unlesioned side. At the border of the ischemic lesion, two areas were distinguished: 1 - 2 days after induction of photothrombosis, pyknotic cells located immediately adjacent to the lesion core displayed nuclear Bcl-X and Bax immunoreactivity. In contrast, large, morphologically intact neurons located more towards the healthy brain parenchyma displayed an increase in cytoplasmic Bcl-2 and Bcl-X proteins. Double staining for each of the Bcl-2 family proteins and TUNEL revealed that DNA strand breaks and nuclear fragmentation seen in cells located in the lesion core were often associated with increased levels of Bax, but not with elevated Bcl-2 or Bcl-X protein levels, suggesting a role for Bax in the induction of apoptotic death in these cells. The upregulation of Bcl-2 and Bcl-X expression in surviving neurons close to the penumbra might reflect an active survival mechanism that protects these neurons from cell death following a sublethal insult.

The identification of neurotrophic factors ameliorating secondary neuronal death in the mammalian CNS has raised hopes for improved treatment strategies in neurodegenerative diseases, CNS trauma and ischemia. Glial cell-line derived neurotrophic factor (GDNF) has potent neuroprotective properties in both the CNS and PNS. We sought to investigate whether GDNF exerts survival promoting effects on axotomized retinal ganglion cells (RGCs) in the adult rat in vivo. Transection of the optic nerve induces delayed retrograde death of approximately 85% of RGCs within 14 days. Intraocular GDNF rescued 21% of the RGCs which would otherwise have died after axotomy (34% of the normal control population), thereby extending the group of neuronal populations responsive to GDNF.

Retinal ganglion cell (RGC) axons from the temporal retina project to the anterior superior colliculus (SC), whereas nasal retinal axons project to the posterior SC. The stripe assay has shown that temporal retinal axons avoid growing on membrane stripes from the posterior SC but nasal retinal axons show no growth preference. Several putative guidance molecules have been identified in target tissues for these axons during development in vertebrates. Regenerating axons from adult rat retinae also possess the capacity to recognize appropriate target cells and to form functional connections in vivo and in vitro. However, the expression of information for axonal guidance and target recognition in mammals and birds seems to be limited to the period when central nervous system projections develop during embryogenesis. Nevertheless, the deafferented adult rat SC re-establishes target information recognizable by embryonic rat retinae, although the re-expression of guidance factors after deafferentation does not seem to be a mere recapitulation of their normal developmental expression pattern. The roles of astrocytes and microglia in axon guidance are also discussed.

Axonal lesions to the optic nerve (ON) induce c-Jun expression in retinal ganglion cells (RGCs) of the rat in vivo. Detailed investigations using retrograde tracers, and double labeling studies for c-Jun and regeneration-associated factors, such as the growth-associated protein GAP-43, have suggested that this upregulation of c-Jun is part of a cell body response in an abortive attempt of affected RGCs to survive and regenerate an axon. On the other hand, prolonged expression of c-Jun protein has in several paradigms of neurodegeneration been linked to the induction of apoptotic cell death. In the present study, we examined the time course and subcellular localization of c-Jun protein by immunocytochemistry on retinal sections after optic nerve crush and carried out double labeling for c-Jun protein and DNA strand breaks to detect apoptosis on the same sections. Several days after ON lesion, a subpopulation of RGCs was detected in which c-Jun protein was not confined to the nucleus, but also located in the cytoplasm. In addition, RGCs were seen that displayed morphological signs of apoptosis, DNA strand breaks, and c-Jun immunoreactivity at the same time. Therefore, c-Jun expression is not confined to intact or regenerating ganglion cells, but also occurs in cells that are destined to die. Our results suggest that the decision to undergo either fate depends on additional signaling events that modulate the transcriptional actions of c-Jun.

Retrograde degeneration of retinal ganglion cells as a consequence of optic nerve lesion has been shown to fulfil the criteria of apoptosis. In the present study, we investigated the time course of ganglion cell apoptosis following intraorbital crushing of the optic nerve in adult rats using morphological criteria and applying a terminal transferase technique (TUNEL) for in situ detection of DNA strand breaks. In addition, we examined expression patterns of the anti-apoptotic proteins Bcl-2 and Bcl-X and the cell death-promoting protein Bax in retinae after crushing the optic nerve. Apoptotic nuclei were detected in the ganglion cell layer in the first 3 weeks after optic nerve crush, with a peak after 6 days. Bcl-2 and Bcl-X proteins were expressed in ganglion cells at low levels. Expression of Bcl-2 decreased further during the days following crush. Bcl-X expression was initially increased, followed by a decline over the following days. In contrast, Bax protein, which was expressed in most ganglion cells at moderate baseline levels, was sharply increased as early as 30 min after crush, reached peak levels after 3 days, and remained up-regulated for at least 1 week thereafter. Double labelling for Bax and TUNEL in retinal sections, however, did not reveal colocalization of the two signals in individual retinal ganglion cells, consistent with the idea that increases in Bax precede apoptosis after optic nerve lesion. Thus, retinal ganglion cell death might be prevented by ablation of Bax protein in these cells, or by up-regulation of Bax-antagonists such as Bcl-2 or Bcl-X.

The collaboration of more than 600 scientists from over 100 laboratories to sequence the Saccharomyces cerevisiae genome was the largest decentralised experiment in modern molecular biology and resulted in a unique data resource representing the first complete set of genes from a eukaryotic organism. 12 million bases were sequenced in a truly international effort involving European, US, Canadian and Japanese laboratories. While the yeast genome represents only a small fraction of the information in today's public sequence databases, the complete, ordered and non-redundant sequence provides an invaluable resource for the detailed analysis of cellular gene function and genome architecture. In terms of throughput, completeness and information content, yeast has always been the lead eukaryotic organism in genomics; it is still the largest genome to be completely sequenced.

Axon interruption elicits a complex neuronal response that leaves neurons poised precariously between death and regeneration. The signals underlying this dichotomy are not fully understood. The transcription factor c-Jun is one of the earliest and most consistent markers for neurons that respond to nerve-fiber transection, and its expression can be related to both degeneration and survival including target re-innervation. In vitro experiments have demonstrated that expression of c-Jun can kill neonatal neurons but, in the adult nervous system, c-Jun might also be involved in neuroprotection and regeneration. The functional characteristics of c-Jun offer a model for the ability of a single molecule to serve as pivotal regulator for death or survival, not only in the response of the cell body to axonal lesions but also following neurodegenerative disorders. In this model, the fate of neurons is determined by a novel transcriptional network comprising c-Jun, ATF-2 (activating transcription factor-2) and JNKs (c-Jun N-terminal kinases).

[GlyA21,ArgB31,ArgB32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of 125I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7 x 10(3) IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC50 (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [AspB10]insulin competed significantly more efficiently for IGF-I binding (IC50: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5 x 10(-7) M) for 16 h in the presence of [3H]thymidine. [Asp(B10)]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P < 0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10(-9) to 10(-8) M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC50 (half-effective concentration) values of 0.7 x 10(-10) and 1.9 x 10(-9) M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.

There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelin-deficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media.

Reflex sympathetic dystrophy (RSD) is a pain syndrome characterized by somatosensory and motor disturbances, as well as by autonomic and trophic changes. The term is used in a descriptive sense and does not imply specific mechanisms of pathogenesis. We report on a patient who fulfilled the clinical criteria of RSD and who also displayed increasing impairment of peripheral blood supply. Angiography revealed a circumscribed stenosis of the abdominal aorta adjacent to the bifurcation. Disturbances in peripheral circulation as a potential cause of RSD are discussed.

During development, retinal ganglion cell axons establish a topographically ordered projection from the retina to the superior colliculus (SC). The putative guidance activities for retinal axons that operate during embryonic development are not detectable in the normal adult SC. However, these cues reappear upon transection of the optic nerve of adult rats. In the present study, we used a modified version of the "stripe assay," in which membranes from either anterior or posterior SC alternated with laminin stripes. Temporal embryonic retinal axons consistently avoid membranes from embryonic posterior SC, but only rarely from adult deafferented SC. However, they are attracted to membranes from both embryonic and adult deafferented anterior SC. Nasal retinal axons only show a significant preference for membranes from posterior SC after deafferentation. When retinal axons were offered a choice to grow on membranes either from their embryonic or their deafferented target regions, they showed a preference for the deafferented SC. On carpets consisting of laminin and membranes from normal SC (not deafferented) or nontarget regions (inferior colliculus), temporal and nasal axons grow either in a random fashion or show preferences for the laminin stripes. Our modified version of the classic stripe assay shows specific growth preferences of embryonic retinal axons for membrane lanes from their appropriate embryonic or deafferented adult target regions. These findings suggest that the deafferentation of the SC in adult rats triggers the reexpression of specific guidance activities for retinal axons. Those "attractive" guidance cues appear to be differentially expressed in the developing and deafferented SC.

Freshly isolated and primary cultured adult rat cardiomyocytes were used to elucidate the mechanism of action of the new oral antidiabetic agent (+/-)-5-[4-(6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-yl-methoxy)benzyl]-2,4-thiazolidinedione (troglitazone) on the heart. Interaction with protein kinase C (PKC) and regulation of glucose transport were evaluated as possible sites of drug action. Acute treatment (30 min) of cardiomyocytes with troglitazone did not affect the phorbolester-induced membrane association of PKC-delta and PKC-epsilon, which represent the major isoforms present in these cells. However, under these conditions the phorbolester-mediated increase in membrane associated PKC activity was inhibited by 43 +/- 4% (n = 4) without affecting the basal distribution of PKC activity. In contrast to these findings, troglitazone had no acute effect on basal or insulin-stimulated glucose transport in freshly isolated cardiomyocytes; even after 120 min treatment an unaltered release of lactate was determine in the presence of the drug. After 20 h in serum-free culture troglitazone induced a dose-dependent increase in 2-deoxyglucose uptake reaching a 40-fold stimulation at 5 mumol/l. This was paralleled by a dose-dependent increase of glucose transporter-1 (GLUT1) and GLUT4 protein expression to 320 +/- 80 and 156 +/- 15% of control, respectively. In addition, chronic exposure to troglitazone increased the GLUT4 abundance in a plasma membrane fraction about twofold. These data show that troglitazone exerts multiple effects on cardiomyocytes involving inhibition of PKC and regulation of glucose transporter expression and distribution. We suggest that an increased glucose supply may be beneficial for the diabetic heart and that modulation of PKC-activity could be relevant for improving insulin action in muscle tissue.

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the "stripe assay", to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons.

So far, it has not been possible to treat many neurological conditions causally. However, in the past few years underlying genetic defects have been characterized for a substantial number of neurodegenerative disorders. Experimental methods have been developed that allow for efficient gene transfer into defined regions of the mammalian CNS. Such techniques can be applied to deliver genes into target cells of a recipient organism or to transfer genetically modified cells into defined regions of the CNS. Candidate genes for gene therapy are those encoding for neurotrophins and neurotransmitters for symptomatic therapy and, in the case of neurodegenerative disorders with localized gene defects, the wild-type allele as a causal treatment approach. In this review article, we describe some of the most widely used strategies for gene transfer to the CNS. We also report on the results obtained with animal models for human disease, and discuss both the chances and problems of gene therapy approaches in clinical medicine.

The present study examined the effect of insulin on Glut4 transcription in isolated ventricular cardiomyocytes. Upon exposure to the hormone (2 x 10(-7) M) the cellular Glut4 mRNA content increased to 224 +/- 46% of control within 3.5 hours. Nuclear run-on analysis indicated a parallel increase of transcription of the Glut4 gene to 208 +/- 49% of control. Direct incubation of cardiac nuclei with insulin resulted in a comparably significant increase of Glut4 transcription. Cross-linkage experiments with 125I-labelled insulin demonstrated the absence of soluble nuclear insulin receptors, which were readily detected in plasma and microsomal membranes. These findings suggest that expression of the cardiac Glut4 gene is subject to regulation by insulin at the transcriptional level, a process possibly involving nuclear association of the hormone.

In the CNS of mammals axonal regeneration is limited by inhibitory influences of the glial and extracellular environment. Myelin-associated inhibitors of neurite growth as well as some properties of so called "reactive astrocytes" which make the environment nonpermissive for axonal growth contribute to the inhibitory nature of the mammalian CNS. In contrast, the PNS is supportive of regeneration and Schwann cell surfaces and Schwann-cell-derived extracellular matrix provide suitable substrates for regenerating axons in vivo and in vitro. However, as the results presented here indicate, myelin derived from normal and axotomized sciatic nerves is a nonpermissive substrate for axonal regrowth. Addition of laminin to either CNS or PNS myelin or freezing of the myelin, however, allows reproducibly axonal growth. Membrane preparations from CNS or PNS tissue on the other hand allow axon outgrowth from retinal explants when adhesive substrates (e.g., polylysin) are available. This suggests that inhibitors of neurite growth are present in myelin from the CNS and PNS. Growth supportive substrates, which are present in large quantities after PNS but not after CNS injury, can overcome nonpermissive substrate properties.

Freshly isolated and primary cultured cardiac myocytes from adult rats were used to elucidate acute and chronic effects of the sulfonylurea drug glimepiride on basal and insulin-stimulated glucose transport and on expression of the transporter isoforms glucose transporter-1 (GLUT1) and GLUT4. A 30-min incubation with glimepiride (100 microM) was unable to modify the initial rates of 3-O-methylglucose transport in freshly isolated cardiocytes, both in the absence or presence of insulin (10(-7) M). Cells were then kept in serum-free culture for 20 h in the presence of glimepiride (10 microM) and a physiological insulin dose. Under these conditions, the sulfonylurea induced an increase in 2-deoxyglucose uptake to 186% of control. This drug effect was dose dependent and could also be demonstrated in the absence of insulin during the culture period. The acute action of insulin on glucose transport was additive to the effect of glimepiride, and the insulin responsiveness of glucose transport remained unaltered in sulfonylurea-treated cultures. Western blot analysis of crude membrane fractions obtained from cultured cardiocytes showed that glimepiride increased the expression of both GLUT1 and GLUT4 to 164% +/- 21% and 148% +/- 5% of control, respectively. We concluded that glimepiride increases cardiac glucose uptake by an insulin-independent pathway, probably involving an increased protein expression of GLUT1 and GLUT4. The increased expression of GLUT4 may have a therapeutic impact on the treatment of insulin-resistant states.

We have directly compared the abilities of astrocytes from newborn and adult rats to support or inhibit the growth of regenerating axons in vitro. Astrocytes prepared from newborn rats were able to promote retinal ganglion cell (RGC) axon growth from embryonic and adult rat and from adult fish retinal explants. Retinal axons from E16 rat retinae grew significantly faster on astrocytes from neonatal rats than those from E18 or adult rat retinae with growth rates comparable to RGC axons from adult fish retinae. RGC regeneration from adult rat retinae was almost completely inhibited on adult rat optic nerve astrocytes. Only axons from adult fish retinae were able to extend onto monolayers from these reactive astrocytes, although their growth rates were significantly reduced. We conclude that the failure of mammalian RGC axons to regrow within the lesioned optic nerve environment is, at least in part, due to nonpermissive aspects of adult "reactive" optic nerve astrocytes. However, the cell intrinsic growth potential of RGCs also appears to influence their ability to extend axons on cellular substrates.

We have recently shown that optic nerve transection is followed by a delayed appearance (7-14 days after lesion) of putative guidance activities for retinal axons in the superior colliculus (SC) of adult rats. In the present study we analysed with immunohistological methods whether there is a correlation between localized reactive changes in the cellular compartment of the SC after deafferentation and the reappearance of putative guidance activities. The optic nerve of adult female Sprague-Dawley rats was transected. After 1 day to 1 year the contralateral SC was sectioned sagitally and examined with antibodies directed against Vimentin and glial fibrillary acidic protein (GFAP) to label astrocytes, myelin basic protein (MBP) to detect oligodendrocytes, OX-42 to stain microglia and SMI-31 to determine degeneration of afferent retinal ganglion cell axons. These stainings were performed in order to detect differences in the cellular composition of macro- and microglial cells between the anterior and posterior regions of the SC. All cell populations we examined in the SC showed reactive changes after optic nerve transection. Microglia activation occurred as soon as 2 days after optic nerve transection in the anterior part of the SC. After 4 days, a homogeneous distribution of activated microglia was seen in the whole SC. GFAP upregulation occurred without a specific regional distribution. Only Vimentin-expressing astrocytes appeared with a delay of 1-2 weeks after deafferentation and were strongly restricted to the afferent optic tract and the very anterior region of the SC. In conclusion, a temporo-spatial correlation between the re-expression of guidance activities and changes in the cellular compartment of the SC exists in the delayed re-expression of Vimentin in astrocytes and their strong spatial restriction. Activated microglia might be involved in triggering regional changes in an anterior-posterior graded fashion in the deafferented SC.