Systemic application of therapeutics to the CNS tissue often results in subtherapeutic drug level... more Systemic application of therapeutics to the CNS tissue often results in subtherapeutic drug levels, because of restricted and selective penetration through the blood-brain barrier (BBB). Here, we give a detailed description of a standardized technique for intrathecal drug delivery in rodents, analogous to the technique used in humans. The intrathecal drug delivery method bypasses the BBB and thereby offers key advantages over oral or intravenous administration, such as maximized local drug doses with minimal systemic side effects. We describe how to deliver antibodies or drugs over several days or weeks from a s.c. minipump and a fine catheter inserted into the subdural space over the spinal cord (20 min operative time) or into the cisterna magna (10 min operative time). Drug levels can be sampled by quick and minimally invasive cerebrospinal fluid (CSF) collection from the cisterna magna (5 min procedure time). These techniques enable targeted application of any compound to the CNS...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2000
The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but th... more The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but their function exerted on intact neurons is still unclear. In the adult CNS these proteins are thought to regulate axon growth processes to confine plasticity within restricted regions and to prevent the formation of aberrant connections. We have recently shown that application of neutralizing IN-1 antibody Fab fragment against NI-35/250 proteins to the adult cerebellum induces the expression of injury/growth-associated markers in intact Purkinje cells. Here, we asked whether these cellular modifications are accompanied by growth phenomena of Purkinje neurites. A single intraparenchymal application of IN-1 Fab fragment to the adult cerebellum induces a profuse sprouting of Purkinje axons along their intracortical course. The newly formed processes spread to cover most of the granular layer depth. A significant axon outgrowth is evident 2 d after injection; it tends to increase at 5 and 7 d...
Proceedings of the National Academy of Sciences, 2011
Whereas the role of NogoA in limiting axonal fiber growth and regeneration following an injury of... more Whereas the role of NogoA in limiting axonal fiber growth and regeneration following an injury of the mammalian central nervous system (CNS) is well known, its physiological functions in the mature uninjured CNS are less well characterized. NogoA is mainly expressed by oligodendrocytes, but also by subpopulations of neurons, in particular in plastic regions of the CNS, e.g., in the hippocampus where it is found at synaptic sites. We analyzed synaptic transmission as well as long-term synaptic plasticity (long-term potentiation, LTP) in the presence of function blocking anti-NogoA or anti-Nogo receptor (NgR) antibodies and in NogoA KO mice. Whereas baseline synaptic transmission, short-term plasticity and long-term depression were not affected by either approach, long-term potentiation was significantly increased following NogoA or NgR1 neutralization. Synaptic potentiation thus seems to be restricted by NogoA. Surprisingly, synaptic weakening was not affected by interfering with NogoA signaling. Mechanistically of interest is the observation that by blockade of the GABA(A) receptors normal synaptic strengthening reoccurred in the absence of NogoA signaling. The present results show a unique role of NogoA expressed in the adult hippocampus in restricting physiological synaptic plasticity on a very fast time scale. NogoA could thus serve as an important negative regulator of functional and structural plasticity in mature neuronal networks.
Initially discovered as a potent neurite outgrowth inhibitor in the central nervous system (CNS),... more Initially discovered as a potent neurite outgrowth inhibitor in the central nervous system (CNS), Nogo-A has emerged as a multifunctional protein. Involvement of this protein has been demonstrated in numerous developmental processes, ranging from cell migration, axon guidance and fasciculation, dendritic branching and CNS plasticity to oligodendrocyte differentiation and myelination. Although initially necessary and beneficial for shaping and later maintaining CNS structure and functionality, the growth restricting properties of Nogo-A can have negative effects on nervous system injury or disease. Hence, correlating with its various neurobiological roles, Nogo-A was implicated in a range of CNS disturbances, including trauma such as spinal cord injury or stroke, neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis or multiple sclerosis, or in schizophrenia. In this review, we summarize the current state of knowledge for Nogo-A's involvement in these nervous system diseases and perturbations and discuss the possible underlying mechanisms. Furthermore, we provide a comprehensive overview on molecular signaling pathways as well as structural properties identified for Nogo-A and point to open questions in the field.
Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in t... more Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediate...
The use of the visual system played a major role in the elucidation of molecular mechanisms contr... more The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological ...
Anatomically incomplete spinal cord injuries are often followed by considerable functional recove... more Anatomically incomplete spinal cord injuries are often followed by considerable functional recovery in patients and animal models, largely because of processes of neuronal plasticity. In contrast to the corticospinal system, where sprouting of fibers and rearrangements of circuits in response to lesions have been well studied, structural adaptations within descending brainstem pathways and intraspinal networks are poorly investigated, despite the recognized physiological significance of these systems across species. In the present study, spontaneous neuroanatomical plasticity of severed bulbospinal systems and propriospinal neurons was investigated following unilateral C4 spinal hemisection in adult rats. Injection of retrograde tracer into the ipsilesional segments C3-C4 revealed a specific increase in the projection from the ipsilesional gigantocellular reticular nucleus in response to the injury. Substantial regenerative fiber sprouting of reticulospinal axons above the injury site was demonstrated by anterograde tracing. Regrowing reticulospinal fibers exhibited excitatory, vGLUT2-positive varicosities, indicating their synaptic integration into spinal networks. Reticulospinal fibers formed close appositions onto descending, double-midline crossing C3-C4 propriospinal neurons, which crossed the lesion site in the intact half of the spinal cord and recrossed to the denervated cervical hemicord below the injury. These propriospinal projections around the lesion were significantly enhanced after injury. Our results suggest that severed reticulospinal fibers, which are part of the phylogenetically oldest motor command system, spontaneously arborize and form contacts onto a plastic propriospinal relay, thereby bypassing the lesion. These rearrangements were accompanied by substantial locomotor recovery, implying a potential physiological relevance of the detour in restoration of motor function after spinal injury.
Certain cell, molecular, and bioengineering strategies for repairing the injured spinal cord are ... more Certain cell, molecular, and bioengineering strategies for repairing the injured spinal cord are showing encouraging results (either alone or in combination) in animal models, with limited recovery of mobility being reported in some cases.
News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 1998
Neurite growth and regeneration in the adult central nervous system (CNS) is extremely limited. A... more Neurite growth and regeneration in the adult central nervous system (CNS) is extremely limited. An important factor contributing to these restrictions is specific growth inhibitory proteins associated with oligodendrocytes and CNS myelin. A major inhibitory factor is the antigen of a monoclonal antibody; the application of this neutralizing antibody to spinal cord- or brain-lesioned adult rats induces long-distance regeneration of lesioned axons, as well as a specific increase in sprouting and rewiring of the cortical output system to the brain stem and the spinal cord. These anatomic changes are paralleled by important functional recoveries of locomotion and precision movements.
The lack of axonal regeneration in the adult central nervous system is in part attributable to th... more The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth-associated proteins Sprr1a and Gap-43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein-expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down-regulation may locally contribute to improve axonal growth at the injury site. In summary, our three-dimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.
Ischemic insults give rise to severe visual deficits after blood vessel occlusion. In this study ... more Ischemic insults give rise to severe visual deficits after blood vessel occlusion. In this study we investigated the effects of retinal stroke on the direction-selective circuit of the inner retina in a new adult mouse model. The inner retinal blood flow was interrupted for 60 minutes by ligating the ophthalmic arteries and veins in the optic nerve sheath. The optokinetic response (OKR) was measured to assess ischemia/reperfusion-mediated functional deficits and structural changes were studied by immunohistochemistry. Ischemia/reperfusion induced reactive gliosis and degeneration of the inner retina. The OKR was almost completely abolished from 7 days after reperfusion, whereas approximately 40% of retinal ganglion cells were still alive. Ischemia led to severe degeneration of the processes of starburst amacrine cells (SAC), which cell bodies are in the ganglion cell layer (ON SACs), and to a lesser extent of the dendrites of SACs, which cell bodies are in the inner nuclear layer (OFF SACs). In addition, the elimination of retinal ganglion cells, direction-selective ganglion cells, and ON SACs was much greater at 10 days and 21 days than that of OFF SACs. After reperfusion, P-Stat3 was transiently activated in ganglion cells, whereas P-Erk1/2 signal was specifically detected in Müller glia. These results show a pronounced destruction of the ON direction-selective circuit in the inner retina that correlated with the irreversible loss of the OKR early after ischemia/reperfusion.
Systemic application of therapeutics to the CNS tissue often results in subtherapeutic drug level... more Systemic application of therapeutics to the CNS tissue often results in subtherapeutic drug levels, because of restricted and selective penetration through the blood-brain barrier (BBB). Here, we give a detailed description of a standardized technique for intrathecal drug delivery in rodents, analogous to the technique used in humans. The intrathecal drug delivery method bypasses the BBB and thereby offers key advantages over oral or intravenous administration, such as maximized local drug doses with minimal systemic side effects. We describe how to deliver antibodies or drugs over several days or weeks from a s.c. minipump and a fine catheter inserted into the subdural space over the spinal cord (20 min operative time) or into the cisterna magna (10 min operative time). Drug levels can be sampled by quick and minimally invasive cerebrospinal fluid (CSF) collection from the cisterna magna (5 min procedure time). These techniques enable targeted application of any compound to the CNS...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2000
The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but th... more The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but their function exerted on intact neurons is still unclear. In the adult CNS these proteins are thought to regulate axon growth processes to confine plasticity within restricted regions and to prevent the formation of aberrant connections. We have recently shown that application of neutralizing IN-1 antibody Fab fragment against NI-35/250 proteins to the adult cerebellum induces the expression of injury/growth-associated markers in intact Purkinje cells. Here, we asked whether these cellular modifications are accompanied by growth phenomena of Purkinje neurites. A single intraparenchymal application of IN-1 Fab fragment to the adult cerebellum induces a profuse sprouting of Purkinje axons along their intracortical course. The newly formed processes spread to cover most of the granular layer depth. A significant axon outgrowth is evident 2 d after injection; it tends to increase at 5 and 7 d...
Proceedings of the National Academy of Sciences, 2011
Whereas the role of NogoA in limiting axonal fiber growth and regeneration following an injury of... more Whereas the role of NogoA in limiting axonal fiber growth and regeneration following an injury of the mammalian central nervous system (CNS) is well known, its physiological functions in the mature uninjured CNS are less well characterized. NogoA is mainly expressed by oligodendrocytes, but also by subpopulations of neurons, in particular in plastic regions of the CNS, e.g., in the hippocampus where it is found at synaptic sites. We analyzed synaptic transmission as well as long-term synaptic plasticity (long-term potentiation, LTP) in the presence of function blocking anti-NogoA or anti-Nogo receptor (NgR) antibodies and in NogoA KO mice. Whereas baseline synaptic transmission, short-term plasticity and long-term depression were not affected by either approach, long-term potentiation was significantly increased following NogoA or NgR1 neutralization. Synaptic potentiation thus seems to be restricted by NogoA. Surprisingly, synaptic weakening was not affected by interfering with NogoA signaling. Mechanistically of interest is the observation that by blockade of the GABA(A) receptors normal synaptic strengthening reoccurred in the absence of NogoA signaling. The present results show a unique role of NogoA expressed in the adult hippocampus in restricting physiological synaptic plasticity on a very fast time scale. NogoA could thus serve as an important negative regulator of functional and structural plasticity in mature neuronal networks.
Initially discovered as a potent neurite outgrowth inhibitor in the central nervous system (CNS),... more Initially discovered as a potent neurite outgrowth inhibitor in the central nervous system (CNS), Nogo-A has emerged as a multifunctional protein. Involvement of this protein has been demonstrated in numerous developmental processes, ranging from cell migration, axon guidance and fasciculation, dendritic branching and CNS plasticity to oligodendrocyte differentiation and myelination. Although initially necessary and beneficial for shaping and later maintaining CNS structure and functionality, the growth restricting properties of Nogo-A can have negative effects on nervous system injury or disease. Hence, correlating with its various neurobiological roles, Nogo-A was implicated in a range of CNS disturbances, including trauma such as spinal cord injury or stroke, neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis or multiple sclerosis, or in schizophrenia. In this review, we summarize the current state of knowledge for Nogo-A's involvement in these nervous system diseases and perturbations and discuss the possible underlying mechanisms. Furthermore, we provide a comprehensive overview on molecular signaling pathways as well as structural properties identified for Nogo-A and point to open questions in the field.
Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in t... more Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediate...
The use of the visual system played a major role in the elucidation of molecular mechanisms contr... more The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological ...
Anatomically incomplete spinal cord injuries are often followed by considerable functional recove... more Anatomically incomplete spinal cord injuries are often followed by considerable functional recovery in patients and animal models, largely because of processes of neuronal plasticity. In contrast to the corticospinal system, where sprouting of fibers and rearrangements of circuits in response to lesions have been well studied, structural adaptations within descending brainstem pathways and intraspinal networks are poorly investigated, despite the recognized physiological significance of these systems across species. In the present study, spontaneous neuroanatomical plasticity of severed bulbospinal systems and propriospinal neurons was investigated following unilateral C4 spinal hemisection in adult rats. Injection of retrograde tracer into the ipsilesional segments C3-C4 revealed a specific increase in the projection from the ipsilesional gigantocellular reticular nucleus in response to the injury. Substantial regenerative fiber sprouting of reticulospinal axons above the injury site was demonstrated by anterograde tracing. Regrowing reticulospinal fibers exhibited excitatory, vGLUT2-positive varicosities, indicating their synaptic integration into spinal networks. Reticulospinal fibers formed close appositions onto descending, double-midline crossing C3-C4 propriospinal neurons, which crossed the lesion site in the intact half of the spinal cord and recrossed to the denervated cervical hemicord below the injury. These propriospinal projections around the lesion were significantly enhanced after injury. Our results suggest that severed reticulospinal fibers, which are part of the phylogenetically oldest motor command system, spontaneously arborize and form contacts onto a plastic propriospinal relay, thereby bypassing the lesion. These rearrangements were accompanied by substantial locomotor recovery, implying a potential physiological relevance of the detour in restoration of motor function after spinal injury.
Certain cell, molecular, and bioengineering strategies for repairing the injured spinal cord are ... more Certain cell, molecular, and bioengineering strategies for repairing the injured spinal cord are showing encouraging results (either alone or in combination) in animal models, with limited recovery of mobility being reported in some cases.
News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 1998
Neurite growth and regeneration in the adult central nervous system (CNS) is extremely limited. A... more Neurite growth and regeneration in the adult central nervous system (CNS) is extremely limited. An important factor contributing to these restrictions is specific growth inhibitory proteins associated with oligodendrocytes and CNS myelin. A major inhibitory factor is the antigen of a monoclonal antibody; the application of this neutralizing antibody to spinal cord- or brain-lesioned adult rats induces long-distance regeneration of lesioned axons, as well as a specific increase in sprouting and rewiring of the cortical output system to the brain stem and the spinal cord. These anatomic changes are paralleled by important functional recoveries of locomotion and precision movements.
The lack of axonal regeneration in the adult central nervous system is in part attributable to th... more The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth-associated proteins Sprr1a and Gap-43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein-expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down-regulation may locally contribute to improve axonal growth at the injury site. In summary, our three-dimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.
Ischemic insults give rise to severe visual deficits after blood vessel occlusion. In this study ... more Ischemic insults give rise to severe visual deficits after blood vessel occlusion. In this study we investigated the effects of retinal stroke on the direction-selective circuit of the inner retina in a new adult mouse model. The inner retinal blood flow was interrupted for 60 minutes by ligating the ophthalmic arteries and veins in the optic nerve sheath. The optokinetic response (OKR) was measured to assess ischemia/reperfusion-mediated functional deficits and structural changes were studied by immunohistochemistry. Ischemia/reperfusion induced reactive gliosis and degeneration of the inner retina. The OKR was almost completely abolished from 7 days after reperfusion, whereas approximately 40% of retinal ganglion cells were still alive. Ischemia led to severe degeneration of the processes of starburst amacrine cells (SAC), which cell bodies are in the ganglion cell layer (ON SACs), and to a lesser extent of the dendrites of SACs, which cell bodies are in the inner nuclear layer (OFF SACs). In addition, the elimination of retinal ganglion cells, direction-selective ganglion cells, and ON SACs was much greater at 10 days and 21 days than that of OFF SACs. After reperfusion, P-Stat3 was transiently activated in ganglion cells, whereas P-Erk1/2 signal was specifically detected in Müller glia. These results show a pronounced destruction of the ON direction-selective circuit in the inner retina that correlated with the irreversible loss of the OKR early after ischemia/reperfusion.
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Papers by Martin Schwab